<%BANNER%>

Detection and Molecular Characterization of Manatee Papillomavirus in Cutaneous Lesions of the Florida Manatee (Trichech...

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101123_AAAAAL INGEST_TIME 2010-11-23T06:31:37Z PACKAGE UFE0011885_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 2014 DFID F20101123_AAAHSB ORIGIN DEPOSITOR PATH woodruff_r_Page_053.txt GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
419d1f65d49dfc515517ff8ff5728123
SHA-1
de8a2331af9424f6e8782788f319198170b59130
2057 F20101123_AAAHRN woodruff_r_Page_038.txt
0bed343ee3577b583809f714a7700b79
6a62230db5c4ab5f6afc7ffc2140c66744c21b42
2073 F20101123_AAAHQY woodruff_r_Page_018.txt
c178f60006e4a1a03f855fd3339824ae
318416c5ae1cfe0e94d729b2c1ab2b3b897ae662
750 F20101123_AAAHSC woodruff_r_Page_055.txt
fd533fdfd85b5ed93a59522006f5ba37
2d015631c3a7324ec79e652d44bccbc39ee40614
1985 F20101123_AAAHRO woodruff_r_Page_039.txt
e1d43eda9123c36f38764e893705bcf3
b6945e30fd3b2336b9a49792ef1a72d680220f0a
1976 F20101123_AAAHQZ woodruff_r_Page_019.txt
97d510996053c03c9332ef8a909561e4
db75c8d4776c8c2d3c993f40999c634931886664
1588 F20101123_AAAHSD woodruff_r_Page_056.txt
eb552e3279079b37616585c59ba3ffe9
c40bd0f3a78fc4ae36224177357997c31b55bfe5
1813 F20101123_AAAHSE woodruff_r_Page_059.txt
cbb6687248fd689ed4b2e7309036fe8a
4a4e042b13a7f7776ab607c36c2829aab80c6d71
2030 F20101123_AAAHRP woodruff_r_Page_041.txt
5782079e78991c6fd297d3e143aadf22
b58ace1cecefd330e7ebacce8a29991c030f250e
1990 F20101123_AAAHSF woodruff_r_Page_060.txt
51493d710a8b6519e90159c0c9289f04
8cf887560cbaed7dccaf2b97c9459c807e16e92e
1885 F20101123_AAAHRQ woodruff_r_Page_042.txt
f3b137959e041cc74bd160c7d5a414fe
dc02a85bc35e49dc7f5a8b5f5a2c6d928d6dc2d9
1855 F20101123_AAAHSG woodruff_r_Page_061.txt
797ea8960e38b8d3e87b33ecfea28aeb
6fda80c31a6e6019dc56eb0d9dc4311c786399c0
1995 F20101123_AAAHRR woodruff_r_Page_043.txt
29736bb7a487dc7b2cbde2318fefb645
920fa9a894cfe077471c513e723c7bfb002a6f03
1953 F20101123_AAAHSH woodruff_r_Page_062.txt
a955a36dfa5652acb99652e57dc341c6
e8db56ff080550807728451ebb16386c4bab29ad
2037 F20101123_AAAHRS woodruff_r_Page_044.txt
4f63bd7600a771380d23fe80e81a8bdf
a0ec258551705905362b5be0c239e8a6aee63eaa
1850 F20101123_AAAHSI woodruff_r_Page_063.txt
d4d58c61b04dc694424a01dc1e13b89c
3d5a523b7c7954f7e1355304a287762182ea7624
1957 F20101123_AAAHRT woodruff_r_Page_045.txt
45437f922de9289512a6925728376f37
87183c9b612507e0e5c60abe83fb2b1f6f68405e
1821 F20101123_AAAHSJ woodruff_r_Page_064.txt
3ce6ed7bdddf5e660cd840362f4a9b7d
299bc71338e1d3a5fbe544648734da7853e9d2ba
2010 F20101123_AAAHRU woodruff_r_Page_046.txt
5c398f4344b423a07c41cadff4477f58
7832eae18791c9d95f9196524508494de83fce2a
1938 F20101123_AAAHSK woodruff_r_Page_065.txt
b3bd214aa8a33b511bb6fe3ef9155e8a
bc533bf6c9a1e4983ed443b956dd85c7b99139e7
1993 F20101123_AAAHRV woodruff_r_Page_047.txt
6ab90ce8fb5e4bc645a2286599441049
bf4d33fd7183570405d72b762814fe842bd70595
1512 F20101123_AAAHSL woodruff_r_Page_066.txt
17dde9effb9b4b6e45ed0acbf5569d71
fb36c65a8a6b9dec77c0930840602586082cc100
1766 F20101123_AAAHRW woodruff_r_Page_048.txt
c042bc0768f61b5bea00ae1549dfa697
2af07b063070fdf5699611f04836acaf61f3f1a4
530 F20101123_AAAHSM woodruff_r_Page_069.txt
fc6fff3539a6a4cf8edb1425fe2098c8
80f561eae9d597f7dc4807f302df5e726b1c4ce5
2109 F20101123_AAAHRX woodruff_r_Page_049.txt
626c51c487aefc57ccf149d2194320d0
dd9a7dfafab7051e63b1467d2a6328e4017d01f1
1810 F20101123_AAAHTA woodruff_r_Page_090.txt
f30d30ea9bcca7341bac565f9cdff71a
e8454402bfdc9b150980972f0869fcbf84d35de0
1106 F20101123_AAAHSN woodruff_r_Page_070.txt
22c4fb4fc363d607b1128c391f75581b
c6f4a37fc80d20da44297840bb41ead69121ee5d
1951 F20101123_AAAHRY woodruff_r_Page_050.txt
c06b6abd89a0b291ddedc6b95d9c02a4
0218acb0ee4d2a16de279db6ac4b1c24635b1e5c
2024 F20101123_AAAHTB woodruff_r_Page_091.txt
4b2e23229e3e5b8601dba29e5025a865
bcab3d332a664bb3d7616e82ccb03cf46624ef0a
1121 F20101123_AAAHSO woodruff_r_Page_075.txt
5b459a0a65bd99682ff2ab0cbcaea554
bf88778eb8d776fcc5d267cd6e3b422c166cf620
1943 F20101123_AAAHRZ woodruff_r_Page_051.txt
24e277971ca03e798a669c53efc2f7bf
2778968da28bdbc221fe3e8671ef3a5ca7bae6c1
1975 F20101123_AAAHTC woodruff_r_Page_092.txt
eb91fd3ac658a7d902efba0f40eec4ba
4a5ea7c27178d967dbf93add7c65353e38f47499
924 F20101123_AAAHSP woodruff_r_Page_076.txt
79e80f9d72b7db5a5fcc2025c3cd417d
9506c0c8afcfa5c2650252910bbdb002ec86f13c
1949 F20101123_AAAHTD woodruff_r_Page_094.txt
238113297bad645898da6fb1545c4545
f0a5c822387aaaa38eaf6f704e017ed14927571e
1955 F20101123_AAAHTE woodruff_r_Page_095.txt
504adb28f5c25e9658c0069db1e5bfba
07939ea803696a4768cde0fc13edbb93856eae82
432 F20101123_AAAHSQ woodruff_r_Page_077.txt
ee22f972e707da0dd327c9ead15e3a59
ffbbecf6bca7d59ca4b8242810bca0f6ff43c9b5
2004 F20101123_AAAHTF woodruff_r_Page_096.txt
1ded7bfa49773f3566377a5ceca9d699
5974a6635df85bb0b6a10976d869027850fc759b
3181 F20101123_AAAHSR woodruff_r_Page_078.txt
7ab679ff53cc5226e022ce67508dc63b
537f8b55681fdaba6eaa244d1e5a1da678b30d1a
F20101123_AAAHTG woodruff_r_Page_097.txt
b7a7c17628a8c7550b571ebf542fd22a
9620436048e8fbf118a49a33f5e0c669db000250
1918 F20101123_AAAHTH woodruff_r_Page_098.txt
4b6b38889c5384b17c77b68d4cd37780
87aa45ab87d33e711a075921c85134d317f03163
567 F20101123_AAAHSS woodruff_r_Page_079.txt
0816890df20f069b9bc7698a3fd043ce
d8a4f2167f4eaca7ea5fc058b8e6887ad0da57c0
1823 F20101123_AAAHTI woodruff_r_Page_099.txt
011d8493cd156675e167955c210c78f1
a188e6dd9da6af44cbd8d97996e336da4c4f53e7
1483 F20101123_AAAHST woodruff_r_Page_080.txt
2d8eb8e6c002228afde2db4f1b319f43
ea6249b30795c8538cf73d2b76de7a30e0816650
2064 F20101123_AAAHTJ woodruff_r_Page_100.txt
bb6a240b204ca566b8a6579c2512043c
67455a24ce00aafb218161c1f4e7b69ed764cc16
1001 F20101123_AAAHSU woodruff_r_Page_081.txt
f6bf998992216c786e50ebe042ba5aa1
f5e40766f5aa440cf1db665bf16d4c90dd3a7a4b
1929 F20101123_AAAHTK woodruff_r_Page_101.txt
362f7fcb63ac3b99f9c8da1c5207b1dc
506edbe6e401dc80fad71fa0c4ad0584d0491968
333 F20101123_AAAHSV woodruff_r_Page_083.txt
0968408653cdff0474992b853cd8de40
8cf24da75a0a9781fe960aa4bc34f1e899b5bcb9
1954 F20101123_AAAHTL woodruff_r_Page_102.txt
8ee43a59df28b37342220009a16d4562
d4a8e97e118e43fa700e21a8d7e3acf6808ba07c
466 F20101123_AAAHSW woodruff_r_Page_085.txt
6589f631ee80b09685f3472d5ca03ec0
c6684ef40255fbba86220130a55e5cd4c23cbc51
8156 F20101123_AAAHUA woodruff_r_Page_025thm.jpg
b025440306493f187ea92ca74dec709a
5948e6c8b545b2f6a7c442e2eb80604396592fb2
1909 F20101123_AAAHTM woodruff_r_Page_103.txt
6059615ef47c2e4c02994e65131422e4
1a95abdcb4726e08ecd866aacf0a4fa508fdfaa9
243 F20101123_AAAHSX woodruff_r_Page_087.txt
8cd81397ec05dccbf9bea14c659f7c1a
e9e1e1689ec06eb88ca2d95f04c11b60b27a6c09
32407 F20101123_AAAHUB woodruff_r_Page_104.QC.jpg
096000f04165de99d04608bea3d1dfdf
7f5678c936b35c0c6c25ed48bc4fef0bd32ccb90
1937 F20101123_AAAHTN woodruff_r_Page_104.txt
b9ae0f1a80952049856fc8c3bc6f6b59
e614936b1a2da796434c9af91089bae7dbb00eb5
3863 F20101123_AAAHSY woodruff_r_Page_088.txt
d485a1e0f33e4badefc69130fa306c3c
05bf0679f79b555216695924816369f0f8c5d0bc
6780 F20101123_AAAHUC woodruff_r_Page_072thm.jpg
17c891e46212c303b5a863e1335a7c18
186f4407dc123c67648c56cf023d2bc9233e657d
1409 F20101123_AAAHTO woodruff_r_Page_105.txt
40d33b1328a8b67665689dc707376c27
992016b1bd9e9d82f642f3db431fc0f30c591891
199 F20101123_AAAHSZ woodruff_r_Page_089.txt
6d278b7acb1a6d7a4023f0e2cabdb9bc
bdb4cb54f602b4e6e39dd7bf84c6e56e5472909e
31719 F20101123_AAAHUD woodruff_r_Page_099.QC.jpg
45642edd21a2fac4763a92ec914ec639
05f643ebde2b2723a7ceedf129fe4ca0415cc2eb
680 F20101123_AAAHTP woodruff_r_Page_106.txt
00260bd677d0eaa28aaaf21c6cd8b259
f07c9b171acf4bcaf7d6bb5768ba50d4a44e10aa
8393 F20101123_AAAHUE woodruff_r_Page_116thm.jpg
5a901504a4adbb415c09fe6f8fa61e27
c02f19f827ee16b287544a3172d0ad10ae4be8ff
2271 F20101123_AAAHTQ woodruff_r_Page_107.txt
79452f7572e6870632346a251576749f
426492579bfc983fd0dea5dd8ba1ba0ae10f680c
17895 F20101123_AAAHUF woodruff_r_Page_073.QC.jpg
21306d831d5db4ea1f81473a1d8dff65
60c3deb1c433e751506f3d64dc7bcc214cfedb10
4919 F20101123_AAAIAA woodruff_r_Page_080thm.jpg
50fdcb513ad8ab83cac759be7d9f4ece
6983b726183c0d76eb2400b749195b2dc9b17dc8
17809 F20101123_AAAHUG woodruff_r_Page_075.QC.jpg
c32ed0b842706013a984ef92dd100343
36c1ce91bbb51019707c8c813f5b4c2d5d7dda60
2388 F20101123_AAAHTR woodruff_r_Page_108.txt
f508677322df1bbbdcd2eb26fe20b8ee
b33b75524ac9cbe5044d0d78474fc924efa95638
20315 F20101123_AAAIAB woodruff_r_Page_082.QC.jpg
fb422fc88a2eaa9be52482e1b7f76b73
17260caa112528f2121b4af24b67826239f00dc9
7994 F20101123_AAAHUH woodruff_r_Page_064thm.jpg
a3b4f6b683697b1dec00fb6bd62115b2
c945b48da1de439f69e2c8dd2733e2e450ebc7d9
2360 F20101123_AAAHTS woodruff_r_Page_109.txt
f9663dd9b06ad10dc92e7f0847772f2e
a48ec3f7bfb06acd759c91c5cdf0e2ac73482479
15522 F20101123_AAAIAC woodruff_r_Page_083.QC.jpg
43e5218d25cc2125ae5d8de9998c8649
0a249a8feb6d42768573b0f123e7f82fbc46cacd
4939 F20101123_AAAHUI woodruff_r_Page_083thm.jpg
aec088f0410e11a5526ed68c668dd7cf
9fd37f89e0c9021d1380d1efffd8de3ee49399b7
2611 F20101123_AAAHTT woodruff_r_Page_110.txt
05eb224520234b6efabf201264e31843
77dd1722f50d445521d00f30e3e580d8d9909d64
21045 F20101123_AAAIAD woodruff_r_Page_084.QC.jpg
99c71ebbbbb341a99ec66fce5357d402
0329d5d01b17b9eacc6f4580d44d09ba4f97aa4c
35791 F20101123_AAAHUJ woodruff_r_Page_021.QC.jpg
df15050d389850c810358481177a6fa8
66ab74ee0718085ab98fa7acad145e89bb82be5a
2592 F20101123_AAAHTU woodruff_r_Page_112.txt
099b4a36a9b007c5255942e5d4ac7239
484681be876c23c72d401f0f23bc7039e8cf2909
15960 F20101123_AAAIAE woodruff_r_Page_085.QC.jpg
56361051df6c23a88910de01543007d7
530a30b0feed52a5e382a1797f63f9b83c540c87
33449 F20101123_AAAHUK woodruff_r_Page_101.QC.jpg
084164b463d97ba2d56a7a5fdd60f709
5160a2718f167e84c74e0591289d2a64a7af6133
2434 F20101123_AAAHTV woodruff_r_Page_113.txt
58630a54ed7d8c605c41f9c64d15d73d
0de6e8412cc891340bde92fdd5cd311ac1865e6b
16685 F20101123_AAAIAF woodruff_r_Page_087.QC.jpg
648ab595c6aa1e83caca75f27c3fbd80
98be5a50a028d74fac591ec873380d5c6bcb40f3
33410 F20101123_AAAHUL woodruff_r_Page_025.QC.jpg
6e033912f65e7e11756c9b5a12b2ecba
5db10fde6247bba520e9a5368bde78304eb86ae4
2653 F20101123_AAAHTW woodruff_r_Page_114.txt
9066221a0e662ad7287d966d8128e5ad
430287884b770ac5264632b11a207831a1a1ee1e
21761 F20101123_AAAIAG woodruff_r_Page_088.QC.jpg
177cb264bc2e5363611e1371343b5334
b54636556899b2e4b353ee8ef7ae88246bbc72ce
8060 F20101123_AAAHUM woodruff_r_Page_020thm.jpg
6af24c0f64b78f85c6ad2649582b0878
5048638616938054afd1086a6e0a6966d4a00116
2644 F20101123_AAAHTX woodruff_r_Page_115.txt
4b66cbf278a8bea4291ef5e79866f6cf
2453088e026feda5e4f2ed53eafe201aa49c987b
7934 F20101123_AAAHVA woodruff_r_Page_012thm.jpg
8b81341424a9e621f15b900001589f5e
d8bb9738b32407d05f50237e49760c12b302c3e3
4637 F20101123_AAAIAH woodruff_r_Page_089thm.jpg
d70ba320fa0baf1b42194eefac1a5bb0
29ddf7c08c5fa6eb65e25234e421e88fb6088248
1843 F20101123_AAAHUN woodruff_r_Page_003.QC.jpg
1b88ec0cfd5165df357c6a44431a0562
fff322e00313f0ec3a98d73c8c89098006aea2a5
2325 F20101123_AAAHTY woodruff_r_Page_116.txt
a795dd42aa9fcd16e20071c38e99b001
455a379f2be49f6e33e80d03d194dfc8f06bab55
8686 F20101123_AAAHVB woodruff_r_Page_113thm.jpg
2f33f584d6aed42fe4b0cbdc5d6f64d9
592699de054d4f629c8ccc68397069a6e578745b
8690 F20101123_AAAIAI woodruff_r_Page_091thm.jpg
895f0e16efcc2e82eed44194d4dfd99d
9d739e2ab1668f0bd14b744b3ab5f3677e171014
35847 F20101123_AAAHUO woodruff_r_Page_060.QC.jpg
9e4fd0a188cd3d815970d4b92a3198b1
defc5130a948c33e437ab9dcc08be1351c8493ac
1966279 F20101123_AAAHTZ woodruff_r.pdf
482b3b28c3b5b0814ad6b62c45b696b1
25a86f499b95bf756a29d84ff52a198ed73e15ac
25916 F20101123_AAAHVC woodruff_r_Page_056.QC.jpg
ce2ab64b896cbae275118efd2239a7ab
e2d9df6bcf00714a7a0bb4654b06fbbf1f261a5c
34409 F20101123_AAAIAJ woodruff_r_Page_091.QC.jpg
4d559f5f1c19b5ff95a514bdab845042
6ce64eb4c6de484f529ee65cebee6842e150fd47
5101 F20101123_AAAHUP woodruff_r_Page_118thm.jpg
5d94b728247049567cc887f3361cca6b
0bf080add733d24a206be60e6b15e122eaf3c058
37518 F20101123_AAAHVD woodruff_r_Page_112.QC.jpg
8a5505f00d690db3b614f044b7c80901
f98a0979f01ff815d8980cb1744210894acb025e
8665 F20101123_AAAIAK woodruff_r_Page_092thm.jpg
655af234b77bb866c40e18aa59cd55ee
68c24e9794b61317500ec772a525c429bbd2911f
8288 F20101123_AAAHUQ woodruff_r_Page_102thm.jpg
49b0c6c8f9a4f5089770874bccd199e9
84af1832430c50e44d57f9f8119f69c0f307192e
1656 F20101123_AAAHVE woodruff_r_Page_054thm.jpg
ac1e677d257ee98b226ed84c84224f85
e8a17b260726010a76d46401c981d2614c36b831
8304 F20101123_AAAIAL woodruff_r_Page_094thm.jpg
cdd7df9026fa156c00dd7374e16be1b2
d0da886562476437c1ebf9e5dc3ca34575609d42
8433 F20101123_AAAHUR woodruff_r_Page_046thm.jpg
9560f52caf5c806a041fb47dad504648
df69b310b2d027043dc3f73f5ad08571da5feab8
14545 F20101123_AAAHVF woodruff_r_Page_079.QC.jpg
7b2680d4b86db208efc729a03ad3cde8
0812a5476a2b27340f1bd77fdb3ef4e0aa1bfc7e
8440 F20101123_AAAIBA woodruff_r_Page_111thm.jpg
559f1b121a0845effbb37461e75805bf
7c3aa8d63bade8adea852a34228bc4277779ed00
8508 F20101123_AAAIAM woodruff_r_Page_095thm.jpg
4e45a37cd35bdf48c2b9f30491e3a056
d5902aeea11a62c5367409c7993700a956f54ff3
8586 F20101123_AAAHVG woodruff_r_Page_021thm.jpg
05a4a560f6812e050bae67dbfb4922ab
42da2f2727a5e263b0119a5ad00013fbe3e5577d
8959 F20101123_AAAIBB woodruff_r_Page_112thm.jpg
41180d0242c20e9a6e5824b729e310fb
33066a77407f5d0ea4d5cf3a76071b74c7d93768
33256 F20101123_AAAIAN woodruff_r_Page_095.QC.jpg
2c1663375c8986fa41a9ef8023b6efc7
5e9acb74a47fdb53c7128864ea7de52455c7e9fa
7828 F20101123_AAAHUS woodruff_r_Page_099thm.jpg
cea239702fa749ec83b5f50f5f402aed
b42e57600e85c90d469399e5d8fe447b560b8e25
8556 F20101123_AAAHVH woodruff_r_Page_017thm.jpg
2cd0d57376a328d1d7a78f3286b4ceaa
927e927427b33aee1cb735d5bf1994c6fb046289
37648 F20101123_AAAIBC woodruff_r_Page_114.QC.jpg
29e86d25996ee68daa5b677e5a29fabe
95bfdfdcae8bb76bea457af897c72d68f6cc6fc9
35377 F20101123_AAAIAO woodruff_r_Page_096.QC.jpg
10b1208a49bb982f4e3c73d3787df0c7
55daa5ed6fd5bb1327a4878f9a15a4db824d7b5f
8402 F20101123_AAAHUT woodruff_r_Page_062thm.jpg
6451fdb76b28b5724faadbfe519431c8
1697927aabd96233bae5f4b1b6f6199c3c63169a
8787 F20101123_AAAHVI woodruff_r_Page_043thm.jpg
71efb18a3592fc23a068b31d266e42c7
a4276ac61494eab5622ed51837f9287e45b46590
4648 F20101123_AAAIBD woodruff_r_Page_117thm.jpg
9910c73c6e3d65895be32fa5bb79bb18
afa111ca92cb17932d5c11886959957b9ea47e88
8525 F20101123_AAAIAP woodruff_r_Page_097thm.jpg
a35eea77b0451be07ddcdec75ce7d1c4
773a0f08deba7ed398ecf39e85d7e5a846c7be81
31974 F20101123_AAAHUU woodruff_r_Page_103.QC.jpg
c00e544b0ecfffb82b6f10e067163e53
101de885058e5b93b428477539fd1f46e3103f4d
6988 F20101123_AAAHVJ woodruff_r_Page_055.QC.jpg
ffdbce62cdffdb6ecd8e331c47c28bff
ad74a7b877f99c602aa698de29bb36f9fca4a1bf
20430 F20101123_AAAIBE woodruff_r_Page_117.QC.jpg
554bb23d8fab8533592119a0462a9822
2f29c2a9e96946b907d52f35c1df42b8a7653576
34158 F20101123_AAAIAQ woodruff_r_Page_097.QC.jpg
39e4c241c3bf9d63f13bc5221bebdaa9
cbebe467fd4087b1c6586739be7ed9d4d224adb9
8682 F20101123_AAAHUV woodruff_r_Page_038thm.jpg
5305c077e4a61c3aab865e55a31bb6fd
c9de5a876e6da0789c853618a6fa3bf28d8cab00
6551 F20101123_AAAHVK woodruff_r_Page_069.QC.jpg
a415d17a5cd922e6dce817506e0c830f
e37adaec22e27389e0d8e4b59e5cfb8010be4a04
19744 F20101123_AAAIBF woodruff_r_Page_118.QC.jpg
f0c973a6d8a7e51ff227d60b4d477f88
0cd4a1e5bfbad78f1dbfd3c004c1c420c607f330
8421 F20101123_AAAIAR woodruff_r_Page_098thm.jpg
8ed1f6ea7907f6241857f866dddbc13a
0d1717d60528c9b48520b770d1d9acd498c93e73
5812 F20101123_AAAHUW woodruff_r_Page_088thm.jpg
0c70ebebe65e8230e19fefa6d7f24b68
ec0c6aff79ca4f38c930913e1daa085d4f84e749
8316 F20101123_AAAHVL woodruff_r_Page_030thm.jpg
9bb466e0fc5afb7b3296015453cd55d5
b4a9a94bc8c1d2fab4059093716ffc33128ebd84
34151 F20101123_AAAIAS woodruff_r_Page_098.QC.jpg
55856ec7601aa1d98ce79abde866fc42
c458ad9370b2fab5ce2ff74395990f95d73c24f5
32800 F20101123_AAAHUX woodruff_r_Page_034.QC.jpg
1eb4c4a432c0a54ac479ba64000bb08d
7dc07f258453fcc257a9cf40ee88a01ec2bfdc54
33461 F20101123_AAAHWA woodruff_r_Page_065.QC.jpg
db486332bfaba32105bac5510c23d85f
04d673ecef6c32d2a9fe8ae6c7dfb61f0fe182d4
8043 F20101123_AAAHVM woodruff_r_Page_103thm.jpg
7bcf1b1ba6d1d97f631e2c5562b608ad
16b310e283318f412c0e12910f78667be2419ddd
F20101123_AAAIAT woodruff_r_Page_104thm.jpg
13ad8d5f9e6c56356beef9af645feb24
47ce5e61c6cc221d679e131b44a4a8662ef47fa2
8619 F20101123_AAAHUY woodruff_r_Page_018thm.jpg
e6edec2b89c2cec93927b9297bea4f3d
c64c4d38c3c29414e7bba56263d4504d5468ed01
8197 F20101123_AAAHWB woodruff_r_Page_063thm.jpg
d04d92d20d96d578f5069280a4933ef4
7e1f80fca8ce0f553b9cfe36add4172cfca15c31
8815 F20101123_AAAHVN woodruff_r_Page_100thm.jpg
0f0cd7edbc491ddda315849c214b37ae
559ca85bf964df5b70bc7571d9bd24a5cfed6850
7536 F20101123_AAAIAU woodruff_r_Page_105thm.jpg
2de96d9ac03e91315e63a500c336a026
ed53b67c730b0a2d7cd38f9add372d22ec75c5ab
7905 F20101123_AAAHUZ woodruff_r_Page_034thm.jpg
3e717791855a13db9d85133610a4c9ee
c6317226199a8a3ba826ffb23aff5f702d70e489
F20101123_AAAHWC woodruff_r_Page_002.QC.jpg
a738408f93dabd480d78bf19fdf8c326
43236741eff2e91d920e5c7a118fff6e462aa492
34297 F20101123_AAAHVO woodruff_r_Page_111.QC.jpg
8ff21e0f80f0f6f5f6722b2bc5dad832
38aca3793b3349d850ef97119e23deaad83fb76f
6938 F20101123_AAAIAV woodruff_r_Page_106thm.jpg
5efca8792a143ea8dd75a7e0488f42f0
52a8a6342400e3e45088bc1b7863f4b36ebeecd7
8322 F20101123_AAAHWD woodruff_r_Page_093thm.jpg
40c3b91749b8740ad82d43c0a907006b
799617a5f28fd03d095328ad58b34c0f781fa571
34003 F20101123_AAAHVP woodruff_r_Page_094.QC.jpg
9597c7c09c209b8b957c054852e47fae
9fc3fb3d1a18cdf06b8c326269cccb9145325c0c
33321 F20101123_AAAIAW woodruff_r_Page_107.QC.jpg
d448b5658ec7ae96a786476c90a134d1
b0901ab65068fe45e53712f96e9a02b967f1a073
8076 F20101123_AAAHWE woodruff_r_Page_031thm.jpg
96bb132832639292ff4e132549d4e27b
388539693dea13d90cf978d44814f669346a9f4b
34979 F20101123_AAAHVQ woodruff_r_Page_045.QC.jpg
a414a40b8df1a6062f7b4f0e426e4938
9cefdd894ddeb9ef48f41031a75147195105a1e9
35465 F20101123_AAAIAX woodruff_r_Page_108.QC.jpg
7a278d43cdc728dc28a27517cb3411da
edd4fbc8ba811d041b369c119659f2f2ca521384
15465 F20101123_AAAHWF woodruff_r_Page_089.QC.jpg
77c9b1cfbbab0703614a5b3b14991151
164e1356b99e9c575e5cea294d5ac04dc416bdd9
5048 F20101123_AAAHVR woodruff_r_Page_081thm.jpg
38f1db85dd4860969c8f06969e37e009
1b3bb9a2fe40a34ac21ea67d06efd249a0a0dc08
F20101123_AAAIAY woodruff_r_Page_109thm.jpg
f15415fff10071cb9f335ec4b5b220b8
9949704908b662335328bde5b2a7aa8b826f0dbb
32186 F20101123_AAAHWG woodruff_r_Page_064.QC.jpg
66841f148192025925b9544791912455
413bb2bb03befa92c563c3650c874d5287edd2dc
35700 F20101123_AAAHVS woodruff_r_Page_040.QC.jpg
da9e422be0d938ff668a0532046f26fe
195524591e97156399bdc9e5c19885c4a14fd4ee
8860 F20101123_AAAIAZ woodruff_r_Page_110thm.jpg
cca7d4ce494b108a05a336b426b3d42d
15e8431d2a5b23175c2268f614e0565afbb7d61a
26687 F20101123_AAAHWH woodruff_r_Page_051.QC.jpg
3124f6bdc232af8e6db0abcfaf80c2da
48cb379611d07da5e98ed8370c5d652a30f3bac2
36782 F20101123_AAAHWI woodruff_r_Page_044.QC.jpg
3c95dc56e8846d31802e398fc32cffaf
169b9430623300ef0ae29bcc63a524df42910f7c
19911 F20101123_AAAHVT woodruff_r_Page_080.QC.jpg
651148272eaddc2e42421ffd0f01fab6
ac5dc1af8e37441539acbd88a2ecfa6cd801f605
7659 F20101123_AAAHWJ woodruff_r_Page_052thm.jpg
f8bc9dd90bbc24efa2aca035f9e9a397
8c08771caa638b18592da51fe18a333b863513b6
8445 F20101123_AAAHVU woodruff_r_Page_022thm.jpg
fcdf80712e36af789f1f33bd09b160ad
83ad24a45d8fd72d73c77f5f14f0e52455618307
28550 F20101123_AAAHWK woodruff_r_Page_013.QC.jpg
82085f27fc07245c240b2bfa804ea269
ea017fdf4cf0e6e1cb4bd1e08e61805774eee421
17872 F20101123_AAAHVV woodruff_r_Page_081.QC.jpg
f8c55d5d69766ef15723372fd3a8e05b
fd99731f9352c7bfe42c00d82421015deb65e401
35907 F20101123_AAAHWL woodruff_r_Page_041.QC.jpg
436ba62f112aaae9717ca3a32767f339
a435c191a86d9a1e246b1d7387aec2f9da1a8a8e
32121 F20101123_AAAHVW woodruff_r_Page_012.QC.jpg
37522a6e2dfc70dc7aaf4151ef9957db
92a25187296019aa1b3c954be3458d6ce58ebf66
8554 F20101123_AAAHXA woodruff_r_Page_026thm.jpg
a703b8f0dec74633720465d4e77de89b
b2ed6b099008259e82e0fde424d924b7ccdaf62b
34053 F20101123_AAAHWM woodruff_r_Page_015.QC.jpg
460fe8ea0a496d7afaca21f4ee862719
92d4f9ee571034a81b4401e45055a49f3e88f2ca
8323 F20101123_AAAHVX woodruff_r_Page_101thm.jpg
2726f4abbe45346dbe9452a9865c2c9b
5aacecf9a14f34cc9266019d32456230da6637c7
8980 F20101123_AAAHXB woodruff_r_Page_001.QC.jpg
d3c2171df5e02d34303135116228f970
191891af08074d0be1bbfbc257e074ae8ec5e74a
13328 F20101123_AAAHWN woodruff_r_Page_078.QC.jpg
28dd46e7443a73344aef23ae97136114
c9a1151e4a9cfa24e832ade077be1d544f411c20
8824 F20101123_AAAHVY woodruff_r_Page_096thm.jpg
215277af1ce1a9c5b0c3a58e484a93cc
75d3d9c352f9e50bd17b63c3a585ce0cf44a5825
8491 F20101123_AAAHXC woodruff_r_Page_077.QC.jpg
2ad74936b7b570605ba86ba0632526ca
af3f0fc45ec6842fb87fb0b63fabac400127798d
4201 F20101123_AAAHWO woodruff_r_Page_070thm.jpg
41427781439c96d8f0de22da098fdef8
a11af9d9160b395e3c538cc3af6a946a8a9e7db1
29718 F20101123_AAAHVZ woodruff_r_Page_090.QC.jpg
9de725bc828c84d5b7b87ceccbced6f9
3364e6b479118ceda8752eae4d3e836f676f8524
5182 F20101123_AAAHXD woodruff_r_Page_087thm.jpg
4a0f2f006906f58a5c791129948c7f30
1acdcdc9a69d9774505be2ed50a9d991641f111c
32572 F20101123_AAAHWP woodruff_r_Page_024.QC.jpg
9d7ed1288d4bb66e6072198d972a8d97
383351b533613c56b3be367b54c69b642f183b6a
29200 F20101123_AAAHXE woodruff_r_Page_105.QC.jpg
c71488536d1a1de4ab85a161ae442b12
8cb8bfa69f7621dd9a507cba69ca83ff25020178
8359 F20101123_AAAHWQ woodruff_r_Page_047thm.jpg
93a955f37a341000c5f01f173f639637
f638e5c70f63c8095fc6074c29e18c4b373756fb
179039 F20101123_AAAHXF UFE0011885_00001.xml FULL
ce4237e6ec2f6894b7250715359a077b
eb6700765c258379bc7b584c8125eb7851546d8a
34013 F20101123_AAAHWR woodruff_r_Page_093.QC.jpg
26c6f48f705b2bee79d6af673ab9a68d
37b25be7b3faae6db768c59583b88056622d14c7
646 F20101123_AAAHXG woodruff_r_Page_002thm.jpg
97146aadecc104e2960b62c58c46cdcd
5b3b610d43027842e93951499c7c4cd3e50227d4
37522 F20101123_AAAHWS woodruff_r_Page_110.QC.jpg
dbb3669480b2376a0b06239fc5597305
bef28345bb7f494e67e7bcc3a5883d38b28a1fe7
728 F20101123_AAAHXH woodruff_r_Page_003thm.jpg
e368d6962e61e3d1bb1f0b7952ee0e67
0bcba982d81ab1fe81578f49f0ef8ed8f1cf87f6
16062 F20101123_AAAHWT woodruff_r_Page_070.QC.jpg
7f1272e7212aa87e1a27b199861cd2be
42274cd3ebda98b62c26857f467199f9834e9b92
6948 F20101123_AAAHXI woodruff_r_Page_004thm.jpg
acaa721576c7d598b2f14dc0dcbfcd67
215aca525f4f4b9e1e5a33d3e55e5f5747042e46
F20101123_AAAHAA woodruff_r_Page_061thm.jpg
66db50bdc15b9ede0155a05437a75fc6
6b83c4b2e3b5672acec89377b921dc5a2ef88933
27215 F20101123_AAAHXJ woodruff_r_Page_004.QC.jpg
984f9edfe16b144b2e018e94805821dd
7e6fb56b4730444e2ac0568e07766d7dd4da8575
7681 F20101123_AAAHWU woodruff_r_Page_048thm.jpg
e79de55629b9091bdd025d9ce36d9008
95a722530ad697f1b06d60b6fe79dc14df32761f
1053954 F20101123_AAAHAB woodruff_r_Page_003.tif
2dd8b51becaa2500d4d3edecfaa004e4
83209893be63850699d037ee5456be5c801ef240
5604 F20101123_AAAHXK woodruff_r_Page_005thm.jpg
52d60fa20117ac51c3209030cd399b34
74738b27695ccf82e599c0a3cc92c4aea60aeb53
35200 F20101123_AAAHWV woodruff_r_Page_046.QC.jpg
98302e026898180967de3609800c700b
84c95f5f70876f99037756843b370a0b45e42a10
25293 F20101123_AAAHAC woodruff_r_Page_088.pro
06ea5267c75f83a0d78d330daa21ef83
f1a50f2e8b6702b57dc76e6b34b479ee768271bc
36485 F20101123_AAAHXL woodruff_r_Page_006.QC.jpg
92fa159a5c9891cd3b30405d0407b620
7311eae055c06ca03f66781dac661b066fe09abc
21230 F20101123_AAAHWW woodruff_r_Page_086.QC.jpg
90c352d1c2a65fcf372f2470a0dc75e2
057cc6646c3bd6f5773dabdd49625eac3eb0d1c4
103333 F20101123_AAAHAD woodruff_r_Page_094.jpg
7fb5c813d70183f8843295ca46e2442f
8efb7bf1ac3f7c206b714396e2ecb8827635adc6
6525 F20101123_AAAHXM woodruff_r_Page_008thm.jpg
b3fd39a3456e173940dfbaada0a6f71b
e296ec4162bf4d457235296ded969c6a0f1bdd5d
31691 F20101123_AAAHWX woodruff_r_Page_057.QC.jpg
df60307383551b394de9d9e719cc4d76
de9c231e73bd1ac961493e6339259e5f8971f485
48243 F20101123_AAAHAE woodruff_r_Page_030.pro
f0639bb53a7afb8c1a31eae222e9c96a
9c53f2a4059027454a004e5057f48b749233cad1
8286 F20101123_AAAHYA woodruff_r_Page_019thm.jpg
550e0967fec7769df1499be358980cb4
26d1f9225120668644bc812c1414132565b62222
26213 F20101123_AAAHXN woodruff_r_Page_008.QC.jpg
5b055b07d56c0c2542216e1559b8f536
7849d894de1b9dfebd062aaf2cfb04b1b4d3903c
2159 F20101123_AAAHWY woodruff_r_Page_069thm.jpg
aeeb5975f21a313f404858e7c1cef40b
a72ae9bdadb6dfedff695545efe166272976b953
103788 F20101123_AAAHAF woodruff_r_Page_030.jpg
4e4093a32cf78e9cb406fa2b6ef27baf
76beaf7a5360bd99357f0af449250413ffce6bef
33090 F20101123_AAAHYB woodruff_r_Page_019.QC.jpg
96715411246820f24ac71625fe6e0305
27164db1e88c281bf945fcbb7cbf98b4df014463
8104 F20101123_AAAHXO woodruff_r_Page_009thm.jpg
e077fb0a35b4190ffc553325292a7f23
4513706b568da956db1b8b4050b699c8f17b38b3
33697 F20101123_AAAHWZ woodruff_r_Page_032.QC.jpg
c1ecbc144ca6d700e6e743e7420b6823
e54252593e1c2a9e21b2e87d0bb4944bee80dd21
3126 F20101123_AAAHAG woodruff_r_Page_071.txt
3ffa9d9a7d8d89b0c2f227ba81492f60
23bc16d66d4254127bf318334740f940f314998f
35486 F20101123_AAAHYC woodruff_r_Page_022.QC.jpg
d559b355af9954ce56f80bdb0ee33a5f
c2de57f20e0e980195cb96f943f6a5e96f6165e8
32195 F20101123_AAAHXP woodruff_r_Page_009.QC.jpg
6216a3d610b7bf645cdc847280db905e
162643414f18960dfd3c41062df8f05412709a8c
8857 F20101123_AAAHAH woodruff_r_Page_049thm.jpg
11914bdfd97b8106dd5ea436661c85d6
787c27589cfbdb4dbdb0bcf467045f5985e85da0
8639 F20101123_AAAHYD woodruff_r_Page_023thm.jpg
59a05bd01f785987d17ac198fac304b0
98187dd96326e29780dc8845c35a1c6b365b9466
35177 F20101123_AAAHAI woodruff_r_Page_038.QC.jpg
0557da42b81055f81618d6fae052aff6
109b620f1e6fc404fb0cc3f58dd07cc5d4a368c0
8345 F20101123_AAAHYE woodruff_r_Page_024thm.jpg
608f7de5ad32de954ae4d1957f5a2799
a677c311280803a9226e6a584e93b885ae93d307
5331 F20101123_AAAHXQ woodruff_r_Page_010thm.jpg
78fb6452f0e806420f9f10284f53a7f6
6a869b3bf8844ad2b15f49117562b4bce83fa6ba
2586 F20101123_AAAHAJ woodruff_r_Page_001thm.jpg
f28b18eab4eeca76c065a4aa05f15b0f
fe870af566f260239e731f9940bc8e3ad50569cb
33957 F20101123_AAAHYF woodruff_r_Page_026.QC.jpg
e90bd99b4731e9f5ce3520050d20af0f
b9198b1f6da7a9ceeb9024c811499d9b6015cf5b
22027 F20101123_AAAHXR woodruff_r_Page_010.QC.jpg
a3add0a45e3adefd9ed4738556320a25
9fbde582ff352b1456c15bf7ef91b5dd5bba3582
37210 F20101123_AAAHAK woodruff_r_Page_056.pro
fb81f42421e68aaa3b6d4a5cd08f3f61
e1ca9a267d063b384348c25aefe344fa5d5c4920
28908 F20101123_AAAHYG woodruff_r_Page_027.QC.jpg
f4f5d342222f434d7bc46ee75d62e01d
bd8d9c095570d331e31596e3bf4accf9a78190b8
26433 F20101123_AAAHXS woodruff_r_Page_011.QC.jpg
3ce4d6217d4eacc8ea86312971fa6b38
d2ea94eeed8471e2d69aa6fd27117defbc777c20
105225 F20101123_AAAHAL woodruff_r_Page_015.jpg
aec8ba0aef19220663a747006b278883
c02752954690a2d4b83510427dad76a3904eca58
7123 F20101123_AAAHYH woodruff_r_Page_028thm.jpg
014131e54bab058f319333790909a9b1
98f7d1f873a44b95387815a3999d352eb6d49278
7279 F20101123_AAAHXT woodruff_r_Page_013thm.jpg
f1735d09f54ae324bfb87364a1e40e56
8674c595c9d0b5c7bf9b960b9bd2edcd6aec808d
79668 F20101123_AAAHBA woodruff_r_Page_056.jpg
9fafd980380b8ff863fdb8e08d1007e9
c3f94a2cd66592f841d22b72515bc3954441c33d
8401 F20101123_AAAHAM woodruff_r_Page_045thm.jpg
2fc346d2acb3e639ee1044549881d063
438d4a1f0659d4ff63f48d7632ea57ea40d90433
28204 F20101123_AAAHYI woodruff_r_Page_028.QC.jpg
90dbd8a221a7ae420a33f31f7a64a28e
df1b5ff51432131f92f5ae1c9520eea0425e24a0
8386 F20101123_AAAHXU woodruff_r_Page_014thm.jpg
f0263ea4c3c64a0499e9fa3fa2d14977
d6fdbd0d912ef649748d5654a7c0abe8767bd8b0
111399 F20101123_AAAHBB woodruff_r_Page_046.jp2
4c67b5e309dfa9b52438c69fb3a040e5
bb667c5a703844bf0e4796f601cd2717b2342bb5
6621 F20101123_AAAHAN woodruff_r_Page_056thm.jpg
0577958304849b5e06f754d586b24735
3ce8e6915b45a6453be3d3f618eb3cafa9f87e25
F20101123_AAAGVH woodruff_r_Page_062.tif
399513013e6f920c1a1b4836ce3e1b13
61ed69d039b16b5e8a755eded332d9e5e6bbb64e
F20101123_AAAHYJ woodruff_r_Page_029thm.jpg
67b759045e3484779c95974991e717e4
0a44a8a5c2db2c11237518043ecaa0fca3a5b133
1837 F20101123_AAAHAO woodruff_r_Page_034.txt
e74d76c89a00df9b571a92e7260a5e97
ecb7db70d4f8147fbf33cdbec77567d03838c327
437083 F20101123_AAAGVI woodruff_r_Page_077.jp2
745fbe49537699d7f189b96225902098
5a53cb432e45f6df7766ff46b82295a925b79984
34764 F20101123_AAAHYK woodruff_r_Page_029.QC.jpg
c12132b730d35ba871ec3b1b6a0d7f5d
746afb47d381a88089a48ff67c9bf04012f269b6
35338 F20101123_AAAHXV woodruff_r_Page_014.QC.jpg
850dadd2f3d6320f9d1c3b5742d8e6a7
736f3b15bb43ee284254ca9c4e7a94e24fc04d85
51561 F20101123_AAAHBC woodruff_r_Page_091.pro
c19dcff890bab4bda66d8d8b561b6f19
ff4a2ff58709527569ce0c46dc2738dd3dfa994c
3098 F20101123_AAAHAP woodruff_r_Page_086.txt
2749f501a2e848218ad0be3c0e374033
c683949d8b20a7e887932c5aad466f701dba895a
39321 F20101123_AAAGVJ woodruff_r_Page_115.QC.jpg
b2e7fa4aa2911472520275a3f9ab01ee
82a0b087fd8d4df41b5543e75e00a04b700da00f
34560 F20101123_AAAHYL woodruff_r_Page_030.QC.jpg
dc3fd895be34fe5ab547319f00fdfa99
b5d3b358645ff25c365cd86a03ea43615693fd70
8443 F20101123_AAAHXW woodruff_r_Page_015thm.jpg
571954387d376e16f417d519985beef5
cd38fb8d00d4cc73dff92927b7b5de2a88484838
49141 F20101123_AAAHBD woodruff_r_Page_101.pro
029617738f8756b1574e86936a2b4f4d
771e98970e547ee2976a90240e775333baacc681
24414 F20101123_AAAHAQ woodruff_r_Page_005.QC.jpg
92de7710c572773f06b0ff202708a6a8
1efa1f5c97e793a071799cb9a3078f6781fa0845
34272 F20101123_AAAHZA woodruff_r_Page_053.QC.jpg
9b2e6b9eab2ab5137d05580540051170
661e7921e251494c257b1a609e95f0801eb3ce5e
2063 F20101123_AAAGVK woodruff_r_Page_040.txt
58893b296d97dea8f0ae7953334e7776
d7d3d2ef9e5a52ad7432b0124e4f5a755f689d40
34462 F20101123_AAAHYM woodruff_r_Page_033.QC.jpg
35ead8ea6c8023c2dda8a2c4cc33014f
9fec2e1f9661bb1d28a74397226d81c4b83a7aba
8618 F20101123_AAAHXX woodruff_r_Page_016thm.jpg
47887fafa60abec443cf169e2a0b6272
1f8f08a3f0115382d114c8a603221f4390a772b0
1787 F20101123_AAAHBE woodruff_r_Page_057.txt
4e49943142f78dc77c223cfcc39608e3
83819d6fb46c106aadf0cf9f5a0c9459cb58f94a
104110 F20101123_AAAHAR woodruff_r_Page_036.jpg
c1d703bd9d565c5e53b27587266a141f
1e15cd8089527f0cd103f03498a669585a0af7aa
6232 F20101123_AAAHZB woodruff_r_Page_054.QC.jpg
38c87dcf7c793b05be8899de11e53556
0f43b3456e1d0f079a58d2b7a0244a74ed7121a4
107557 F20101123_AAAGVL woodruff_r_Page_101.jp2
617c138d1f753c0b214e83c65b5c73e4
84096e70ec41b827f5a0f5328a2dcb50f813a15e
F20101123_AAAHYN woodruff_r_Page_036thm.jpg
b53e03eb1bc5bacd6fa8cebefb98737a
2941bb2a775d3c2664e3f9df08e7cf66cab699c6
35591 F20101123_AAAHXY woodruff_r_Page_017.QC.jpg
5f76184365627a693773003e22844e85
f7369ef3d4ce9352d47ac395a1fe8a1ef613d2ed
50781 F20101123_AAAHBF woodruff_r_Page_047.pro
08f78e5b19b38eded5b91a5164b7803a
0f6e74d625326ad46f46a45069daca50f3bda296
3497 F20101123_AAAGWA woodruff_r_Page_082.txt
a5a37444f8223515c4162dd2d8f1ddd3
cad89edc416df9a162844530471e20e15cfaa037
6693 F20101123_AAAHAS woodruff_r_Page_011thm.jpg
3c2d652393a2486adb299970d9b72856
df8ffc00813d53d1446f5ee95615dcb8f55e8bae
1839 F20101123_AAAHZC woodruff_r_Page_055thm.jpg
ddfdf9aedda069de9b2fb6630cb63bbf
572c16fcf758cc24ecf0f464a1217e54e3b89819
118765 F20101123_AAAGVM woodruff_r_Page_107.jpg
3a24f5f7780fe7003ee0f0e5e7e3625f
3224b4dd5be93edc51d63a028013a0dc8288ccc9
35006 F20101123_AAAHYO woodruff_r_Page_036.QC.jpg
5f2a18c4aeb1d482578d237959195165
de68f8f0dd5519acff3910ae9289833698525b1b
35146 F20101123_AAAHXZ woodruff_r_Page_018.QC.jpg
374a2e98827aea1d4bb85a3b893bfb19
1156992528ed3f695ed258ed8201568dbc9b8135
138280 F20101123_AAAHBG UFE0011885_00001.mets
825a8bdd663ef8a450a0ce9600de3c4c
632152cdfb7553e23caa6e98ca705cefbd4fdd32
F20101123_AAAGWB woodruff_r_Page_096.tif
78dd709d6e15827123f050c28d3f7589
e4ec106de631e831708c3c997fb27f95ce778c7f
36874 F20101123_AAAHAT woodruff_r_Page_007.jpg
1825817116e1e5b93bedd61c239ae01c
ddc3fd320f18a4fecba161cc1a6e9c0b0e3d8f09
8117 F20101123_AAAHZD woodruff_r_Page_057thm.jpg
cc853cf7cef0ccac0d2776022cbc403a
9c7d6227d300da17d37d2b83a9a46e3daf18f9d5
58390 F20101123_AAAGVN woodruff_r_Page_052.pro
a22378d951898385d55df1b8fe26ad61
0154dea5eb36874f1f87bf6f31ac51571490c6e5
36415 F20101123_AAAHYP woodruff_r_Page_037.QC.jpg
33e4b210b6897bd16ba29b4d221ba27b
9ccc2466d79ec36fd7321ef9f2d392f57d4321f9
8668 F20101123_AAAGWC woodruff_r_Page_037thm.jpg
5bddf2f423474bcdf53c7de7a477065c
75f2745d752123bc2c679ff159e9dff689fc3e9c
118564 F20101123_AAAHAU woodruff_r_Page_009.jpg
34ebf405af1abdc87960cc3d0c0e04ab
7ebcacb25eea236a7f6fab67605a23cb35ff0ebe
7873 F20101123_AAAHZE woodruff_r_Page_058thm.jpg
7728933133b5c6d5024d2f6ec8958a72
6df59168476bda70b83eebe275dffbc0b422450b
1104 F20101123_AAAGVO woodruff_r_Page_073.txt
26f97c0c29b316cca2bc2d50d12f0957
f21e0977c8355596c69c32e727a3a55cdbfa6972
8330 F20101123_AAAHYQ woodruff_r_Page_039thm.jpg
2d0572c664ec176c85e18e47b0be510b
85f3fee1376457c0b009411b2f4a23c084eedec8
4999 F20101123_AAAGWD woodruff_r_Page_085thm.jpg
49538fb81e3ebdfe6192d6686c1a8d37
34af9af7966422fba93b6605d69448664db03383
97197 F20101123_AAAHAV woodruff_r_Page_071.jp2
7a6e8df54278148b1e19b6cc5b6f9254
7c5eab0608db675306af61fef8a4d7bae0ee834a
31744 F20101123_AAAHZF woodruff_r_Page_058.QC.jpg
5826b6108c8b4b6cdc9079821728cfdf
a28eb2007e41ecfe4261399777c05ebc657bea04
28327 F20101123_AAAGVP woodruff_r_Page_082.pro
eca62f9116f69cfabdd497b0f00efadb
f6bd8bb4eb78a4ef4859cdb38d141452c7bd3179
34136 F20101123_AAAHYR woodruff_r_Page_039.QC.jpg
8d7863a40363ebef259e62496b515973
4ca8ef16307e54014d1b6f7d372f2a337aecc4c5
5129 F20101123_AAAHBJ woodruff_r_Page_002.jpg
6ba1cc7fd6e0b3ea1c943c218bad24df
76c67cabdea095d764495dd18cba5a272c4a2a78
36496 F20101123_AAAGWE woodruff_r_Page_023.QC.jpg
d5e91570cbbc3b90814e7cbce0775bbc
895c52e6c613b06c11592832a821dfcf8774de85
7870 F20101123_AAAHAW woodruff_r_Page_027thm.jpg
05a5fec1aab733d7daee99b4e7cc4ce5
fbfb8adc4db6928be194e5f3db1d48efc8e095e7
8142 F20101123_AAAHZG woodruff_r_Page_059thm.jpg
80b0f9526e6632c6c21c547ea9c834ba
5eae9e9121a2b810c6a66bfdbce7aaa71251b542
2117 F20101123_AAAGVQ woodruff_r_Page_023.txt
62469c0e94af7f3e669a09ea4015bbcc
40cefc35ae4d70e458d4a75fefaee4fa51f7ed91
8602 F20101123_AAAHYS woodruff_r_Page_041thm.jpg
889c056c01c99140b5625eb277a0b6ed
698b0b2a99eb5aa5a7893064d892d2ff5d3f0f06
7336 F20101123_AAAHBK woodruff_r_Page_003.jpg
e52bb7f5c83df0fe3b21f065f125d62e
09f7b0a10ecda9367a92b6c0398e06799363f739
97375 F20101123_AAAGWF woodruff_r_Page_090.jp2
37a73ec9cce1c208f5df177d80ebad93
2cb1b0b0da4bc4c5ea30fd44992773a8e333e214
112164 F20101123_AAAHAX woodruff_r_Page_022.jp2
bc67faad3d9ad372a28c498d1b5ac78a
83e31f968946a94b9d554c94b67096b452eab8bf
31835 F20101123_AAAHZH woodruff_r_Page_059.QC.jpg
6040b07641a5639e8045a8c4c6d6c6c7
6f4fd2bc692d636a64143bf44025e63f666b073b
107460 F20101123_AAAGVR woodruff_r_Page_016.jpg
167a0385518b12eaa0badfe3481cab07
b9d04117d3fec7f0a63de7f0cff617569d74b722
8041 F20101123_AAAHYT woodruff_r_Page_042thm.jpg
d6bbbd7b0afffff1dcfb99fa92e57ab1
ba53a2d88ba5e57c3fd7830fe2bf708f2f7a4a5a
84123 F20101123_AAAHBL woodruff_r_Page_004.jpg
fbbe05f7fb91dc435eb30143c2fa5e73
789c511e0f66015eebfe434d22a66d07bd595dd8
121207 F20101123_AAAGWG woodruff_r_Page_053.jpg
ba5665fdebbf51e70f03f2d15caf3830
38fe15a231e2fdd5f6fd2c0b3ce2a611b0f94486
109355 F20101123_AAAHAY woodruff_r_Page_060.jpg
ece0e2fc151e3f9d5ab6b9fbc54b6d5d
f4b0349f7109d7e1a7452cfa51c578875c8f26b1
8680 F20101123_AAAHZI woodruff_r_Page_060thm.jpg
c00822997c188d4f6c06c72ef5cbf064
1af1f78b92371fdd24e9ef4265f7508f2321bdb3
8569 F20101123_AAAGVS woodruff_r_Page_044thm.jpg
2d10738c8be3dd48a75f6b416d1b60c6
a4ff31844e96c711859342b84f89eafa7d4fe8c9
35030 F20101123_AAAHYU woodruff_r_Page_047.QC.jpg
a3a4de37f46789d4623184bd68fe424b
5f48e82c24c6ea2e510a975bffc5f53f94bb843e
104044 F20101123_AAAHCA woodruff_r_Page_026.jpg
f8323d02ca66befe9a2a76dd166acbd0
dc7ee875ff0d8f4cb7801bd69f87fb01683190a1
108151 F20101123_AAAHBM woodruff_r_Page_005.jpg
b9ae59c9251280ff402349ab53195054
40647845e54f401ab8d986612195e80678b939a1
6814 F20101123_AAAGWH woodruff_r_Page_071thm.jpg
df4b250cbe31fe90973596e07465406c
19a804953f8f55ca340bce2449d7582edc7ceb78
114774 F20101123_AAAHAZ woodruff_r_Page_021.jp2
6e71b1e8023717cc9388f16fcb4c96a0
89dff7e51ffdb2038f60fb34ddc087bfdd1e67d0
35640 F20101123_AAAHZJ woodruff_r_Page_062.QC.jpg
ce142c557113e653e452db64f25e0487
3984eab9df67733989c8d8e7d216e666f22390d9
47470 F20101123_AAAGVT woodruff_r_Page_042.pro
d6ff2ba667cc8f7f6d026823fbd59e3a
6f92b35f6ad2cafb69ce65ee86476d4bcb49ca72
30755 F20101123_AAAHYV woodruff_r_Page_048.QC.jpg
eb917a86078e4fe3b5d70ca2e19f1005
c7473c101137df8e02dc7a3726fa88c38c4801d1
84575 F20101123_AAAHCB woodruff_r_Page_027.jpg
6c1a7e11f59e77bd4cb11e1f081aacb5
385f6513d5eff99e092aafd59130d204fceb6eed
163022 F20101123_AAAHBN woodruff_r_Page_006.jpg
191910ed5c483b968c9f6fd88c6c0983
4c0ce9b95d9a08b7a2771ca7d6711610ef109f2d
1051979 F20101123_AAAGWI woodruff_r_Page_105.jp2
9b136bd12ae0ea83b29153e59498dcb7
485a6a7c1dda91930de2c31c6a6f0c210887d887
33749 F20101123_AAAHZK woodruff_r_Page_063.QC.jpg
b1b6d84b777b7d815cb608be50da62f6
7c2c43ed635894757a73dba4b72434dcbb8ed83e
88217 F20101123_AAAHCC woodruff_r_Page_028.jpg
dad84c5157225c1eb9b7f0beebcec7d8
f637eeb8da2842d9b1151106ca732f86d86374a4
94572 F20101123_AAAHBO woodruff_r_Page_008.jpg
0c80e05a15797236d92c8b31e6fea6b4
c71a634686c6da15f873e338cd617624bf247416
F20101123_AAAGWJ woodruff_r_Page_056.tif
63f10dcd0011c7b928af1d9d441ed178
b094bc26872682b6b885fb6f4c594db8b68bf27a
6898 F20101123_AAAHZL woodruff_r_Page_066thm.jpg
8e30e16317295089d21cc06ab1528b2a
a13b148d0256a075dbc2a5ddec58654873a15d76
2122 F20101123_AAAGVU woodruff_r_Page_008.txt
0997338d9cca718ca9ae72984124ab4d
a5e7191b8474aee99e5aa267147cdd241fcefc8a
37168 F20101123_AAAHYW woodruff_r_Page_049.QC.jpg
7c055ae70b29ab16465526c1c1b164e7
878c7a5f23dcba97f1c7e370c3dccc82120a828d
103747 F20101123_AAAHCD woodruff_r_Page_029.jpg
b55c7520070e4f6ea7f50107502c8a74
d8d806c2815089b414700a97550593a77e4ba382
77421 F20101123_AAAHBP woodruff_r_Page_010.jpg
0473bd78b50c1b57ec2cfe4efb7ca765
659bf86c5fe6f876eea0c4574c60308b14b62b55
35237 F20101123_AAAGWK woodruff_r_Page_100.QC.jpg
c6f74f8962103523c6fd4e9549704a13
684c95944828dc325bb2c58e9b3b359ff3505b3b
29423 F20101123_AAAHZM woodruff_r_Page_066.QC.jpg
686ccad93d2f91cd8efd53f03a467706
f9c68d75d54f00439af4490e03513384d64e107d
1620 F20101123_AAAGVV woodruff_r_Page_004.txt
5fddc89e5a7307e721856f2614c4c81a
dc4d48121e5c39ebccbd1c4abaac515fa072ce60
8400 F20101123_AAAHYX woodruff_r_Page_050thm.jpg
1e1b6f74ccc578f0e19dcd3ec47599c1
4742fe282ed6532c680ac98fa08710d9d0748629
99545 F20101123_AAAHCE woodruff_r_Page_031.jpg
7a1d25b9b57a0ddadbf112cc2daf5485
7c32fe0ba0489ff02b7354e7f64c2593cbdd3a17
86776 F20101123_AAAHBQ woodruff_r_Page_011.jpg
da0a84d3f7e26eca1da341f6fc02cd8d
0510492008c6e2325f364d582afd4e31c0a76381
13871 F20101123_AAAHZN woodruff_r_Page_067.QC.jpg
4e038eee870be951c5910cf9d96b93bf
7a10983798a1bb522e33f1d48ea585c9b66b6509
2441 F20101123_AAAGWL woodruff_r_Page_007thm.jpg
457e1dc47318823f6cc4f877586282c2
d145c102c22d724f8c47979774688016654fa571
107761 F20101123_AAAGVW woodruff_r_Page_038.jpg
516e643c3cbd8e91a8a650aa9ad39201
90bd4cd36cdee55ac1b6b11da0b4703cb80d56ba
31828 F20101123_AAAHYY woodruff_r_Page_052.QC.jpg
2b4d30d121d6827a0d3a3f6ca8dcbfb4
e67cb996ec63039571782b7a1474624a2f121c7d
101160 F20101123_AAAHCF woodruff_r_Page_032.jpg
6996c0c78dbc37fe6d44477bd6e75119
37241913328d2246a6bb268cc72641d5241f52b5
88902 F20101123_AAAHBR woodruff_r_Page_013.jpg
aa1eaf5819e0dfb9c770edd5d69a6513
d08bd26799d923f504bbe170d7874a5f5cd5f106
4136 F20101123_AAAHZO woodruff_r_Page_068thm.jpg
c426908a5b8b0e904b33757625afa704
1cd0b54c0d4888ed68e7f5a1b54f35a33ea8a285
125718 F20101123_AAAGWM woodruff_r_Page_109.jp2
3c7b9fd42f2ccd8e32c45d108c2d5558
3ccc5886cafc5087c2939cdb28f7194d5899aae8
112750 F20101123_AAAGVX woodruff_r_Page_100.jp2
637c10af7c52fc6a544928bd10d3eabf
90d774c08db128fd4f193dc25a419d57c0c12858
8486 F20101123_AAAHYZ woodruff_r_Page_053thm.jpg
ab90be7c0b0fae1ec9edede9c30bf9be
a69240955087c009c5dba5e143c75c092b1bd4a6
102831 F20101123_AAAHCG woodruff_r_Page_033.jpg
0ab7b417f774aad824c6f2a3ca0e0d86
59dd83aded201efb0a45b0c63115491a34866c3b
F20101123_AAAGXA woodruff_r_Page_054.tif
3fd73d219f16c7f6a7acfb45af4fc4b8
f474d3bf9d1fa11a2dc2ad9a3e5448395c92ce54
106773 F20101123_AAAHBS woodruff_r_Page_017.jpg
bea4cd9364d4fd527166242d68e60ff0
c98e1705060925d67930811542b23b9cb377144c
15266 F20101123_AAAHZP woodruff_r_Page_068.QC.jpg
c8d6b12104075aa2142a69b1361459ec
313e5b8a271293717c94172d945ec20dcb79b6d7
1917 F20101123_AAAGWN woodruff_r_Page_032.txt
5379fe7ec5c7852d32c67cfd55778f82
2b076373dc70fe16212557da66a9bcc2d7c85558
1291 F20101123_AAAGVY woodruff_r_Page_117.txt
e8a507b2824031f95ead508939fb321a
9cb14c384197638541f97543e5216b3f8de154e9
94902 F20101123_AAAHCH woodruff_r_Page_034.jpg
0fc1c53b599d5b94246433aa33170a48
d817c2af8879a59506c7d313d4f62af6dd7367c1
33046 F20101123_AAAGXB woodruff_r_Page_102.QC.jpg
4fff8cd343f43a229578e94b7c481a3e
56e3cb23bd890dcffcb616817187fa6bbc0b6046
105924 F20101123_AAAHBT woodruff_r_Page_018.jpg
7647beaed12c0a391777cb166f6ae604
9daf294c19ff8e0f1dd39701c5a62969e636fa3c
27908 F20101123_AAAHZQ woodruff_r_Page_071.QC.jpg
33cd280d9b063b5a37b09db995a2b245
d8ae8dd6573fec7ed9701fbf8d3bb11d85750884
6015 F20101123_AAAGWO woodruff_r_Page_082thm.jpg
76d2c1fd0b68baff7fcd1012bd29001c
957b6e87c7463825bcdbcf9b7ea9d6500352391b
8791 F20101123_AAAGVZ woodruff_r_Page_040thm.jpg
267c5bc5435ab6fa3a8f36b1e54299d0
ab5b3d41bcef52b22c8cdf2733c3601d8818fd67
106538 F20101123_AAAHCI woodruff_r_Page_035.jpg
9ac4c9a6b3b64e8db217c98183e9a8ed
c623d328c352d6a0a914d2fd8775637ff467871d
F20101123_AAAGXC woodruff_r_Page_100.tif
66d3be5fa85bd2676a92ccdb51ecad59
d4766bfba36f54cd7a13762a63030164ceee4dc7
101611 F20101123_AAAHBU woodruff_r_Page_019.jpg
791659d99ae3f6d42e14d39798e3a24b
69e4f0fd8f25abcc8051ae84d5ec9ce43a54eeee
26970 F20101123_AAAHZR woodruff_r_Page_072.QC.jpg
06b90fd51eace0c9b64b78ffda64e883
391641091f221af23ba1a994d50fe2fbce4d055d
8232 F20101123_AAAGWP woodruff_r_Page_033thm.jpg
e64d390359f3839254b2d22e82bb9195
3f7dd6adb5fe9be18528476de26f47d254cf3582
109631 F20101123_AAAHCJ woodruff_r_Page_037.jpg
c0c8c6bb2c6a827a4f5d0dbd9783dcba
1f7e94cafe2f7e9addd6e99944647548f9dd31f5
9031 F20101123_AAAGXD woodruff_r_Page_007.QC.jpg
66006ffb2ebc9a5b5d609da484f27b80
6bfe73d9085ad863b719b9bf6211e25c8bd96c8d
100371 F20101123_AAAHBV woodruff_r_Page_020.jpg
7ab9da9f77294455c528bb987653ac06
f703c3323f7a57567060c174e28e38ad2d094e87
4578 F20101123_AAAHZS woodruff_r_Page_073thm.jpg
32ce013be4364c4a5555c124b6ba3165
745c53b1949f2d88530ad25f8df190557ad43108
106528 F20101123_AAAGWQ woodruff_r_Page_092.jpg
b70824e75acb91cecdeb8d8052fb1a71
6d12f677126bcd97094796fa53c0b05cf1b6db91
102925 F20101123_AAAHCK woodruff_r_Page_039.jpg
d75f8386c8296b32ec5ac008e38bef5c
c89bb309f256dcf36cc7973f8d671ad4cd9740a7
25265604 F20101123_AAAGXE woodruff_r_Page_078.tif
a54e372ab23e28f47184a7e5849c2e7d
4d35c82a294a27958f3d3dc0b9803dd0d7b6663a
107418 F20101123_AAAHBW woodruff_r_Page_022.jpg
91d4673f8a9f4270847fc973c468ed2f
58ffe7f41fc7c7f547e89f80eeb6657f624cfe4e
5258 F20101123_AAAHZT woodruff_r_Page_074thm.jpg
97d34e477d5ea5f36ed095970a66c0ea
877387beb622520c3d677e54f3952577e3bcb915
1054428 F20101123_AAAGWR woodruff_r_Page_055.tif
83b824e897d2ab21112464542341de7f
e5cd09c82434cb58d07b373e325a7d4b25b5886a
98201 F20101123_AAAHDA woodruff_r_Page_057.jpg
a6f5cbf31e613cc947bb1c77b98d29a7
211b08a7cee6aabcb9d1c5ab41a1c35e928a2db5
109331 F20101123_AAAHCL woodruff_r_Page_040.jpg
472177f96eca07af701cf735d0845fa4
6d9f6f2639f4cbac136d42b5a05c200c1bcbd437
8319 F20101123_AAAGXF woodruff_r_Page_032thm.jpg
898262b22ff12952e10c5e629b511027
c91bafb17755121b39260374ea2bc37aa64e4e23
110172 F20101123_AAAHBX woodruff_r_Page_023.jpg
a3e03d0f33b7749b270bff499db99cc9
4d3ee54b467af4b13c3f2ddf1650efcbcfa96339
18276 F20101123_AAAHZU woodruff_r_Page_074.QC.jpg
358128ebd9ec77bb92f99c4cc154e727
782f32655ece9e987c9c338bdcc73766b7d93071
31168 F20101123_AAAGWS woodruff_r_Page_001.jpg
1e9a9d4a67e20fffe0f2f388b988cf5f
51719eca19b0ec8113e0c2816fd1d50c0cd819ca
109744 F20101123_AAAHCM woodruff_r_Page_041.jpg
b42bdcb9b73d7865f92ae3d58497b549
983c6a918b4186ce4f406b1aaf8738d850901bd6
35794 F20101123_AAAGXG woodruff_r_Page_016.QC.jpg
f3ccc223198f774c7a58a01d08b37b25
86376cf689b7271751bc2d499dbd4d6430657dcf
101775 F20101123_AAAHBY woodruff_r_Page_024.jpg
3ef7e723353c3d81347f2c739f1d7c49
875a19f1498e1a13c103b50fd69d0ed769b1de6f
5120 F20101123_AAAHZV woodruff_r_Page_075thm.jpg
343a761588c90715708848c4f8958464
3c0a5faa68a7d8cc414329fff54d76b281d2f0f2
1932 F20101123_AAAGWT woodruff_r_Page_020.txt
8400ac602eef27736f1f5a05cdcfa8f7
77cc1980321e567fc352c682891df37429ec7063
95132 F20101123_AAAHDB woodruff_r_Page_058.jpg
c68173bd149b419524076e627a9fb876
f8cae1114659779295fe9594363a4ddd98dc8296
98819 F20101123_AAAHCN woodruff_r_Page_042.jpg
cc77785d9217f981879f65b040ad1425
0afc0570a865045d3e9b2d74f8da22ca0ea075d3
109006 F20101123_AAAGXH woodruff_r_Page_021.jpg
cdc280fa7e6cd3694882c95d0112b468
dbfcd283903302995069f09eb2457e20d3348481
101325 F20101123_AAAHBZ woodruff_r_Page_025.jpg
cb2e03792e14b93813bc5d594b7487a5
3f7bf927148d6f1e3ebeaf144b7dc63cc1b055b1
4519 F20101123_AAAHZW woodruff_r_Page_076thm.jpg
1b5e833de71979ccd62fa5cc4f6b3619
e5853feb6f80666583499ae983f36a49ffb5d0e1
517 F20101123_AAAGWU woodruff_r_Page_001.txt
3efaefcbf2c4ee3216c5840c6760d00d
7f71f8572aa0f6fb3c598e6e5c9d8d57ff190940
99451 F20101123_AAAHDC woodruff_r_Page_059.jpg
01b8f4c5489e3ddf3c6cb7ca46a5cf26
c4d6c11813cc067df1e73b6972a8e1d911f28a61
109232 F20101123_AAAHCO woodruff_r_Page_043.jpg
eb18022bf975d2b86445beeda8b99b74
52f11165184e0a3b428076d67909ddbf5b1ba43e
F20101123_AAAGXI woodruff_r_Page_099.tif
b1cfe311c35937a75724312daafecbbc
0da284954437b665ffcc07d2e16fcfa70e711acc
100509 F20101123_AAAHDD woodruff_r_Page_061.jpg
79d4bdf318979f83c16e3f758a1563f8
ac15f54029e84320136e50065f335e12dc9a7e3a
109366 F20101123_AAAHCP woodruff_r_Page_044.jpg
98b208a66f8fe5bdd41675a136f7d2e5
dc19ac458947c5c3ed458a2f556d6a9166258ff3
64180 F20101123_AAAGXJ woodruff_r_Page_110.pro
057faeb58c99dccd41588352837ee8ae
a7a42238f560998d4b941e359781105c0e8d251c
15746 F20101123_AAAHZX woodruff_r_Page_076.QC.jpg
9f95482c1d1d4e8f068c85cf70337f33
1fe23699cb06c36158601c8467454f941b469dd1
48862 F20101123_AAAGWV woodruff_r_Page_098.pro
e7ed4700ac72fd10f45a5f4e69560048
dfec95c6f5e393d2320ab170c67c1a5db6b393c9
102936 F20101123_AAAHDE woodruff_r_Page_063.jpg
ceaec6fa1fdf5622755497e36d7b9563
9981de9448d985bf4e631d14bb83206d1b5fd957
104622 F20101123_AAAHCQ woodruff_r_Page_045.jpg
4316daea5ec1115e013a884b1e028c6e
1cb80e71f0542e064d31df0ed05d572e0a9f3f46
25271604 F20101123_AAAGXK woodruff_r_Page_074.tif
f0a8b234ddab69c774a2fc291f22a773
0e3d1250fbbc7b2c17f67fdc829a76026e79bdcd
2537 F20101123_AAAHZY woodruff_r_Page_077thm.jpg
4c2da48b9d00a8fb966720bb1caad995
382474f3ef505d0bbbc11d2eb13639419472661b
338 F20101123_AAAGWW woodruff_r_Page_054.txt
e0e99bcc9a6cb6920ad2fce872454fc1
78d518831853df0edc9a1068284a8f9c05e2a659
103332 F20101123_AAAHDF woodruff_r_Page_065.jpg
f817a59cf01274d30f094aec674d4d9d
4a7253c5bb816a05517eebffa2e5e1113aa36842
104635 F20101123_AAAHCR woodruff_r_Page_046.jpg
ef2fe31fe57960bbc7f8be13933d4a62
c997e28884038e4c7b3426498bc60ee04aab9009
104248 F20101123_AAAGXL woodruff_r_Page_103.jp2
84ee876086e09ac37feb118f986cdc54
e5eeec0ca3170e23322846dc4d858fa647aaa01f
3031 F20101123_AAAHZZ woodruff_r_Page_078thm.jpg
961946121d74510e26f7fa80aff998c8
fed6df3b791f1cc6d183ce53c91fcb6fa7675e77
8227 F20101123_AAAGWX woodruff_r_Page_003.jp2
d823134144897e9ae6c15a71180d3f44
8c52e08a1730a8452d67ace6a35b14af2955bb91
89329 F20101123_AAAHDG woodruff_r_Page_066.jpg
ae7e6d6129e57f6ba5c8a73b34fc4a2c
ea8a5dc8245e5f0adc8a3a52a7466929b5d3b3c4
51831 F20101123_AAAGYA woodruff_r_Page_044.pro
3cf924f704d71846071047974162fbec
5fc17302bfc2b2dd4777860f976c50fd35a2b5bf
107055 F20101123_AAAHCS woodruff_r_Page_047.jpg
7b740d592ec4673acc41fbf92de648f9
cde7b9afe5cce53217436cf1fd337ac683f5105f
128901 F20101123_AAAGXM woodruff_r_Page_109.jpg
7bd421704945a74500b7289a6d4df593
d0e1c12d2a7a5c0fea0e4b8377fede10880e01bd
35129 F20101123_AAAGWY woodruff_r_Page_035.QC.jpg
ecfc6bb33516b2cd9461a329efb3d82b
57a944bda383cf162e6988045b74f97f48e1e18c
45365 F20101123_AAAHDH woodruff_r_Page_067.jpg
61ad021361d0f18057e8d936ebd68cbb
25b080ac7affd85a4f44d29b9d2a4a7fc31b8419
F20101123_AAAGYB woodruff_r_Page_112.tif
6877451c6f34dc93b6bc68baef628026
b803cca7475a5563b7ddc278c26fcabbb0b2d887
92978 F20101123_AAAHCT woodruff_r_Page_048.jpg
88c70b63768ac199600fb28c81541485
4d10bdc02c61a18fd0da93ca7f4c80b0a14aa3d0
1751 F20101123_AAAGXN woodruff_r_Page_058.txt
010eeb47ce2110f427af113f125a0ff0
0f7040156305ed3b93113a28ffbabf261772e7ee
5873 F20101123_AAAGWZ woodruff_r_Page_084thm.jpg
71aaddf581ab6fa0c54a8a0e63fcfc01
fe5dc43b78dfeba0f910278f18db323658e9c939
55666 F20101123_AAAHDI woodruff_r_Page_068.jpg
89b97852ae37fd00b6c9fc7e9f932605
720cd1e6d070ea50f3584965495a6fd42d3f45a5
2118 F20101123_AAAGYC woodruff_r_Page_016.txt
1106c8cc38b60928c58799a391dd016b
3421296c9f841877496231542cca87920da0e3c8
112710 F20101123_AAAHCU woodruff_r_Page_049.jpg
e8be9b07d8a34a06077ecd3209437895
677d15f27d62c1382c4c9d738ba2a9b0b387013c
3004 F20101123_AAAGXO woodruff_r_Page_072.txt
a0c680afde79c6b8ca87cd9409b0940a
fc3dcea7e6673ac2fe820ffeeb0a3cc389ae2cf4
19983 F20101123_AAAHDJ woodruff_r_Page_069.jpg
be2fc9435a32454dbd5ea81042eeee22
a9862c15581af8fcfa10496f507e2dab8e37c812
4323 F20101123_AAAGYD woodruff_r_Page_079thm.jpg
a318bfe8dac964e380b4299c76529889
b30e5b3793ca4ffacd865b875c6dc4f0dd285b17
105822 F20101123_AAAHCV woodruff_r_Page_050.jpg
87fee5f95d973b8e0a3bfe68ae278ff5
90cd99d303d8c17722b73afac60d95deaf3f855c
F20101123_AAAGXP woodruff_r_Page_091.tif
f7af90423b7b2ffddd9237ea742ef1c7
310a013c0313f9b2681c0b0e686d9f2af34cd79d
49151 F20101123_AAAHDK woodruff_r_Page_070.jpg
efdd7092d714093933c2941c01e3d684
8eee2492307a122d71129ef0ade559c75c9267d1
24461 F20101123_AAAGYE woodruff_r_Page_106.QC.jpg
ddb61ee04bd4abac8a68262ab62d3c9f
6525ac89bfa2c185457d6573a5693c7187239532
89147 F20101123_AAAHCW woodruff_r_Page_051.jpg
c0a24ecb43834e7c004915c5046c5d98
a570ad85c213c8d509eff65009d4f7a09b70fcc1
8837 F20101123_AAAGXQ woodruff_r_Page_114thm.jpg
ac804a79b9a887a72a4da01b79a47eff
c8a107cbb264c5cfa8fb8112970d292b4d42c5e5
93082 F20101123_AAAHDL woodruff_r_Page_071.jpg
5fa53cb25b45c4ab8ccf2ec6f4401f1a
e28e2376a003ce7dd6b0388696085e24f71cecaa
146 F20101123_AAAGYF woodruff_r_Page_003.txt
294794574ac58a912180cf687575608a
f2fcf854fdb86ed8ef8bbf44be237bef7427baf5
113013 F20101123_AAAHCX woodruff_r_Page_052.jpg
0d28215b8522a99133061a0e06747e9e
b87ab24f7aff58ead12fa3f7edbe0c0d4dcdc98e
8080 F20101123_AAAGXR woodruff_r_Page_065thm.jpg
309f0e597441cd52808c266b310a5602
69ea6b32ec27ef59f091187dfd1ec5eb3f9b1188
70294 F20101123_AAAHEA woodruff_r_Page_086.jpg
7cc8ca9e72fc6b4aeb014b91c40f92a0
47a1a9327f4d53746bfca0e1e9eb7ed59f894440
95437 F20101123_AAAHDM woodruff_r_Page_072.jpg
869b7ed307e612d5666669ea8cd3b1a5
8b39d329079429e2aff81ad5d5804c0b9c9c8f12
8909 F20101123_AAAGYG woodruff_r_Page_115thm.jpg
e858df1aa92a1bb9d6f0eca4bfee7687
edb183a7a2d6433def6660079e420571d8e11269
20957 F20101123_AAAHCY woodruff_r_Page_054.jpg
888351975f602436b2f514ad31381348
51dd0584a7edfdcdccf4ff53dda6a121c03f84d8
5805 F20101123_AAAGXS woodruff_r_Page_086thm.jpg
43f356f60e07f924b4f0f2a14497996e
b0396fbf3e09e69cb01dcc2f11168683e4283ffe
46513 F20101123_AAAHEB woodruff_r_Page_087.jpg
1c9cbc78877f13ff36e99f653a83cfdb
bad71dcee390d9b5d83bb004d0e5497a084faef6
61022 F20101123_AAAHDN woodruff_r_Page_073.jpg
3b2325c4dde853a2a3dcbde4848639ca
5da1234e755387e15f8d759608eafd49bc2a225d
112620 F20101123_AAAGYH woodruff_r_Page_041.jp2
698f3dcf39bd09929f658966b2ab595c
10940cb27a38e6647372fefbb71080588825f6c4
20627 F20101123_AAAHCZ woodruff_r_Page_055.jpg
4b468e746a665980a34bb21a54e1cd6a
56bee6c54304468f78553709ec6eea849f4709c1
3925 F20101123_AAAGXT woodruff_r_Page_083.pro
d21698463e44304f665a6c2819fb7c21
8aa1d94d045bd377261d1a84b9228914d7a4d746
65566 F20101123_AAAHDO woodruff_r_Page_074.jpg
4ef7d18ebf6472a1c851ffc27a480970
bc0269f3e44546dbd6d09f914601a85b9a930653
F20101123_AAAGYI woodruff_r_Page_052.tif
0fa2b9deba10fb23305f7738c8c82d70
8e2b9c722e7280a22de0e192940d26d7cb54c3e6
1922 F20101123_AAAGXU woodruff_r_Page_093.txt
22645e39d09f53f3b88db670925573f3
8013cc89870ceade05e39eb6c2330ad514db0150
70781 F20101123_AAAHEC woodruff_r_Page_088.jpg
ae829e5010339af5063aa5cd9de2a943
d3c7ac2d47ff58420ec25df084cff71d9ae44cc2
65838 F20101123_AAAHDP woodruff_r_Page_075.jpg
f7cfcdd09cb474e94ca31232473595de
8f2171c3e6153e386643605f3bea272f7b8aeacf
109288 F20101123_AAAGYJ woodruff_r_Page_096.jpg
476c005215c3c1897f2ae8b7efb0b144
2f1179eb420102c87303b9627823a58fcda5b09a
3780 F20101123_AAAGXV woodruff_r_Page_067thm.jpg
6639f41bc8a6d0ca2c1b35daeef76246
d8219b3b877f1ac51210c636da4cb2d2fd8bf47c
43467 F20101123_AAAHED woodruff_r_Page_089.jpg
c093daffa2b7bf9eeb261f7fa69a05b0
ff78fd4b1ad0ee1f022a6f58de00471898640c6f
57341 F20101123_AAAHDQ woodruff_r_Page_076.jpg
0babaa8594ea2aef389c1f8fd2678b4f
e242dbd72a2a410215b69a421a4dd8697786cc0a
1013 F20101123_AAAGYK woodruff_r_Page_074.txt
c650761106633a47d430ba8443673ae6
9e2b3cf0af38ce1ec04895110dbf462b8a237071
92453 F20101123_AAAHEE woodruff_r_Page_090.jpg
4503fc3b572dc75ae63124a07378f005
f0ffcb94a744cba10f7ff8c1177f8736bc57e6b1
31748 F20101123_AAAHDR woodruff_r_Page_077.jpg
6f39cfe4e04de788424feb55a592a008
e4c14c997c2697135207930d5e0e5d956d8f01fe
2318 F20101123_AAAGYL woodruff_r_Page_111.txt
7dd64be09f942cfd73a11a93f4844bca
529dc4ad2d09942971f0d32ac2515b3e5e9b43fb
36982 F20101123_AAAGXW woodruff_r_Page_113.QC.jpg
257ed93755fe3aab6d80786a24d92ad3
7950392983e98165f7d4c95821875614be0051b8
106729 F20101123_AAAHEF woodruff_r_Page_091.jpg
2def97d7a1cbcbb7b540703aca93ac7b
831383e59491c00a4c1053f0e830ab01331d1d50
44964 F20101123_AAAGZA woodruff_r_Page_057.pro
123aefec51017b7babb88ca4d34060df
aa611e7a99ea21099654797d37eb27f6d082c7e7
53430 F20101123_AAAHDS woodruff_r_Page_078.jpg
60cf662ead404a78e44352d7f9e77a8a
02a49262757241ee743cba12e2b5a36eb22eeabc
112422 F20101123_AAAGYM woodruff_r_Page_038.jp2
2a5f4d828162e6f77293e5cb50d2a5a6
d3cc82c4474b2a9d2c06561b266d3b24b9b2c42d
36235 F20101123_AAAGXX woodruff_r_Page_109.QC.jpg
ddf5a06e1adadf2da5f4453373621be6
4c7c8ad0e163dfa96934eef33ebb4f8b18b787d0
103650 F20101123_AAAHEG woodruff_r_Page_093.jpg
b6929b22ab20869153f2e38c8ebe1640
4ecfb1412ecee6d1a85211587f0b693842e7bd40
25451 F20101123_AAAGZB woodruff_r_Page_073.pro
7ae2e7b0692e34a1b3d7e40f44d8f5be
7ec866b80b5f530741bc58125c805ef8ca3a1c15
48148 F20101123_AAAHDT woodruff_r_Page_079.jpg
bf9e381f4467b3d6e912896422ecc2e3
643d578660783a527e89a0aee25c5a9dd7f31f6e
F20101123_AAAGYN woodruff_r_Page_114.tif
52c30b07cf49bb8420a4857dd6a518c0
0476acc1342dbd0c63b10c56f2ae095a7d731620
F20101123_AAAGXY woodruff_r_Page_073.tif
8ed109c14df1f71519c4b89224c5ff85
140729dd96c9aaf8fc98e97eddae0679bbceb8ed
102653 F20101123_AAAHEH woodruff_r_Page_095.jpg
8f66c2e0517442cce74b95a224112061
be162e7928763c039622407af16b5e2290972d88
77234 F20101123_AAAHDU woodruff_r_Page_080.jpg
ca946d747b489c3084729e048ba98e94
32046aa386a93a30b859fc7068e6db3259f79e63
108382 F20101123_AAAGYO woodruff_r_Page_019.jp2
79e685a001cf51bac6bbe44b8cd1d15c
245116d5198ba29d75fb9b69a8a41b40568c5d29
803 F20101123_AAAGXZ woodruff_r_Page_084.txt
8c06f01398ab1ed9b6d54e41418840a7
c1875717f7da47233e0e111156b7183fb76dc549
104674 F20101123_AAAHEI woodruff_r_Page_097.jpg
2a6e6a6b4424a18c4c291a6d19fc2e4c
0f140f0c3a5b816beab0d8ddb33476d5ddd861fb
34499 F20101123_AAAGZC woodruff_r_Page_092.QC.jpg
bc1d62d53a74b8190595bec1c93457c5
0736dc809a5060f66f1dff52e6fc4c0a9f99b278
64839 F20101123_AAAHDV woodruff_r_Page_081.jpg
e53bc495d4a43ec23089d4f862ddeb9c
4af899b98604c77b6450834a041a1f3093a52db3
98122 F20101123_AAAGYP woodruff_r_Page_064.jpg
4a659ca4976ded9a4e1889c02b3a0aa8
12099604aded584243af1dd7515e884224e3c1e3
103591 F20101123_AAAHEJ woodruff_r_Page_098.jpg
28884b14a620a7197718a138c29c71af
9676f7f68a12260cd50625a19add869389fe8674
34373 F20101123_AAAGZD woodruff_r_Page_116.QC.jpg
2bbd1f64b2c061b4bad1d7046088846d
d847d35e6133eca01514e28e4dc4b943e1cd627b
68914 F20101123_AAAHDW woodruff_r_Page_082.jpg
e4cb112bfa165de80c660ef2f8c8fc04
b9c473b7c8aa1ebff2da00f92c7d4cfc059b9427
106150 F20101123_AAAGYQ woodruff_r_Page_020.jp2
7e3c15c7bdb965bb67d013d86208d622
5a712e7ce5dbfdb1243fc66b6609c9f431d4a432
97032 F20101123_AAAHEK woodruff_r_Page_099.jpg
2da8d7a9b67875790f9f1bbabe2b2f86
fd44415da1c5d8a9d279074e4ab4bdfe70b03537
8088 F20101123_AAAGZE woodruff_r_Page_006thm.jpg
f1b5697acf5906afd7e36ccfe00add09
7bd367789a0b4f94af446d7045ed44f002bbd7a3
43245 F20101123_AAAHDX woodruff_r_Page_083.jpg
6478bd2a0816aaf14c7e40818f9fc9df
4a8d877545835f50630bcf0f898eed505a1baca7
8590 F20101123_AAAGYR woodruff_r_Page_108thm.jpg
97460150e15d9c120035530939a2be6c
32896a96736ed7e6bd0e10ad38f4f1c65e7999b5
70296 F20101123_AAAHFA woodruff_r_Page_117.jpg
4c034abf7b83e3d3a0916f104af4fc0a
fc007096e5147585119ae6abbe8eea61c6cf56d4
107876 F20101123_AAAHEL woodruff_r_Page_100.jpg
dbb67eead4f48b4822e8f9a9b7942ed0
7cd399a89cde83894a7172808620a742f4d5c8dc
1150 F20101123_AAAGZF woodruff_r_Page_118.txt
8db584cfd39e9400e37fe8c324247af7
8ae19439d01fe722c5f370c7d966419baf47a9dd
72012 F20101123_AAAHDY woodruff_r_Page_084.jpg
899987235c78003b1d25c23066e92e33
c754c9fec7ac57a674a9a88367d4ab20990dd4ff
F20101123_AAAGYS woodruff_r_Page_117.tif
257139d6868f0b2e5d129dec4a4b692d
a7efd4288f3c2d3161d9b0151c123a3b62795d6b
61287 F20101123_AAAHFB woodruff_r_Page_118.jpg
6cc233a6fb5bb54db2554327a4816cb4
10571050befe47597ee9fa5e0709f8167a72d9ad
103524 F20101123_AAAHEM woodruff_r_Page_101.jpg
3b27b45c9d7a4644709087f61e0b56eb
41e5adb279c7475f27d4266735a5e4ede679341d
8599 F20101123_AAAGZG woodruff_r_Page_035thm.jpg
bd2d77f22b0a11305d975c9ee56b27da
8145768e244ed215a0e0b31de7df27db95d8a445
45048 F20101123_AAAHDZ woodruff_r_Page_085.jpg
8374c00b5212840ef6ccb860e884fe6b
1cd1b0d707c4d2d6d88ec55fc5c8c76b48823be4
49839 F20101123_AAAGYT woodruff_r_Page_062.pro
b89341f093ad18897e0fd24d9f8ebece
2a7f35fdb3890cebd6a071f48a541016cc82d7d6
27803 F20101123_AAAHFC woodruff_r_Page_001.jp2
71b81d5b57e61f55f0df4ea2c3cf18fc
6fd038e731b168ac3cfffaa777376f7cb92da330
102624 F20101123_AAAHEN woodruff_r_Page_102.jpg
973e5d0066a393626da1b3d50aa5de0d
55da058ed968b26f89d335172551c96dea0de2be
33126 F20101123_AAAGZH woodruff_r_Page_061.QC.jpg
e2099301b5a00836aa4dd61dc2e0fe0b
8a87900c92ba5fca7f2cb215ed9db16feb772cc5
6781 F20101123_AAAGYU woodruff_r_Page_051thm.jpg
a45dd3a8f50a83532eb62e9e4aae9708
3f7c0a1b336dc14a6d1dfd0393c266a596bc610e
98504 F20101123_AAAHEO woodruff_r_Page_103.jpg
755be2a073847ed9b356768f365bc53a
412e5613e958bed65b755941f03b689b138f13ab
107567 F20101123_AAAGZI woodruff_r_Page_014.jpg
40b6c3ab1d7b31eeb317a19301ffa4cb
2f23ba8c35ef51dcad6641af3cee216a4e36aa7a
34775 F20101123_AAAGYV woodruff_r_Page_050.QC.jpg
b2a658598e7c1a0550e01069d9f765f8
24b133e94f1949e336aeff4661fe78be00c98193
6152 F20101123_AAAHFD woodruff_r_Page_002.jp2
78155b4684bffefc4149f540762929eb
5114cafaf2213a788d91c91d4697aded027982c3
101511 F20101123_AAAHEP woodruff_r_Page_104.jpg
63dfca8b9231e352f52f112550b52f9d
7571e6630489025ac4bc3cbccd60724e3de8aaf9
F20101123_AAAGZJ woodruff_r_Page_110.tif
4ac3f0c5a00b9f29bf87a983b3b96f7a
a93016760e3c4329ad1e902000f3b142d5da9360
F20101123_AAAGYW woodruff_r_Page_016.tif
7560186e66c1d3cf2a9a3aca97be294f
55d8bf21edc32c083340d63961da0803af86cdd9
88320 F20101123_AAAHFE woodruff_r_Page_004.jp2
e94787422e23e3b3e0e60136caed9ad3
11b990ffe46e467314f88edac57cc81f7f041dc8
93836 F20101123_AAAHEQ woodruff_r_Page_105.jpg
1263601bc5d782e412df05a255878855
4f92024c49d8a4d5ccf191012e1771ee1d3a28ea
33572 F20101123_AAAGZK woodruff_r_Page_042.QC.jpg
440c73d83e79eb6f940cf4b824ed99dc
3db60a95b5df128e3ac094019dbe6e54e0eb76e0
1051983 F20101123_AAAHFF woodruff_r_Page_005.jp2
748698f1a04c6c11cd982a84634c1697
2e15889747d574a5b1042780d6efa089d57a8a80
82237 F20101123_AAAHER woodruff_r_Page_106.jpg
97cb4d0cb10ea231bb5ddc5ed624ceea
23978e895939a7ecf850d35aba961e9b09c6b8c9
128063 F20101123_AAAGZL woodruff_r_Page_113.jp2
787d081a96442ea998299665c3957a36
a662d3ea9f374eb6b459bfb0e239dd98e0a7eab9
7821 F20101123_AAAGYX woodruff_r_Page_107thm.jpg
47591d2d7eb3dc1c7056a0a797d6bb2a
b588ea3b0fca9364b8b7bf31785b938d8d331efa
1051986 F20101123_AAAHFG woodruff_r_Page_006.jp2
0dfe356603f1323a7614de288b13c8a7
1a9143db48b8b4eadb930d9c719a09348b2ba91a
126020 F20101123_AAAHES woodruff_r_Page_108.jpg
620196c94ddf17e073e306003fe3843e
5ce671d02d7d009767fb06870a0daf203e9f64d4
7241 F20101123_AAAGZM woodruff_r_Page_090thm.jpg
88e39193ffc48ae3eb51b2f5ebc86b78
725dd50a43ce88156b4545b82a3259a0afc090fc
33081 F20101123_AAAGYY woodruff_r_Page_020.QC.jpg
bf01ff7ae1239b85c7a8034d152ce9c4
bc6ec98174e9a441219ec2bcea3dd0b984b5e86a
652452 F20101123_AAAHFH woodruff_r_Page_007.jp2
f4eeef9f292010914ddf3116988f15bf
97715db4248e01c995285bbb647058b200db5eb7
134344 F20101123_AAAHET woodruff_r_Page_110.jpg
e82bc28b7d596ad7353dede4ecd98c71
492bfb6d6a36c693e38587a34cc24c2806f5e050
1051970 F20101123_AAAGZN woodruff_r_Page_008.jp2
7ee71af784b46a615e1e8d0a2fdb51af
12e45de51781cac24f4a2fe64dedfc68ac21bbe3
1580 F20101123_AAAGYZ woodruff_r_Page_067.txt
14c02dc0b01a8a208f0a9e7c31afe2ac
552b0cf745b6c5d17e93a4c4914fa7d5ce831182
F20101123_AAAHFI woodruff_r_Page_009.jp2
098559f0aeec8fd77604182d81b50830
0521aa4f811e8e1ac33f50377268ac58489eab9d
122072 F20101123_AAAHEU woodruff_r_Page_111.jpg
aec2bb25c37a4617f21d1d3f5475d0e5
9dfeca7fd4843d5ffc07ad281a800aa823b73fbd
64927 F20101123_AAAGZO woodruff_r_Page_114.pro
60f0b1db235cf1de5326c19fe011a179
9e15c0b0a735a471bd45734f696ec5110ddd1c77
1051980 F20101123_AAAHFJ woodruff_r_Page_010.jp2
2f1cec9ef8e16c1ee3779dbe4008a662
f8a686586cd8dcfbcb5677606d84d18e57d220bc
136577 F20101123_AAAHEV woodruff_r_Page_112.jpg
6f625a18f3da43ae853f140d20d3f17f
1635d12c4a744228dcf41055c9c0264ec5f6834d
49818 F20101123_AAAGZP woodruff_r_Page_095.pro
bd20363c9965a7767ef3ce7388621300
a0a292158387a8affb8104d352981e418cf25554
88632 F20101123_AAAHFK woodruff_r_Page_011.jp2
c68f870ca84315a34cb9fb182ad9a16d
8a00cd3dd6a7d2f60a9cda35e6ed0404cc10493a
132861 F20101123_AAAHEW woodruff_r_Page_113.jpg
714487ed58f3652c95a8a94484854fa8
5fcef1969490b2d793d23426b324eaf86c268488
F20101123_AAAGZQ woodruff_r_Page_105.tif
e3854fff79b4141c60e59a5f9660c5d3
21d280770a91b8c90da2c87950a885474f1d7177
101940 F20101123_AAAHFL woodruff_r_Page_012.jp2
f2c3d38a6b53669ba6c5ef7b562f82ec
8918a339c963fd38dd1ac7ce9a6b9ad3af181a05
139720 F20101123_AAAHEX woodruff_r_Page_114.jpg
245484fadb64f693515def8d724aed1f
84200b7c82cc4194be4107fcec9f8f15fc8dc5f7
F20101123_AAAGZR woodruff_r_Page_024.txt
53d84e485af34b861efcb2b27da769de
dd3fd4117ea4624fe85c2c682322d8306272ba2d
104151 F20101123_AAAHGA woodruff_r_Page_031.jp2
6f19ab5937c255f961f0122b12426c14
9aa1ea5906198f0586d5e7b95b5932c914498656
94851 F20101123_AAAHFM woodruff_r_Page_013.jp2
83716731e8e8bef5f1fd6459cd9a322c
b43f7c178f42218682f33d6b4bac0520ce9fcb4c
145053 F20101123_AAAHEY woodruff_r_Page_115.jpg
18a7117046da5d9ac6ef6f31ed853f27
3bc3504783947bbef17b209c63d2dc80e48501e8
36072 F20101123_AAAGZS woodruff_r_Page_043.QC.jpg
0c1c23ee5eba2ce409d6773de66bdb58
70cfa1ca02b33cefadead827fbaad697ad3fb92f
105266 F20101123_AAAHGB woodruff_r_Page_032.jp2
e68c1a7fe293cddf4df4487844a71341
d53c446869e054ae4d6241a1f89cf3d1093775a9
113666 F20101123_AAAHFN woodruff_r_Page_014.jp2
bd9bbfc64c434b0643f2570f403cdf7f
e6a600d25a8b36e49818f1a9c2b9f8f22806e042
123172 F20101123_AAAHEZ woodruff_r_Page_116.jpg
895096ca79658315d2e94bcfade5583b
0b2bf9cae2f1013e7f2473d6a27e5b09a1435cd6
23241 F20101123_AAAGZT woodruff_r_Page_075.pro
b578cf9e156e6b051d7e20b97086a19d
7e82ce8c0c807125eb725e67b095d807ac7c76fe
106986 F20101123_AAAHGC woodruff_r_Page_033.jp2
6d960f581576c6ea90fde699a4107870
294d8c794b04afd82a08b6410b6d9d1267b13ce7
108838 F20101123_AAAHFO woodruff_r_Page_015.jp2
f293a4c49262e90ec7f7c4fae9ed5cc3
ef415cbed7f3d71b524f9ba27dfbc05838ab27a7
53134 F20101123_AAAGZU woodruff_r_Page_021.pro
f7f3249a74b540600d1c03053cd70c53
d9d300f4a992de7f49d4c7c3e03478cd3b9cfe09
100240 F20101123_AAAHGD woodruff_r_Page_034.jp2
2df69517f26b1cf34bab351af6a0cac0
a1875397ac731851c43d8b52a24f82425f93512e
112595 F20101123_AAAHFP woodruff_r_Page_016.jp2
b61da65849929bd0039a279a6a134500
bad417b9b3a7df0a6a33f1e910b382d4bb4fe85f
97855 F20101123_AAAGZV woodruff_r_Page_012.jpg
36525fcef18e226037982a89146cb84a
a3034c983446f78091b61825bc2a9199a3b3e6bd
112181 F20101123_AAAHFQ woodruff_r_Page_017.jp2
046b9a954d3b79e8b172d1d6c42fd442
5abff3bac6132b190c637a91f3c40e2a1dc215ec
33347 F20101123_AAAGZW woodruff_r_Page_031.QC.jpg
12875d5cf56bc28d4b6db890fb4edbb7
536c18cd66defaacc14694e7f9d49fc272837d6d
110885 F20101123_AAAHGE woodruff_r_Page_035.jp2
10b0dcd98ef6985ee84cef34d13b47da
30e9aeb4aa6e5559eb852b0e62ba2a7eb43054d5
112482 F20101123_AAAHFR woodruff_r_Page_018.jp2
1e378be1a00d4311072d62ebb6e196dd
abe487378ab9ab064f59b63e1c36da2ca930c302
F20101123_AAAGZX woodruff_r_Page_014.tif
8ebd8a3e9dc268f27620ced279da26c0
f885adfed4ee172bb6113996e1edeaae69b38d56
107978 F20101123_AAAHGF woodruff_r_Page_036.jp2
2710b2a0cd84a0d9dbceaf212e30b7a8
639a7244c05235fecd0f527192cc10b789d23228
114746 F20101123_AAAHFS woodruff_r_Page_023.jp2
9652c17167bd704b2de386412444df12
5c536be0ef9bd0eefcf7daeb9e04cc60c08533aa
112338 F20101123_AAAHGG woodruff_r_Page_037.jp2
c28359b836b77348fa104ba7f428f7a7
92f3479c230910eb660dc9d81c0b8d4a78c32875
107241 F20101123_AAAHFT woodruff_r_Page_024.jp2
b49fc7c42eba79616a969d8d531e259e
3ac96464b64e6f01a909f687dd632803813a055c
107910 F20101123_AAAGZY woodruff_r_Page_062.jpg
bc698c00decdf2c2ad3d9eecd316c511
32691e15a0dbd659b9b4ca6af6c954fcb88359fe
108327 F20101123_AAAHGH woodruff_r_Page_039.jp2
47e505ee4a139fd6a9581b6f60cc80ca
186985916dc31bf3921f9f4661ac94084f6ce1f4
108465 F20101123_AAAHFU woodruff_r_Page_025.jp2
1f629f11fbc4f49b2fb093cd6814a271
ac778df980f755bd65aae1c9bbc8c80de1cf1773
1735 F20101123_AAAGZZ woodruff_r_Page_068.txt
9187c98a259ad081f60112b4bbe5b5ee
feded9dc321a4026e3c2c4ee180ca0b1ffcba334
114470 F20101123_AAAHGI woodruff_r_Page_040.jp2
8d846b05b1cef20d326eca3ae05fef0e
3a54ac0e0d1dfd5fca5ee1f1a9d0c4340263f0db
110446 F20101123_AAAHFV woodruff_r_Page_026.jp2
8bb10a42bfd6fef356fad92e947b7116
e8b50a4ad9687d99125440774c5d47b2f9d29f39
105293 F20101123_AAAHGJ woodruff_r_Page_042.jp2
39ebf9a8ecdcffe77be10b445ce21a5f
29e36a94b1724710b70a0625b1046e0f0d276f3b
927210 F20101123_AAAHFW woodruff_r_Page_027.jp2
95026f2c25b85122909f8b2e3e767654
a5414173cbc1bfdf681cfd1eae211f68ac186d82
1051949 F20101123_AAAHGK woodruff_r_Page_043.jp2
89984ce8b48e51e95927e4e12590418a
7ccc3993852313394e5314d453133e639070cada
92418 F20101123_AAAHFX woodruff_r_Page_028.jp2
eb4be3270c3e7251bb9ca73209ccfac5
4c62bb9857946a2d4180109ab19f713164fab928
113405 F20101123_AAAHHA woodruff_r_Page_060.jp2
927c0f313e55bd500377952352e29e12
44280bf85d41d3173f57390513f292cea462b656
114568 F20101123_AAAHGL woodruff_r_Page_044.jp2
bf7bcf1a0878de32898e6494747ec7c6
793bc4cf29b796b2f54d94f43756671268223ea3
109432 F20101123_AAAHFY woodruff_r_Page_029.jp2
b9fade39178b18c55dab67b069d45de2
fd8ac51c28a28bab51425c814d6325cf2433b202
106783 F20101123_AAAHHB woodruff_r_Page_061.jp2
5c7cacec9c7de9e306c9a8371b19d910
52d0ba0aecc34ca99338b377157b4779a9c42d0c
109319 F20101123_AAAHGM woodruff_r_Page_045.jp2
a8f79034622dd3ba3e2c7815fcd999b9
94d529bef147350c020411808f04cd432761df27
106905 F20101123_AAAHFZ woodruff_r_Page_030.jp2
8d08df9215dce2e6b215b232326cd783
cf6f4820db82a878aef1873f471ccc69b1fc5c64
112080 F20101123_AAAHHC woodruff_r_Page_062.jp2
b4f4a33df81071a688c1c3f6170638d6
4b9cc0a4544a0b4e0d2cb30dad242e274bdb37fb
111141 F20101123_AAAHGN woodruff_r_Page_047.jp2
d1d59da64d27919575c84be317efc72f
46212b47e400df42b3b396bb7380693258229a68
106146 F20101123_AAAHHD woodruff_r_Page_063.jp2
772a2ca68d8fed1c55ccc2a39b38cf6b
71877146c73261729407c8f0a18d6990e51799e6
98325 F20101123_AAAHGO woodruff_r_Page_048.jp2
6398875847099a76acf7e0cae38f28b7
58290e5cefba36022c9c9d6a8c72b59607eb62a3
102643 F20101123_AAAHHE woodruff_r_Page_064.jp2
a152fbcaffdaca8575c026bb46bc2ed9
a4c4ed0f1a016291c0f5af60822fb82c54119c4c
117213 F20101123_AAAHGP woodruff_r_Page_049.jp2
134121fabadf0017a08070889aaef1e6
e6060f071d7fb106f14463e86c3021facd131469
110899 F20101123_AAAHGQ woodruff_r_Page_050.jp2
5c275bc8febbc7c16c030bad2c93ccad
69d8d16856eb5295fd46bfb52629689b97d94828
107929 F20101123_AAAHHF woodruff_r_Page_065.jp2
c0b2f5a2d348be059ac5ddb06ab14b1a
97128f8498fc86ac7b2346834857e88f3d45d3dc
93323 F20101123_AAAHGR woodruff_r_Page_051.jp2
7d36e35e96743b0e8a50a0ff7c664c18
d769fae2e0f82d1901662dfff9604d6c62c0809a
91288 F20101123_AAAHHG woodruff_r_Page_066.jp2
80c01033121efa60aa96322a3f0f688c
81dea5dc30ed2adfa5a9bb5eb927dcfb0b8afe8b
109726 F20101123_AAAHGS woodruff_r_Page_052.jp2
5f8357b7f1790b91a8cd8024968b5f27
735ee879e047277b26a611727c1d23bebcc64a81
71175 F20101123_AAAHHH woodruff_r_Page_067.jp2
fcbb81de5ae74a4a8ff74f43c80c313c
35f607000ed9936ed9afef62b9dbea0b0d129ef4
113417 F20101123_AAAHGT woodruff_r_Page_053.jp2
2288330039f3c0b4e47c2fa786b93982
49fedafaed7976f45f5bf87940c2f83806c519cb
82872 F20101123_AAAHHI woodruff_r_Page_068.jp2
a54e783108cfabf26cf05cd8770dd0a1
ccb1ada2d6076be4d7aff6387147fcf7d1ed96cc
19164 F20101123_AAAHGU woodruff_r_Page_054.jp2
f367845065e60e1173f308ec045a0899
5e53676cd83c8b43601f1a9f9243a160ca529eae
31097 F20101123_AAAHHJ woodruff_r_Page_069.jp2
7b66c8e2e98edd7c28b6a0bf57ff584c
81aa2fac2c6114237947db66de8520372173e63e
31288 F20101123_AAAHGV woodruff_r_Page_055.jp2
de2992bd712e000f0b253983a7253eb6
3db7e2fb745650e3d9e08858da08a3650062521b
54125 F20101123_AAAHHK woodruff_r_Page_070.jp2
1dc28e0f10b5a7ece48e2ab7f8af7d27
ac4050a4e1d2f86a2e6f0143c18d3e2a2c4c9db9
84289 F20101123_AAAHGW woodruff_r_Page_056.jp2
3033a2988cbb24449d022fe127f91a8f
a023dd02eb9c19668496d2010e176caebe600c7e
781589 F20101123_AAAHIA woodruff_r_Page_088.jp2
869d797c07e96020ac0e1f1ea221b026
de6f40a3a61956e940d44029da68d394c8be9965
97567 F20101123_AAAHHL woodruff_r_Page_072.jp2
5c4e3ecb27af06f4bc3b919e597f46f2
eaf0dfa42504dda384174779415883d0f7cf896e
102141 F20101123_AAAHGX woodruff_r_Page_057.jp2
1f4e99a6a25dba903518da4d376c863f
a4d4cf7b6000cc7a4dc579ee24e0e9ad2bf545b4
405674 F20101123_AAAHIB woodruff_r_Page_089.jp2
0d75bd9c9a2cc4d23c89000058e14c17
65ad7af3a27146616420b4726bd0059bd9cdd7bc
63328 F20101123_AAAHHM woodruff_r_Page_073.jp2
6cddca3c8fd255399337a267fea36e18
910b3ab3d23332bfd33bfe3117f7dfb565a85562
99088 F20101123_AAAHGY woodruff_r_Page_058.jp2
a9f5d25e7fa35098db504f26533cbe49
90f4c97df6943ce99535ae98c306626609e45f96
112325 F20101123_AAAHIC woodruff_r_Page_091.jp2
f1cd9236ea8f7d0aa6cf7c4a53b8697f
606fcca4e479952da7639a9e14961d7bc4a0c096
949072 F20101123_AAAHHN woodruff_r_Page_074.jp2
915996acea6512d0b127532eddbadd39
84b56ec9c42cb622b25e091671c17f4578eb8fec
103069 F20101123_AAAHGZ woodruff_r_Page_059.jp2
f7bda6d95e3d81c05db60f23cf904864
f08cc0a21c320b69a75833c0ee14bc72c0bb9d0f
112147 F20101123_AAAHID woodruff_r_Page_092.jp2
d958eaeef5cdf3f5d8a39ca348f232e2
1ee88f1af77716673e4c509050923902a3ef408c
937476 F20101123_AAAHHO woodruff_r_Page_075.jp2
8c0edc81f08e151873ff9bb32f06f4fd
5af4f99fc59648fb611b37b38ed2692c4bae18c4
109715 F20101123_AAAHIE woodruff_r_Page_093.jp2
4d31922d0f3a22f12bbd7fc0485055de
fa5ca5383efbe97eed2bfbf70e060c15134278c3
828000 F20101123_AAAHHP woodruff_r_Page_076.jp2
94345b53390a38e828a19a29b4e743bc
77178e6180712e3a4979c750decf313c30ea8659
110053 F20101123_AAAHIF woodruff_r_Page_094.jp2
1a746b29771f487d1af904bee0709a0e
008fbf3834805be8f29857ff52f75ddb2643b5bd
776471 F20101123_AAAHHQ woodruff_r_Page_078.jp2
fd1e57fb70f5acef679893dc46d2e462
3debd98b3937cbfc9be2b3945adf64ed53e47d67
729085 F20101123_AAAHHR woodruff_r_Page_079.jp2
6887eb095d7a86b8d4106b589979e903
16e07c076ba1e9a3a86ed476bfd71187e577ef12
110222 F20101123_AAAHIG woodruff_r_Page_095.jp2
599e6539ee0a93351f7cdd59b21a86ac
4dde5ddfbfd44ec7d31b237ff3a18b72c0ea5882
953305 F20101123_AAAHHS woodruff_r_Page_080.jp2
fdd7d46d113461591050a802a38bddce
82b2898ad406943a382e2b0fc45209641b0611a5
113559 F20101123_AAAHIH woodruff_r_Page_096.jp2
daf858345d31629466118f8324076c42
8ca8ace6b707d5ab838b9d8d0d94a73df5218d71
940798 F20101123_AAAHHT woodruff_r_Page_081.jp2
c668ee9c5c9adbaaf203c5aeb2ed5ad1
275a647fa1fd888cc95df138cd0ecfafc1cd5bbb
111646 F20101123_AAAHII woodruff_r_Page_097.jp2
60a6441fe9b10d83ce5459317324c772
79e6c539a2e7d82394a75127f5e15693f66a4efa
841939 F20101123_AAAHHU woodruff_r_Page_082.jp2
e2753a7b80a34d226b4f73c919839074
9d07e9caf454fdc400f3bcac194dc140fb6af0c3
107656 F20101123_AAAHIJ woodruff_r_Page_098.jp2
d1bad7d05ac2c04f0df1fe4672b33d8c
84ef2e525ea4729d7885d92a95dae8d5c267bc95
412958 F20101123_AAAHHV woodruff_r_Page_083.jp2
d5ac263f260f3e51094d16232a5a010e
464315acff401c5aabb055218a3476fc18aa6177
101391 F20101123_AAAHIK woodruff_r_Page_099.jp2
433a45d197212231e2828e67618e9a52
e20588f83107ffb73fe8f11a3fc883360323217d
817984 F20101123_AAAHHW woodruff_r_Page_084.jp2
069a1ee94bd8226af8c025cea2e1568a
315173f8bc16e2bd7850c47c0fafdd02c27d9760
108733 F20101123_AAAHIL woodruff_r_Page_102.jp2
380a5ce2bd50ad45f8fe06ceef077564
54d7292f4d72bc781d1456e79e93a2658ebab634
425600 F20101123_AAAHHX woodruff_r_Page_085.jp2
94b61a2a32cc68efe00ed3be4f7fa7ed
90957144e7b16443969f6f1677a4c839c743c4c8
F20101123_AAAHJA woodruff_r_Page_004.tif
06d027dc1cfadd1abf27b5cc31f0b7a4
57c33576b68a12fa825ab194ef8c430d63dda488
107827 F20101123_AAAHIM woodruff_r_Page_104.jp2
6c2ccd6b3339e2528396e4dee9150bc2
77802c3a2d9c70a68c24fda93042381cbc367f5d
842373 F20101123_AAAHHY woodruff_r_Page_086.jp2
6f8f0c53a0221ac76cd2ce05d11481c9
b717ff8ba84a33eee2febae0da22e620e1ae4ca1
F20101123_AAAHJB woodruff_r_Page_005.tif
c3a55cf875277e7dd0766901188d24cc
e52b52000d9722acca21dd7d553d1d1d7ae33759
F20101123_AAAHIN woodruff_r_Page_106.jp2
464a26d0a56774504dc9f23a7b78e433
98b6565393645ac36a91907a400455ccf0112720
432843 F20101123_AAAHHZ woodruff_r_Page_087.jp2
02d1e039d086ffce1d0bf428e3ed9b51
c79456f215c29eda36875e2384d0738ef5c03820
F20101123_AAAHJC woodruff_r_Page_006.tif
ba616040a7d60ada077261c712af83da
d90b205fd485d43bcbc29b84648a7f77c3253fed
119707 F20101123_AAAHIO woodruff_r_Page_107.jp2
ec6fb94f71bce42c95dc79de5205433b
34780a80a5952768477ee9d84bd4dcdfc66cedf5
F20101123_AAAHJD woodruff_r_Page_007.tif
05ca092e2b00236cf1a09a53afaa5846
5124c1dd659a629b61410f8a51e2cefef7c04f4b
125539 F20101123_AAAHIP woodruff_r_Page_108.jp2
c562bebd0363077a0bb1f0091da37b83
e00c9bb77a111c78c9f565df67dd04f03fe32f78
F20101123_AAAHJE woodruff_r_Page_008.tif
37499945d4cde7643ecd9b0d77132bfd
322b6acf6f6bbf48b0e86bcc58a9c7e4aa116e9d
135777 F20101123_AAAHIQ woodruff_r_Page_110.jp2
cf314bfe7a0b71d48a856e8e3a944716
ecb5360895f475fed535ac6b8c049451244a38cd
F20101123_AAAHJF woodruff_r_Page_009.tif
646468a94bc6a3a776c4ab1c89695696
caa47336e9c666d58d73be7ba758d911a8e8af8d
121441 F20101123_AAAHIR woodruff_r_Page_111.jp2
eccc3741917c645703cf9a5c65030cf7
e10405d3bcc257b98e8b0cff607aa8dbf504d84c
F20101123_AAAHJG woodruff_r_Page_010.tif
9120b48c7f4aa881639b1a84478f82a3
93f394a396717f0eb3b30cfb397a407a5768cc54
135308 F20101123_AAAHIS woodruff_r_Page_112.jp2
4a176502ebdf69bdf9b5677f92005a7c
e96acef7d9bb5667e693bdcfce2c21990be16a2e
135394 F20101123_AAAHIT woodruff_r_Page_114.jp2
618bb3d135581e63f2464cc2bce40ebe
b64a7342f28ba86676637d898c38d3709fcdb605
F20101123_AAAHJH woodruff_r_Page_011.tif
b97670ede8a3e8db823c463eb92e1898
f84596648da0c7a4a034cb78b97d7e6678f1940e
138599 F20101123_AAAHIU woodruff_r_Page_115.jp2
b7a8b4bcf9a5b8e6ca2f17d45d47a246
e3aafa5633fc9e7cd1213c2b5d64d9a6bf89e208
F20101123_AAAHJI woodruff_r_Page_012.tif
3d9406e2b4d19daf2f1b5dc0562ece36
ef630356913e279bd1498fcdcbf246cf37885e43
122255 F20101123_AAAHIV woodruff_r_Page_116.jp2
73297431688533f45e989dc0a2d497af
4e95cd56b27417ea93b5a0e9b4466a3222abd605
F20101123_AAAHJJ woodruff_r_Page_013.tif
a91a32f1ac08a513d32fa9ec56051165
1fa5f4b56b38bc2481197f0544c154d3b013316c
70590 F20101123_AAAHIW woodruff_r_Page_117.jp2
ace550e31f0ead5d638655604a35b5db
b771092b73c35966fed2708b96fcf721f2ea8fea
F20101123_AAAHJK woodruff_r_Page_015.tif
7715a3ef85bea4b8222d404473dfab11
9d12ae7bfebc4627d5ac770b8d303330a3483b3e
64222 F20101123_AAAHIX woodruff_r_Page_118.jp2
323dc320a2b0117fd0efde38ba39dd05
4c585a9afb3b18eebaf13e4c693ae90c054a1234
F20101123_AAAHKA woodruff_r_Page_032.tif
1edd75098d2ae2b266ae16897270764a
45fe5a1fbb82612b51b58480dfec87f44ea05c99
F20101123_AAAHJL woodruff_r_Page_017.tif
dd4309d2ead970ab75e18bf7c199894c
57aa4b3bda519e6324ebdf69a2b8b9c7500b8c34
F20101123_AAAHKB woodruff_r_Page_033.tif
1b043fe57ce9327c1dfcd7f3477f0abd
dad43393245d011276a221753f9102d724bd6678
F20101123_AAAHJM woodruff_r_Page_018.tif
c60de4e6652b2b2929c42cc6518fcef2
4ac97ab4e5708835b9941ebfc900a336c81a99be
F20101123_AAAHIY woodruff_r_Page_001.tif
402f5b301016a986c5c036c8afebf25b
b5891ed558b2f0f1934d2dc94b47dbb02895e8a3
F20101123_AAAHKC woodruff_r_Page_034.tif
9ecb779a804c4024ae3c8199bc89a7d9
5e6c58ef2c4b980cc565a63f30bcfaab5c087710
F20101123_AAAHJN woodruff_r_Page_019.tif
db1e32dc4288648752bb2eacb9114925
e62a7f5291e75371e2bb80dcd454617bb4273665
F20101123_AAAHIZ woodruff_r_Page_002.tif
1b284133923acde70a3ec440f3e2e48c
7355013aae23788950c7dea7341496906796faff
F20101123_AAAHKD woodruff_r_Page_035.tif
11dc8fa8326a6e7cc44dccbb32e1f0b8
95f45f459649f1eb589314431b34e880ba0ed21f
F20101123_AAAHJO woodruff_r_Page_020.tif
21b53c80c5b661c503f0387f8b8898ea
0e905f60dc3843ccae13aef2d91bb124837f6946
F20101123_AAAHKE woodruff_r_Page_036.tif
a7cabd34e78bebfda70ff32cc8795c24
fe40f6295e0e4f5398311ebec16bc9c65425c339
F20101123_AAAHJP woodruff_r_Page_021.tif
f5febad54bcb6e8f7cd792911c09cae1
110e8c46e8b19cb402ee11710d82b960b225154e
F20101123_AAAHKF woodruff_r_Page_037.tif
281109ab68ed82fb737aced8b506b146
8b13cedfabca7468ff4c5b4ccc900714157d5752
F20101123_AAAHJQ woodruff_r_Page_022.tif
69f1d2a81d13784f3ec9d731589908ba
c63c67c49fdf5944e4d3927ea8dc995932f94fd8
F20101123_AAAHKG woodruff_r_Page_038.tif
d2683782de7f335d50c1ea17f63115b7
8a74ed9b05f06da702d59f6ba65b0f7fc5d21159
F20101123_AAAHJR woodruff_r_Page_023.tif
8a73ef2432aafce91f37eac6f2ab7b99
a86d7d876d5a706f99462c1bcc8c4e217fd78c83
F20101123_AAAHKH woodruff_r_Page_039.tif
f0107a2be73d115eed75f1a14c527e29
a6c0e8763850de9014c84613fd5e8903fbfafcfb
F20101123_AAAHJS woodruff_r_Page_024.tif
a6a7054a2f34e99fdad9e570c24637de
285cb6fa17a8bfd9cd2449a097817ccc00ff944e
F20101123_AAAHJT woodruff_r_Page_025.tif
e305fe659ad7377fd819608d1fdd2694
de390ddeb5a850bf3c117861403812e113cf0e8e
F20101123_AAAHKI woodruff_r_Page_040.tif
f0d781ca45bacaf8fe5e521d56bf42e0
f14b91d50316e368b67dc7125800e93e6be2d20f
F20101123_AAAHJU woodruff_r_Page_026.tif
fad384da55464a5cd079f5e8630d6564
0eb351ab9dab660f7bf680016944edacf5563849
F20101123_AAAHKJ woodruff_r_Page_041.tif
2f450a951ca3d4ab78bcc5dee13b759e
cf1fee337f302bb207b5674ef89a7699a9d59d2d
F20101123_AAAHJV woodruff_r_Page_027.tif
8c9bf6f87df831b23d400b21b44dba96
ac60b5971700542d1b072af972580587c455c7ba
F20101123_AAAHKK woodruff_r_Page_042.tif
b487a4ac51dbb622a9d9ab8a752d34ed
b18694440d4aa3a3636170f4898f592a7cfe072c
F20101123_AAAHJW woodruff_r_Page_028.tif
c8886d24fed180149ca3ddf70a34c18d
8d0d780bb5590499a2f1e3e40a351131a5815398
F20101123_AAAHKL woodruff_r_Page_043.tif
87db6530faf7112086fe78fe21354189
aa7c1eca9f8c0cad9f8a9bb1792c45072dbaeee7
F20101123_AAAHJX woodruff_r_Page_029.tif
2987b6d61cd8238e8abd25e95a217c36
19accc52ca62961975c6f1d23f40eb9b1b37c28c
F20101123_AAAHLA woodruff_r_Page_063.tif
555c27ee0f1f7a1cf02d33aa9c25396c
aec249f7122bc1426c59d1b4d00c545fafc183f3
F20101123_AAAHKM woodruff_r_Page_044.tif
4c53469b818e99e4e717a1e302ffdf27
5a215e8bb92af906a4fb5eff76eff397ae15cafc
F20101123_AAAHJY woodruff_r_Page_030.tif
26cff06ea4e8652e504e02571c26239a
d7afd5ccc008b0ce9d1ced608a89be110921b51c
F20101123_AAAHLB woodruff_r_Page_064.tif
bea990cbb91e09c76132e944e825d5f6
6c5a0f2eb9c222d136ae4ed5ddda5ba5eb0271d8
F20101123_AAAHKN woodruff_r_Page_045.tif
15f30bf38918c2a2546c2224775a7bbc
e21821a0c82a5efb94d5bb2665fd8257d15953a4
F20101123_AAAHJZ woodruff_r_Page_031.tif
c4b94520a7432868bc36f04c6370115b
d05a453f995b124ab970091d5df07774d3d3a950
F20101123_AAAHLC woodruff_r_Page_065.tif
89e1826efdfee8e8714601b2bfaaad09
c0d6fb81a61685035ca7c27386c718adf3591f50
F20101123_AAAHKO woodruff_r_Page_046.tif
3a77e4e48d3ab3e6138e68d1ee90d1e1
011c2d599d2b2620667ab538cb056d4a87373cdb
F20101123_AAAHLD woodruff_r_Page_066.tif
cf15568fc4dd7bded914ab79994e2d85
c63fd62c23266399d4fb0a64660135390449290f
F20101123_AAAHKP woodruff_r_Page_047.tif
c98d06ade5d0e60f97cdf4dae1912041
4a9d0e5b621039f0d36cd7631d31fc5ff53a12f5
F20101123_AAAHLE woodruff_r_Page_067.tif
79e03bc84de705e48186d0463e81aab6
fce1f8c75628bf80756088cc21039d7ee7325b22
F20101123_AAAHKQ woodruff_r_Page_048.tif
4d4f043b29b4dafc8fade8ce2cc1550d
e5e30b2c88bcadb61f156d528edde067bec6b935
F20101123_AAAHLF woodruff_r_Page_068.tif
3a7371fc584f8a3448e14cc633b6abff
57625dc1b1c1c73cc16196fa1421a27b9de65379
F20101123_AAAHKR woodruff_r_Page_049.tif
d0a1ed1363a09058ff36151df456f218
41b5a3ccac97837abf69b3f98c7ea1a877fe3703
F20101123_AAAHLG woodruff_r_Page_069.tif
f8e9be65d8581458172715b145029c18
85cd80ece4aea76f341cdd5417b3715e56e3c93e
F20101123_AAAHKS woodruff_r_Page_050.tif
3dda1e3317109669b9f9c1b0c0b29fe5
16da44f2d26a3a507e029017368a9dbea38a52cd
F20101123_AAAHLH woodruff_r_Page_070.tif
ff82d3aaa01a9364128e73fa13f2ea4f
bf7bb803341affd894ffc1dec42226aee22d0e20
F20101123_AAAHKT woodruff_r_Page_051.tif
051e98d6460ecbc9dd3ac48b4e0ee1d4
7c06e62953a5225a1835dc04abf6d3f47b059f0a
F20101123_AAAHLI woodruff_r_Page_071.tif
cd9df1dec0e53d598e7326a2b241fa32
bbeea32813dc6cb5288b23c5349a9eb7673541f3
F20101123_AAAHKU woodruff_r_Page_053.tif
26f526a3da1b2a500963633ed61b96ef
530a2317a8d3e4dcb9be40e2714afd5639a6dd41
F20101123_AAAHKV woodruff_r_Page_057.tif
6285be96f306cbd5809528ed56dd5029
fc5e55cc701e5a336b4876225d5df3e2266b0a9e
F20101123_AAAHLJ woodruff_r_Page_072.tif
27f58d025260edc68d435b72fe3f6bbf
7b4177f18337e77badb461f6893fa6e24b1a9685
F20101123_AAAHKW woodruff_r_Page_058.tif
70fcc24698019b60026e95bf4130c607
9f02bde0673436c71879022500212b8d4a624561
F20101123_AAAHLK woodruff_r_Page_075.tif
d749a803901a3096b4b6757d6d63585e
77f1245ef52bb4d305dafc593fa89cc0bbe6b74f
F20101123_AAAHKX woodruff_r_Page_059.tif
13b503616bed56f58ad2a565d58587ac
e75cb2ec849f33db3a96d755e95206d792cdb0ab
F20101123_AAAHMA woodruff_r_Page_093.tif
3511dd155512b924a007c298909fa38a
3a4d18b26222ae7deef995ea99649786034752f2
F20101123_AAAHLL woodruff_r_Page_076.tif
c61837573959eaa1583c0c7aa1c34989
4fa29fbbb0c7a65db6124626c16ebf3cd9cbe785
F20101123_AAAHKY woodruff_r_Page_060.tif
ca58cec6682df147bdcc425f99e6c75d
ae113a714db6e28cbf6886e1de6e9e35c6796cc7
F20101123_AAAHMB woodruff_r_Page_094.tif
30c2f877657862eb880ac8cf5aed74fa
b182d0b753dcb4ac922eb3cefd71f973e36e729e
F20101123_AAAHLM woodruff_r_Page_077.tif
931f17caf016d524f96c0b6026b41633
57e1815686fdc4fbc426590e711d54ab2226b0f4
F20101123_AAAHKZ woodruff_r_Page_061.tif
835d9c9c7e8dd85d48c1666218573e7f
d835ddfdda3d036a7bbccf423f120db4a28c93b4
F20101123_AAAHMC woodruff_r_Page_095.tif
4a16f4d9f2fa3434d648d2b27035ae98
07c36c5084b14d01d581208f23be158dca639aeb
F20101123_AAAHLN woodruff_r_Page_079.tif
34ddffbbcc266d62be72173866c1920e
c36c677a8d0fcd9b155351c27e3777ee5727abca
F20101123_AAAHMD woodruff_r_Page_097.tif
a71c74a4cd55b108b958e40ddf12ea79
71f055ad34696465a8c025a137c3d16cdb981fc9
F20101123_AAAHLO woodruff_r_Page_080.tif
298e9c941f73226349aae234c2537d15
2fa8635739af41f1865d74c756678d13e30a4e91
F20101123_AAAHME woodruff_r_Page_098.tif
855eedbc7b8ae4119d141f193cffbb02
09fb233e4ee10750654510cacc431411b6c38711
F20101123_AAAHLP woodruff_r_Page_081.tif
8472a841c8259092805163268f7f7746
47e9a45469d42d14a62d9fa8958d9e1a800724be
F20101123_AAAHMF woodruff_r_Page_101.tif
accc3821dcf796c47f19ac660c843d01
128c4c7abe83a0d63e4cbe51af2c6a1978849a0e
F20101123_AAAHLQ woodruff_r_Page_082.tif
1d3b6596754b08fae12ac688e0183925
2a382fc447308d4fbaa813066a54ae573a06f106
F20101123_AAAHMG woodruff_r_Page_102.tif
e0502b9c533a3b50c6ba9a93c090d143
0b4d89e59cb6eb17a32144074281f4901fee2e50
F20101123_AAAHLR woodruff_r_Page_083.tif
3ccb8befbcd8f972414d84273b09ead5
b2ce203489b2867ce92e230e590be17ed1dc781e
F20101123_AAAHMH woodruff_r_Page_103.tif
87e37bbbc1f243248b5d1bafabdbdc0e
065640bc24b1c4509aef8796ce83332b2d8334f0
F20101123_AAAHLS woodruff_r_Page_084.tif
0f4b5a27fb11559d09fe43ccf72f5851
069e04f68892569c1d326d81f2a49691fce3304e
F20101123_AAAHMI woodruff_r_Page_104.tif
e87d8abacf5416a28cbdf46b168568cd
4776ecc835b203029f54f6d0e9eb0cac00089a71
F20101123_AAAHLT woodruff_r_Page_085.tif
e2fd50434e4ad8bda2776c2ae2fb57b2
2b4d73ae921284d0e88e0528511d45807ca720fd
F20101123_AAAHMJ woodruff_r_Page_106.tif
9ebfb99a95eb8765f0f3485293ef7c9e
1f3481c9fc96c8a9442c279aa1d864482a4890ee
F20101123_AAAHLU woodruff_r_Page_086.tif
807352ea79ae77761185bb7f933f5b58
f05c677fcd1258565d21cc286ddf60c092d55601
F20101123_AAAHLV woodruff_r_Page_087.tif
a2efe9d47c8d2c36a5fa2792d761bdce
100031570b85de7d8313b7bc6cc1872d0aff33e0
F20101123_AAAHMK woodruff_r_Page_107.tif
eed87199808233596dbe4cd82b3284e2
b2761274b1e663b016c94930d4175b72351ba0f4
F20101123_AAAHLW woodruff_r_Page_088.tif
5739522d863d37fdd94ec17b9312154b
8e2c1c6f6cd7f892b963724e8670c8dd258282a1
63049 F20101123_AAAHNA woodruff_r_Page_009.pro
f685e98e252123fc08b05e6f277ecca7
b448317fa1e988d6fcc74429d6406761961d8d0f
F20101123_AAAHML woodruff_r_Page_108.tif
ed65aae88af64fa32970434080a0699e
f5d999ff8c0e6a3f2e66680ea3f95522fe0501fc
F20101123_AAAHLX woodruff_r_Page_089.tif
7b2e0cdcfce3e33647f94f9e3afdfa2b
3ebc48468fb176af3572a471c3cd3c8bf4458db6
34766 F20101123_AAAHNB woodruff_r_Page_010.pro
0faa467e729614643615fcab867253f1
f49f31ff1cf7f64a2ceea371330dc3e5ebb50d33
F20101123_AAAHMM woodruff_r_Page_109.tif
6876c089e562b5738c1c535ed7610f7f
1583795a6c6a7be541629cb25c47795b5e0ad7fd
F20101123_AAAHLY woodruff_r_Page_090.tif
7efc1b8065eb1c7d45df3eeabb445572
644f66ceb74cdea889cd10ee89360c6f6d46ff4a
39172 F20101123_AAAHNC woodruff_r_Page_011.pro
b0195530a35d8cd37a9e395c604ee208
83e4cbb92637eff0b2ba3b0b52339629002bb265
F20101123_AAAHMN woodruff_r_Page_111.tif
1748e3aa4812257b7796bd7edd3bc7c0
1a27749327b21db72aaad6c80c65d8d564332a50
F20101123_AAAHLZ woodruff_r_Page_092.tif
aba49b310503d5b306af91dfb6fcf61c
96dee3b0d654c9f763fd4c337fe8d80bcab8ff38
45652 F20101123_AAAHND woodruff_r_Page_012.pro
1312a9d2104a796ae3a3afdc1d57bffd
1f90e76e6a6cd90042d701df481fb13f3cb952bf
F20101123_AAAHMO woodruff_r_Page_113.tif
e6a4a36b12abcb7b6c6317232bbf7250
da4a7c760c7372a8be3ca432f1b3e07c525eca44
42444 F20101123_AAAHNE woodruff_r_Page_013.pro
202c01dc783847ee7d5483a782032fdb
53e85cf42d65cc389ccd97ed018db8fe8f511ba5
F20101123_AAAHMP woodruff_r_Page_115.tif
4df3da0ca18cc0bd2806dc128f103854
ebc6d83601ac21bd148e811004a59ff436330746
52042 F20101123_AAAHNF woodruff_r_Page_014.pro
4dec9c663b4dd33cda532531969f094e
8afc01e09651a7a40150d543450f4f96c92bb148
F20101123_AAAHMQ woodruff_r_Page_116.tif
ae6e4ac53a8e54341c2147cff7af18d7
a05f3e932e825395e07ac7b14b6c6e0734420b8e
49740 F20101123_AAAHNG woodruff_r_Page_015.pro
7b09b5fef31e09c133c0bf6ad6582cb5
20c479b444ed535d909f359c4303ebea53735f0c
F20101123_AAAHMR woodruff_r_Page_118.tif
2d645cb098113c3f308dc4395574bb2a
0390c9c8b03ac1c72dd52d14e4da65df01e02e18
53265 F20101123_AAAHNH woodruff_r_Page_016.pro
8eb4433d7570101017088e1ad881dc04
1108395a2f77d06dc2e70fc7f7cfea9947c7e39e
9738 F20101123_AAAHMS woodruff_r_Page_001.pro
0558aeeea88a8635184e67ca826b3217
0317d2b849931bd18f7a3194f3a9d656213b1205
52741 F20101123_AAAHNI woodruff_r_Page_017.pro
b7c71813ca3631c9260e5215bd7a8de0
3eed60cf5291cf39f5bc33cbd7726015b7159a12
1306 F20101123_AAAHMT woodruff_r_Page_002.pro
637dd6074d58dc5392bda6721280ae23
54e94c4d12fa6df348f04b88c7b94923de935229
52150 F20101123_AAAHNJ woodruff_r_Page_018.pro
042116aa8f32017932ea3e5ab1c17d8c
544a4081dc7fda955c351c5149904c76fb33596e
2394 F20101123_AAAHMU woodruff_r_Page_003.pro
6a7bc5ef1a27f34ed2ecd9a5b42a6a97
7904f07c41b77c9abc218adb671271e127ca6a83
50006 F20101123_AAAHNK woodruff_r_Page_019.pro
3c27ecf13820ac9cd26c5131b9145929
acaf65004719695f98a29e4ee5067cf91e99b616
40006 F20101123_AAAHMV woodruff_r_Page_004.pro
b9df219e3315e52f3212d6e6ba43cc46
fbea457cb11aac31fcd4e57722286807eadb659d
79581 F20101123_AAAHMW woodruff_r_Page_005.pro
2646bf51fb66de25b4fcd037fddb2113
bce4c459ce9cae293cdd32afa316f54265b81bf8
48521 F20101123_AAAHNL woodruff_r_Page_020.pro
e5234421876f7ec35addc8889357d732
7d6b7a30deb48cfae5ccdd3dd21e81e85d08a5ce
111048 F20101123_AAAHMX woodruff_r_Page_006.pro
54fcdedffd3081568d1a19a8c81b2da9
f1264c9f710c6bbde99835f1ef0e331ce1b042af
51653 F20101123_AAAHOA woodruff_r_Page_037.pro
7b14a39987429e8bc6ef5acb21688579
f135ff482cd8da11f6c0531349ce6dfbd8ae719b
51922 F20101123_AAAHNM woodruff_r_Page_022.pro
bcaf48035cb16b4c23cac207787fa8a2
7061fe097edbc79e38201e9a0b6a6bfa257f9cf4
24366 F20101123_AAAHMY woodruff_r_Page_007.pro
13596569d2189e76591f5fa0e25a8a2d
374d2448d0e9ea1ea87c01a1420ba370a9a72ee8
52417 F20101123_AAAHOB woodruff_r_Page_038.pro
d436c54e07d49d5e26ba81b82cca55a5
b73b62888176df1c90e1300ed05d7fb4d58c4e1a
53461 F20101123_AAAHNN woodruff_r_Page_023.pro
985a3d55921498d59f7acf488644bf5d
517b791f60b9108a393c5c29c1f1bb42bbeb2aaf
50721 F20101123_AAAHMZ woodruff_r_Page_008.pro
120b29cea8c7392ce6fce9f3ecfe4cfe
986d7053394bd011f8454cb509b240588a553b92
49941 F20101123_AAAHOC woodruff_r_Page_039.pro
e0071df40e42dae4371ecf12d8a30740
18950e253c2252b3eb230a871b8d01ce70df52b2
49157 F20101123_AAAHNO woodruff_r_Page_024.pro
2bdf217ac3d909297b4ef58abac9b928
081025edcfcf09d016a0c862f7488a5e6d13bc35
52753 F20101123_AAAHOD woodruff_r_Page_040.pro
bfdca3e623c6b1848afd738fb910ab0f
26a611666e8df674c78115b8fdecc9abee893b32
49011 F20101123_AAAHNP woodruff_r_Page_025.pro
b26ae18e865bd941685c3074c46f53ff
660ee2b4b4094181ffb1e32acb6d7af6ca1da49c
51796 F20101123_AAAHOE woodruff_r_Page_041.pro
6de38a935ca523c4b89909d6221ce761
b35646d3c23fdf057533c4d79567d3d9256ffb44
50542 F20101123_AAAHNQ woodruff_r_Page_026.pro
e8c6ffc0620507610a3d52ace908ce01
feaaf8a2bad37bcb021af13eafc402a3f06d0714
50325 F20101123_AAAHOF woodruff_r_Page_043.pro
5f07ac199990b9914c8f70d257e2aeff
52c49fc723442f6614a8929f4319dacd579439d8
33976 F20101123_AAAHNR woodruff_r_Page_027.pro
035489fb09b51961e844e0e0db425c9e
49fb6fe999e9e14aef77599cb9c2aa893c115ccb
49848 F20101123_AAAHOG woodruff_r_Page_045.pro
53c91b87ab4258ce12836bbf15e49e6d
b217782d01c594f738fa51e692e28cd8fda5b9bd
41227 F20101123_AAAHNS woodruff_r_Page_028.pro
09eb634cc58bac02f7bc953a892db77b
b94e870383e6844078220960e4a123114070f86a
51082 F20101123_AAAHOH woodruff_r_Page_046.pro
4d0689fadec5972a749aca50045dbb5b
6eafd736397b8a596644d3600cb10fab8d592154
49312 F20101123_AAAHNT woodruff_r_Page_029.pro
5a584e7396b60c04c22273cf4efd5e69
46986b4dbb5da2a49ce62d26d5c852b86a278f4e
43945 F20101123_AAAHOI woodruff_r_Page_048.pro
c52155495eca222e320653c73f85a8b9
1b861580b5dd1a8a72dd5d608caaf01e901f9631
47616 F20101123_AAAHNU woodruff_r_Page_031.pro
4da2352420489c7db471f2f18762981f
4b323721c35fa4b442f5798b1d138a1fc547a16e
53912 F20101123_AAAHOJ woodruff_r_Page_049.pro
6c46556655113103aa8403080363287e
e50f35a18f273a857721f3276c42b13a72d0222f
49631 F20101123_AAAHOK woodruff_r_Page_050.pro
9cb1af9171c5eb70ccc567daab58b33b
237cb9a25a3ee5e3f1c6fb4d7a22778bb5494145
47022 F20101123_AAAHNV woodruff_r_Page_032.pro
d6c1631eebde362b5ac2be9612a51f46
1067179ef967cb257645f646419337a638e1ebbd
44485 F20101123_AAAHOL woodruff_r_Page_051.pro
5130982bb2f6023436a0d8fd6b77fe63
2fa2c077b5c1012cac1c388e9338c03ff8aa98d8
47596 F20101123_AAAHNW woodruff_r_Page_033.pro
edffd7701a5f81864ddf055fd6db719f
33a3a9128403e959a1a9724e73a77d3369bd8a82
19292 F20101123_AAAHPA woodruff_r_Page_070.pro
c6fa7444ac71c66ff317e797a51ea968
2009670fd03ac5a4617444d2bd8032fe8b8790a8
44478 F20101123_AAAHNX woodruff_r_Page_034.pro
d97d58384258a623f89c75413b760315
7c7e699ceb039c915a445e37b66ea33504567a79
61324 F20101123_AAAHPB woodruff_r_Page_071.pro
dd88c43368512d7b721b365387b1e4fc
08b0df588eaf0ab0da71512bef5c4f71185c208b
49964 F20101123_AAAHOM woodruff_r_Page_053.pro
bffaff7ef2dd343337009f26a049b501
64dfb53e4347aa68e9204457799d63c5999e90a6
50658 F20101123_AAAHNY woodruff_r_Page_035.pro
5f555e94f4722fd0c0e2a1ab6fc7f113
72455571e87cccfdd029a2e9483e9ae339df82c2
59010 F20101123_AAAHPC woodruff_r_Page_072.pro
b14fa0853dc692d24ece10dfa90f5c0a
24990ae5490288b40f7a47595eacc776e8b18bbd
7069 F20101123_AAAHON woodruff_r_Page_054.pro
8f09524f659de1d449b511c11118dc96
00f5b4a4460d2a001f4327302ab1b80907fcf61d
49620 F20101123_AAAHNZ woodruff_r_Page_036.pro
ef8fb6b5905ce1744994a00fe659918e
d9fbe303bf05bdfcef9402fc35300982f3d95880
22629 F20101123_AAAHPD woodruff_r_Page_074.pro
a695b7dd98f5988ce8c34802d5c2f814
221467f2bd21d078b46b3d455fcda113788fb78e
14352 F20101123_AAAHOO woodruff_r_Page_055.pro
7d854c98ab6f7ea38c74eb6a997b561c
8cdf26097b2ae24dfa8e8f0af23caf546de51d36
19962 F20101123_AAAHPE woodruff_r_Page_076.pro
f6084c1387ad45ea1d6b6edd801fc1aa
fae0513cafd51c7288506a9d1a3e84c1cfc71fb0
43535 F20101123_AAAHOP woodruff_r_Page_058.pro
6812014846286e689bf28639608a4956
fe8fd60a9c1f91ee5a3cb733571014b39d777f42
9464 F20101123_AAAHPF woodruff_r_Page_077.pro
84c6aeef41cb00da51db598b8d5b6996
c6d8b03bbd42b608afe19e9fa5e9a568d0688629
46017 F20101123_AAAHOQ woodruff_r_Page_059.pro
2135b692bd45cdf3e92fe04cd402ca8f
763310de7c32d0fb8b2d7001fbf84aea409205f2
54267 F20101123_AAAHPG woodruff_r_Page_078.pro
38e40d613649805985c7a99b8490de05
228c6e5493981d6022e5a236e93c6a57ee93edf1
50776 F20101123_AAAHOR woodruff_r_Page_060.pro
3f0e82c63e6b2dcfc66e4d7367f10b10
08aef43c9e61ae346d90365a6dfbecc4f0970542
9311 F20101123_AAAHPH woodruff_r_Page_079.pro
bd6897a15bb6e3e5a6a9da66c19dff6b
af200e280fb069a5d168ea6bb6aca70fdafa89bc
47082 F20101123_AAAHOS woodruff_r_Page_061.pro
954bc20329d584c1dac84709bd046e09
5914f0d6b1098f6505098a715e11249c5d1a0004
33020 F20101123_AAAHPI woodruff_r_Page_080.pro
ee6715d65f54dfb4bfd75a2d94bda66c
28a2c62e51e8c803f3228bb8ce9cc589ea818f56
46820 F20101123_AAAHOT woodruff_r_Page_063.pro
a20bf9ac560365592839352f560dd870
c77a2a4575b5dde73c83774bff97bfefcfa5ca52
21140 F20101123_AAAHPJ woodruff_r_Page_081.pro
b8ba363639b742431be7e11d63d72516
8166bc0e58b14eb17d782d9963c1b6ca024d2429
45702 F20101123_AAAHOU woodruff_r_Page_064.pro
3f294c096df99aab0ccb49bfed844c58
9a06b91a7fe8d8851b374972001f9ae244b643c3
17762 F20101123_AAAHPK woodruff_r_Page_084.pro
7c472289aa9cb29b4b10aacec3203790
406dd68a9be5090b20eaf3ac9d15b10dec6e767b
48455 F20101123_AAAHOV woodruff_r_Page_065.pro
efdb73e20acc6c09dfe916a3be643a7b
826e523060de8bc87c1f397b311afa57771d5de7
8214 F20101123_AAAHPL woodruff_r_Page_085.pro
9f9143c5b06cdf28e54838fb5236ac4d
974db810c282ddfbca2e7e7c594ba59b471ada8a
38025 F20101123_AAAHOW woodruff_r_Page_066.pro
366bbc56153ad5444b5866827d2f9483
4af9bf5d66b4e78d2f3e298d982957f01a1d448e
25768 F20101123_AAAHPM woodruff_r_Page_086.pro
7d2a41ed26e55e3743fc5939a69922c3
1c18f4f1b4ca6fc12428b205713973e3e2ddb750
28814 F20101123_AAAHOX woodruff_r_Page_067.pro
b7576dedb629256fe7e999539aab85a1
01c0555de3eed88a54ea3935c6d4393d0d33e34e
31870 F20101123_AAAHQA woodruff_r_Page_105.pro
962fc851941f49306486fa0a2306ca7f
a3d2cde0ef24126ae03cff861df4e8839ba5e010
36277 F20101123_AAAHOY woodruff_r_Page_068.pro
5b9f0f279cf4a163596169f86ea2216a
6c0b857ea5edaebd093bd320c0b73a5f2972501f
14221 F20101123_AAAHQB woodruff_r_Page_106.pro
3409f3dcc5eff12b315c35155ac338a6
0b5133177edbb2b6624547973fe0853477c3c3b8
4683 F20101123_AAAHPN woodruff_r_Page_087.pro
257e4b7cacfb9a71194738cdde305c53
c1f0d6f29e966f6e434c515fdc405ab327dd3463
11021 F20101123_AAAHOZ woodruff_r_Page_069.pro
199b05300524713b5e0ac1f41487a042
557f9a70ba8d1281737b644d4931499e40f79eaa
55603 F20101123_AAAHQC woodruff_r_Page_107.pro
5b91cd2ee7c2bb49381a702ab0f40a3b
b903f083482b4b9f4b6c398bc1c08384d2ed9014
3347 F20101123_AAAHPO woodruff_r_Page_089.pro
2c8037e0a95420189b23173745c33861
4f3753dad566f7b32203c55f77b7e1fa8ea5b831
58378 F20101123_AAAHQD woodruff_r_Page_108.pro
f17bb77d1b44fae49f80d551534905d0
6c6c1f32b869980f417edc22e8781d9c036067ba
44090 F20101123_AAAHPP woodruff_r_Page_090.pro
712786767702d7272c2520234291f163
1b56e15d1a86d5ca91d2f9a4e50ea705dd18de63
57904 F20101123_AAAHQE woodruff_r_Page_109.pro
7701a61db4dd09b8217df0d1596d4c6e
a69f6f8151deb7385fb13e0e0a9afd4790699559
50444 F20101123_AAAHPQ woodruff_r_Page_092.pro
e854a3672a03c7f1ee6f86dd40118518
bebcb0f61c50044945a0d89082e8eac6c8f26a82
56548 F20101123_AAAHQF woodruff_r_Page_111.pro
ab8d188cdbcfad247824e124741b60b3
a24c3ffa72a366a65e3567080479f9a0fed74492
48927 F20101123_AAAHPR woodruff_r_Page_093.pro
f92e9e1f7c687a59e4eeb050d8debe81
93cb1f2336c7cda6ca8e69ea68e8788bd548d970
63603 F20101123_AAAHQG woodruff_r_Page_112.pro
29c5774d25334c03d2ffe85c6ecd794e
5fb8695adf04376f82821fcf6e274a5c0f84065e
49645 F20101123_AAAHPS woodruff_r_Page_094.pro
75283fd02077e73c42533f2177fde1cf
2cb3eced8ad34b6dbd792d259a7e7761ec1f9e75
59919 F20101123_AAAHQH woodruff_r_Page_113.pro
5c4bf96f6964e4cfabc7c3ba2d430e85
16fd05174c32bf424d618514c53dc5d58c3cf504
51219 F20101123_AAAHPT woodruff_r_Page_096.pro
431f801e8ed31538197f57313afedd01
ad46d4211fbddbcc6780e4ffcdecf3d155b7ed42
65437 F20101123_AAAHQI woodruff_r_Page_115.pro
12ed8b9a5ad3c7a8bedfbe551e524388
60934402fc7e6d30229b3b223c127725d11137d9
50588 F20101123_AAAHPU woodruff_r_Page_097.pro
ca6e7e7fe41e9c090b49e994f3c6e5f8
02fb0c9da73be541bd76c80851d2c1e6cfbcfc98
56814 F20101123_AAAHQJ woodruff_r_Page_116.pro
e33344dc49ee5d74872cb538509de01e
5175af203804d5035dae821d0b4dd7b287ea6151
46152 F20101123_AAAHPV woodruff_r_Page_099.pro
91f553312bee96a7feb6e39f7e7fa7b2
80ab4fa32a57367d314ba813a35a0267f60b1097
31484 F20101123_AAAHQK woodruff_r_Page_117.pro
fad58224836ab9200d7bc3538fe2176c
222905a2122ad25ef1b59dd305626749ffd044ed
52480 F20101123_AAAHPW woodruff_r_Page_100.pro
505b9a1acd5b1dc85a6fb056f11215b5
5407ad37e62d9d178d06c819f325742f9109637b
27784 F20101123_AAAHQL woodruff_r_Page_118.pro
966b8d14a9726fdc0ba717255b8d264a
ffd7080b76d8b6252d798dd3d36dc189278df7fb
49778 F20101123_AAAHPX woodruff_r_Page_102.pro
1135e4db0201b69ae12eb13438d6ba7e
04effe391e20839093b71cbfa1814f06a342a2bd
2091 F20101123_AAAHRA woodruff_r_Page_021.txt
b3696fa9663effc2851c43c5673e8430
d98e10d37a33aa974f61a82d5d81fc8607f50117
121 F20101123_AAAHQM woodruff_r_Page_002.txt
407c7cee1393039c0742397a31eb7ead
fdbdb41a9cbd545f943dbbdab1c058b342c6a3a1
48495 F20101123_AAAHPY woodruff_r_Page_103.pro
294f00ef8c1f0b097f9c32deeb55a9dd
92a0456143a900759a683248733b34c548cf4e5c
2075 F20101123_AAAHRB woodruff_r_Page_022.txt
718f1d3d953732858b0f4af8a1a43560
ac7fce8bfb7c3e70ed90adec66ec5f19d929e233
3302 F20101123_AAAHQN woodruff_r_Page_005.txt
60cb46d38e9d36e5ef2c60e832d89105
dfedbc003051e11021526acbefcf7ccb450bc0ab
49231 F20101123_AAAHPZ woodruff_r_Page_104.pro
2988d1e7e048b51a837b73f6b3548e1b
1bb4605a5a3d2d031e374abb1b3c6835c77bac9a
1966 F20101123_AAAHRC woodruff_r_Page_025.txt
ee22f65c44c5b8043ade6dc8c4bbb232
86c79eb26824394971ef0a06647d1a96218a82f1
1991 F20101123_AAAHRD woodruff_r_Page_026.txt
e9b788eb3e52508630c186adb23e7ac4
3d21f1940a02612d4d33f3cfcc56a6d7520c3bfe
4543 F20101123_AAAHQO woodruff_r_Page_006.txt
be58a3147fc88dc02b262177f242f6d9
1121522c1dfbfaa29240dc6d42e5014e48ddbbfc
1491 F20101123_AAAHRE woodruff_r_Page_027.txt
1a978311add51834beacc943bd1fa5a2
52b62e2aa921d472694c131658fb16ed4c731a0b
990 F20101123_AAAHQP woodruff_r_Page_007.txt
823feb9944456336ece4c2c5fd596dcf
8ba09ec5ddef08ea796d404bf39336875d5e6e63
1761 F20101123_AAAHRF woodruff_r_Page_028.txt
11f52d525991cf608d086f706ab26a0f
a22e561d7dd811871ef767b9d0650a310938aae6
2616 F20101123_AAAHQQ woodruff_r_Page_009.txt
d96f337ac02edeb8cc2ef570e62e5afb
3eb7694ba97c6d4bffe6f9146c2df9e1af896c2f
1958 F20101123_AAAHRG woodruff_r_Page_029.txt
4a2d49de8f62dc4f00f0fb299fa0d797
36cbefc2fac06c50c9f6f99f28b999a1496e87ca
1442 F20101123_AAAHQR woodruff_r_Page_010.txt
493c87fb685ac0cfb27e06a028d08f5a
67143efd8d0817d1cbadc486ff89e805f4dbae8d
1924 F20101123_AAAHRH woodruff_r_Page_030.txt
eba4bbd28ddcffd773df9831b0883c42
be5101566fb7b0184b0a91727a24601ceca80b45
1729 F20101123_AAAHQS woodruff_r_Page_011.txt
41ac13afcf62ddac874201b199761ef6
74591ac97adf6deda10709b2292ac58e283c4808
1874 F20101123_AAAHRI woodruff_r_Page_031.txt
84c28b2546d173def95dbf2303d7a2c9
1ca9f75cc5423f03dd2a6784a4221e688a180da3
1798 F20101123_AAAHQT woodruff_r_Page_012.txt
98f403f9c70777719b4cd5b99bf05727
4fde3fd885a9e0b82fd56712b12a4727f41390d4
F20101123_AAAHRJ woodruff_r_Page_033.txt
c759d97222180c3bb0366809fdc01614
9741a9b06048f874395f49f32fb5e16af20b29ef
1772 F20101123_AAAHQU woodruff_r_Page_013.txt
652e801dcfe097c0a102c33e4b978670
598ba72114ebaaef1cb97b3bc430e4a9b2a4fbfa
F20101123_AAAHRK woodruff_r_Page_035.txt
e76282734f5a4f88d6d0912c44bfeedd
b3f3837c1e0e85afd117594a8580bfcd99dc45b6
2055 F20101123_AAAHQV woodruff_r_Page_014.txt
d55d68a3a6f09f3a91a5bb86f648a2ad
e91545c868f01c1315c6d67c44286ec1adfabaeb
1959 F20101123_AAAHRL woodruff_r_Page_036.txt
dab0336ed5d2c069a6deef4b3cf4297a
6e1f90c32a7445d21c3cad8f569dc3da53095cc4
F20101123_AAAHQW woodruff_r_Page_015.txt
18ee81447fc768a39a390c24f7ad4499
dc935547997eb969bd8b647098cdcc360595c2ff
2614 F20101123_AAAHSA woodruff_r_Page_052.txt
d6a3f468a535c2d9dfe21cf404c8ce48
57ab36690bcf3b36ee5a30a9126b16beb5ac0eaf
2069 F20101123_AAAHRM woodruff_r_Page_037.txt
49f134d5f1ed2187d50c5d2531f7cdf1
707ffb540b755d143f36e2156d38a31c9d7018a1
F20101123_AAAHQX woodruff_r_Page_017.txt
6c2757535895057a2af46e7d3d64455c
5c29e86bb9071f1113c11851b817f935093088fe



PAGE 1

DETECTION AND MOLECULAR CH ARACTERIZATION OF MANATEE PAPILLOMAVIRUS IN CUTANEOUS LESIONS OF THE FLORIDA MANATEE (Trichechus manatus latirostris ) By REBECCA ANN WOODRUFF A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

PAGE 2

Copyright 2005 by Rebecca Ann Woodruff

PAGE 3

This document is dedicated to the graduate students of the University of Florida.

PAGE 4

iv ACKNOWLEDGMENT I would like to extend my greatest appreciation and th anks to my mentor, Dr. Carlos Romero, for allowing me to join hi s laboratory and enjoy many opportunities that I would not have been able to elsewhere. I would also like to thank my committee members, Dr. Peter McGuire and Dr. Ayalew Mergia, for their time, assistance, and suggestions. I would like to thank Dr. Elli s Greiner for first introducing me to the pathobiology graduate program at the University of Florida. I extend great thanks to Bob Bonde, not only for obtaining all of the manatee samples used in this study, but also for providing his expertise and know ledge of manatees. This project was funded by the Florida Fish and Wildlife Commission th rough the Marine Mammal Animal Health Program of the College of Veterinary Medicine at the University of Florida, and would not have been possible without the U. S. Ge ological Survey (USGS) Department of the Interior, contributions of collaborators affiliated with numerous zoological parks, and those involved with manatee capture release st udies. I would also like to thank my fellow lab-mates, Alexa Bracht, Kara Smolarek-Benson, Shasta McClenahan, and Rebecca Grant, for their much valued friendships. I am greatly appreciative of my best friend, David Kottke, for supporting me unconditionally and following me to pursue my dreams, even if it meant putting his own aside. I w ould also like to thank my family for their constant encouragement during these past two years.

PAGE 5

v TABLE OF CONTENTS page ACKNOWLEDGMENT....................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION........................................................................................................1 Classification of Papillomaviruses...............................................................................1 Papillomavirus Genome Organization and Characterization.......................................2 Papillomavirus Replication...........................................................................................4 Papillomavirus Transmission........................................................................................5 Papillomavirus Pathogenesis........................................................................................6 Diagnosis of Papillomavirus Infection.........................................................................8 Treatment and Prevention of Papillomavirus Infection................................................9 Human Papillomaviruses............................................................................................10 Non-Human Papillomaviruses....................................................................................11 Marine Mammal Papillomaviruses.............................................................................13 The Florida Manatee (Trichechus manatus latirostris)...............................................13 2 MATERIALS AND METHODS...............................................................................16 Clinical Data...............................................................................................................16 Source of Samples......................................................................................................16 DNA Extraction from Papillomatous Lesions............................................................17 General Methods of PCR............................................................................................18 Taq Polymerase Reactions..................................................................................18 Accuprime Taq DNA Polymerase Reactions..................................................19 Expand High Fidelity Plus Reactions..................................................................19 PCR Targeting the L1 458-bp Fragment....................................................................20 PCR Targeting the L1 TmlPV 458-bp Fragment.....................................................20 PCR Targeting the TmlPV L1-E1 Region..................................................................20 PCR Targeting the Complete TmlPV E6 Gene..........................................................21 PCR Targeting the Complete TmlPV E7 Gene..........................................................21

PAGE 6

vi PCR Targeting the TmlPV L1-L2 Region..................................................................21 PCR Targeting the Complete TmlPV L1 Gene..........................................................22 PCR Targeting the Complete TmlPV L2 Gene..........................................................22 Gel Electrophoresis.....................................................................................................22 General Cloning of PCR Products..............................................................................23 Cloning into pCR 2.1 TOPO T/A Vector........................................................23 Cloning into P-Target Mamm alian Expression Vector...................................24 Cloning into pcDNA .1 Directio nal TOPO Expression Vector.................24 Analyzing Recombinants............................................................................................25 Sub-cloning of Purified Recombinants.......................................................................27 Sub-Cloning into pcDNA 3.1/ Zeo+ Expression Vector......................................27 L1 Gene Sub-Cloning into pFastBac Vector.................................................29 L1 Gene Sub-Cloning into pBlueBac 4.5............................................................30 Sequencing of PCR-amplif ied and Cloned Products..................................................30 Transfection of Insect Cell Cultures...........................................................................31 Culturing Insect Cells..........................................................................................31 Transformation of MAX Efficiency DH10Bac Competent E. coli..............32 Transfection of Insect Cells with Bacmid DNA Recombinants..........................33 Harvest of Recombinant Baculovirus Stocks......................................................34 Reverse Transcription PCR (RT-PCR) of Infected Cell Cultures.......................34 Generation of Recombinant Baculovirus............................................................35 Transfection of Mammalian Cells..............................................................................36 Culturing African Green Monkey Kidney (COS-7) Cells...................................36 Electroporation of COS-7 Cells with Recombinants for Immunofluorescence Assays..............................................................................................................37 Culturing Florida Manatee Respiratory Epithelial Cells.....................................38 Transfection of TmlRE Cells with DNA Recombinants for Immunofluorescence Assays...........................................................................39 3 RESULTS...................................................................................................................44 PCR Results................................................................................................................44 PCR Targeting the Papillomavirus L1 458-bp Fragment....................................44 PCR Targeting the L1 TmlPV Fragment..........................................................45 PCR Targeting the TmlPV L1-E1 Region...........................................................45 PCR Targeting the Complete TmlPV E6 Gene...................................................45 PCR Targeting the Complete TmlPV E7 Gene...................................................45 PCR Targeting the TmlPV L1-L2 Region...........................................................46 PCR Targeting the Complete TmlPV L1 Gene...................................................46 PCR Targeting the Complete TmlPV L2 Gene..........................................................46 Sequencing Results and Genetic Analyses.................................................................46 TmlPV L1 458-bp Fragments..............................................................................46 L1-E1 TmlPV Region..........................................................................................48 TmlPV E6 Gene..................................................................................................49 TmlPV E7 Gene..................................................................................................49 TmlPV L1-L2 Region..........................................................................................50 Complete TmlPV L1 Gene from Captive Manatee Lorelei................................51

PAGE 7

vii Complete TmlPV L2 Gene..................................................................................51 Immunofluorescence and Ge ne Expression Assays...................................................52 Mammalian Expression Systems.........................................................................52 Bac-to-Bac Baculovirus Expression System.......................................................52 Bac-N-Blue Baculovirus Expression System.............................................................53 Phylogenetic Analysis................................................................................................53 4 DISCUSSION.............................................................................................................78 LIST OF REFERENCES...................................................................................................95 BIOGRAPHICAL SKETCH...........................................................................................106

PAGE 8

viii LIST OF TABLES Table page 2-1 Samples obtained from skin lesions of captive Florida manatees.......................39 2-2 Samples obtained from skin lesions of free-ranging Florida manatees...............40 2-3 PCR primers designed to target manatee papillomavirus sequences..................40 2-4 Sequencing primers used to obtain th e complete sequence of PCR amplified TmlPV gene fragments........................................................................................41 3-1 PCR results of DNAs obtained from captive manatee skin lesions tested for the presence of TmlPV infection.........................................................................55 3-2 PCR results of DNAs obtained from free-ranging manatee skin lesions tested for the presence of TmlPV infection...................................................................56 3-3 Summary of PCR results.....................................................................................57 3-4 Accession numbers of ma natee papillomavirus sequen ces deposited into the GenBank tool of the NCBI website.....................................................................58 3-5 Pair-wise comparisons of the amino aci d sequences of the L1, L2, E6, and E7 gene fragments of manatee papillomavi rus (TmlPV) with several human and non-human papillomaviruses...............................................................................59 3-6 Pair-wise comparisons of the amino aci d sequences of the L1, L2, E6, and E7 gene fragments of manatee papillomavi rus (TmlPV) with several human and non-human papillomaviruses...............................................................................60 3-7 Summary of the pair-wise comparis ons of the amino acid sequences of manatee papillomavirus gene fragments with several human and non-human papillomaviruses..................................................................................................61

PAGE 9

ix LIST OF FIGURES Figure page 1-1 Illustration demonstrating the genetic organization of a typical papillomavirus genome.......................................................................................15 2-1 Linear representation of the open re ading frames (ORFs) of the circular manatee papillomavirus (TmlPV) genome with the relative positions of the PCR primers used to amplify TmlPV DNA........................................................16 3-1 Agarose gel electrophoresis of PCR amplified 458-bp fragments of the L1 capsid protein gene of manatee papillomavirus..................................................62 3-2 Agarose gel electrophoresis of PCR amplified 2,772-bp fragment of the L1E1 region of manatee papillomavirus..................................................................62 3-3 Agarose gel electrophoresis of PCR amplified 587-bp fragments of the E6 gene of manatee papillomavirus..........................................................................63 3-4 Agarose gel electrophoresis of PCR amplified 489-bp fragments of the E7 gene of manatee papillomavirus..........................................................................63 3-5 Agarose gel electrophoresis of PCR amplified 3,208-bp fragments of the L1 gene plus the L2 gene of manatee papillomavirus..............................................64 3-6 Agarose gel electrophoresis of P CR amplified 1,712-bp fragments of the complete L1 gene of ma natee papillomavirus.....................................................64 3-7 Agarose gel electrophoresis of P CR amplified 1,660-bp fragments of the complete L2 gene of manatee papillomavirus.....................................................65 3-8 Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the L1 gene fragment of manatee papillomavirus identified in cutaneous lesions of captive and free-ranging Florida manatees........................66 3-9 Electron micrograph of Sf21 ins ect cell cultures transfected with pFastBac1/TmlPV L1 gene recombinant............................................................67 3-10 Agarose gel electrophoresis demonstr ating the PCR amplification of the manatee papillomavirus complete L1 gene fragment from cDNAs obtained from infected Sf21 cell cultures..........................................................................68

PAGE 10

x 3-11 Agarose gel electrophoresis demonstra ting restriction enzyme digests of the manatee papillomavirus E6 gene and E7 gene recombinants.............................69 3-12 Agarose gel electrophoresis demonstra ting restriction enzyme digests of the manatee papillomavirus L1 complete gene recombinant....................................69 3-13 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the complete L1 gene of several human and non-human papillomaviruses........70 3-14 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the complete L2 gene of several human and non-human papillomaviruses........72 3-15 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the complete E6 gene of several hum an and non-human papillomaviruses ......74 3-16 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the complete E7 gene of several human and non-human papillomaviruses........76 4-1 Papillomatous skin lesions on the flipper of a captive Florida manatee (Trichechus manatus latirostris) housed at Homosassa Springs State Wildlife Park, Homosassa, Florida....................................................................................93 4-2 Typical papillomatous skin lesions of free-ranging Florida manatees................44

PAGE 11

xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Master of Science DETECTION AND MOLECULAR CH ARACTERIZATION OF MANATEE PAPILLOMAVIRUS IN CUTANEOUS LES IONS OF THE FLORIDA MANATEE (Trichechus manatus latirostris ) By Rebecca Ann Woodruff August 2005 Chair: Carlos H. Romero Major Department: Veterinary Medicine Papillomaviruses are widespread and succe ssful pathogens associated with the development of benign warts and malignant ne oplasia in humans and domestic and wild animals. Phylogenetic comparisons have classified human and non-human papillomaviruses into 16 genera within the Papillomaviridae family. With the advent of PCR and molecular cloning, si gnificant advances have been made in understanding human papillomaviruses; however, l ittle is known about marine mammal papillomaviruses. DNA extracted from skin lesions of captive and free-ranging manatees was assayed by polymerase chain reaction (PCR) to detect papillomavirus infection. Initially, primers were based on the widely used MY11 and MY09 human papillomavirus (HPV) primer set, which target a highly c onserved region of the L1 capsid protein. The MY11 and MY09 primers directed the amplif ication of a 458-bp fragment from DNA extracted from captive and free-ranging Florida manatee (Trichechus manatus latirostris ) skin lesions. Sequences of this fragment led to the development of L1 PCR primers,

PAGE 12

xii which specifically target the L1 capsid pr otein of Florida manatee papilloma virus (TmlPV). The L1 and L1 DNA fragment sequences were 100% identic al, suggesting that there is only one genot ype of manatee PV present in captive and free-ranging manatees around Homosassa Springs State W ildlife Park. PCR primers were also designed to specifically target the comple te TmlPV E6, E7, L1, and L2 genes. Amplification of the TmlPV E6 gene product yielded a 589-bp fragment, which included the complete TmlPV E6 gene, and amplifica tion of the TmlPV E7 gene product yielded a 487-bp fragment, which included the complete TmlPV E7 gene. Targeting the TmlPV L1 capsid protein gene yielded amplifica tion of a 1,712-bp fragment which included the complete TmlPV L1 gene, and targeti ng the TmlPV L2 capsid protein yielded amplification of a 1,660-bp fragment, which included the complete TmlPV L2 gene. The complete TmlPV L1, E6, and E7 genes were successfully cloned into mammalian and insect expression vectors, allowing for the future production of recombinant genes and the development of serologic assays, such as ELISA. The described assays based on PCR and direct sequencing of amplicons have allowed for the molecular detection of TmlPV in captive and free-ranging Florid a manatees and have allowed for the phylogenetic comparisons of TmlPV to severa l human and non-human papillomaviruses. The TmlPV is a unique papillomavirus base d on phylogenetic analyses of the deduced amino acid sequences of the L1, L2, E6, and E7 proteins, quite distinct from another marine papillomavirus of Burmeisters porpoise s, most closely related to HPVs that cause common skin warts, and leas t related to high-risk HPVs involved in malignancy.

PAGE 13

1 CHAPTER 1 INTRODUCTION The papillomaviruses (PVs) are a group of small DNA viruses that cause benign warts and malignant neoplasia in humans (Walboomers et al., 1999) and domestic and wild animals (Sundberg, 1987). To date, more than a hundred human PV (HPV) types have been partially identified and a wide vari ety of PV types have also been detected in mammals and birds (de Villiers et al., 2004). Papillomavirus infection has also been detected in genital and cutaneous lesions of several species of marine mammals (Bossart et al., 2002; Kennedy-Stoskopf, 2001; Van Bresse m et al., 1996) and studies have shown that the healthy skin of humans and animal sp ecies can harbor sub-cl inical PV infections in the absence of overt lesions (Antonss on and Hansson, 2002, and Antonsson et al., 2000). Classification of Papillomaviruses Papillomaviruses were originally grouped together with the polyomaviruses in the family Papovaviridae based on their small size, nonenveloped capsids, and circular, double-stranded genomes. However, a lack of overall homology of particle size, genome organization, and sequence similarities be tween the two viral genomes led to the recognition of two separate families, Polyomaviridae and Papillomaviridae (Howley and Lowy, 2001). Viruses within the Papillomaviridae family are defined by genomic properties, rather than serology, and are therefore described as PV genotypes, not serotypes. Papillomavirus ge notypes are classified by analysis of only part of the viral genome that encompasses the combined nucleo tide sequences of the E6, E7, and L1 open

PAGE 14

2 reading frames (ORFs) (Chan et al., 1995). Recent phylogenetic comparisons of the L1 ORF nucleotide sequences of 96 HPVs and 22 animal PVs have further classified PV types into genera and species. Higher-orde r phylogenetic clusters (major branches) of PV types have now been classified into gene ra, sharing less than 60% identity in the L1 ORF nucleotide sequences. Each genus within the 16 genera identified is defined by its biological properties and genome organization. Lower-order clusters (minor branches) of PV types, or PV subtypes, have now been termed species. Species within each genus share 60-70% identity at the nucleotide leve l (de Villiers et al., 2004). Nucleotide sequence analyses of the L1 ORF of various HPV types have been used to construct phylogenetic trees that display clusters of PV types with similar tissue tropisms and oncogenic potentials (Chan et al., 1995). Papillomavirus Genome Organization and Characterization Papillomaviruses have double-stranded, circular DNA genomes of about 8,000 base pairs (bp) in size. The papillomavirus part icles (52 to 55 nm in diameter) contain the viral genome within a spherica l capsid composed of 72 capsome rs. All ORFs are located on one strand, indicating that transcription occurs on only one stra nd. Transcription is regulated by the differentiati on state of the infected cells and is complex, due to the presence of multiple promoters, alternate and multiple splice patterns, and differential production of mRNA in different cel ls (Howley and Lowy, 2001). A PV genome usually contains seven majo r ORFs that code for five early (E) proteins and two late (L) cap sid proteins, plus an upstream regulatory region (URR), or long coding region (LCR) (Tachezy et al., 2002). The early region of the PV genome encodes the viral regulatory proteins E1, E2, E4, E6, and E7, which are necessary for initiating viral DNA replication. The late region encapsulates the genome and encodes

PAGE 15

3 the L1 and L2 capsid proteins (Howley a nd Lowy, 2001). The LCR does not contain any ORFs, but does contain the origin of viral DNA replication. Elements present in the LCR regulate viral DNA replication and transcri ption (Desaintes and Demeret, 1996). Unusual ORFs have been described in severa l types of PVs (Tachezy et al., 2002). The genomes of European elk (Odocoileus alces ) PV, white-tail deer (Odocoileus virginianus ) PV, and reindeer (Odocoileus hemionus ) PV contain a transformi ng E9 gene (Eriksson et al., 1994). The African grey parrot (Psittacus erithacus timneh ) papillomavirus (PePV) genome does not contain the typical E6 or E7 ORFs (Tachezy et al., 2002) and the Burmeisters porpoise (Phoecena spinipinnis ) papillomavirus (PsPV-1) genome also lacks the E7 ORF (Van Bressem et al., 1996). Two ORFs with unknown functions, E3 and E8, may also be present in a PV ge nome (Lowy and Howley, 2001). An illustration of a typical PV genome is shown in Figure 1-1. The PV genome is contained within the capsid region which consists of the major and minor structural proteins, L1 and L2, re spectively. The L1 ORF is the most highly conserved ORF within the papillomavirus genom e (de Villiers et al., 2004) and represents 80% of the total viral prot ein (Howley and Lowy, 2001). The L1 protein can selfassemble into virus-like part icles (VLPs) or in combination with L2, although the L2 protein is not required (Hag ensee et al., 1993). The L2 pr otein may enhance packaging (Stauffer et al., 1998) and inf ectivity (Roden et al., 2001). The early genes E6 and E7, and in some PV types, E5, contain oncogenic properties that can modulate the transforma tion process (Baker and Howley, 1987). The E6 protein of bovine papilloma virus type 1 (BPV-1) and of the oncogenic HPVs is believed to function through bi nding cellular target s (Howley and Lowy, 2001), such as

PAGE 16

4 binding and degrading the p53 tumor suppressor protein (Scheffner et al., 1993). The E7 protein of the oncogenic HPVs binds a number of important cellular regulatory proteins, such as the retinoblastoma tumor suppre ssor protein (pRB) and the related pocket proteins, p107 and p130 (Dyson et al., 1989). Th e papillomaviruses may activate cellular genes necessary for the replication of thei r own DNA through the binding of the viral E6 and E7 proteins to cellular f actors (Howley and Lowy, 2001). The early genes E1 and E2 are regulatory proteins that modulat e transcription and replication (Baker and Howley, 1987). The E1 and E2 proteins are essential for viral DNA replication (Chiang et al., 1992). The E1 protein has been shown to bind a number of cellular proteins, including the cellular DNA polymerase -primase, thus recruiting the cellular DNA replication initiation machinery to the viral origin of re plication (Park et al., 1994). The multifunctional E2 protein can activate or repress viral promoters, has critical roles in viral DNA replication (Ham et al., 1991), and targets the E1 protein to the replication origin (Sedman and Stenlund, 1995). Although the E4 ORF is located in the early region of the PV genome, it is expresse d as a late gene (Howley and Lowy, 2001). The precise role of the E4 protein is unc lear, but studies have shown E4 expression coincides with the onset of viral geno me amplification (Peh et al., 2002). Papillomavirus Replication Key life cycle events seem to be simila rly regulated in both human and non-human PVs (Peh et al., 2002). The PV life cycle is strictly depe ndent on differentiation of the epithelial tissue (Barksdale and Baker, 1993) an d PV replication can be divided into three stages (McBride et al., 2000). First, the PV virion must bind to a basal keratinocyte, although studies have shown that the PV viri ons can bind a wide variety of cell types (Muller et al., 1995). During this stage, the viral genome is maintained as an episome

PAGE 17

5 within the nucleus (McBride et al., 2000). Th e viral genome is then amplified and the viral copy number is increased up to 1,000 per haploid cell genome (Lepik et al., 1998). As the basal cells differentiate, the viral DNA is maintained as a stable plasmid (Howley and Lowy, 2001). During this second, maintena nce stage, the viral ge nome replicates in synchrony with the host cell chromosome (G ilbert and Cohen, 1987). The PVs rely on cellular replication factors a nd enzymes (Muller et al., 1994), such as replication protein A (RPA) (Mannik et al., 2002), in order to repl icate their genomes from a single origin of replication (Melendy et al., 1995) The earliest PV DNA synthesis is within the fragment containing the PV replication origin and synt hesis proceeds in both directions from the replication origin (Melendy et al., 1995). The third replicat ion stage takes place in the terminally differentiated epith elial cells of the papilloma (Howley and Lowy, 2001). In this next layer of stratified epithelium, the stratum granulos um, late viral gene expression, synthesis of capsid proteins, vegetative vira l DNA synthesis, and assembly of virions occur (McBride et al., 2000). The PV DNA is thought to remain in the basal epithelial cells and to be reactivated when levels of immune system monitoring decline (Doorbar, J. 2005). Papillomavirus Transmission Papillomavirus enters infected skin via skin surface abrasions, allowing the virus access to the proliferative cells of the skin (Egawa, 2003), where it promotes cellular proliferation and accelerated epithelial growth (Silverberg, 2004). Infection with HPV is primarily found on the extremities, face, and body, and moist skin is more likely to allow viral transmission (Silverberg, 2004). Transm ission of high-risk mucosal HPVs occurs predominately through sexual contact, but horizon tal and vertical rout es have also been identified. Vertical transmi ssion of mucosal HPVs may be acquired from the mother in

PAGE 18

6 utero, across the placenta, intr apartum, during birth through an infected birth canal, or post partum (Cason and Mant, 2005). In 1989, Sedlacek et al. described the first confirmed vertical transmissi on of HPV infection in nasoph aryngeal samples of infants born to HPV positive mothers. Papillomavirus Pathogenesis Papillomas (warts) are induced in the skin and mucosal epithelia at specific sites (de Villiers et al., 2004) and di ffer in their tissue specificity and the associated disease (McMurray et al., 2001). The highly tissue-specific papilloma viruses can be divided into two groups: one group is primarily found in cuta neous epithelia (skin), in which there is thickening of the epidermis, and the other group is predominantly present in mucosal epithelia (Smits et al., 1992), involving th e oral pharynx, esophagus, or genital tract (Howley and Lowy, 2001). Warts are diagnos ed by physical examination and are defined by morphology, location, and host immune response. The three main types of lesions observed are: common warts (r ough plaques of skin), mosaic warts (groups of common warts), and flat warts (smooth, flesh-colore d papules). Mucosal warts may appear as single plaques or as a group with a grap elike appearance (Silverberg, 2004). The genital mucosal HPV types are defined as either high-risk or low-risk based on their involvement with lower genital tract can cers (McMurray et al ., 2001). In the lowrisk types, such as HPV-6 or HPV-11, benign wa rts proliferate. In the high-risk types, such as HPV-16 or HPV-18, the virus deregu lates checkpoints that normally monitor the fidelity of chromosome replication and segreg ation, leading to the development of cancer (Galloway, 2003). Anogenital carcinomas caused by HPV infection include penile (Rubin et al., 2001), vaginal (Daling et al., 2002), vulvar (Trimble et al., 1996), and anal cancers (Krzyzek et al., 1980). The high-risk HP V types are also associated with cervical

PAGE 19

7 dysplasia (Kurman et al., 1982), uterine can cer (Parkin et al., 1999, and zur Hausen, 1996), and cervical cancer, one of the mo st common cancers of women world-wide (Schiffman, 1992, and zur Hausen, 1991). The E6 and E7 genes of the high-risk types are expressed in cervical cancer (Schwarz et al., 1985). The roles of E6 and E7 of the low-risk types are unclear, but studies suggest that they may act in a similar manner as observed in the high-risk E6 and E7 proteins (Oh et al., 2004). Risk factors associated with cervical cancer and ot her anogenital tumors include cigarette smoking, sexual factors, and, possibly, genetic sus ceptibility (Daling et al., 2002). Both the low-risk and high-risk HP V types cause low-grade squamous intraepithelial lesions (LSIL) of the cervix, but the high-risk HPV types cause high-grade SIL (HSIL), carcinoma in situ, or invasive cancer. The steps leading to cervical carcinogenesis include infecti on with an oncogenic (high-risk) HPV, development of HSIL, progression of HSIL to carcinoma in s itu, and, then, invasive cancer (Baseman and Koutsky, 2005). Frequent integration of th e HPV genome into the host genome usually occurs in the case of high-risk PVs (Mannik et al., 2002). At least 25 types of HPVs have also been detected in oral lesi ons on the lips, hard palate, and gingiva (Syrjane n, 2003). The high risk HPV types 16 and 18 are highly associated with oropharyngeal and laryngeal squamous cell ca rcinomas (Kreimer et al., 2005) while the low risk HPV types 6 and 11 are predominant in benign lesions of the oral mucosa (Syrjanen, 2003). Infection w ith HPV is a significan,t independent risk factor for oral squamous cell carcinoma (Miller and Johnstone, 2001). The pathogenesis of HPVs differs for viruse s that are considered high risk or low risk group members. In the case of epidermodysplasia verruciformis (EV), the disease

PAGE 20

8 behaves like a genetic cancer of viral or igin, which may result from an abnormal recessive gene (Jablonska, 1991). Clinical forms of EV may be benign or malignant. The benign form is associated with HPV-3 and/or HPV-10 and induces flat, wart-like lesions over the trunk and limbs. The maligna nt form is associated with EV-HPV and induces reddish, polymorphous lesions, flat wa rt-like lesions, and pr e-malignant lesions disseminated over the body. Epidermodysplasia verruciformis is the first human genetic condition in which cutaneous cancer is asso ciated with HPV infection (Majewski and Jablonska, 1995). There is an increased risk of developing cutaneous HPV-associated disease if the virus is not cleared from the skin (Silverb erg, 2004). Cutaneous HPV types, such as HPV-5 and HPV-8, may contribute to skin cancer development in immunosuppressed individuals (Stockfleth et al ., 2004). In renal transplant studies, 90% of recipients developed HPV-induced warts (Blessing et al., 1989) and up to 40% of recipients developed nonmelanoma skin cancer within 15 years after transplantation (Birkeland et al., 1995). HPV-5 and HPV-8 were also pr edominately detected in squamous cell carcinoma of patients diagnosed with EV (Orth, 1987). Immunosuppression may increase the activity of HPV, which may lead to the development of cancer (Stockfleth et al., 2004). Diagnosis of Papillomavirus Infection Testing for the presence of HPV viral DNA includes methods such as Southern blots, dot blots, in situ hybridization, polymerase chai n reaction (PCR), and solution hybridization (hybrid capture assay) (Trofatter, 1997). Detection of HPV by PCR is more sensitive than the other methods, enab les the detection of a single genome copy per cell for HPV DNA that has integrated (Sha manin et al., 1994), and allows for the

PAGE 21

9 detection of a broad spectrum of genital HPVs (Ting and Manos, 1990). The widely used consensus PCR primers, MY09 and MY11, are based on sequences obtained from the highly conserved PV L1 capsid protein gene (Manos et al., 1989). The primers were designed from homologous regions 20 to 25 base pairs (bp) in length that were identified in genital HPV types 6, 11, 16, 18, and 33 (T ing and Manos, 1990). These primers are known to amplify a 458 base pair (bp) fragment when used with DNA from most types of genital HPVs (Bernard et al., 1994 ) Human papillomavirus DNA can be detected by method of PCR in fresh or frozen cervical biopsies (Li et al., 1988; Manos et al., 1989), condylomata acuminata (genital warts) tissu es (Brown et al., 1999), cutaneous wart tissues (Harwood et al., 1999), and in swab samples taken from the top of lesions (Forslund et al., 2004). Ha rwood et al. (1999) have described a degenerate nested PCR that is capable of detecting cutaneous, mucosal, and EV HPV types. A new method of HPV detection using high-density DNA microarrays is able to detect single and multiple mucosal HPV infections (K laassen et al., 2003). Treatment and Prevention of Papillomavirus Infection The host response to HPV infection is a co mplex process of skin barrier protection, innate immunity, and acquired immunity (Sil verberg, 2004). Warts generally will regress over time and after six months of infecti on, 30 percent of warts will clear on their own (Messing and Epstein, 1963). Following immu ne regression, PV DNA persists in a latent state, with only a few cells, if any, capable of supporting th e productive cycle that occurs during epithelia l cell differentiation (Doorbar, 2005). A possible approach to controlling the level of HPV-a ssociated disease is to prevent HPV infection (McMurray et al., 2001); however, the PV life cycle requires a differentia ted stratified epithelium to replicate and this has been difficult to genera te in cell culture (McB ride et al., 2000).

PAGE 22

10 Due to the fact that PVs could not previ ously be propagated in cell cultures, the development of a capsid-directed vaccine wa s hindered for a long time (Biemelt et al., 2003). A recently described raft system now allows for the genetic analysis of the complete viral life cycle of BPV-1. Using a combination of organotyp ic raft cultures and xenografts on nude mice, BPV-1 DNA can be amplified and capsid antigens and infectious BPV-1 virus particles can be produced (McBride et al., 2000). Several animal models of PV infection ha ve shown that neutralizing antibodies can block new infection (Galloway, 2003). Vacci nation against PV infections using viruslike particles (VLPs) based on the L1 capsid pr otein or the L1 plus the L2 protein is currently being developed (Leder et al., 2001). Vaccines based on VLPs are desirable because they retain repetitive, highly immunogenic epitopes found on the surface of infectious virions, but lack the potentially harmful PV ge nomes. Three types of HPV VLP-based vaccines are currently being developed. The first, most basic type is designed to prevent genital HPVs by inducing virus-ne utralizing antibodies against the L1 major capsid protein. The second type of vaccine is based on chimeric VLPs which incorporate polypeptides of other viral a nd cellular proteins into the VLPs. These vaccines induce cell-mediated responses to nonstructural viral proteins, such as the HPV E7 protein. The third type of vaccine incorpor ates self-peptides into the ou ter surface of the VLPs and is designed to induce antibodies against central self-antigens (Schiller and Lowy, 2000). Vaccination with HPV VLPs has been well tole rated, induces high titers of antibodies, and shows evidence of T-cell responses (Galloway, 2003). Human Papillomaviruses Papillomaviruses are one of the most impor tant viruses associated with benign and malignant neoplasia in humans (Chan et al., 199 5). Papillomaviruses were first isolated

PAGE 23

11 almost 30 years ago (Orth et al., 1977) and the first HPVs fully sequenced were HPV-1 (Danos et al., 1982), HPV-6 (Schwarz et al ., 1983), and HPV-16 (Seedorf et al., 1985). To date, more than 100 types of human papi lloma viruses (HPVs) have been identified, of which 96 have been cloned and characterized (deVilliers et al., 2004). More than 50 types of HPVs have been found to infect th e genital tract (Gallo way, 2003). Low-risk (benign) genital HPVs include: t ypes 6, 11, 40, 42-45, 53-55, 57, 67, 69, 71, and 74. High-risk (oncogenic) genital HPVs include: types 16, 18, 31-35, 51, 52, 56, 58, 66, 68, 70, and 73 (deVilliers et al., 2004 and McMurr ay et al., 2001), with types 26, 53, and 66 being probably oncogenic (Munoz et al., 2003) The HPV types 59, 61, and 82 may be associated with benign or malignant lesions (deVilliers et al., 2004) Infection with a single HPV type or infection with multiple HPV types can occur (Munoz et al., 2003). Non-Human Papillomaviruses Warts in animals have been recognized for centuries. Equine papillomas were described as early as in the 9th century A. D. and the first experimental transmission of animal papillomas occurred in 1898 (Lan caster and Olson, 1982). Warts in wild cottontail rabbits were the fi rst animal papillomas thoroughly examined for properties of transmissibility, etiology, and histology. The activities and char acteristics of the papilloma-producing agent in co ttontail rabbits classified it as a virus (Shope, 1933). Additional non-human PVs initi ally characterized include: BPV (Lancaster and Olson, 1978), equine PV (Fulton et al., 1970), canine oral PV (Chambers and Evans, 1959), deer fibromavirus (Shope et al., 1958), and chaffi nch PV (Lina et al., 1973). Presently, 22 animal PVs have been fully characterized and classified into genera and species based on the L1 ORF sequences (de Villiers et al., 2004). As many as 53 putative new animal PV types have been identified by polymerase ch ain reaction (PCR) in 7 animal species,

PAGE 24

12 including chimpanzees, gorillas, spider monke ys, long-tailed macaques, domestic cattle, aurochs, and European elk (Antonsson and Hansson, 2002). Animal papillomas can be divided into four groups based on tissue tropism and histology of lesions. These groups comprise animal PVs that can induce neoplasia of cutaneous stratified epithelium, fibromas with a minimally hyperplastic cutaneous epithelium, cutaneous papillomas and fibropapillomas (an underlying fibroma of connective tissue), and hyperplasia of either normal non-stratified squamous epithelium or metaplastic squamous epithelium. Infection with non-human PVs is generally contained to the skin or mucous membrane s of the host species (Lancaster and Olson, 1982); however, canine oral PV can also infect the eyelid, conjunctival epithelium, and skin around the nose and mouth (Chambers and Evans, 1959). Some animal PV types, such as BPV, cottontail rabbit PV, and Europ ean harvest mice PV, have been implicated in cancers (Antonsson and Hansson, 2002), w ith BPV being the most oncogenic of the PVs (Lancaster et al., 1977). The E5 protein of BPV type 1 (BPV-1) transforms cells and functions by altering the activ ity of cellular membrane prot eins that are involved in proliferation (DiMaio et al., 1986). Most PVs are species-specific or may inf ect closely related animals within the same genus (Sundberg et al., 2000), although BPV-1 and BPV-2 can induce fibroblastic tumors in a strain of inbred mice (Boir on et al., 1964), hamsters (Cheville, 1966), and rabbits (Breitburd et al., 1981). The pres ence of BPV-specific DNA has also been detected in both naturally occurring tu mors and BPV-induced tumors of horses (Lancaster et al., 1977). An oral PV that infected one coyote a nd three dogs has also been described (Sundberg et al., 1991). St udies have shown that many domestic and

PAGE 25

13 wild species of mammals and birds can be in fected by one or more PVs (Sundberg et al., 2000). Marine Mammal Papillomaviruses Viruses and viral diseases have long been identified in marine mammals (Smith and Skilling, 1979) and new PVs in marine ma mmals have recently been described (Van Bressem et al., 1996, and Bossart et al., 2002). Papillomavirus-like particles have been observed in association with genital le sions of male sperm whales (Physeter catodon ) (Lambertsen et al., 1987), dusky dolphins (Lagenorhynchus obscurus ), and Burmeisters porpoises (Phocoena spinipinnis ) (Van Bressem et al., 1996). The high prevalence of papillomatous lesions in se veral small cetaceans (L. obscurus P. spinipinnis Delphinus capensis Tursiops truncatus ) indicates a possible venereal transmission of the disease (Van Bressem et al., 1996). Squamous papill omas and fibropapillomas have also been identified on the skin, the surface of the penis, and the tongue of mysticetes and odontocetes (Geraci et al., 1987) and gastric papillomas contai ning PV-like particles have been observed in a significant am ount of beluga (Delphinapterus leucas ) inhabiting the St. Lawrence River (M artineau et al., 2002). The Florida Manatee (Trichechus manatus latirostris ) The Florida manatee is a marine mammal that is primarily found in the southeastern United States waters and the Gulf of Mexico and is li sted as endangered at both the state and federal levels (U. S. Fi sh and Wildlife Service, 2001). The manatee immune system appears to be highly devel oped and it has been hypothesized that natural disease in manatees is uncommon (Bossart et al., 2002). Environmental diseases that may represent emerging problems for the Flor ida manatee include brevetoxicosis and cold stress syndrome (Bossart, 2001). Recen tly, it has been shown that exposure to

PAGE 26

14 multiple stressors, such as cold weat her and harmful algal blooms (Karenia brevis ), may have synergistic effects on the immune f unction of manatees (Walsh et al., 2005). Papillomas in Florida manatees were initially identified in 1997 in a captive population maintained at Homosassa Springs State Wildlife Park (HSSWP), Homosassa, Florida. These seven captive manatees developed multiple, cutaneous, pedunculated papillomas located on the pectoral flippers, upper lips, external nares, and periorbital regions. Approximately three y ears later, four of the manat ees developed papillomas that were clinically distinct from the previously observed lesions. These lesions were sessile, firm, and more diffuse and numerous than those biopsied in 1997. Based on histological, ultrastructural, and immunohistochemical findings PV was considered to be a theoretical causative agent in both outbreaks. El ectron microscopy evaluation of the lesions revealed the presence of round to hexa gonal 45to 50-nm virions that were ultrastructurally identical to those of know n PVs. Positive immunohistochemical staining was demonstrated with polyclonal antibodies against BPV-1. This was the first viral infection described in Florida manatees (Bossa rt et al., 2002). Similar papillomatous skin lesions have since been observed in free -ranging Florida manatees inhabiting two locations in Florida waters (W oodruff et al., in press). The first molecular detection of PV inf ection in Florida manatees was performed by amplifying PV DNA from papillomatous le sions of captive and free-ranging manatees using the degenerative HPV primers MY09 and MY11. These primers amplified a 458bp DNA fragment of the highly conserved L1 capsid protein gene of manatee papillomavirus (TmlPV) (Woodruff et al., in pr ess). Recently, the entire TmlPV genome has been completely sequenced and characterized. The complete TmlPV genome

PAGE 27

15 (TmPV-1) contains 7,722 bp and consists of seven major ORFs that encode five early proteins and two late capsid proteins (Rector et al., 2004) Sequences of nine L1 fragments previously described by Woodruff et al. (2005) were 100% identical to the corresponding L1 region of the published ma natee papillomavirus, TmPV-1, suggesting that there is only one type of manatee PV that causes skin le sions in manatees (Rector et al., 2004). Although the exis tence of a manatee papillomavirus has been well documented, further work is required in orde r to better understand th e overall impact and possible oncogenic potential of papillomavir us infection in th e already endangered Florida manatee. The goals of this study were to determine if more than one TmlPV is associated with papillomatous lesions in captive and free-ranging manatees, to genetically characterize the TmlPV genome(s), a nd to develop serological assays with the potentially oncogenic E6 and E7 protei ns and with the L1 capsid protein. Figure 1-1. Illustration de monstrating the genetic organization of a typical papillomavirus genome. The HPV-16 genome is divided into early (E) and late (L) regions depending on th e timing of protein expression.

PAGE 28

16 CHAPTER 2 MATERIALS AND METHODS Clinical Data Nine adult female Florida manatees (Trichechus manatus latirostris ) comprised the captive population dwelling at Homosassa Springs State Wildlife Park (HSSWP) located in Homosassa, Florida. Ma natees at HSSWP were housed in Homosassa Springs, a natural freshwater spring at the headwaters of the Homo sassa River, Citrus County, Florida. An underwater fence placed at the junction of the spring and the river confined the manatees to an area of approximately 2 acres (Bossart et al., 2002). Free-ranging manatees were occasionally observed at the ou ter perimeter of the unde rwater fence. The free-ranging manatees found in this area are winter residents that use the springs for thermoregulation (Woodruff et al., in press). Source of Samples Between January, 2003, and February, 2005, sk in lesions from captive and freeranging manatees were brought to our laborator y fresh, on ice, or fixed in either 10% non-buffered formalin (NBF) or Dimethyl Sulfoxide (DMSO). Papillomatous skin lesions from captive Florida manatees were received from various parks, including HSSWP, Florida, and other marine parks in Florida and California. Papillomatous skin lesions were also obtained from free-ranging Florida manatees inhabiting several bodies of Florida waters, including Cr ystal River, Homosassa River, Port of Isles, and Tampa Bay, and from free-ranging Antillean manatees (Trichechus manatus manatus ) inhabiting the offshore waters of the Drowned Keys, Belize. One papillomatous penile lesion

PAGE 29

17 preserved in 10% NBF was obtained from a free-ranging manatee carcass examined at the Fish and Wildlife Research Institute, St. Petersburg, Florida, and an uninfected, normal manatee liver was obtained from th e Marine Mammal Pathology Laboratory, St. Petersburg, Florida. Samples biopsied fr om HSSWP captive manatees between July, 1998, and January, 2000, were preserved in 10% NBF or DMSO and samples biopsied from free-ranging manatees in April, 2004, and August, 2004, were preserved in 10% NBF. All other manatee tissue samples (captive and free-ranging) arrived fresh or on ice. Sample description and information ar e located in Table 2-1 and Table 2-2. Blood serum from captive manatees hous ed at HSSWP was brought to our laboratory on dry ice, courte sy of Bob Bonde, U. S. Geol ogical Survey, Gainesville, Florida. Serum samples were obtained fr om the following captive manatees (date obtained): Holly (July 9, 1998), Betsy (Febru ary, 1999), Oakley (Feb 25, 1999; June 20, 2002), Willoughby (January 13, 2000), Amanda (Jan 13, 2000), and Lorelei (Jun 20, 2002). Purified anti-manatee IgG monoc lonal antibody (1.2 mg/ml) was obtained courtesy of Dr. Peter McGuire, Department of Biochemistry, University of Florida, and Ms. Linda Green, Hybridoma Core Laboratory, University of Florida, Gainesville, Florida. DNA Extraction from Papillomatous Lesions Total DNA was extracted from all tissue samples using the DNeasy tissue kit (Qiagen Inc, Valencia, California, USA) acco rding to the protocol recommended by the manufacturer. Working in a laminar flow cabinet equipped with an HEPA filter, approximately 25 mg of each papillomatous skin lesion was minced with a sterile surgical blade and placed and ground in a sterile 1.5-ml micro centrifuge tube. The tissues were incubated overnight at 55C in a mixture containing 180l of digestion

PAGE 30

18 buffer ATL and 20 l proteinase K (20 mg/ml) until lysis was complete. Then, 200 l of buffer AL and 200 l of 100% molecular grad e ethanol were added to precipitate the DNA. The solution was centrifuged in a DNeasy Spin Column to bind the DNA to the membrane and the membrane was washed with 500 l of buffers AW1 and AW2 for 1 minute each time. A final centrifugation step was performed to eliminate residual ethanol remaining in the membrane. The DNA was el uted in 200 l of buf fer AE and evaluated for yield and purity by spectrophotometr y using the Ultros pec 3000 (Amersham Biosciences Corp., Piscataway, New Jerse y, USA). A negative tissue sample from uninfected manatee skin or uninfected manatee liver was extracted along with each set of tissue samples for use as a negative contro l in PCR analyses. The eluted DNA samples were stored in 1.5-ml scre w-cap tubes at -80C. General Methods of PCR Taq Polymerase Reactions The PCR reaction in a 0.2 ml tube contained: 200 nM of each primer (IDT, Coralville, IA, USA), 2 mM MgSO4, 100 M of each deoxynucleoside triphosphate (dNTP), 20 mM Tris-HCl (pH 8.4), 10 mM KCl, 0.1 % Triton X-100 (pH 8.8), 10 mM (NH4)2SO4, 1 unit of Taq DNA polymerase (New England BioLabs, Beverly, Massachusetts, USA), 0.5-1.0 g of template DNA, and ultrapure H2O in a final volume of 50 l. A total of 40 PCR cycles were performed in a PTC-100 thermal cycler (MJ Research, Inc., Waltham, Mass achusetts, USA) for the amp lification of the manatee papillomavirus (TmlPV) L1 fragments using the modified MY11/MY09 primers (CR333/CR332) and the L1 TmlPV-specific primers (CR490/CR491). Following an initial denaturation step at 94C for 1 min, r eactions were subjected to 39 cycles of: a denaturation step at 94C for 1 min, an a nnealing step at 48C for 1 min, and an

PAGE 31

19 elongation step at 72C for 2 min. An elongation step at 72C for 10 min was incorporated in the final cycle. Accuprime Taq DNA Polymerase Reactions The PCR reaction in a 0.2 ml tube cont ained: 400 nM of each primer, 200 mM Tris-HCl (pH 8.0), 500 mM KCl, 15 mM MgCl2, 2 mM of each dNTP, 2 units of Accuprime Taq DNA polymerase (Invitrogen, Carlsbad, California, USA), 0.2-0.5 g of template DNA, and ultrapure H20 in a final volume of 50 l. Cycling conditions for the amplification of the complete TmlPV E6 gene fragment included: an initial denaturation step at 94C for 2 min, then, 39 cycles of: a denaturation step at 94C for 30 sec, an annealing step at 53C for 30 sec, and an ex tension step at 68C for 1 min. Cycling conditions for amplification of the L1 fragments and the complete E7 complete gene were similar, except that the annealing temperatures were set at 59C and 51C, respectively. Expand High Fidelity Plus Reactions The PCR reaction in a 0.2 ml tube contai ned: 400 nM of each primer, 200 M of each dNTP, 1.5 mM MgCl2, Expand High Fidelity Plus Reaction Buffer diluted to 1.5 mM MgCl2 (Roche Applied Science, Mannheim, Germany), 2.5 units of Expand High Fidelity Plus Enzyme Blend (Roche App lied Science), 0.5 g of template DNA, and ultrapure H2O in a final volume of 50 l. Cyclin g conditions for amplification of the TmlPV L1-E1 region were: an initial denaturation at 94C for 2 min, followed by 39 cycles of 94C for 30 sec, 50C for 30 sec, and 72C for 3 min, w ith a final extension step at 72C for 7 minutes. Cycling conditi ons for amplification of the TmlPV L1-L2 capsid region were similar, except that the annealing temperature was set at 59C and the extension step was performed at 68C for 3.5 min. Cycling conditions for amplification

PAGE 32

20 of the TmlPV complete L1 capsid gene were similar, but the extension steps were performed at 72C for 1.5 min. For amplifi cation of the TmlPV complete L2 capsid gene, cycling conditions were similar and th e annealing temperatur e was set at 53C and the extension step performed at 72C for 2 min. PCR Targeting the L1 458-bp Fragment The MY11 and MY09 L1 consensus primers th at amplify a 458-bp fragment of the L1 capsid protein gene of several human papillomaviruses (HPVs) (Manos et al., 1989) were modified in our laboratory to contai n deoxyinosines at positions of nucleotide degeneracy [forward primer (FP) CR333 and re verse primer (RP) CR332] (Table 2-3). DNA obtained from a lesion from captive ma natee Oakley was used as the positive control. A blank sample (water) and DNA extracted from a liver of an uninfected manatee were used as negative controls. PCR Targeting the L1 TmlPV 458-bp Fragment Based on newly generated nucleotide se quences of the amplified TmlPV L1 fragment obtained in our la boratory, the MY11/MY09 HPV L1 primers (Manos et al., 1989) were modified to contain TmlPV L1-speci fic nucleotides at pos itions of nucleotide degeneracy. The L1TmlPV primers, FP CR490 and RP CR491 (Table 2-3), target a 458-bp sequence contained within the L1 capsid gene of TmlPV. DNA obtained from a lesion from captive manatee Oakley was used as the positive control. A blank sample (water) and DNA from a negative tissue (manatee liver) were used as negative controls. PCR Targeting the TmlPV L1-E1 Region Using a PCR method described by Forslund and Hansson (1996), oligonucleotide primers were designed to target a region of the TmlPV genome that spans an area from within the 3 end of the TmlPV L1 ORF to an area within the 5 end of the TmlPV E1

PAGE 33

21 ORF. A plus-strand oligonucleotide prim er designed from the TmlPV L1 458-bp fragment nucleotide sequence was used in a PCR reaction together with a minus-strand HPV consensus primer from the E1 ORF (Smits et al., 1992). The TmlPV L1 FP CR442 and the HPV E1 RP CR441 (Table 2-3) direct the amplification of an approximately 3,000-bp fragment. DNA obtained from a lesi on from captive manatee Oakley was used as the positive control. A blank samp le (water) and DNA from a negative tissue (manatee liver) were used as negative controls. PCR Targeting the Complete TmlPV E6 Gene Based on the nucleotide sequences of th e TmlPV L1-E1 region obtained by us, oligonucleotide primers FP CR498 and RP CR499 (Table 2-3) targeting the complete TmlPV E6 gene were designed for cloning and expression. DNA obt ained from a lesion from captive manatee Oakley was used as the positive control. A blank sample (water) and DNA from a negative tissue (manatee liver ) were used as negative controls. PCR Targeting the Complete TmlPV E7 Gene Based on the nucleotide sequences of th e TmlPV L1-E1 region obtained by us, oligonucleotide primers FP CR500 and RP CR501 (Table 2-3) targeting the complete TmlPV E7 gene were designed for cloning and expression. DNA obt ained from a lesion from captive manatee Oakley was used as the positive control. A blank sample (water) and DNA from a negative tissue (manatee liver ) were used as negative controls. PCR Targeting the TmlPV L1-L2 Region Based on nucleotide sequences of the complete TmlPV genome (AY609301) (Rector et al., 2004), a plus strand oligonuc leotide primer FP CR572 (Table 2-3) was designed from an area upstream of the L2 ORF. Used together with an L1 minus strand primer RP CR574 (Table 2-3), designed by us these primers target a region of the

PAGE 34

22 TmlPV genome that spans an area upstream of the 5end of the TmlPV L2 ORF to an area downstream of the 3end of the TmlPV L1 ORF. DNA obtaine d from a lesion from captive manatee Oakley was used as the posi tive control. A blank sample (water) and DNA from a negative tissue (manatee liver) were used as negative controls. PCR Targeting the Complete TmlPV L1 Gene Based on nucleotide sequences of the L1-L2 fragment obtained in our laboratory, a plus-strand oligonucleotide prim er FP CR573 (Table 2-3) was designed upstream of the L1 start codon to be used together with the L1 FP CR574 for am plification of the complete L1 gene for cloning and expression. DNA obtained from a lesion from captive manatee Oakley was used as the positive co ntrol. A blank sample (water) and DNA from a negative tissue (manatee liver) were used as negative controls. PCR Targeting the Complete TmlPV L2 Gene Based on nucleotide sequences of the L1-L2 fragment obtained in our laboratory, a minus-strand oligonucleotide primer RP CR622 (Table 2-3) was designed downstream of the L2 stop codon to be used together with the L2 FP CR572 for amplification of the complete L2 gene. DNA obtained from a le sion from captive manatee Oakley was used as the positive control. A blank samp le (water) and DNA from a negative tissue (manatee liver) were used as negative contro ls. A linear representation of the circular TmlPV genome with the relative positions of th e PCR primers used in this study is shown in Figure 2-1. Gel Electrophoresis Between 20-30 l of PCR products were re solved by horizontal electrophoresis in 1.0% agarose gels containing ethidium brom ide (0.5 g/ml). Amplified DNA fragments

PAGE 35

23 were visualized under ultr aviolet light and photographe d using a gel documentation system (Bio-Rad Laboratories, In c., Hercules, California, USA). General Cloning of PCR Products As described under gel electrophoresis PCR products containing amplified fragments of only the expected size were purified for use in cloning reactions. PCR products containing additional amplified fragments were resolved in 1.2% low-melting point (LMP) agarose and the band of the a ppropriate size was exci sed and purified for use in cloning reactions. PCR products and excised gel pieces were purified using the Wizard SV Gel and PCR Clean-Up System (P romega Corporation, Madison, Wisconsin, USA) according to the protocol provided by the manufacturer. Br iefly, an equal volume of membrane binding solution was added to the PCR product, the prepared PCR product was added to the SV minicolumn assembly, and the minicolumn was washed twice with the membrane wash solution. The purified DNA was eluted in 50 l of nuclease-free water and stored at -80C until further use in cloning reactions. Cloning into pCR 2.1 TOPO T/A Vector Purified PCR products were cloned into the pCR 2.1 TOPO vector (Invitrogen Life Technologies, Carlsbad, California, USA) for sequencing analysis. In a 0.2 ml tube, ligation reactions contained: 1 l of salt solution (1.2 M NaCl, 0.06 M MgCl2), 1l of the TOPO T/A vector (Invitrogen), 50 ng of purifie d PCR or gel product, and ultrapure H2O in a final volume of 6 l. Reactions were incubated at room temperature for 1 hour; then, 3l of the reaction were adde d to one vial (50l) of DH5 or TOP-10 chemically competent Escherichia coli cells (Invitrogen). Tubes were placed on ice for 1 hour, heat shocked for 30 sec at 42C in a water bath, a nd returned to ice. After adding 250 l of S.O.C. medium (Invitrogen), the tubes were shaken horizontally at 220 rpm at 37C for 1

PAGE 36

24 hour. Reactions (100-200 l) were spread onto bacterial agar plates containing ampicillin (100 g/ml) (Roche Applied Science) and bl ue/white selection me dium [75 l 2XYT: 16g Bacto-tryptone, 10 g Bacto-Yeast Extract, 15 g Bacto-Agar (Beckton Dickinson, Franklin Lakes, New Jersey, USA), 5g enzyme grade NaCl (Fisher Scientific International Inc., Hampton, New Hampshire, USA) in 1 L of ultrapure H2O, 20 l IPTG (100mg/ml) (Invitrogen), and 5 l UltraPure Bluo-gal (100mg/ml) (Invitrogen)] spread on the agar plates surface one hour before the plates were inoculated with cloning reactions. Plates were in cubated overnight at 37C. Cloning into P-Target Ma mmalian Expression Vector Purified PCR products were directly cloned into the P-Target mammalian expression vector (Promega) to test for protein expre ssion after tran sfection and immunofluorescence. In a 0.5 ml tube, the li gation reaction contained: 1l of T4 DNA ligase constituents, 1 l of T4 DNA ligase, 1 l of pTarget cloni ng vector (ProMega), 5l of purified PCR product, and ultrapure H2O in a final volume of 10 l. The ligation reaction was incubated at 4C overnight in a re frigerated block. Five l of the ligation reaction were added to one vial (50l) of JM109 high efficiency competent cells (Promega) and incubated on ice for 20 min. Th e cells were heat shocked for 45 sec at 42C in a water bath and returned to i ce for 2 min. After adding 450l of S.O.C. medium, the tubes were shaken at 150 rpm at 37C for 1.5 hrs. The transformation reaction was spread in 100l volumes onto bacterial agar plates containing ampicillin (100g/ml) and blue/white selection medium. Cloning into pcDNA .1 Directional TOPO Expression Vector In order to pair with the -GTGGoverhang of the pcDNA 3.1 Directional TOPO Expression Vector, the TmlPV E7 FP CR 500 was modified at the 5end to

PAGE 37

25 contain a corresponding 4-bp CA CC sequence. To amplify the complete TmlPV E7 gene with the TOPO overhang, the modified E7 FP CR 530 (Table 2-3) was used in a PCR reaction with the TmlPV E7 RP CR501. The P CR reaction in a 0.2 ml tube contained: 200 nM of each primer, 300 mM Tris-SO4 (pH 9.1), 90 mM (NH4)2SO4, 10 mM MgSO4, 200M of each dNTP, 2 units of Elongase enzyme mix (Invitrogen), 0.5-1.0 g of template DNA, and ultrapure H2O to a final volume of 50 l. Cycling conditions for the amplification of the E7 TOPO PCR products were: an initia l denaturation step at 94C for 2 min, then 39 cycles of a denaturation st ep at 94C for 30 sec, an annealing step at 51C for 1 min, and an extension step at 68C for 1 min. Purified E7 TOPO PCR products were directly cloned into th e pcDNA 3.1 Directional TOPO expression vector for use in immunofluorescence assays. In a 0.2 ml tube, the ligation reaction contained: 1l of salt solu tion (50 mM NaCl, 2.5 mM MgCl2), 1l of pcDNA 3.1 TOPO vector (Invitrogen), 50 ng of purified E7 TOPO PCR product, and ultrapure H2O in a final volume of 6l. Ligation reacti ons were incubated at room temperature for 1 hour; then, 3l of the reaction were added to one vial (50l) of One-shot TOP10 chemically competent E. coli cells (Invitrogen). Tubes were placed on ice for 1 hour, heat shocked for 30 sec at 42C in a water bat h, and returned to ice. After adding 250 l of S.O.C. medium (Invitrogen) the tubes were shaken horizontally at 220 rpm at 37C for 1 hour. Transformations (100-200 l) we re spread onto bacterial agar plates containing ampicillin (100 g/ml) and the plat es were incubated overnight at 37C. Analyzing Recombinants Using sterile toothpicks, bacterial colonies were selected from agar plates and added to 10 ml sterile glass tubes containi ng 3 mL 2XYT and 3l ampicillin (100g/ml) and the tubes were shaken overnight at 275 rpm at 37C. DNA was extracted from

PAGE 38

26 approximately 1 ml of overnight culture usi ng the 10-minute Mini-Prep Protocol (Zhou et al., 1990). Briefly, overnight cultures were centrifuged for 10 sec in a 1.5 ml capped tube. The supernatant was discarded, 300l of TENS was added to the cell pellet, and the tube was vortexed for 2-5 sec. Then, 150 l of 3.0 M sodium acetate (pH 5.2) (Gibco Life Technologies, Carlsbad, California, USA) were added and the tube was vortexed and centrifuged. The supernatant was transferred to a fresh 1.5 ml capped tube and mixed thoroughly with 0.9 ml of 100% mo lecular grade ethanol which had been pre-cooled to 20C. After centrifugation, the plasmid DNA pe llet was washed twice with 1 ml of 70% ethanol, allowed to dry, and resuspended in 50 l of TE buffer (pH 8.0). Recombinants were analyzed by restriction enzyme dige stion using endonucleases HindIII, ApaI, BamHI, EcoRI, and the combination of ApaI and BamHI (Invitrogen). Restriction digest reactions contained: 0.3 l of restriction enzyme, 3.0 l of the appropriate enzyme buffer, 1.0 l of mini-prep DNA, and ultrapure H2O in a final volume of 30 l. Reactions were incubated at 37C for 1 hour in a dry block and analyzed by gel electrophoresis. Recombinants containing inserts of the a ppropriate size were fu rther propagated in competent E. coli cells and purified with the Auru m Plasmid Mini Kit (Bio-Rad Laboratories, Inc., Hercules CA, USA) for sub-cloning a nd sequencing. Following the protocol provided by the manufacturer, 1 ml of overnight culture was added to a 2.0 ml tube, centrifuged, and the supernatant decanted. Then, 250 l of resuspension solution and 250l of lysis solution were added, the tu be was inverted 6-8 times, and the mixture was allowed to incubate at room temperature for 5 min to ensure lysis was complete. After adding 350 l of neut ralization solution, the tube was inverted 6-8 times and centrifuged. The cleared lysate was transferre d to a mini spin column and the column

PAGE 39

27 was washed once with 750 l of wash solutio n. The DNA was eluted in 50 l of elution solution and evaluated for yield and purity by spectrophotometry. Sub-cloning of Purified Recombinants In order to obtain a high yield and purity of vector and recombinant DNAs for subcloning reactions, midi-prep DNAs were prepar ed with the Qiagen plasmid purification kit (Qiagen, Hilden, Germany), according to the manufacturers protocol. Transformation reactions cont aining 1 l of DNA were pr opagated overnight in 100 ml of 2XYT medium (100l ampicillin), the overnight cultures were centrifuged, and the supernatants were removed. The bacterial pell ets were resuspended and vortexed in 4 ml of buffer P1 and mixed gently in 4 ml of buf fer P2. The lysis reactions were allowed to proceed at room temperature for 5 min and 4 ml of chilled buffer P3 were added. The reactions were mixed gently, incubated on ice for 15 min, and centrifuged for 1 hour until the supernatant was clear. Each supernatant wa s added to an equilibr ated Qiagen-tip 100 and allowed to enter the resin by gravity flow The Qiagen-tip was washed twice with buffer QC and the DNA was eluted in 5 ml of buffer QF. The DNA was precipitated in 5 ml of isopropanol, washed with 2 ml of 70% ethanol, and allowed to dry for 15-20 min. The DNA pellet was redissolved in 200 l of buffer EB and evaluated for yield and purity by spectrophotometry. Sub-Cloning into pcDNA 3.1/Zeo+ Expression Vector The complete TmlPV E6 and L1 capsid genes were sub-cloned from pCR 2.1 TOPO T/A into pcDNA 3.1/Zeo+ expression vector at positions of compatible restriction sites present in the vector multiple cloning sites. The TmlPV E6 recombinants were subcloned into the expression vector at the Ec oRI site, and the TmlPV complete L1 gene recombinants were sub-cloned into the expression vector at the HindIII and XbaI sites.

PAGE 40

28 Purified recombinant DNAs and purified pcDNA 3.1/Zeo+ DNA were cut with identical restriction enzyme(s) in separate digest re actions. Restriction digests of pcDNA 3.1/Zeo+ contained: 3.0 l of restri ction enzyme, 6.0 l of the corresponding buffer (Invitrogen), 3.0 l of bovine serum albumin (Invitrogen), 1 g of purified pcDNA 3.1/Zeo+ vector DNA, and ultrapure H2O in a final volume of 60l. Re striction digests of TOPO T/A recombinants were similar, except that they contained 3g of purified recombinant DNA. Digest reactions were incubated at 37C in a dry block for 2 hours and resolved by horizontal electrophoresis in 1.2% low-melting point (LMP) agarose gels containing ethidium bromide (0.5 g/ml). The bands of the appropriate size for the digested pcDNA 3.1/Zeo+ vector and for the digested recombinants were excised from the LMP gel and purified using the Wizard SV gel and PCR cl ean-up system. DNA from the purified gel products was used in the ligation reaction that contained: 4 l of 5X T4 ligase buffer, 1.5 l of T4 ligase (Invitrogen), a 1l of gel purified recombinant DNA, 3l of gel purified pcDNA 3.1/Zeo+, and ultrapure H2O in a final volume of 20l. Ligation reactions were incubated overnight at 14C in a refrigeration block. Ten l of the ligation reaction were added to one vial (50 l)of DH5 competent E. coli cells, incubated on ice for 1 hour, heat shocked at 42C for 30 sec, and returned to ice. Then, 600 l of 2XYT containing 50 mM glucose were added and the vial was shaken horizontally at 220 rpm at 37C for 1 hour. Reactions (200l) were spread onto agar plates containing ampicillin, and plates were incubated overnight at 37C. Bacterial colonies were selected from the plates and propagated in 2XYT medium containing am picillin. Recombinant DNA was purified using the 10 minute mini-prep protocol and in serts of the appropriate size cloned into the pcDNA 3.1+ vector were confirmed by restricti on digest using 6-ba se cutter enzymes

PAGE 41

29 NsiI, known to cut the TmlPV E6 fragment on ce, and SpeI, known to cut the TmlPV L1 fragment sequence once. L1 Gene Sub-Cloning into pFastBac Vector The TmlPV complete L1 gene was subcloned from pCR 2.1 TOPO T/A into the pFastBac1 vector (Invitrogen) at positions of compatible restriction sites present in the vector multiple cloning site for use in the Bac-to-Bac B aculovirus expression system (Invitrogen). Purified L1 recombinant DNA and purified pFastBac vector DNA were cut with identical restriction enzyme(s) in sepa rate digest reactions. Restriction digests of pFastBac contained: 3.0 l of restriction enzyme EcoR I (Invitrogen), 6.0 l of the corresponding 10X buffer III (Invitrogen), 3.0 l of bovine serum albumin (Invitrogen), 1 g of purified pFastBac ma xi-prep DNA, and ultrapure H2O in a final volume of 60l. Restriction digests of the TOPO T/A TmlPV L1 complete gene recombinant were similar, except that they contained 3g of purified maxi-prep recombinant DNA. Digest reactions were incubated at 37C in a dry block for 2 hours and resolved by horizontal electrophoresis in 1.2% low-melting point (L MP) agarose gels containing ethidium bromide (0.5 g/ml). Bands of the appropria te size for the digested pFastBac vector and the digested TOPO T/A TmlPV L1 comple te gene recombinant were excised from the LMP gel and purified using the Wizard SV gel and PCR clean-up system. The ligation and transformation reac tions of the L1 complete gene into pFastBac were set up similar to the reactions pr eviously described for sub-cl oning into the pcDNA 3.1+/Zeo expression vector, except that transformati ons were performed in TOP-10 competent E. coli cells (Invitrogen). Individua l bacterial colonies were se lected from the plates and propagated in 2XYT medium containing ampi cillin (100g/ml). DNA was extracted from the overnight cultures using the 10 minute mini-prep protocol (Zhou et al., 1990)

PAGE 42

30 and pFastBac L1 recombinants were iden tified by restriction digest with enzymes EcoRI, Hind III, and BamHI, a 6-base cu tter enzyme known to cut the TmlPV L1 sequence at one site. L1 Gene Sub-Cloning into pBlueBac 4.5 The TmlPV complete L1 gene was su b-cloned from pCR 2.1 TOPO T/A after digesting with enzymes XbaI and SstI into the pBlueBac 4.5 vector (Invitrogen), which was digested with the same enzymes. Th e ligation and transformation reaction of the TmlPV L1 gene into the pBlueBac vector was set up similar to the described sub-cloning reaction into the pFastBacI vect or. Individual bacterial coloni es were selected from agar plates and propagated in 2XYT medium containing ampicillin (100g/ml). DNA was extracted from overnight cu ltures using the 10 minute mini -prep protocol (Zhou et al., 1990) and pBlueBac L1 recombinants were identi fied by restriction digest with XbaI and SstI enzymes. Sequencing of PCR-amplified and Cloned Products As described under gel electrophoresis, amp lified PCR products of the expected size were purified and sequenced directly us ing the PCR primers diluted 1:5 in sterile H2O. Cloned PCR products were sequenced with the corresponding vector sequencing primers diluted 1:10 in sterile H2O. Additional sequencing pr imers (1:5) were used to obtain the complete sequence of cloned DNA fragments that were greater than 600 nucleotides in length. A list of sequencing pr imers is contained in Table 2-4. Between 50 fmol of purified PCR products or purif ied recombinants were sequenced in duplicate using specific forward and revers e primers in the Beckman-Coulter CEQ 2000XL sequencing instrument (Beckman-Coulte r Inc., Fullerton, California, USA). Chromatograms were checked manually for errors in nucleotide sequences using the

PAGE 43

31 Chromas 2.3 software (Technelysium Pty Ltd., Tewantin, Queensland, Australia), and the assembled sequences were analyzed using the seqed, gap, translate, and multiple alignment functions of the University of Wisconsin P ackage Version 10.2 (Genetics Computer Group [GCG], Univ ersity of Wisconsin, Madi son, Wisconsin, USA). The Basic Local Alignment Search Tool (BLA ST) function of the National Center for Biotechnology Information (NCBI) website ( http://www.ncbi.nlm.nih.gov/ ) was used to identify papillomavirus sequences most cl osely related to sequences obtained from TmlPV fragments. Neighbor-Joining phyloge netic trees were generated by PAUP 4.0 (Sinauer Associates, Sunderland, Massachuse tts, USA) software, using Clustal W slow and accurate function using Gonnet residue weight table, gap penalty of 35 and gap extension penalty of 0.75 for pairwise alignment parameters, and gap penalty of 15 and gap extension penalty of 0.3 fo r multiple alignment parameters. Phylogenetic trees were constructed with the deduced amino acid sequ ences of the TmlPV L1 ORF, L2 ORF, E6 ORF, and E7 ORF DNA fragments. Trees we re based on the amino acid sequences of human and non-human PVs obtained from th e GenBank database through the NCBI website and from the HPV database of the Los Alamos National Laboratory Theoretical Biology and Biophysics website ( http://hpv-web.lanl.gov /stdgen/virus/hpv/ ). Transfection of Insect Cell Cultures Culturing Insect Cells Sf21 (Spodoptera frugiperda ) insect cells (Invitrogen) we re used as indicator cell cultures to generate recombinant baculovi ruses. Sf21 cells were cultured in 75 cm2 flasks (TPP, Switzerland) in serum free Sf-900 II medium (Invitrogen) containing: 200 g/ml of antibiotic/antimycotic [penicillin (1X104 units/ml)], streptomycin sulfate (10 mg/ml), amphotericin B (25 g/ml)] (Gibco), 200 g/ml of gentamicin (100 g/ml) (Gibco), and

PAGE 44

32 10% fetal bovine serum (Gibco). Insect ce lls were incubated at 27C in a humidified incubator. After one week, th e cell monolayer was scraped w ith a sterile, disposable cell scraper (Greiner Bio-One, Kremsmuenster, Australia), resuspende d in 10 ml of Sf-900 II/10% FBS, and counted with a hemocytome ter (Reichert Scientific Instruments, Buffalo, NY). Approximately 1 X 106 Sf21 cells in 2 ml of Sf-900 II/10% FBS medium were added to 35 mm dishes and incubated at 27C. All steps were performed in a laminar-flow cabinet under sterile conditions. Transformation of MAX Efficiency DH10Bac Competent E coli In order to transfect insect cell cultures a nd generate a recombin ant baculovirus, the pFastBac L1 constructs were firs t transformed into MAX Efficiency DH10Bac competent E. coli (Invitrogen) that contain a bacul ovirus shuttle vector (bacmid) and a helper plasmid. Briefly, 1 l of purified pF astBac recombinant (1ng/l) was added to 50l of DH10Bac E. coli incubated on ice 30 min, heat shocked at 42C for 45 sec, and returned to ice for 2 min. The reaction was transferred to a 15 ml screw-cap conical tube (Sarstedt Inc., Newton, North Carolina, USA) and 900 l of S.O.C. medium were added. The reactions were shaken at 225 rp m at 37C for 4 hours and transformations were spread onto bacterial agar plates c ontaining kanamycin (50g/ml), tetracycline (10g/ml), gentamicin (7 g/ml) (Gibco), a nd blue/white selection medium. Plates were incubated overnight at 37C. Individual bact erial colonies were selected and propagated in 3ml 2XYT medium containing kanamycin, tetracycline, and gentamicin and shaken overnight at 275 rpm at 37C. Overnight cultures were purifie d using the 10 minute mini-prep protocol and recombinant bacmid s were confirmed by PCR analysis. The Bacto-Bac M13 primers, forward primer CR 613: 5-CCC AGT CAC GAC GTT GTA AAA CG-3 and reverse primer CR 614: 5-AGC GGA TAA CAA TTT CAC ACA GG-

PAGE 45

33 3, were used together in a PCR reaction th at contained: 200 nM of each primer, 2 mM MgSO4, 100 M of each dNTP, 20 mM Tris-HCl (pH 8.4), 10 mM KCl, 0.1 % Triton X100 (pH 8.8), 10 mM (NH4)2SO4, 1 unit of Taq DNA polymerase (New England BioLabs), 1l of mini-prep DNA, and ultrapure H2O in a final volume of 50l. For amplification of bacmid DNAs, cycling conditi ons were: an initial denaturation step at 93C for 3 min, followed by 29 cycles of dena turation at 94C for 45 sec, annealing at 51C for 45 sec, and extension at 72C for 5 min. An elongation step at 72C for 7 min was incorporated into the final cycle. Bacmid DNA recombinants containing the L1 complete gene were expected to be a pproximately 4,000-bp in size (2,300 bp bacmid DNA plus 1,712 bp L1 complete gene) a nd bacmid DNA amplicons that were not recombinants were expected to be approxi mately 300-bp in size. PCR products were resolved by horizontal electropho resis in 1.0% agarose gels containing ethidium bromide (0.5 g/ml) and visualized under UV light. Recombinant bacmids were further confirmed to contain the pFas tBac L1 gene by PCR analysis targeting the TmlPV L1 complete gene. Transfection of Insect Cells with Bacmid DNA Recombinants In a 10 ml glass bacteriological tube (Fis her Scientific), 1 g of L1 bacmid DNA was added to 100l of unsupplemented Graces ins ect cell culture medium (Gibco). In a separate glass tube, 6 l of Cellfectin Reagent (1mg/ml) (Invitrogen) were added to 100l of unsupplemented Graces insect cell culture medium. The mixtures were combined and the tube was incubated at room temperature for 45 min. Prior to transfecting Sf21 insect cells, the cell m onolayer was washed with 2 ml of Graces medium and incubated for 5 min at room temperature. The medium was removed and 800 l of Graces medium were added gen tly and directly to the cells. The

PAGE 46

34 Bacmid/Cellfectin transfection mix was th en added dropwise ont o the cells and the cultures were incubated at 27C for 5 hours. At this time, the transfection mixture was removed and 2 ml of unsupplemented Graces me dium was added to the transfected cells. Cultures were incubated at 27C in a humi dified incubator. Sf21 cells fed with unsupplemented Graces medium (without bacu lovirus DNA) and Sf21 cells transfected with purified baculovirus DNA (w ithout insert) were used as negative controls. All steps were performed in a laminar-flo w cabinet under sterile conditions. Harvest of Recombinant Baculovirus Stocks After the transfected cell cultures were incubated for approximately 96 hrs, the cell monolayer and supernatant (~2 ml) were collect ed with a sterile pipette and inoculated onto Sf21 cultures in 60 mm tissue culture dishes One ml of the ce ll supernatant plus 1 ml of Sf-900 II/5% FBS medium were added to fresh 60 mm dishes containing approximately 2 X 106 Sf21 cells. The cells were incubated at 27C for 1.5 hrs, the inoculum was removed, and the cultures were transfected with 3 ml of unsupplemented Graces medium. Infected cultures were incubated at 27C for approximately 96 hrs and sub-cultured again into fresh cultures. The infected cultures were sub-cultured a total of 5 times and the final harvest of cells and supernatants were obtained from 150 mm dishes. All steps were performed in a la minar-flow cabinet under sterile conditions. Harvests from infected ce ll cultures were analyzed by electron microscopy, by Mr. Woody Frazer, from the Florida Animal Disease Diagnostic Laboratory, Florida Department of Agriculture and Consum er Services, Kissimmee, Florida. Reverse Transcription PCR (RT-PCR) of Infected Cell Cultures RNA was extracted from infected Sf21 cell cultures transfected with the pBlueBac plasmid containing the L1 capsid gene to determine whether messenger RNA (mRNA)

PAGE 47

35 encoding the TmlPV L1 capsid protein was pres ent. To use as negative controls, RNA was extracted from untransfected Sf21 cell cu ltures and from Sf21 ce lls transfected with purified parental pBlueBac vect or. Cells and supernatants were harvested 96 hrs after transfection, centrifuged at 4,000 rpm at 10C for 10 min, the supernatant was removed, and RNA was extracted from the cell pellets using the Aurum total RNA mini kit (BioRad). The RNA samples were treated with twice the recommended amount (160l) of DNase I solution for 30 min, eluted from a mi ni column with 100l of elution solution, and treated again with 160l of DNase I so lution. The alcohol pr ecipitated RNA pellet was resuspended in 60l of RNase-free H2O and analyzed for yield and purity by spectrophotometry. Total RNA was used in reve rse transcription reactions in order to obtain a first-strand cDNA product for use in PCR analysis. Synthesis of cDNA was performed with SuperScript II (Invitrogen) acco rding to the manufacturers protocol. Reactions contained random hexamer primers (Invitrogen) or a TmlPV L1 gene-specific primer (RP CR574) designed by us. The revers e transcription assays were performed in duplicate with or without the incorporation of SuperScript II reverse transcriptase enzyme (Invitrogen) in order to ensure that the DNA had been completely degraded by the DNase I treatment during the RNA extrac tion process. The cDNAs were used as templates for amplification in TmlPV L1 PCR assays according to the PCR protocol targeting the TmlPV complete L1 gene. Generation of Recombinant Baculovirus Recombinant vector pBlueBac 4.5 containi ng the complete TmlPV L1 gene under the control of the polyhedron promoter and the Bac-N-Blue baculovirus DNA (Invitrogen) (0.5g DNA in 10l volume) were incubated at room temperature for 10 min. Then, 1 ml of unsupplemented Graces insect medium (Invitrogen) was added to

PAGE 48

36 the tube, followed by 20l of Cellfectin reag ent (Invitrogen). The reaction was gently mixed for 10 sec and allowed to incubate at room temperature for 15 minutes. Prior to transfecting the Sf21 in sect cultures (3X106) seeded in 60 mm dishes, the medium was removed and the cell monolayer was rinsed gently twice with 2 ml of fresh, unsupplemented Graces insect medium wit hout FBS. The transfection mixture was added dropwise onto the cells and incubate d at 27C for six hours in a humidified incubator. After the incuba tion period, 2 ml of complete TNM-FH medium (Invitrogen) containing gentamycin (10g/ml) and FBS (10%) were added to each dish. The dishes were incubated at 27C for 10 days, at which time cells and medium were harvested and stored at 4C prior to screen ing and purification of the reco mbinant viruses. A second dish containing Sf21 cell cultures was transfec ted with the transfer vector pBlueBac 4.5 containing the L1 capsid gene, but no bacul ovirus DNA, and treated similarly. A third culture of untransfected Sf21 cells served as a negative control. After 72 hrs, 500l of the medium was harvested from each dish a nd transferred to a st erile 15 ml screw-cap tube to which 2l of Bluo-gal substrate (200 g/l in DMSO) was added. The tubes were incubated at 27 and monitored for the development of a blue color. Transfection of Mammalian Cells Culturing African Green Monkey Kidney (COS-7) Cells African green monkey kidney (COS-7 ) cells were propagated in 75 cm2 flasks in Dulbeccos modified eagle medium (D MEM) (Gibco) containing gentamycin, antibiotic/antimycotic, and 10% FBS. Cells were incubated at 37C in a humidified incubator.

PAGE 49

37 Electroporation of COS-7 Cells with Recombinants for Immunofluorescence Assays COS-7 cells were dispersed with tr ypsin/EDTA (0.25% Trypsin/1mM EDTA) (Gibco), resuspended in 10 ml DMEM/5% FBS in a 15 ml screw cap tube, and counted with a hemocytometer. The cells were cen trifuged at 1,500 rpm for 10 min at 10C in a refrigerated centrifuge (Jouan, Unterhachi ng, Germany). The medium was aspirated from the cells and the cell pellet was resuspe nded in cold, sterile 1X PBS (Gibco). Then, 0.4 ml of cell suspension (1 X 107 cells) and 5g of purified recombinant plasmid DNA were added to a 0.4 cm cuvette placed inside the Gene Pulser II unit shocking chamber (Bio-Rad Laboratories). COS7 cell electroporation assays we re performed using either the E6 complete gene in pcDNA 3.1+/Zeo or the L1 complete gene in pcDNA 3.1+/Zeo vector. Cells were electroporated in the Gene Pulser II Electroporation System (Bio-Rad Laboratories) for 0.64 msec with the low capacito r set at 25F and the voltage set at 0.6 KVolts. Then, 200 l of electroporated cells were added to each chamber of a four chamber glass slide (Lab-Tek, Nalge Nunc Intl., Naperville, Illinois, USA), each containing 1 ml of DMEM/10 % FBS and zeoci n, and the chambered coverglass sides were incubated at 37C. Recombinant plasmid DNAs with the genes cloned in the wrong orientation and parental plas mid DNA (without insert) were used as negative controls. Cells in each chamber of the slide were fixe d at four different times; 12 hrs, 24 hrs, 36 hrs, and 48 hrs. Briefly, the medium was as pirated from the chamber and the cells were rinsed with 1ml of 1X Hanks balanced salt solution (Gibco). The cells were then fixed for 1 min using 1 ml of cold acetone: meth anol (1:1) solution, the fixative was removed, and the chamber slide was stored at -20C until use. Immediately before use, to equilibrate the cells, 1 ml of PBS/ 3% BSA wa s added to each slide chamber, allowed to soak the monolayer for 5 minutes at room temperature, and removed. Serum obtained

PAGE 50

38 from captive manatees (Holly, Betsy, Oakl ey, Willoughby, Amanda, and Lorelei) with a history of papillomavirus infection was diluted 1:20 in PBS/3% BSA solution and 500 l of diluted serum were added to the appropriate chamber. The cells were incubated at 37C for 1 hr, the serum was removed, and the cells were washed three times with 1 ml PBS/0.025% Tween 20 (Fisher Scientific, Fairla nd, New Jersey, USA) solution. Purified anti-manatee IgG monoclonal antibody (1.2mg/ ml) was diluted 1:20 in PBS/3% BSA solution and 500 l of diluted monoclonal an tibody were added to each chamber. The chambers were incubated at 37C for 1 hr, the monoclonal antibody was removed, and the cell monolayer was washed three times with 1 ml PBS/0.025% Tween 20 solution. Fluorescein-labeled protein G conjugat e (250g/ml) (Sigma-Aldrich, Steinheim, Germany), was diluted 1:40 in PBS/3% BSA, 200 l were added to each slide chamber, and the chamber slides were incubated at 37C for 1 hr. The monolayers were gently rinsed three times with 1 ml PBS/0.025% Tw een 20 solution, the cove r and lining of the chamber were removed, and ProLong gold anti-fade reagent (Molecular Probes, Carlsbad, California, USA) was added dropw ise to the monolayer. Cover slips were placed on the anti-fade reagen t and the obtained monolayers were evaluated for specific immunofluorescence using a fluorescent micr oscope (Zeiss Axiovert 25). Similar immunofluorescence assays were also perf ormed using fluorescein-labeled Protein A (Sigma-Aldrich), instead of Protein G, according to the methods described. Culturing Florida Manatee Respiratory Epithelial Cells Florida manatee respiratory epithelial (TmlRE) cell cultures were obtained from Dr. Mark Sweat (Fish and Wildlife Research Institute, St. Petersburg, Florida) and propagated in 75 cm2 flasks in Dulbeccos modified eagle medium (DMEM) (Gibco)

PAGE 51

39 containing antibiotic/antimycotic, gentamycin, and 10% FBS. Cells were incubated at 37C in a humidified incubator. Transfection of TmlRE Cells with DNA Recombinants for Immunofluorescence Assays Methods for transfecting TmlRE cells were the same as the methods described for transfecting COS-7 cells. TmlRE cell transfection assays were performed with E7 pcDNA 3.1+ TOPO and the L1 complete gene pcDNA 3.1+/Zeo recombinant plasmids. Table 2-1. Samples obtained from skin lesions of captive Florida manatees. DNA extracted from lesions was tested by P CR for the presence of papillomavirus infection. HSSWP: Homosassa Springs St ate Wildlife Park, FL: Florida, SD: San Diego, CA:California. Sample I.D. Manatee I.D. Location Date Obtained CR130 Oakley HSSWP, FL July, 2002 V368 Betsy HSSWP, FL July, 1998 V369 Holly HSSWP, FL July, 1998 V370 Betsy HSSWP, FL July, 1998 V371 Amanda HSSWP, FL January, 2000 V372 Amanda HSSWP, FL January, 2000 V373 Willowby HSSWP, FL January, 2000 V374 Willowby HSSWP, FL January, 2000 V375 Lorelei HSSWP, FL January, 2000 V376 Lorelei HSSWP, FL January, 2000 V377 Lorelei HSSWP, FL January, 2000 V684 Rosie HSSWP, FL October, 2003 V685 Lorelei HSSWP, FL October, 2003 V686 Betsy HSSWP, FL October, 2003 V687 Amanda HSSWP, FL October, 2003 V909 SW04031 SD, CA January, 2004 V910 SW04031 SD, CA January, 2004 V989 TM0334 Orlando, FL January, 2004 V991 Stubby Orlando, FL January, 2004 V995 TM0341 Orlando, FL January, 2004 V996 TM0341 Orlando, FL January, 2004

PAGE 52

40 Table 2-2. Samples obtained from skin lesi ons of free-ranging Fl orida manatees. DNA extracted from lesions was tested by PCR for the presence of TmlPV infection. CR: Crystal River, FL: Florida, HR: Homosassa River, DK: Drowned Keys, BZ: Belize, POI: Po rt of Isles (Everglades City), TB=Tampa Bay,*: manatee penile le sion, **: uninfected manatee liver. Sample I. D. Manatee I. D. Location of Manatee Date Obtained V378 RKB-1029-17 CR, FL January, 2003 V389 RKB-1035-31 CR, FL February, 2003 V390 RKB-1036-19 HR, FL February, 2003 V396 RKB-1040-23 HR, FL February, 2003 V397 RKB-1039-13 HR, FL February, 2003 V408 RKB-10474 CR, FL February, 2003 V556 BZ01M16 DK,BZ May, 2003 V1329 TNP-29 POI, FL April, 2004 V1330 TNP-29 POI, FL April, 2004 V1331 TNP-29 POI, FL April, 2004 V1332 TNP-31 POI, FL April, 2004 V1333 TNP-32 POI, FL April, 2004 V1334 Unidentified POI, FL April, 2004 V1335 Unidentified POI, FL April, 2004 V1343 BZ04M64 DK, BZ May, 2004 V1437* MEC0449 TB, FL August, 2004 V1351 BZ04M58 DK, BZ May, 2004 V1774 THR-02 HR, FL April, 2005 V1776 THR-03 HR, FL April, 2005 V1777 THR-04 HR, FL April, 2005 V1855** MEC-0515 TB, FL February, 2005 Table 2-3. PCR primers designed to targ et manatee papillomavirus sequences Target PCR Primer PCR Primer Sequence L1 458-bp FP CR333 5GCI CAG GGI CAT AAI AAT GG-3 Fragment RP CR332 5CGT CCI AII GGA IAC TGA TC-3 FP CR490 5CAG GGG CAT AAG AAT GGT ATT G -3 RP CR491 5GAG GGG AGA CTG ATC GAG TTC TG-3 L1--E1 FP CR442 5CCT GCT GAA AAT GAT GAT CC -3 Region RP CR441 5TTA TCA IA T GCC CAI TGT ACC AT -3 Complete E6 FP CR498 5CAA CCA TCT TCT ACA TGC TTA GT-3 Gene RP CR499 5CGT ATT CTT GGA TAT GTG GTG -3 E6 TOPO FP CR529 5CAC CCA AC C ATC TTC TAC ATG CTT AGT-3 Fragment RP CR499 5CGT ATT CTT GGA TAT GTG GTG -3

PAGE 53

41 Table 2-3. Continued Target PCR Primer PCR Primer Sequence E7 TOPO FP CR530 5-CAC CTT AGA AGAC ACA GCA CGT ATC-3 Fragment RP CR501 5ATC TGT TGT ATC CGA GTC AC -3 L1L2 FP CR572 5TAA CCG CA T TTA ATG GGC AAT TTG -3 Region RP CR574 5AAT AAA ATG ATG CAC AGT GCC AG -3 Complete L1 FP CR573 5-CAC CTA CAA TCC TTA TTG ATT TTC AAT C -3 Gene RP CR574 5AAT AAA ATG ATG CAC AGT GCC AG -3 Complete L2 FP CR572 5TAA CCG CAT TTA ATG GGC AAT TTG -3 Gene RP CR622 5-TTC GG T ATT GAG GAT GCG GG-3 Table 2-4. Sequencing primers used to obtain the complete sequence of PCR amplified TmlPV gene fragments. Target Sequencing Primer Primer Sequence L1 458-bp FP CR333 5GCI CAG GGI CAT AAI AAT GG -3 Fragment RP CR332 5CGT CCI AII GGA IAC TGA TC -3 FP M13 5GTA AAA CGA CGG CCA G -3 RP M13 5CAG GAA ACA GCT ATG AC -3 L1E1 T7 Promoter 5T AA TAC GAC TCA CTA TAG GG -3 Region RP CR216 5TAC AAG ACA GGT TTA AGG AGA C -3 FP CR579 5TGC GCA TAG TTA CTT CTG AG -3 RP CR478 5CAG TGT ACC ATT GAA GAT AAG TC -3 FP CR492 5ATG TAT GAA GTA TAA ATA GCA C -3 RP CR493 5CAA CTC TAC CTG TAC GTT CC -3 Complete E6 FP M13 5GTA AAA CGA CGG CCA G -3 Gene RP M13 5CAG GAA ACA GCT ATG AC -3 T7 Promoter 5TAA TA C GAC TCA CTA TAG GG -3 RP CR532 5-TAG AAG GCA CAG TCG AGG-3 Complete E7 FP M13 5GTA AAA CGA CGG CCA G -3 Gene RP M13 5CAG GAA ACA GCT ATG AC -3 T7 Promoter 5TAA TA C GAC TCA CTA TAG GG -3 RP CR532 5-TAG AAG GCA CAG TCG AGG-3 L1L2 FP M13 5GTA AAA CGA CGG CCA G -3 Region RP M13 5CAG GAA ACA GCT ATG AC -3 FP CR583 5AGA TTA CAC CAG AGG CTC C -3 RP CR580 5ATT CAT TGT ATG TAT GTG GG -3 FP CR588 5CCC TAT CTT TGA CAA TTC TG -3 RP CR601 5GAG CGT CTG CTT TCG TGT GT -3 FP CR 592 5-GGG AGA CT C CAC TGA TAC CA-3

PAGE 54

42 Table 2-4. Continued Target Sequencing Primer Primer Sequence Complete L1 FP M13 5GTA AAA CGA CGG CCA G -3 Gene RP M13 5CAG GAA ACA GCT ATG AC -3 FP CR579 5TGC GCA TAG TTA CTT CTG AG -3 RP CR580 5ATT CAT TGT ATG TAT GTG GG -3

PAGE 55

43 E6 FP CR498 E6 RP CR499 E6ORF E7 ORF E1 ORF NC R L1 ORF L2 ORF NC R E 7 FP CR500 E 7 R P CR500E1 R P CR441 L2ORF FP CR572 L2OR F RP CR622 L1OR F FP CR573 L1ORF RP CR574 L1 FP CR333/ L1 FP CR490 L1 RP CR332/ L1 RP CR491 L1FP CR442 Figure 2-1. Linear representati on of the open reading frames (ORFs) of the ci rcular manatee papillomavirus (TmlPV) genome with the relative positions of the PCR primers used to amp lify TmlPV DNA. FP=Forward primer, RP=Reverse primer.

PAGE 56

44 CHAPTER 3 RESULTS PCR Results Total DNA extracted from 13 skin lesions of captive Florida manatees (Trichechus manatus latirostris ) and six skin lesions of free-ra nging Florida manatees from the vicinity of Homosassa Springs State Wild life Park (HSSWP) amplified DNA fragments of the expected size in PCR assays for the de tection of manatee papillomavirus (TmlPV). DNA fragments of the expected size could not be amplified from DNA extracted from three skin lesions of free-ra nging Antillean manatees (T. manatus manatus ) in PCR assays and, therefore, served as negative controls in subsequent PCR assays. DNA extracted from one manatee liver was also inco rporated into the PCR in order to serve as a negative control and to validate the results in subsequent PCR assays. PCR results of manatee skin lesions assayed for the presen ce of TmlPV DNA are shown in Tables 3-1, 3-2, and 3-3 (summary). PCR Targeting the Papillomavirus L1 458-bp Fragment Oligonucleotide primers MY11 and MY09 (M anos et al., 1989), known to amplify a 458-bp fragment within the L1 capsid gene of several human papillomaviruses and modified by us to contain deoxyinosines at pos itions of nucleotide degeneracy (primers CR333 and CR332), amplified DNA fragments of identical size from five captive manatee skin lesions, out of 11 tested, and fr om four free-ranging manatee skin lesions (near HSSWP), out of seven tested.

PAGE 57

45 PCR Targeting the L1 TmlPV Fragment The L1 TmlPV oligonucleotide primers CR4 90 and CR491, designed to amplify a 458-bp fragment within the L1 capsid gene of TmlPV, amplified DNA fragments of identical size from 11 captive manatee skin le sions, out of 15 assayed, and from five freeranging manatee skin lesions, out of 15 tested for the presence of papillomavirus infection. Total DNA from four skin lesions from which no amplification of the 458-bp DNA fragments could be ach ieved using the MY11 and MY09 HPV L1 primer set amplified DNA fragments of the expected size (458-bp) using the TmlPV-specific CR490 and CR491 primer set (Figure 3-1). PCR Targeting the TmlPV L1-E1 Region Oligonucleotide primers CR442 and CR441, kno wn to amplify an approximately 3,000 bp fragment of the L1-E1 region of the HPV-70 genome (Forslund and Hansson, 1996), amplified a fragment of similar size fr om one captive manatee skin lesion that was tested with the CR442/CR441 primers (Figure 3-2). PCR Targeting the Complete TmlPV E6 Gene Oligonucleotide primers CR498 and CR 499, designed to amplify a 587-bp fragment of the TmlPV genome that contai ns the complete TmlPV E6 gene ORF, amplified DNA fragments of the expected size from five captive manatee skin lesions (Figure 3-3), out of eight tested for the presence of papillomavirus infection. Amplification was not obtained from 12 fr ee-ranging manatee skin lesions tested. PCR Targeting the Complete TmlPV E7 Gene Oligonucleotide primers CR500 and CR 501, designed to amplify a 489-bp fragment of the TmlPV genome that contai ns the complete TmlPV E7 gene ORF, amplified DNA fragments of the expected size from five captive manatee skin lesions

PAGE 58

46 (Figure 3-4), out of five tested for the pres ence of papillomavirus. Similar fragments were not amplified from any of the seven free-ranging manatee skin lesions tested. PCR Targeting the TmlPV L1-L2 Region Oligonucleotide primers CR572 and CR574, designed to amplify a 3,208-bp fragment of the TmlPV genome that contai ns the complete L1 gene ORF plus the complete L2 gene ORF, amplified DNA frag ments of the expected size from two skin captive manatee skin lesions (Figure 3-5), out of two tested for the presence of papillomavirus infection. PCR Targeting the Complete TmlPV L1 Gene Oligonucleotide primers CR573 and CR574, designed to amplify a 1,712-bp fragment of the TmlPV genome that contai ns the complete L1 capsid gene ORF, amplified DNA fragments of the expected si ze from three captive manatee skin lesions (Figure 3-6), out of three tested for the presence of papillomavirus infection. PCR Targeting the Complete TmlPV L2 Gene Oligonucleotide primers CR572 and CR622, designed to amplify a 1,611-bp fragment of the TmlPV genome that contai ns the complete L2 capsid gene ORF, amplified DNA fragments of the expected size from two captive manatee skin lesions (Figure 3-7), out of two tested for the pr esence of papillomavirus infection. Sequencing Results and Genetic Analyses TmlPV L1 458-bp Fragments Sequencing of papillomavirus L1 capsid gene fragments amplified with the modified HPV primers MY11 and MY09 (CR3 33/CR332) revealed that the fragments were 458-bp in length, s upporting the universal ity of the MY11 and MY09 primers, which are known to amplify a 458-bp DNA fragme nt from most types of genital HPVs

PAGE 59

47 (Bernard et al., 1994). Sequencing of papillo mavirus L1 capsid gene fragments amplified with the L1 TmlPV-specific primers revealed th at the fragments were also 458-bp in length. Nucleotide sequences of the 458-bp Tm lPV L1 gene fragments were entered into the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information Website (NCBI Bethesda, Maryland) to identify papillomavirus homologues that had the highest similarity and identity to TmlPV L1. This demonstrated that the amplified TmlP V fragments were contained within a highly conserved domain of the L1 capsid protein ge ne of papillomaviruses. Nine TmlPV L1 fragment sequences, obtained from the DNA of three captive manatee lesions and six free-ranging manatee lesions, were submitted to the GenBank database of the NCBI website (Table 3-4). The TmlPV L1 gene fragments translated correctly from the first nucleotide of forward primer (FP) MY11 and FP L1TmlPV (CR 490) into protein fragments consisting of 152 amino acids. The GAP f unction of the GCG Genetic Package (GCG, University of Wisconsin, Madison, Wisconsi n, USA) showed that the nine TmlPV L1 fragment sequences were 100% id entical at the nucleotide an d the amino acid level. The multiple alignment of the deduced amino aci d sequences of the TmlPV L1 fragments demonstrated the identity of the TmlPV L1 sequences (Figure 3-8). Comparisons of the TmlPV L1 amino acid sequence with homologues from human and non-human papillomaviruses demonstrated identities th at ranged between 36% and 57% (Table 3-5) and similarities that ranged between 57% and 67% (Table 3-6) Af ter removal of the L1 primer sequences, the L1 fragment sequence was 100% identical to the corresponding L1

PAGE 60

48 region of the TmPV-1 sequence obtained by another research group and recently deposited in the GenBank database (AY 609301) and published (Rect or et al., 2004). L1-E1 TmlPV Region Sequencing of the approximately 3,000-bp papillomavirus DNA fragment amplified from one lesion of a captive mana tee (V369, Holly) revealed that the fragment was 2,772 nucleotides in length. The ORF(Op en Reading Frame) Finder tool of the NCBI website revealed that the 2,772-bp seque nce contained partial sequences of the L1 ORF, the complete E6 ORF, the complete E7 ORF, and partial sequenc es of the E1 ORF. Further genetic analysis of the 2,772-bp sequence showed that it contained: partial sequences of the 3 end of the TmlPV L1 ORF (261-bp, including the L1 stop codon), followed by a large non-coding region (816bp), the TmlPV E6 ORF (414-bp, including the E6 stop codon), then, separated by tw o nucleotides, the TmlPV E7 ORF (348-bp, including the E7 stop codon), a small non-codi ng region (334-bp), and partial sequences of the 5 end of the TmlPV E1 ORF (599bp). The 2,772-bp sequence that spans the L1E1 region of the TmlPV genome was submitted to the GenBank database of the NCBI website (Table 3-4). Nucleotide sequences of the TmlPV E6 ORF translated into a protein consisting of 137 amino acid residues. Comparisons of the TmlPV E6 137 amino acid sequence with homologues from hu man and non-human papillomaviruses demonstrated identities that ranged between 22% and 32% (Table 3-5) and similarities that ranged between 28% and 42% (Table 36). The TmlPV E6 ORF sequence obtained from sequencing the 2,772-bp fragment was submitted to the GenBank database of the NCBI website (Table 3-4). Nucleotide sequenc es of the TmlPV E7 ORF translated into a protein consisting of 115 amino acid residues. Comparisons of the TmlPV E7 115 amino acid sequence with homologues from hu man and non-human papillomaviruses

PAGE 61

49 demonstrated identities that ranged between 23% and 41% (Table 3-5) and similarities that ranged between 31% and 51% (Table 3-6). Pair-wise co mparisons were not performed with Phocoena spinipinnis PV (PsPV-1) because the PsPV-1 genome does not encode an E7 ORF. The complete TmlPV E7 ORF sequence obtained from sequencing the 2,772-bp fragment was submitted to the GenBank database of the NCBI website (Table 3-4). TmlPV E6 Gene Sequencing of amplified DNA fragments co ntaining the complete TmlPV E6 gene from four lesions of captive manatees revealed that the fragments were 587 nucleotides in length. Genetic analysis of the 587-bp TmlP V E6 gene fragment revealed that the sequence contained the complete TmlPV E6 ORF (414-bp, including the E6 stop codon) that translated into a protein consisting of 137 amino acid residues. Comparisons of the four obtained TmlPV E6 sequences showed th at the nucleotide iden tity ranged from 99.5100.0% and the amino acid identity was 100.0% (not shown). The TmlPV E6 gene sequence obtained from the positive cont rol manatee DNA (CR130) demonstrated 100% identity and similarity to the corresponding region of the TmPV-1 genome described by another research group (Rector et al., 2004). The TmlPV E6 ORF sequences were submitted to the GenBank database of the NCBI website (Table 3-4). TmlPV E7 Gene Sequencing of amplified DNA fragments co ntaining the complete TmlPV E7 gene from four lesions of captive manatees revealed that the fragments were 489 nucleotides in length. Genetic analysis of the 489-bp TmlP V E7 gene fragments revealed that the sequences contained the comp lete TmlPV E7 ORF (348-bp, including the E7 stop codon) that translated into a protein consisting of 115 amino acid residues. Comparisons of the

PAGE 62

50 four obtained TmlPV E7 ORF sequences showed that the nucleotide identity ranged from 99.4% to100.0 % and the amino acid identity ra nged from 99.1% to 100.0% (not shown). The TmlPV E7 sequence obtained from positive control manatee DNA (CR130) had 100% identity and similarity to the corresponding region of the recently published TmPV1 genome (Rector et al., 2004). The TmlPV E7 ORF sequences were submitted to the GenBank database of the NCBI website (Table 3-4). TmlPV L1-L2 Region Sequencing of the amplified TmlPV L1-L2 gene fragments from two papillomatous lesions of captive manatees Oakley and Ho lly revealed that the fragments were 3,208 nucleotides in length. Genetic analysis re vealed that the fragments contained the complete TmlPV L1 ORF (1,518-bp, including the L1 stop codon) plus the complete TmlPV L2 ORF (1,536-bp, including the L2 stop codon). The start codon of the TmlPVL1 ORF sequence was contained within the TmlPV L2 ORF sequence, and the TmlPV L1 and TmlPVL2 ORFs had an overla pping region of 20 nucleotides. Nucleotide sequences of the TmlPV L1 ORF translated in to a protein of 505 amino acid residues. Comparisons of the TmlPV L1 505 amino aci d sequence with homologues from human and non-human papillomaviruses demonstrated identities that ranged between 31% and 57% (Table 3-5) and similari ties that ranged between 41% and 68% (Table 3-6). The complete TmlPV L1 sequence obtained from a lesion of manatee Oakley (CR130) demonstrated 100% identity and similarity to the corresponding region of the TmPV1 genome deposited in the GenBank database by another research group (AY609301). The two TmlPV L1 ORF sequences obtained from lesions of captive manatees Oakley and Holly shared 99.7% nucleotide identity and 99.8% amino acid identity (not shown). The TmlPV L1 ORF sequences were submitted to the GenBank database of the NCBI website

PAGE 63

51 (Table 3-4). Nucleotide sequences of the Tm lPV L2 ORF translated into a protein of 511 amino acid residues. Comparisons of th e TmlPV L2 511 amino acid sequence with homologues from human and non-human papillom aviruses showed identities that ranged between 35% and 42% (Table 3-5) and simila rities that ranged between 43% and 51% (Table 3-6). The complete TmlPV L2 sequence obtained from positive control manatee DNA (CR130) demonstrated 100% identity and si milarity to the corresponding region of the TmPV1 genome (AY609301). The two TmlP V L2 ORF sequences obtained from lesions of captive manatees Holly and Oa kley shared 99.9% nucleotide identity and 99.6% amino acid identity (not shown). The TmlPV L2 ORF sequences were submitted to the GenBank database of the NCBI website (Table 3-4). Complete TmlPV L1 Gene from Captive Manatee Lorelei Sequencing of the amplified fragment c ontaining the complete TmlPV L1 gene fragment from one lesion of captive manatee Lorelei (V685) revealed that the fragment was 1,712 nucleotides in length. Genetic analys is revealed that the fragment contained the complete TmlPV L1 ORF sequence that tr anslated into a protein of 505 amino acid residues. The complete TmlPV L1 ORF sequence was shown to be 99.7% and 99.4%99.7% identical to the previous ly obtained TmlPV L1 ORF sequences at the nucleotide and amino acid levels, respectively (not show n). The TmlPV L1 ORF sequence (Lorelei) was submitted to the GenBank database of the NCBI website (Table 3-4). Complete TmlPV L2 Gene Total DNA extracted from two lesions of captive manatees Oakley and Holly from which the complete TmlPV L2 gene (1,611-bp) was amplified was also positive for amplification of the TmlPV L1-L2 fragment (3,208-bp). This fragment had previously

PAGE 64

52 been sequenced; therefore, sequencing of the complete TmlPV L2 genes from these DNAs (V369 and CR130) was not repeated. Immunofluorescence and Gene Expression Assays Mammalian Expression Systems Fluorescence was not detected in im munologic assays utilizing monkey kidney (COS-7) cells transfected with TmlPV L1 or TmlPV E6 genes cloned in eukaryotic vectors under the control of the CMV promot er. Likewise, specifi c fluorescence was not observed in assays using Florida manatee resp iratory epithelial (TmlRE) cells transfected with the eukaryotic vectors containing th e TmlPV L1 gene under control of the CMV promoter. A few TmlRE cells transfected with the E7 gene displayed a faint halo of fluorescence; however, fluorescence was cons idered subjective and ambiguous and did not provide conclusive eviden ce of protein expression. Rest riction digests of the TmlPV E6 and E7 pcDNA3.1+/Zeo recombinants are shown in Figure 3-11. Bac-to-Bac Baculovirus Expression System Supernatant and cell harvests from Sf21 in sect cell cultures transfected with TmlPV L1 capsid protein bacmid DNA were anal yzed by electron microscopy by Mr. Woody Frazer, Florida Animal Disease Diagnostic Laboratory, Florida Department of Agriculture and Consumer Services, Kissimmee, Florida for the pres ence of virus-like particles (VLPs). Amorphous clumps were observed after negative staining that resembled capsid protein particles; however, pa pillomavirus-like particles of the expected size (50-55 nm) were not observed (Figure 39). The cDNAs obtained by RT-PCR of RNAs extracted from infected cell cultures showed that mRNA expressing the TmlPV L1 gene was being produced in the cell cultures (Figure 3-10). Restriction digests of the

PAGE 65

53 TmlPV L1 pFastBac1 recombinant used in the transfection experi ment are shown in Figure 3-12. Bac-N-Blue Baculovirus Expression System After ninety-six hours of incubation at 27 C, supernatants were harvested from Sf21 cell cultures transfected with the recombinant pBlueB ac 4.5 L1 vector DNA. The baculovirus DNA developed blue color after 48 hrs of incubation (27C) in the presence of the -galactosidase substrate Bluo-gal (Invi trogen). This finding indicated that recombination had occurred between the hom ologous sequences in the baculovirus DNA (Bac-N-Blue DNA) and the transf er vector (pBlueBac 4.5 L1 plasmid), reconstituting the essential sequences necessary for replication of the newly generated recombinant virus. Supernatants corresponding to the cultures transfected with the pBlueBac 4.5 L1 vector (no baculovirus DNA) and those from the unt ransfected cultures did not express the galactosidase enzyme, as judged by the lack of development of blue color. Although no direct proof has yet been obt ained on the expression of the L1 capsid protein by the generated baculoviruses, it is speculated at this point that unless the L1 capsid gene has been inadvertently mutagenized, protein expres sion after the isolati on and purification of recombinant baculovirus will be demonstrated by Western blot analysis. Phylogenetic Analysis Multiple sequence alignments and th e construction of phylogenetic trees demonstrated the genetic relatedness of th e TmlPV amino acid sequences to the amino acid sequences of several human and non-human papillomaviruses. Trees were constructed using the following PVs (with their GenBank accession numbers): Human papillomavirus type 1a HPV-1a (V 01116), HPV-2a (X55964), HPV-3 (X74462), HPV-4 (X70827), HPV-5 (M17463), HPV-6 (AF 092932), HPV-7 (X74463), HPV-9 (X74464),

PAGE 66

54 HPV-11 (M14119), HPV-13 (X62843), HPV15 (X74468), HPV-16 (K02718), HPV-18 (X05015), HPV-20 (U31778), HPV-21 (U31779), HPV-26 (X74472), HPV-27 (X74473), HPV-30 (X74474), HPV-32 (X74475), HPV-33 (M12732), HPV-34 (X74476), HPV-41 (X56147), HPV-51 (M62877), HPV-63 (X70828), HPV-65 (X70829), HPV-92 (NC_004500), HPV-95 (AJ 62010), Bovine PV type 1 BPV-1 (X02346), BPV-2 (M20219), Canine oral PV COPV (L22695), Cottontail rabbit PV CRPV (AJ243287), White-tail deer PV DEERPV (M11910), Equus caballus PV ECPV (NC_003748), European elk PV EEPV (M15953), Ovine PV type 1 OPV-1 (U83594), OPV-2 (U83595), Rhesus monke y PV RhMPV (M60184), Phocoena spinipinnis PV PsPV1(AJ238373), and Manatee papillomaviru s type 1 TmPV1 (AY609301). Florida manatee PV (TmlPV) sequences obtained fr om positive control manatee DNA (CR130) were used in all phylogenetic analyses. The L1 capsid pr otein gene phylograms indicated that the TmlPV sequence formed a unique branch, distinct from known human and animal papillomavirus L1 sequences (Figur e 13). The TmlPV complete L2 capsid protein gene also formed a separate branch in the L2 capsid protein phylograms (Figure 14), indicating the uniqueness of this virus. The E6 protein gene based phylograms indicated that TmlPV was the sole member of a unique branch (Figure 3-15). Similarly, the E7 protein gene claded by itself to form a branch that is closely rooted to the papillomaviruses of hoofed animals (Figure 3-16).

PAGE 67

55Table 3-1. PCR results of DNAs ob tained from captive manatee skin lesions tested for the presence of TmlPV infection. POS: positive, DNA fragment s of the expected size were amplified; NEG: negative, DNA fragments of the expected size were not amplified; X: assay not performed, ORF: open reading frame. Sample I. D. No. L1 458-bp Fragment TmlPV L1 Fragment L1-L2 Region L1 ORF L2 ORF L1-E1 Region E6 ORF E7 ORF CR130 POS POS POS POS POS X POS POS V368 NEG X X X X X X X V369 POS X POS POS POS POS POS POS V370 NEG POS X X X X X POS V371 NEG POS X X X X NEG X V372 NEG X X X X X X X V373 NEG X X X X X X X V374 POS X X X X X X X V375 POS POS X X X X POS X V376 POS POS X X X X X X V377 NEG X X X X X X X V684 X POS X X X X X X V685 X POS X POS X X POS POS V686 X POS X X X X POS POS V687 X POS X X X X X X V909 X NEG X X X X X X V910 X POS X X X X NEG X V989 X NEG X X X X X X V991 X NEG X X X X X X V995 X POS X X X X NEG X V996 X NEG X X X X X X

PAGE 68

56Table 3-2. PCR results of DNAs obtained from free-ranging manat ee skin lesions tested for the pr esence of TmlPV infection. PO S: positive, DNA fragments of the expected si ze were amplified; NEG: negative, DNA fragments of the expected size were not amplified; X: assay not performed, ORF: open reading frame, *= penile sk in lesion, **= normal manatee liver DNA Location of Animal Animal I. D. L1 458-bp Fragment TmlPV L1 Fragment L1-L2 Region L1 ORF L2 ORF L1-E1 Region E6 ORF E7 ORF CR, FL V378 POS X X X X X X X CR, FL V389 NEG POS X X X X NEG X HR, FL V390 NEG POS X X X X NEG X HR, FL V396 POS POS X X X X NEG X HR, FL V397 POS POS X X X X NEG X CR, FL V408 POS POS X X X X NEG X DK, BZ V556 NEG NEG X X X X X X POI, FL V1329 X NEG X X X X NEG NEG POI, FL V1330 X NEG X X X X NEG NEG POI, FL V1331 X NEG X X X X NEG NEG POI, FL V1332 X NEG X X X X NEG NEG POI, FL V1333 X NEG X X X X NEG NEG POI, FL V1334 X NEG X X X X NEG NEG POI, FL V1335 X NEG X X X X NEG NEG DK, BZ V1343 X NEG X X X X X X DK, BZ V1351 X NEG X X X X X X TB, FL V1437* X NEG X X X X X X HR, FL V1774 X NEG NEG NEG NEG X X X HR, FL V1776 X NEG NEG NEG NEG X X X HR, FL V1777 X NEG NEG NEG NEG X X X TB, FL V1855** X NEG X X X X X X

PAGE 69

57Table 3-3. Summary of PCR results. DNAs obt ained from skin lesions of captive and fr ee-ranging manatees were tested for the presence of manatee papillomavirus infection. Tissues CR332/CR333 Fragment L1 Fragment L1-L2 Region L1 ORF L2 ORF L1-E1 Region E6 ORF E7 ORF No. +ve 5 11 2 3 2 1 5 5 No. tested 11 15 2 3 2 1 8 5 No. +ve 4 5 0 0 0 X 0 0 CAPTIVE MANATEES FREE-RANGING MANATEES No. tested 7 20 3 3 3 X 12 7

PAGE 70

58 Table 3-4. Accession numbers of manatee papillomavirus se quences deposited into the GenBank tool of the NCBI website. Amplicon Animal I.D. No. GenBank Accession No. TmlPV L1 458-bp Fragment V369 AY455940 V396 AY455941 V389 AY496568 V390 AY496569 V397 AY496570 V408 AY496571 V375 AY496572 V378 AY496574 CR130 AY496575 TmlPV L1-E1 Region V369 DQ099425 TmlPV Complete E6 Gene CR130 DQ099425 V369 AY830703 V685 AY830704 V686 AY830705 TmlPV Complete E7 Gene CR130 DQ099427 V369 AY830706 V685 AY830707 V686 AY830708 TmlPV L1-L2 Region CR130 DQ099423 V685 DQ099424 TmlPV Complete L1 Gene CR130 AY994164 V369 AY994166 V686 DQ099422 TmlPV Complete L2 Gene CR130 AY994165 V369 AY994167

PAGE 71

59 Table 3-5. Pair-wise comparisons of the ami no acid sequences of the L1, L2, E6, and E7 gene fragments of manatee papillomavi rus (TmlPV) with several human and non-human papillomaviruses. Numbers represent percent identity to corresponding TmlPV sequences obtained from Oakleys DNA. X=no sequence available. PV Type L1 Fragment L1 ORF L2 ORF E6 ORF E7 ORF HPV1a 55.0 53.0 37.0 32.0 40.0 HPV2a 51.0 52.0 37.0 30.0 36.0 HPV3 57.0 53.0 37.0 29.0 39.0 HPV4 57.0 56.0 40.0 28.0 32.0 HPV5 53.0 56.0 41.0 26.0 35.0 HPV6 55.0 54.0 37.0 25.0 40.0 HPV7 50.0 54.0 37.0 27.0 27.0 HPV9 54.0 56.0 40.0 29.0 40.0 HPV11 56.0 54.0 36.0 23.0 38.0 HPV13 56.0 55.0 37.0 22.0 39.0 HPV15 50.0 54.0 41.0 26.0 41.0 HPV16 51.0 52.0 37.0 30.0 35.0 HPV18 43.0 52.0 38.0 22.0 31.0 HPV20 57.0 58.0 39.0 31.0 35.0 HPV21 57.0 57.0 40.0 30.0 38.0 HPV26 55.0 54.0 38.0 26.0 28.0 HPV27 51.0 51.0 39.0 28.0 35.0 HPV30 56.0 55.0 39.0 23.0 30.0 HPV32 52.0 52.0 40.0 31.0 31.0 HPV33 54.0 31.0 38.0 30.0 28.0 HPV34 53.0 52.0 36.0 25.0 32.0 HPV41 55.0 51.0 36.0 31.0 28.0 HPV51 55.0 54.0 37.0 28.0 32.0 HPV63 55.0 51.0 36.0 31.0 27.0 HPV65 57.0 57.0 39.0 28.0 28.0 HPV92 51.0 55.0 40.0 30.0 38.0 HPV95 57.0 57.0 40.0 32.0 24.0 BPV1 49.0 50.0 36.0 28.0 28.0 BPV2 50.0 50.0 35.0 28.0 27.0 OPV1 49.0 50.0 36.0 25.0 31.0 OPV2 51.0 50.0 35.0 22.0 28.0 CRPV 53.0 57.0 37.0 32.0 25.0 DeerPV 47.0 46.0 35.0 22.0 23.0 ECPV 55.0 51.0 40.0 29.0 32.0 EEPV 50..0 51.0 37.0 25.0 21.0 RhMPV 54.0 53.0 38.0 27.0 39.0 COPV 53.0 54.0 41.0 27.0 34.0 PsPV1 53.0 51.0 42.0 26.0 X TmPV1 100.0 100.0 100.0 100.0 100.0

PAGE 72

60 Table 3-6. Pair-wise comparisons of the ami no acid sequences of the L1, L2, E6, and E7 gene fragments of manatee papillomavi rus (TmlPV) with several human and non-human papillomaviruses. Numbers represent percent similarity to corresponding TmlPV sequence obtained fr om Oakleys DNA. X=no sequence available PV Type L1 Fragment L1 ORF L2 ORF E6 ORF E7 ORF HPV1a 65.0 63.0 47.0 42.0 51.0 HPV2a 60.0 62.0 47.0 36.0 40.0 HPV3 66.0 63.0 45.0 36.0 44.0 HPV4 66.0 65.0 49.0 36.0 43.0 HPV5 64.0 66.0 51.0 40.0 44.0 HPV6 64.0 63.0 46.0 34.0 50.0 HPV7 61.0 63.0 47.0 35.0 36.0 HPV9 65.0 66.0 49.0 39.0 50.0 HPV11 63.0 62.0 47.0 33.0 48.0 HPV13 64.0 64.0 45.0 32.0 45.0 HPV15 63.0 64.0 51.0 39.0 49.0 HPV16 60.0 61.0 48.0 36.0 43.0 HPV18 66.0 62.0 47.0 32.0 41.0 HPV20 66.0 62.0 49.0 42.0 44.0 HPV21 67.0 68.0 50.0 41.0 45.0 HPV26 66.0 64.0 47.0 33.0 40.0 HPV27 61.0 62.0 48.0 37.0 40.0 HPV30 64.0 64.0 46.0 30.0 38.0 HPV32 63.0 63.0 48.0 42.0 40.0 HPV33 64.0 41.0 48.0 37.0 36.0 HPV34 62.0 62.0 47.0 32.0 42.0 HPV41 65.0 62.0 46.0 41.0 36.0 HPV51 65.0 64.0 46.0 37.0 43.0 HPV63 65.0 62.0 46.0 41.0 36.0 HPV65 66.0 65.0 48.0 37.0 40.0 HPV92 64.0 65.0 50.0 40.0 45.0 HPV95 67.0 67.0 50.0 38.0 36.0 BPV1 63.0 61.0 44.0 36.0 31.0 BPV2 64.0 61.0 44.0 34.0 31.0 OPV1 60.0 61.0 43.0 33.0 34.0 OPV2 61.0 60.0 44.0 31.0 31.0 CRPV 65.0 66.0 46.0 36.0 34.0 DeerPV 57.0 56.0 46.0 28.0 31.0 ECPV 62.0 60.0 51.0 36.0 37.0 EEPV 60.0 61.0 46.0 32.0 32.0 RhMPV 63.0 62.0 46.0 38.0 51.0 COPV 62.0 64.0 51.0 40.0 41.0 PsPV1 62.0 61.0 50.0 36.0 X TmPV1 100.0 100.0 100.0 100.0 100.0

PAGE 73

61 Table 3-7. Summary of the pair-wise comp arisons of the amino acid sequences of manatee papillomavirus gene fragments with several human and non-human papillomaviruses. TmlPV Fragment Most Similar PV Type (%) Least Similar PV type (%) HPV-21 (67.0%) DeerPV (57.0%) L1 458-bp Fragment HPV-21 (67.0%) OPV-1 (60.0%) HPV-20 (66.0%) EEPV (60.0%) HPV-65 (66.0%) HPV-2a (60.0%) HPV-21 (68.0%) HPV-33 (41.0%) Complete L1 ORF HPV-95 (67.0%) DeerPV (56.0%) HPV-5 (66.0%) OPV-2 (60.0%) HPV-9 (66.0%) ECPV (60.0%) Complete L2 ORF HPV-5 (51.0%) OPV-1 (43.0%) HPV-15 (51.0%) OPV-2 (44.0%) COPV (51.0%) BPV-1 (44.0%) ECPV (51.0%) BPV-2 (44.0%) Complete E6 ORF HPV-1a (42.0%) DeerPV (28.0 %) HPV-20 (42.0%) HPV-30 (30.0%) HPV-32 (42.0%) OPV-2 (31.0%) HPV-63 (41.0%) HPV-18 (32.0%) Complete E7 ORF HPV-1a (51.0%) BPV-1 (31.0%) RhMPV (51.0%) BPV-2 (31.0%) HPV-15 (49.0%) OPV-2 (31.0%) HPV-11 (48.0%) DeerPV (31.0%)

PAGE 74

62 Figure 3-1. Agarose gel electrophoresis of PCR amplified 458-bp fragments of the L1 capsid protein gene of ma natee papillomavirus. MM: 1 Kb Molecular marker, Lane 1: V375, Lane 2: V376, Lane 3: V389, Lane 4: V390, Lane 5: V396, Lane 6: V397, Lane 7: V408, Lane 8:V370, Lane 9:V371, Lane 10:V556, negative tissue; Lane 11: negative tube, water; Lane 12: Positive control captive manatee Oakley (CR130). C: Captive manatee, FR: Free-ranging manatee Figure 3-2. Agarose gel electrophoresis of PCR amplified 2,772-bp fragment of the L1E1 region of manatee papillomavirus. MM: 1 Kb Molecular marker, Lane 1: Captive manatee Holly (V369), Lane 2: negative tube, no DNA; Lane 3: negative tissue control (V1855). MM1 23456789101112 CCFRFRFRFRFRCC MM123

PAGE 75

63 Figure 3-3. Agarose gel electrophoresis of PCR amplified 587-bp fragments of the E6 gene of manatee papillomavirus. MM: 1 Kb Molecular marker, Lane 1: Captive manatee Holly (V369), Lane 2: Captive manatee Betsy (V686), Lane 3: Captive manatee Lorelei (V685), Lane 4: Positive contro l captive manatee Oakley (CR130), Lane 5: negative tube, no DNA; Lane 6: negative tissue control (V1855). Figure 3-4. Agarose gel electrophoresis of PCR amplified 489-bp fragments of the E7 gene of manatee papillomavirus. MM: 1 Kb Molecular marker, Lane 1: Captive manatee Holly (V369), Lane 2: Captive manatee Betsy (V686), Lane 3: Captive manatee Lorelei (V685), Lane 4: Positive contro l captive manatee Oakley (CR130), Lane 5: negative tube, no DNA; Lane 6: negative tissue control (V1855). MM123456 MM12345 6

PAGE 76

64 Figure 3-5. Agarose gel electrophoresis of PCR amplified 3,208-bp fragments of the L1 gene plus the L2 gene of manatee papillomavirus. MM: 1 Kb Molecular marker, Lane 1: Captive manatee Holly (V369), Lane 2: Positive control captive manatee Oakley (CR130), Lane 3: negative tube, no DNA; Lane 4: negative tissue control (V1855). Figure 3-6. Agarose gel electrophoresis of PCR amplified 1,712-bp fragments of the complete L1 gene of manatee papilloma virus. MM: 1 Kb Molecular marker, Lane 1: Captive manatee Holly (V369) Lane 2: Captive manatee Lorelei (V685), Lane 3: Positive control capti ve manatee Oakley (CR130), Lane 4: negative tube, no DNA; Lane 5: ne gative tissue control, (V1855). MM 12345 MM1234

PAGE 77

65 Figure 3-7. Agarose gel electrophoresis of PCR amplified 1,660-bp fragments of the complete L2 gene of manatee papillo mavirus. MM: 1 Kb Molecular marker, Lane 1: Captive manatee Lorelei (V6 85), Lane 2: Positive control captive manatee Oakley (CR130), Lane 3: nega tive tube, no DNA; Lane 4: negative tissue control (V1855). MM1234

PAGE 78

66 1 51 100 V408 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... V397 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... V396 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... V390 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... V389 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... V378 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... V375 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... V369 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... P31 .......... .......... .......... .......... .......... .......... .......... .......... .......... .......... Consensus AQGHKNGIAW QNQLFVTILD NTRGTNMTVS VSTQNALVVD HYDDNDYAQY LRHAEEFELS FVFQLCKVQL TTEALAHIHT MNPKILEDWH IGLRPPPSAS 101 151 V408 .......... .......... .......... .......... .......... .. V397 .......... .......... .......... .......... .......... .. V396 .......... .......... .......... .......... .......... .. V390 .......... .......... .......... .......... .......... .. V389 .......... .......... .......... .......... .......... .. V378 .......... .......... .......... .......... .......... .. V375 .......... .......... .......... .......... .......... .. V369 .......... .......... .......... .......... .......... .. P31 .......... .......... .......... .......... .......... .. Consensus VEDQYRYIQS LATRCPPKEV PAENDDPYKT KKFWVVDLST RFSTELDQSP LG Figure 3-8. Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the L1 gene fragment of manatee papillomavirus identified in cutaneous lesions of captive and free-ranging Florida manatees.

PAGE 79

67 Figure 3-9. Electron micrograph of Sf21 insect cell cultures transfected with pFastBac1 TmlPV L1 gene recombinant. Amorphous viral clumps that resemble capsid protein particles and rodshaped Baculovirus (non-recombinant) particles were found present. Photo s upplied by Mr. Woody Fraser. 200 n m Rod-shaped Baculovirus particles Amorphous viral clumps

PAGE 80

68 Figure 3-10. Agarose gel electrophoresis de monstrating the PCR amplification of the manatee papillomavirus complete L1 gene fragment from cDNAs obtained from infected Sf21 cell cultures. R= Random hexamer primers used in RTPCR reactions, S= TmlPV L1 gene-s pecific primers used in RT-PCR reactions; MM=Molecular marker, 1 KB ladder; Lanes 1 and 2=Sf21 cell cultures (negative control); Lanes 3 and 4=Sf21 cell cultures infected with bacmid DNA (no recombinant); Lanes 5 and 6=Sf21 cell cultures infected with TmlPV L1 gene recombinant; Lanes 7 and 8= Sf21 cell cultures infected with TmlPV L1 gene recombinant (no SuperScript); Lane 9=blank sample (H2O), negative PCR control; Lane 10=TmlPV L1 plasmid DNA, positive PCR control. All of the samples were treated with DnaseI solution, but the samples in Lanes 7 and 8 were not trea ted with the SuperScript enzyme and were not transcribed to cDNA. This de monstrates that the Dnase I solution effectively degraded the DNA in these samples and validated the results of this assay. Samples in lanes 5 and 6 were treated with the DnaseI solution and also with SuperScript enzyme, therefore the TmlPV L1 DNA fragment was able to be amplified from the cDNA. MM 1R 2S 3R 4S 5R 6S 7R 8S 9 10 MM

PAGE 81

69 Figure 3-11. Agarose gel electrophoresis demons trating restriction enzyme digests of the manatee papillomavirus E6 gene and E7 gene recombinants. MM: 1 Kb Molecular marker, Lane 1: pcDNA 3.1+/Zeo E6 recombinant, Hind III; Lane 2: pcDNA 3.1+/Zeo E6 recombinant, EcoRI; Lane 3: pcDNA 3.1+/Zeo E7 recombinant, Hind III; Lane 4: pcDNA 3.1+/Zeo E7 recombinant, EcoRI Figure 3-12. Agarose gel electrophoresis demons trating restriction enzyme digests of the manatee papillomavirus L1 complete gene recombinant. MM: 1 Kb Molecular marker, Lane 1: pcDNA 3.1+/Zeo L1 recombinant, Hind III; Lane 2: pcDNA 3.1+/Zeo L1 recombinant, HindIII plus XhoI; Lane 3: pFastBac1 L1 recombinant, Hind III; Lane 4: pFastBac1 L1 recombinant, EcoRI. MM12 34 MM1 2 3 4

PAGE 82

70 Figure 3-13. A. Neighbor-Joining phylogeneti c tree of the deduced amino acid sequences of the complete L1 gene of severa l human and non-human papillomaviruses. The tree was generated by Clustal W slow and accurate function using Gonnet 250 residue weight table, gap penalty of 35 and gap extension penalty of 0.75 (pairwise alignment parameters), gap pe nalty of 15 and gap extension penalty of 0.3 (multiple alignment parameters). In the rectangular cladogram format, numbers represent the percent confiden ce of 1000 bootstrap replications. B. In the radial format, the 0.1 divergence s cale represents 0.1 substitutions per site A

PAGE 83

71 Figure 3-13. Continued 0.1 HPV3 HPV2a HPV27 HPV26 HPV41 HPV51 HPV18 HPV30 HPV13 HPV6 HPV11 HPV7 HPV32 HPV16 HPV33 RhMPV HPV34 PsPV1 HPV95 HPV4 HPV65 HPV5 HPV21 HPV20 HPV92 HPV9 HPV15 ECPV BPV1 BPV2 OPV1 OPV2 DEERPV EEPV TmlPV TmPV1 CRPV COPV HPV63 HPV1a B

PAGE 84

72 Figure 3-14. A. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of the complete L2 gene of severa l human and non-human papillomaviruses. The tree was generated by Clustal W slow and accurate function using Gonnet 250 residue weight table, gap penalty of 35 and gap extension penalty of 0.75 (pairwise alignment parameters), gap pe nalty of 15 and gap extension penalty of 0.3 (multiple alignment parameters). In the rectangular cladogram format, numbers represent the percent confiden ce of 1000 bootstrap replications. B. In the radial format, the 0.1 divergence scal e represents 0.1 substitutions per site. A

PAGE 85

73 Figure 3-14. Continued 0.1 HPV2 a HPV27 HPV1 8 HPV2 6 HPV 5 1 HPV30 HPV 3 HPV16 HPV 33 HPV34 RhMPV HPV 3 2 HPV7 HPV13 HPV 6 HPV11 BPV1 BPV2 DEERPV EEPV OPV1 O PV2 P s PV1 E C PV HPV41 HPV95 HPV4 HPV65 HPV 9 2 HPV5 HPV20 HPV21 HPV9 HPV15 TmlPV TmPV1 CRPV COPV HPV63 HPV1a B

PAGE 86

74 Figure 3-15. A. Neighbor-Joining phylogeneti c tree of the deduced amino acid sequences of the complete E6 gene of several human and non-human papillomaviruses. The tree was generated by Clustal W slow and accurate function using Gonnet 250 residue weight table, gap penalty of 35 and gap extension penalty of 0.75 (pairwise alignment parameters), gap pe nalty of 15 and gap extension penalty of 0.3 (multiple alignment parameters). In the rectangular cladogram format, numbers represent the percent confiden ce of 1000 bootstrap replications. B. In the radial format, the 0.1 divergence scal e represents 0.1 substitutions per site. A

PAGE 87

75 Figure 3-15. Continued 0.1 HPV41 CRPV TmlPV TmPV1 HPV95 HPV4 HPV65 COPV HPV1a HPV92 HPV5 HPV20 HPV21 HPV9 HPV15 ECPV BPV1 BPV2 OPV1 OPV2 DEERPV EEPV HPV18 HPV16 HPV34 RhMPV HPV30 HPV26 HPV51 PsPV1 HPV32 HPV7 HPV13 HPV6 HPV11 HPV3 HPV27 HPV2a HPV33 HPV63B

PAGE 88

76 Figure 3-16. A. Neighbor-Joining phylogeneti c tree of the deduced amino acid sequences of the complete E7 gene of severa l human and non-human papillomaviruses. The tree was generated by Clustal W slow and accurate function using Gonnet 250 residue weight table, gap penalty of 35 and gap extension penalty of 0.75 (pairwise alignment parameters), gap pe nalty of 15 and gap extension penalty of 0.3 (multiple alignment parameters). In the rectangular cladogram, numbers represent the percent co nfidence of 1000 bootstrap replications. B. In the radial format, the 0.1 divergence scale represents 0.1 substitutions per site. A

PAGE 89

77 Figure 3-16. Continued 0.1 HPV1a HPV63 ECPV BPV1 BPV2 DEERPV EEPV OPV2 OPV1 TmlPV TmPV1 CRPV HPV41 HPV95 HPV4 HPV65 HPV5 HPV20 HPV21 HPV92 HPV15 HPV9 RhMPV HPV34 HPV16 HPV33 HPV30 HPV26 HPV51 HPV18 HPV32 HPV3 HPV2a HPV27 HPV7 HPV13 HPV6 HPV11 COPV B

PAGE 90

78 CHAPTER 4 DISCUSSION Papillomatous lesions harvested from the skin of captive and free-ranging manatees contained manatee papillomavirus (TmlPV) DNA and were morphologically similar to the previously described papillomatous lesi ons of captive Florida manatees (Trichechus manatus latirostris ) housed at Homosassa Springs State Wildlife Park (HSSWP), Homosassa, Florida (Bossart et al., 2002). Lesions were ei ther flat and sessile or pedunculated and distributed over the manat ee body, including the f lippers, nares, and contact regions of the anterior body (Figure 4-1 and Figure 4-2). Conclusions could not be made about the stage of the infection ba sed on the appearance of the lesions; however, the TmlPV L1 fragment sequence (GenBa nk Accession number AY496572) isolated from a pedunculated skin lesion in the pres ent study was 100% identical to the manatee papillomavirus type 1 (TmPV-1) sequence that had been deposited in the GenBank (AY609301) from another research group and had been isolated from a sessile skin lesion. These findings strongly suggest that the same virus may cause both types of papillomatous skin lesions in manatees, and is also suggested by thos e authors (Rector et al., 2004). Until recently, papillomavirus studies dealt mainly with transmission experiments, virus ultrastructure and chemical composition, and pathological desc ription of lesions. The main impediment for the analysis of thes e viruses has been the lack of a reproducible cell culture system permissive for their replication (Lancaster and Olson, 1982). The study of PVs has been limiting, as only a few HP V types have been purified in quantities

PAGE 91

79 sufficient for structural analysis, due to the low virus load of many lesions and the inability to develop tissue culture systems for large scale propagation of the virus (Rommel et al., 2005). However, the papi llomavirus field has advanced recently, especially in the areas of molecular bi ology techniques and molecular cloning, which have made these viruses more amenable to study (Lancaster and Olson, 1982). Despite the absence of advanced molecular tools, immunohistochemical data from previous studies of papillomatous lesions in manatees indicated the presence of a species-specific PV. The immunologic data also suggested th at manatee PV (TmlPV) infection might be latent and, possibly, that it might have been activated after imm unosuppression (Bossart et al., 2002). While electron microscopy ev aluation and immunohistochemistry staining of lesions showed the presence of a PV, these assays did not provide genetic information about the type of PV involve d. The primary objectives of th is study were: 1.) To develop a molecular diagnostic assay for the detection of TmlPV infection in skin lesions, 2.) to molecularly characterize the TmlPV genome and define some of its organization, and 3.) to develop a serological assay based on viruslike particles (VLPs) absorbed to an ELISA plate for the detection an d quantitation of antibodies against TmlPV. The steps for developing a molecular di agnostic assay included: extracting DNA from papillomatous lesions; designing PCR primers and PCR protocols; and sequencing amplified products to confirm th e identity of the virus invo lved. Due to the lack of sequence data available on marine mammal papillomaviruses, specifically, of the manatee, PCR assays for the molecular det ection of TmlPV DNA were initially based on the MY11 and MY09 primer set that targets a highly conserved region of the human PV (HPV) L1 ORF. Our PCR protocol utili zed annealing temperatures 10 below the

PAGE 92

80 primers melting temperatures, in order to ma ximize the chances of amplifying the TmlPV L1 DNA fragment, and incorporat ed deoxyinosines at positions of nucleotide degeneracy in the primer sequences. After testing the modified MY11 and MY09 primers (CR333/CR332) with positive TmlPV DNA, the annealing temperatures of the primers were gradually increased until amplification of a single band of the appropriate size was obtained. Using these primers in PCR assays, five TmlPV positive lesions were identified from captive manatees and four positive skin lesions from free-ranging manatees. These results confirmed the usef ulness of the widely used MY11 and MY09 consensus primers and provided an improve d method for the detection of TmlPV in cutaneous lesions. Based on the sequences ge nerated from the TmlPV L1 fragment using the MY11 and MY09 primers, these primers were modified to contain TmlPV L1specific sequences at positions of nucleotide degeneracy. These TmlPV L1 specific primers effectively amplified DNA fragments of the expected size from DNA samples extracted from lesions of 11 captive and five free ranging manatees. Four manatee DNA samples that had been negative for L1 frag ment amplification of fragments of the expected size using the modified MY11 and MY09 primers were shown to contain TmlPV DNA fragments of the expected size using the more TmlPV L1-specific primers (CR490/CR491). These results demonstrated the robustness of the TmlPV L1-specific primers and improved the use of PCR as a tool for the detection of TmlPV in skin lesions. Nucleotide sequences and deduced amino acid se quences of L1 fragments amplified with the MY11/MY09 primer set and with the Tm lPV L1-specific primer set were 100% identical (Figure 3-8), indicat ing that only one genotype of TmlPV was detected in the captive manatees of HSSWP and a few of th e free-ranging animals that swim around

PAGE 93

81 HSSWP. Three skin lesions (V372, V 373, V377) obtained from captive manatees preserved in DMSO were negative for am plification of the TmlPV L1 fragment; however, DMSO is known to crosslink DNA, making it difficult to amplify DNA fragments from these samples. These manatees may have been infected with TmlPV, but due to the method of sample preservation, the TmlPV DNA fragments we re not able to be amplified from these samples. This demonstrates the need for fresh tissue samples in the diagnosis of TmlPV infection and has im plications on the methods of sample preservation. Positive TmlPV skin lesions, identified by PCR amplification of DNA fragments of the appropriate size, were purified and se quenced directly or purified, cloned, and sequenced. Nucleotide sequences of cloned prod ucts and of directly sequenced products were compared to confirm the nucleotide iden tity of the sequences. In a study by Saiki et al. (1988), an overall error frequency of 0.25% was observed in the sequences of 239-bp amplified products after 30 cycles of amplification of the fr agment. In our PCR assays, whenever long DNA fragments (>1 Kb) were amplified, high fidelity enzymes that are endowed with proofreading activ ity were used in order to minimize possible nucleotide incorporation errors made by the polymerase during DNA amplification. Furthermore, at least two clones from each recombinant DNA were completely sequenced and analyzed and, often, both the purified mini-prep r ecombinant DNAs and the purified maxi-prep recombinant DNAs of cloned products were sequenced, as the b acteria could have possibly introduced nucleotide copying errors during transformations. Nucleotide sequence differences were observed betw een the TmlPV E6 ORF sequences (99.5100.0% identity), E7 ORF sequences (99.4-100.0 % identity), L1 ORF sequences (99.7%

PAGE 94

82 identity), and L2 ORF sequen ces (99.9% identity). These mi nor sequence variations may have been due to nucleotide errors made ei ther by the polymerase in PCR assays or by the bacteria after transformation with plasmi d DNA. Comparisons of the E6 ORF, E7 ORF, L1 ORF, and L2 ORF sequences obtai ned from the TmlPV DNA (CR130), used as positive control throughout this study, with the corresponding sequences of the TmPV-1 genome recently published by another group (Rec tor et al., 2004) revealed that the sequences were almost 100% identical at th e nucleotide and amino acid levels. These results indicated the truthfulness of the se quences obtained from the positive control TmlPV DNA (CR130) and also validated the use of this DNA (CR130) for all future genetic and phylogenetic analyses. Additionally, the identities shared by the manatee PV sequences obtained from two different mana tee papilloma isolates supports the presence of only one type of manatee PV in skin lesions from captive and free-ranging manatees around HSSWP. Our results confirmed the presence of TmlP V in skin lesions of captive manatees and extended the knowledge to encompass skin lesions from a few free-ranging manatees inhabiting Crystal River and Homosassa River, Florida, the nearby waters of HSSWP. Free-ranging manatees are often seen at th e perimeter of the underwater fence that separates the known TmlPV-pos itive captive HSSWP manatees from the free-ranging manatees in this area. DNA samples obtaine d from skin lesions of free-ranging manatees inhabiting more distant bodies of water in Florida (Port of the Isles, Tampa Bay) and Belize (Drowned Keys) were always negativ e for TmlPV DNA. Therefore, it is thought that a few of the free-ranging manatees that swim in the vicinity of the infected population at HSSWP may have acquired the infection by direct contact through this

PAGE 95

83 underwater fence. Manatee papillomavirus DNA was detected in skin lesions that were harvested from free-ranging manatees during winter months (Table 2-2). Low water temperatures have been suspected as a poten tial factor in immunol ogic suppression in the known TmlPV-infected HSSWP manatees (Bossa rt et al., 2002), and exposure to cold water has been directly correlated with im pairment of the immune function in freeranging Florida manatees (Walsh et al., 2005) Cold stress syndrome (CSS), induced by prolonged exposure to cold water, followed by nutritional, metabolic, and immunologic disturbances culminating in multi-systemic, life-threatening opportunistic infectious disease (Bossart et al., 2003), has been documented as a freque nt (18%) cause of death in Florida manatees (Bossart et al ., 2004). It is likely, then, that when the manatee immune system is compromised by ill-defined imm unosuppressive factors, such as CSS, the papilloma virus may become activated, i nvasive, and produce cu taneous lesions. Exposure to harmful algal blooms (Karenia brevis ) can also impair immune function in manatees (Walsh et al., 2005) and may be involved in TmlPV activation and production of lesions. In an attempt to obtain the entire nucle otide sequence of the TmlPV genome, PCR primers were designed to amplify overlapp ing PCR products that, if obtained, would together span the complete TmlPV genome. PCR primers effectively amplified the first amplimer that spans the L1-E1 region of the TmlPV genome; however, additional PCR primers that were based on the overlappi ng PCR product method (Forslund and Hansson, 1996) did not amplify the subsequent amplim ers. Problems encountered in amplifying the overlapping PCR products included: the l ack of sequence data of marine mammal PVs available for primer design, low quantitie s and/or poor quality of starting extracted

PAGE 96

84 DNA, and, leastly, significant se quence variation between the targeted regions of TmlPV and homologous regions in the HPVs. Seque nces obtained from the first amplimer allowed for the partial characterization of the TmlPV genome, which included partial sequences of the L1 ORF, the complete sequ ence of the E6 ORF, the complete sequence of the E7 ORF, and partial sequence of the E1 ORF. Sequences obtained from the first amplimer also allowed for the design of PCR primers that specifically targeted the TmlPV complete E6 and E7 ORFs, widening the targets for the diagnosis of papillomas of manatees. Primers targeting the TmlPV E6 ORF and E7 ORF effectively amplified DNA fragments of the expected size from five DNA samples from HSSWP captive manatees. These results expanded the use of molecular tools for the detection of TmlPV infection and provided a method for complete gene amplification for gene expression assays. However, the TmlPV E6 and E7 ORF primers did not effectively amplify fragments of the expected size from five free-ranging manatee DNA samples and two captive manatee DNA samples that had previously been positive for amplification of the L1 458-bp fragment. These captive manatees we re housed at marine parks (other than HSSWP) in Florida and Califor nia and the TmlPV E6 and E7 ORF primers were specific for TmlPV sequences obtained from the HS SWP captive manatees. The E6 and E7 ORFs are less conserved among PV types than the L1 ORF, the most hi ghly conserved region of the PV genome (deVilliers et al., 2004), and sequence vari ation within the E6 and E7 ORFs may have lowered the efficiency of the E6 and E7 primers. Also, the primers that targeted the E6 and E7 ORFs were located upstream and downstream of the ORFs, within the non-coding regions of the TmlPV genome; unlike the TmlPV L1 fragment primers that targeted a very highly conserved region within the L1 ORF. The quality and quantity

PAGE 97

85 of the manatee DNA samples may have adversel y affected the efficiency with which the TmlPV E6 and E7 primers amplified DNA fragments of the expected size. The DNA samples were frozen and thawed several times for the development of PCR assays, which may have caused the DNAs to degrade. Th ese results demonstrate the importance of sequencing more than one genome of TmlP V. A reverse primer based on our own sequences and a forward primer based on the se quence revealed in a recent publication of the TmPV-1 genome (Rector et al., 2004) eff ectively amplified the complete L1 and L2 capsid protein genes from three captive manatee DNA samples and from two captive manatee DNA samples, respectively. These re sults also expanded the use of molecular tools for the detection of TmlPV infecti on and provided a method for complete gene amplification for gene expre ssion assays. Sequences obtaine d from the L1-E1 amplimer plus the sequences obtained from the complete L1 and complete L2 amplimers allowed for further characterization of the TmlPV genome. Pair-wise comparisons of the TmlPV se quences with the corresponding sequences of several human and non-human PVs revealed amino acid identities (Table 3-5) and similarities (Table 3-6) of these viruses. Id entities refer to the extent to which two amino acid sequences are invariant and similarities refer to changes at a specific position of an amino acid sequence that preserve the physic o-chemical properties of the original residue. Sequence identities a nd similarities also give an idea of the overall similarity (homology) of the TmlPV sequence to each vi rus to which it was compared, but do not provide phylogenetic data on the genetic relate dness of the viruses. Comparisons of the TmlPV L1 fragment with seve ral human and non-human PV types suggested that the TmlPV L1 fragment sequence was more similar to the sequences of cutaneous HPV types

PAGE 98

86 (HPV-3, HPV-4, HPV-20, HPV-21, HPV-65, HPV-95) than to se quences of the high risk genital mucosal HPV types (HPV-16, -18) and ungulate PV types that induce fibropapillomas (Deer PV, OP V-1, EEPV). Comparisons of the TmlPV complete L1 ORF with several human and non-human PV types suggested that the TmlPV L1 ORF sequence was more similar to the sequences of the cutaneous PV types (HPV-20, HPV65, HPV-95) than to the high-risk ge nital mucosal HPV-33 sequence and the fibropapilloma-inducing Deer PV sequence. These L1 ORF sequence comparison results were as expected, as TmlPV infection has so far only been associated with the presence of benign skin lesions and not with genital mu cosal lesions or aggres sive fibropapillomas. Since the L1 ORF is the most highly cons erved region among all members of the PV family (deVilliers et al., 2004) results of the L1 ORF comparisons are, possibly, more reliable than results of comp arisons made with less conserve d, more variable regions of the PV genome. Comparisons of the TmlPV L2 ORF with several human and non-human PV types shows that the TmlPV L2 ORF was more sim ilar to another genetically characterized marine mammal PV sequence (PsPV-1) isolat ed from a Burmeisters porpoise (Phocoena spinipinnis ) and to sequences of cutaneous HPV types (HPV-5, HPV-15), than to the sequences of the fibropapilloma PV types (B PV, OPV, Deer PV). The TmlPV L2 ORF sequence would be expected to be more simila r to cutaneous PV types than to mucosal or malignant papillomas, as TmlPV infection has so far only been associated with the presence of skin lesions. The TmlPV L2 ORF sequence showed the greatest similarity to the Burmeisters porpoise (Phocoena spinipinnis ) papillomavirus (PsPV-1) (Van Bressem et al., 1996), the only other papillomavirus of marine mammals that has been molecularly

PAGE 99

87 characterized. Although marine in nature, th ese viruses do not seem to share a common ancestor, as PsPV-1 and TmlPV clade independe ntly of each other in phylogenetic trees. Also, PsPV-1 is known to have caused genita l warts in cetaceans (V an Bressem et al., 1996), whereas the TmlPV has so far only been a ssociated with cutaneous lesions. These results demonstrated the overall similarity of the TmlPV L2 ORF sequence to that of the L2 ORF sequences of several PV types, but did not provide an accurate phylogenetic relationship of the viruses. Comparisons of the TmlPV E6 ORF with several human and non-human PV types suggested that the TmlPV E6 ORF sequence wa s similar to sequences of a wide variety of PV types, including a ra bbit PV, a high risk mucosal ge nital PV type (HPV-32), and cutaneous PV types (HPV-1a, HPV-20, HPV-95). Inferences could not be made from these results, as they suggested that the Tm lPV E6 ORF sequence is similar to both nononcogenic (HPV-1a, HPV-20, HPV-95) and oncoge nic (HPV-32) PV types. However, the oncogenic potential of TmlPV has not yet been determined, and it is possible that the oncogenic determination of the TmlPV may be a consequence of a fine balance between the E6 and E7 regions and unidentified seque nces in a different region of the TmlPV genome. This is partially the case in other types of terrestrial PVs, such as deer PV, European elk PV, and reindeer PV, which cont ain a novel, transforming E9 gene in their genomes (Erikkson et. al, 1994). The result s of the E6 ORF sequence comparisons showed more variability than the comparisons of the highly conserved L1 and L2 regions, as the E6 ORF is a segment with little sequence conservation among PV viruses (de Villiers et al., 2004).

PAGE 100

88 Comparisons of the TmlPV E7 ORF with several human and non-human PV types suggested that this sequence is most similar to sequences of cutaneous PV types (HPV-9, HPV-15) and a benign mucosal PV type (HPV-6). Results from these comparisons were not surprising, as TmlPV, so far, has only been associated with benign cutaneous lesions. Cladistic phylogenetic diagrams refl ect hypotheses about the evolutionary relationships of organisms. A clade is formed by all species which share derived ancestral characters and a most common ancestor (Chan et al., 1995). The bootstrapped cladograms and the radial divergence trees representing the L1 ORF, L2 ORF, and E6 ORF sequences demonstrated that TmlPV forms a single, distinct branch, or constitutes its own clade. Since no PVs have been charac terized from species closely related to the manatee, it is not surprising that TmlPV cl ades by itself in these phylograms (Figure 313, -14, -15, -16). These results indicated that TmlPV is a unique virus, distinct from the known human and non-human PVs. The single branch formed by the TmlPV in the L1 ORF phylograms may be associated with the type of cell surface receptors used by the virus. The PVs enter a wide range of cells and the tropism of infection by the different PVs is controlled by events downstream of th e initial binding and upt ake (Muller et al., 1995). Studies have shown that the L1 major capsid proteins of low-risk HPV-11 binds to the Kap alpha-2 adapter and the Kap beta-2 import receptor and also interacts with the Kap beta-3 import receptor ( Nelson et al., 2003), while the L1 capsid protein of high-risk HPV-45 interacts with Kap alpha-2 beta-1 hete rodimers (Nelson et al., 2000). It has not yet been determined what the specific r eceptor of the TmlPV L1 capsid protein is; however, if these receptors differ from those described in other PV types, it may explain why the TmlPV L1 protein is phylogenetically ch aracterized as a single, distinct branch.

PAGE 101

89 The bootstrapped cladograms representing the E6 ORF and E7 ORF sequences displayed the TmlPV branch in a multi-species clade that also contained cottontail rabbit PV (CRPV). The E6 ORF and E7 ORF radial divergence phylograms (Figure 3-15 and Figure 3-16) also showed that the TmlPV and CRPV branches arose from a close point of origin. The terrestrial an cestry of the manatee may s upport the results of this phylogenetic relatedness. The closest modern relatives of manatees are hyraxes, furry mammals that are similar to ra bbits (Dawson, 1967). It is possible, then, that the manatee and rabbit may have evolved from a common placental ancestor and that the manatee PV and cottontail rabbit PV may have diverged as they evolved with thei r host species. The E7 ORF cladogram demonstrated that TmlPV is contained within a clade that includes the ungulate PVs (Figure 3-16); however, manat ee PV induces cutaneous lesions, rather than the fibropapillomas that are seen in th e ungulate PV types. A possible explanation for this difference in lesion morphology may be due to the fact that many host-specific PVs have specifically adapted to different eco logical niches (Chan et al., 1995). Since the PsPV-1 genome lacks the E7 ORF, the PsPV -1 and the TmlPV most likely do not share a common ancestor. In the L2 ORF bootst rapped cladogram (Figure 3-14), the two characterized marine mammal PVs, PsPV-1 a nd TmlPV, seem to be the outliers of a clade that includes several benign, cutaneous HPV types (HPV-4, -9, -15, -20, -21-92, 65). Data from the E6 and E7 radial di vergence trees (Figure 3-15 and Figure 3-16) suggested that the described TmlPV genotype found in cutane ous lesions of captive and free-ranging manatees might not have the complete genetic potential to induce the invasive malignancies observed in huma ns infected with high-risk oncogenic

PAGE 102

90 papillomaviruses, such as HPV-16, -18, -30, 33, -34, and -51, that are evolutionarily distant from TmlPV. Virus-like particles (VLPs) have be en produced using recombinant vaccinia viruses, baculoviruses, yeast, or Escherichia coli that express the major L1 capsid protein (Rommel et al., 2005). In our study, insect cells transfected with the TmlPV L1 recombinant bacmid DNA examined by el ectron microscopy were found to contain amorphous clumps resembling capsid particle s, but definitive papilloma virus-like particles of the appropriate si ze (50-55 nm diameter) could not be seen (Figure3-9). However, RT-PCR and PCR analyses performe d on RNAs extracted from the infected cell cultures showed the presen ce of mRNA encoding the TmlPV L1 capsid protein gene. This indicates that the message was being made for the expression of the TmlPV L1 capsid protein; but, for reasons unknown, the expressed protein in the form of VLPs could not be seen. Problems encountered in the detection of VLPs were: possible degradation of protein, protein toxicity to cells, and incorrect multiplicity of infection (MOI) used. A major problem of VLPs is th eir instability at low protein concentrations and their tendency to aggregate at high concentrations, both negatively affecting the performance of the immunologi cal assay (Rommel et al., 200 5). Even though L1 in RNA was produced in Sf21 insect cell cultures tr ansfected with the recombinant vector pFastBac/Bacmid L1, protein expression could not be detected when supernatants from apparently virus-containing cultu res were assayed by ELISA. In view of this setback, the pBlueBac 4.5 expression system, which in corporates blue/white screening of recombinant virus, was explored as a next altern ative. Using this system, we were able to detect the generation of reco mbinant virus that expressed -galactosidase and, if properly

PAGE 103

91 engineered, should express the TmlPV L1 capsi d protein from the polyhedron promoter. Using blue/white selection criteria, this system has provided a positive result for the detection of recombinant virus. Lack of specific fluorescence in the immunofluorescence assay may have arisen from the lack of similar assays used with marine mammal sera. Problems encountered with immunofluorescence detection assays ar ose from the lack of data available for marine mammal papilloma viruses. The immune response to TmlPV is not yet understood, and it is not known if the manatee develops specific l ong-lasting antibodies against E6, E7, or L1 after natu ral infection that can be dete cted with serological assays, such as an ELISA. Sera from animals that had skin lesions and were positive by PCR to TmlPV were assayed as the source of primar y antibodies to detect protein expression in the transfected cell cultures. However, no sp ecific fluorescence was observed. It is not known at this time what the reasons for th e lack of protein ex pression are. The cytomegalovirus (CMV) promoter of the expr ession vector is known to drive high-level expression in a wide variety of mammalian cells, especially in the case of COS-7 cell cultures. On the other hand, it is not known if the CMV prom oter could efficiently drive the expression of the TmlPV proteins in the manatee respiratory epithelial cells used in these assays, since similar work has not been done on this type of cell culture. As suggested before, it is conceivable that the levels of antibodies developing after TmlPV infection were not high. This, plus the re lative sensitivity of the immunofluorescence assay may have contributed to the negative results. Finally, the genes cloned into the eukaryotic expression vectors were not seque nced to confirm their integrity in the

PAGE 104

92 mammalian vector. It is possi ble that a deletion(s) may have occurred that made it impossible for proper protein expres sion in the transfected cells. It is unlikely that PVs have recently been transmitted to manatees; therefore, it is assumed that manatees have been latently in fected with PV on their healthy skin for a long time and that the appearance of PV le sions may have been triggered by impaired immunity or immunosuppression (Rector et al., 2004). Latency has been demonstrated in several animal papillomavirus infections, such as: Mastomys natalensis PV in inbred rodents (Amtmann et al., 1984), bovine PV (B PV) in cattle (Campo et al., 1994), canine oral PV (COPV) in domestic dogs (Nicholls et al., 1999), and several feline PVs (FPVs) in immunosuppressed domestic cats (Sundberg et al., 2000). Many PVs appear to occur preferentially in a latent stage and studies ha ve shown that the appare ntly healt hy skin of humans and animals may harbor multiple PV types in the absence of overt lesions (Antonsson and Hansson, 2002, and Antonsson et al., 2000). Under normal conditions, these PV infections do not exhibit any c linical symptoms, but in immunosuppressed individuals, the amount of virus increases and warts develop (Antonsson and Hansson, 2002). The pathogenesis of PV in marine mammals has not been extensively studied. It has been proposed that the type of squa mous epithelium necessary to protect these mammals from the harsh aquatic environmen t could make them susceptible to PV infection from other marine species (Bossart et al., 2002). Several re ports have described the presence of squamous papillomas and fibropapillomas in other marine mammals, specifically whales, dolphins, and porpoise s (Geraci et al., 1987). The PCR assays developed as part of this study targeted seve ral genes of TmlPV and will aid in the rapid

PAGE 105

93 diagnosis and identification of papilloma vi rus infections of manatees. Cloning and expression of TmlPV genes into mammalian and insect expression vectors will allow the development of new assays, such as ELISA, most likely using recombinant L1 antigens in the form of VLPs. This advance will make possible the detection of antibodies in seroepidemiological surveys in order to determine the prevalence of the infection in different geographical locations. Although the developm ent of a vaccine is within reach, its usefulness and efficacy, at this point, are que stionable, as the infection has only been detected around the HSSWP in an affected popula tion. Further efforts need to be made to better understand the underlying transformation pot ential of manatee papillomavirus. If papillomatous skin lesions became more wide spread in the natura l environment of the manatees, they could pose a more serious health problem for these already endangered animals. Figure 4-1. Papillomatous skin lesions on the flipper of a captive Florida manatee (Trichechus manatus latirostris ) housed at Homosassa Springs State Wildlife Park, Homosassa, Florida. Sessile skin lesion Pedunculated skin lesion

PAGE 106

94 Figure 4-2. A. Typical papillom atous skin lesions of free-ra nging Florida manatees. One lesion (sample V389) was harvested fr om a male calf in Crystal River, Florida, the nearby waters of Homosa ssa Springs State Wildlife Park, and tested for the presence of manatee papillomavirus infection. B. One lesion (sample V396) was harvested from an adult male in Homosassa River, Florida, the nearby waters of Homosa ssa Springs State Wildlife Park, and tested for the presence of manatee papillomavirus infection. B A

PAGE 107

95 LIST OF REFERENCES AMTMANN, E., M. VOLM, AND K. WAYSS. 1984. Tumour induction in the rodent Matomys natalensis by activation of endogenous papi lloma virus genomes. Nature 308: 291-292. ANTONSSON, A., O. FORSLUND, G. EKBERG, G. STERNER, AND B.G. HANSSON. 2000. The ubiquity and impressi ve genomic diversity of human skin papillomaviruses suggest a commensalic na ture of these viruses. Journal of Virology 74: 11636-11641. ANTONSSON, A., AND B. G. HA NSSON. 2002. Healthy skin of many animal species harbors papillomaviruses which are closely related to their human counterparts. Journal of Virology 76: 12537-12542. BAKER, C. C., AND P. M. HOWLEY. 1987. Differential promoter utilization by the papillomavirus in transformed cells a nd productively infected wart tissues. European Molecular Biology Or ganization Journal 6: 1027-1035. BARKSDALE, S., AND C. BAKER. 1993. Di fferentiation-specific expression from the bovine papillomavirus type 1 P2443 and late promoters. Journal of Virology 67: 5605-5616. BASEMAN, J. G., AND L. A. KOUTSKY. 2005. The epidemiology of human papillomavirus infections. Journa l of Clinical Virology 32S: S16-S24. BERNARD, H. U., S. Y. CHAN, M. M. MANOS, C. K. ONG, L. L. VILLA, H. DELIUS, C. L. PEYTON, H. M. BAUER, AND C. M. WHEELER. 1994. Identification and assessment of known and novel human papillomaviruses by PCR amplification, restriction frag ment length polymorphism, nucleotide sequence, and phylogenetic algorithms. Journal of Infectious Diseases 170: 1077-1085 BIEMELT, S., U. SONNEWALD, P. GAL MBACHER, L. WILLMITZER, AND M. MULLER. 2003. Production of human papillo mavirus type 16 virus-like particles in transgenic plants. Journal of Virology 77: 9211-9220. BIRKELAND S. A., H. H. STORM, L. U. LAMM, L. BARLOW, I. BLOHME, B. FORSBERG, B. EKLUND, O. FJELDBORG M. FRIEDBERG, L. FRODIN, E. GLATTRE, S. HALVORSEN, N. V. HOLM, A. JAKOBSEN, H. E. JORGENSEN, J. LADEFOGED, T. LINDHOLM, G. LUNDGREN, AND E. PUKKALA. 1995. Cancer risk after renal transplantation in th e Nordic countries, 1964-1986. International Journa l of Cancer 60: 183-189.

PAGE 108

96 BLESSING, K., K. M. MCLAREN, E. C. BE NTON, B. B. BARR, M. H. BUNNEY, I. W. SMITH, AND G. W. BEVERIDGE. 1989. Histopathology of skin lesions in renal allograft recipients: an assessmen t of viral features and dysplasia. Histopathology 14: 129-139. BOIRON, M., J. P. LEVY, M. THOMAS, J. C. FRIEDMAN, AND J. BERNARD. 1964. Some properties of bovine papilloma virus. Nature (London) 201: 423. BOSSART, G. D. 2001. Manatees, Chapter 43. In CRC Handbook of Marine Mammal Medicine, L. A. Dierauf and F. M. D. Gulland (eds.), CRC Press, Boca Raton, Florida. pp. 939-960. BOSSART, G. D., R. Y. EWING, M. LO WE, M. SWEAT, S. J. DECKER, C. J. WALSH, S.-J. GHIM, AND A. B. JENSON. 2002. Viral papillomatosis in Florida manatees (Trichechus manatus latirostris ). Experimental and Molecular Pathology 72:37-48. BOSSART, G. D., R. MEISNER, S. A. ROMMEL, S. GHIM, AND A. B. JENSON. 2003. Pathological features of the Florida manatee cold stress syndrome. Aquatic Mammals 29:9-17. BOSSART, G. D., R. A. MEISNER, S. A. ROMMEL, J. D. LIGHTSEY, R. A. VARELA, AND R. H. DEFRAN. 2004. Pat hologic findings in Florida manatees (Trichechus manatus latirostris ). Aquatic Mammals 30: 434-440. BREITBURD, F., M. FAVRE, R. ZOOROB, D. FORTIN, D. AND G. ORTH. 1981. Detection and characterizati on of viral genomes and search for tumoral antigens in two hamster cell lines derived from tumors induced by bovine papillomavirus type 1. International Journal of Cancer 27: 693-702. BROWN, D. R., J. M. SCHROEDER, J. T. BRYAN, M. H. STOLER, AND K. H. FIFE. 1999. Detection of multiple human papillomavirus types in condylomata acuminata lesions from otherwise healt hy and immunosuppressed patients. Journal of Clinical Microbiology 37: 3316-3322. CAMPO, M. S., W. F. JARRETT, W. O NEIL, AND R. J. BARRON. 1994. Latent papillomavirus infection in cattle. Re search in Veterinary Science 56: 151-157. CASON, J., AND C. A. MANT. 2005. Hi gh-risk mucosal human papillomavirus infections during infancy and childhood. Journal of Clinical Virology 32S: S52S58. CHAMBERS, V. C., AND C. A. EVANS. 1959. Canine oral pa pillomatosis. Virus assay and observations of the various stages of the experimental infection. Cancer Research 19: 1188-1195.

PAGE 109

97 CHAN, S.-Y., H. DELIUS, A. L. HALPER N, AND H.-U. BERNARD. 1995. Analysis of genomic sequences of 95 papillomavi rus types: uniting typing, phylogeny, and taxonomy. Journal of Virology 69: 3074-3083. CHEVILLE, N. G. 1966. Studies on connect ive tissue tumors in the hamster produced by bovine papilloma virus. Cancer Research 26: 2334-2339. CHIANG, C.-M., M. USTAV, A. STENLU ND, T. G. HO, T. R. BROKER, AND CHOW, L. T. 1992. Viral E1 and E2 pr oteins support replication of homologous and heterologous papillomavirus origins. Proceedings of the National Academy of Sciences of the United Stat es of America 89: 5799-5803. DALING, J. R., M. M. MEDELEINE, S. M. SCHWARTZ, K. A. SHERA, J. J. CARTER, B. MCKNIGHT, P.L. PORTER, D.A. GALLOWAY, J. K. MCDOUGALL, AND H. TAMIMI. 2002. A population-based study of squamous cell vaginal cancer: HPV and cofactors. Gynecologic Oncology 84(2): 263-270. DANOS, O., M. KATINKA, AND M. YANIV. 1982. Human papillomavirus 1a complete DNA sequence: a novel type of genome organization among Papovaviridae. European Mol ecular Biology Journal 1: 231-236. DAWSON, M. R. 1967. Fossil history of the families of recent mammals, order Sirenia. In Recent Mammals of the World. A Synopsis of Families, S. Anderson and J. K. Jones (eds.), Ronald Press Co., New York. 453 pp. DESAINTES, C. AND C. DEMERET. 1996. Control of papillomavirus DNA replication and transcription. Semi nars in Cancer Biology 7: 339-347. DE VILLIERS E. M., C. FAUQET, T. R. BROKER, H. U. BERNARD, AND H. ZUR HAUSEN. 2004. Classification of pa pillomaviruses. Virology 324:17-27. DIMAIO, D., D. GURALSKI, AND J. T. SCHILLER. 1986. Translation of open reading frame E5 of bovine papillomavirus is required for its transforming activity. Proceedings of the National Academy of Scie nces of the United States of America 83: 1797-1801. DOORBAR, J. 2005. The papillomavirus life cy cle. Journal of Clinical Virology 32S: S7-S15. DYSON, N., P. M. HOWLEY, K. MUNGER, AND E. HARLOW. 1989. The human papillomavirus-16 E7 oncoprotein is able to bind the retinoblastoma gene product. Science 243: 934-937. EGAWA, K. 2003. Do human papillomavir uses target epidermal stem cells. Dermatology 207: 251-254.

PAGE 110

98 ERIKSSON, A., A. C. STEWART, J. MORENO-LOPEZ, AND U. PETTERSSON. 1994. The genomes of the animal papillomaviruses European elk papillomavirus, deer papillomavirus, and reindeer papillomavirus contain a novel transforming gene (E9) near the early polyadenylation site. Jour nal of Virology 68: 8365-8373. FORSLUND, O. AND B. G. HANSSON. 1996. Human papillomavirus type 70 genome cloned from overlapping PCR products: complete nucleotide sequence and genomic organization. Journal of Clinical Micr obiology 34: 802-809. FORSLUND, O., B. LINDELOF, E. HRAD IL, P. NORDIN, B. STENQUIST, R. KIRNBAUER, K. SLUPETZKY, AND J. DI LLNER. 2004. High prevalence of cutaneous human papillomavirus DNA on the top of skin tumors but not in stripped biopsies from the same tumors Journal of Investigative Dermatology 123: 388-394. FULTON, R. E., F. W. DOANE AND L. W. MACPHERSON. 1970. The fine structure of equine papilloma and the equine papilloma virus. Journal of Ultrastructure Research 30: 328-343. GALLOWAY, D. A. 2003. Papillomavirus v accines in clinical trials. The Lancet, Infectious Diseases 3: 469-475. GELDER, C. M., O. M. WILLIAMS, K. W. HART, S. WALL, G. WILLIAMS, D. INGRAMS, P. BULL, M. BUNCE, K. W ELSH, S. E. F. MARSHALL, AND L. BORYSIEWICZ. 2003. HLA class II polymorphisms and susceptibility to recurrent respiratory papillomatosis. Journal of Virology 77: 1927-1939. GERACI, J. R., N. C. PALMER, AND D. J. ST. AUBIN. 1987. Tumors in cetaceans: analysis and new findings. Canadian Journa l of Fisheries and Aquatic Sciences 44: 1289-1300. GILBERT, D. M. AND S. N. COHEN. 1987. Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle. Cell 50: 59-68. HAGENSEE, M. E., N. YAEGASHI, AND D. A. GALLOWAY. 1993. Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid pr oteins. Journal of Virology 67: 315-322. HAM, J., N. DOSTATNI, J. M. GAUTHIER, AND M. YANIV. 1991. The papillomavirus E2 protein: a factor with many talents. Trends in Biochemical Sciences 16: 440-444. HARWOOD, C. A., P. J. SPINK, T. SURENTHERAN, I. M. LEIGH, E. M. DE VILLIERS, J. M. MCGREGOR, C. M. PROBY, AND J. BREUER. 1999. Degenerate and nested PCR: a highly sensitive and specific method for detection of human papillomavirus infection in cuta neous warts. Journal of Clinical Microbiology 37: 3545-3555.

PAGE 111

99 HOWLEY, P. M. AND D. R. LOWY. 2001. Pa pillomaviruses and Their Replication, Chapter 65. In Fields Virology, D. M. Knipe and P. M. Howley (eds.). Lippincot Williams and Wilkins, Philadelphia, Pennsylvania. pp. 2197-2229. JABLONSKA, S. 1991. Epidermodysplasia verruciformis. In Cancer of the Skin, Riedman, R. J., Rigel, D. S ., Kopf, A. W., Harris, M. N. and Baker D. (eds.). W. B. Saunders, Philadelphia. pp. 101-113. KENNEDY-STOSKOPF, S. 2001. Viral diseases. In CRC Handbook of Marine Mammal Medicine, L. A. Dierauf and F. M. D. Gulland (eds.), CRC Press, Boca Raton, Florida. pp. 285-307. KLAASSEN, C. H., C. F. M. PRINSEN, H. A. DE VALK, A. M. HORREVORTS, M. A. F. JEUNINK. AND F. B. J. M. T HUNNISSEN. 2003. DNA microarray format for detection and subtyping of human pa pillomavirus. Journal of Clinical Microbiology 42: 2152-2160. KREIMER, A. R., G. M. CLIFFORD, P. BOYLE, AND S. FRANCESCHI. 2005. Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systematic review. Cancer Epidemiology Biomarkers and Prevention 14: 467-475. KURMAN, R. J., L. E. SANZ, A. B. JENS ON, S. PERRY, AND W. D. LANCASTER. 1982. Papillomavirus infection of the cervix I. Correlation of histology with viral structural antigens and DNA sequences. In ternational Journal of Gynecological Pathology 1: 17-28. KRZYZEK, R. A., S. L. WATTS, D. L. AND ERSON, A. J. FARAS, AND F. PASS. 1980. Anogenital warts contain several distinct species of human papillomavirus. Journal of Virology 36: 236-244. LAMBERTSEN, R. H., B. A. KOHN, J. P. SUNDBERG, AND C. D. BUERGELT. 1987. Genital papillomatosis in sperm whal e bulls. Journal of Wildlife Diseases 23: 361-367. LANCASTER, W. D., C. OLS ON, AND W. MEINKE. 1977. Bovine papilloma virus: Presence of virus-specific DNA sequences in naturally occurring equine tumors. Proceedings of the National Academy of Scie nces of the United States of America 74: 524-528. LANCASTER, W. D. AND C. OLSON. 1978. De monstration of two distinct classes of bovine papilloma virus. Virology 89: 371-379. LANCASTER, W. D. AND C. OLSON. 1982. Animal papillomaviruses. Microbiological Reviews 46: 191-207.

PAGE 112

100 LEDER, C., J. A. KLEINSCHMIDT, C. WIETHE, AND M. MULLER. 2001. Enhancement of capsid gene expression: pr eparing the human papillomavirus type 16 major structural gene L1 for DNA v accination purposes. Journal of Virology 75: 9201-9209. LEPIK, D., I. L. ILVES, A. KRISTJUHA N, T. MAIMETS, AND M. USTAV. 1998. p53 protein is a suppressor of papilloma virus DNA amplificational replication. Journal of Virology 72: 6822-6831. LI, C.-C. H., R. V. GILDEN, S. D. SHOWALTER, AND K. V. SHAH. 1988. Identification o the human papillomavirus E2 protein in geni tal tract tissues. Journal of Virology 62: 606-609. LINA, P. H. C., M. M. VAN NOORD, AND F. G. DEGROOT. 1973. Detection of virus in squamous papillomas of the w ild bird species Fringella coelebs Journal of the National Cancer Institute 39: 55-65. LOWY, D. R. AND P. M. HOWLEY. 2001. Papillomaviruses, Chapter 66. In Fields Virology, D. M. Knipe and P. M. Howley (eds.), Lippincot Williams and Wilkins, Philadelphia, Pennsylvania. pp.2231-2264. MAJEWSKI, S. AND S. JABLONSKA. 1995. Epidermodysplasia verruciformis as a model of human papillomavirus-induced genetic cancer of the skin. Archives of Dermatology 131: 1312-1318. MANNIK, A., K. RUNKORG, N. JAANSON, M. USTAV, AND E. USTAV. 2002. Induction of the bovine papillomavirus orig in onion skin-type DNA replication at high E1 protein concentrations in vi vo. Journal of Virology 76: 5835-5845. MANOS, M. M., Y. TING, D. K. WRIGHT, A. J. LEWIS, T. R. BROKER, AND S. M. WOLINSKY. 1989. The use of polymerase chain reaction amplification for the detection of genital human papillomavir uses. Cancer Cells, Chapter 7. In Molecular Diagnostics of Human Cancer, M. Greaves (ed.), Cold Spring Harbor Laboratory, Woodbury, New York. pp. 209-214. MARTINEAU, D.K., A. LEMBERGER, P. DALLAIRE, T. P. LABELLE, P. LIPSCOMB, P. MICHEL, AND I. MIKAELIAN 2002. Cancer in wildlife, a case study: beluga from the St. Lawrence estu ary in Quebec, Canada. Environmental Health Perspectives 110: 285-292. MCBRIDE, A. A., A. DLUGOSZ AND C. C. BAKER. 2000. Production of infectious bovine papillomavirus from cloned viral DNA by using an organotypic raft/xenograft technique. Proceedings of the National Academy of Science, USA 97: 5534-5539. MCMURRAY, H. R., D. NGUYEN, T. F. WESTBROOK, AND D. J. MCANCE. 2001. Biology of human papillomaviruses. In ternational Journal of Experimental Pathology 82: 15-33.

PAGE 113

101 MELENDY, T., J. SEDMAN, AND A. STENLUND. 1995. Cellular factors required for papillomavirus DNA replication. Journal of Virology 69: 7857-7867. MESSING, A. M. AND W. L. EP STEIN. 1963. Natural history of warts: a 2 year study. Archives of Dermatology 87: 306-310. MILLER, C. S AND B. M. JOHNS TONE. 2001. Human papilloma virus as a risk factor for oral squamous cell carcinoma: a me ta-analysis, 1982-1997. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontics 91: 622-635. MULLER, F., Y. -S. SEO, AND J. HUR WITZ. 1994. Replication of bovine papillomavirus type 1 origin-containing DNA in crude extracts and with purified proteins. The Journal of Bi ological Chemistry 269: 17086-17094. MULLER, M., L. GISSMANN, R. J. CRISTIANO., S. Y. SUN, I. H. FRAZER, A. JENSON, A. ALONSO, H. ZENTGRAF, A ND J. ZHOU. 1995. Papillomavirus capsid binding and uptake by cells from differ ent tissues and species. Journal of Virology 69: 948-954. MUNOZ, N., X. BASCH, S. DE SANJOSE, R. HERRERO, X. CASTERLLSAGUE, K. V. SHAHP, J. F. SNIJDERS, AND C. J. L. M. MEIJER. 2003. Epidemiologic classification of human papill omavirus types associated with cervical cancer. The New England Journal of Medicine 348: 518-527. NELSON, L. M., R. C. ROSE, L. LE ROUX, C. LANE, K. BRUYA, AND J. MOROIANU. 2000. Nuclear import and DNA binding of human papillomavirus type 45 L1 capsid protein. Journal of Cellular Biochemistry 79: 225-238. NELSON, L. M., R. C. ROSE, AND J. MO ROIANU. 2003. The L1 major capsid protein of human papillomavirus type 11 interacts with Kap beta2 and Kap beta3 nuclear import receptors. Virology 306: 162-169. NICHOLLS, P. K. AND M. A. STANLEY. 199 9. Canine papillomavirus-A centenary review. Journal of Comparative Pathology 120: 219-233. OH, S. T., M. S. LONGWORTH, AND L. A. LA IMINS. 2004. Roles of the E6 and E7 proteins in the life cycle of low-risk hu man papillomavirus type 11. Journal of Virology 78: 2620-2626. ORTH, G., M. FAVRE, AND O. CROISSANT. 1977. Characterization of a new type of human papillomaviruses that causes skin warts. Journal of Virology, 24: 108-120. ORTH, G. 1987. Epidermodysplasia verruciformis. In The Papovaviridae, the Papillomaviruses, Salzman, NP and Howley, PM (eds.), Plenum Press, New York. pp 199-235.

PAGE 114

102 PARK, P., W. COPELAND L. YANG, T. WANG, M. R. BOTCHAN, AND I. J. MOHR. 1994. The cellular DNA polymerase -primase is required for papillomavirus DNA replication and associ ates with the viral E1 helicase. Proceedings of the National Academy of Sc ience of the United States of America 91: 8700-8704. PARKIN, D. M., P. L. PISANI, AND J. FE RLAY. 1999. Estimates of the worldwide incidence of 25 major cancer s in 1990. International Journal of Cancer 80: 827841. PEH, W. L., K. MEDDLETON, N. CHRIS TENSEN, P. NICHOLLS, K. EGAWA, K. SOTLAR, J. BRANDSMA, A. PERCIVAL, J. LEWIS, W. J. LIU, AND J. DOORBAR. 2002. Life cycle heterogene ity in animal models of human papilloma-virus associated disease. Journal of Virology 76: 10401-10416. RECTOR, A., G. D. BOSSART, S. J. GHIM, J. P. SUNDBERG, A. B. JENSON, AND M. VAN RANST. 2004. Characterization of a novel close-to-root papillomavirus from a Florida manatee by using multiply primed rolling-circle amplification: Trichechus manatus latirostris papillomavirus type 1. Journal of Virology 78: 12698-12702. RODEN, R. B. S., P. M. DAY, B. K. BRONZO, W. H. YUTZY Y. YANG, D. R. LOWY, AND J. T. SHILLER. 2001. Positiv ely charged termini of the L2 minor capsid protein are necessary for papillomavirus infection. Journal of Virology 75: 10493-10497. ROMMEL, O., J. DILLNER, C. FLIGGE C. BERGSDORF, X. WANG, H.-C. SELINKA, AND M. SAPP. 2005. Heparin sulfate proteoglycans interact exclusively with conformationally intact HPV L1 assemblies: Basis for a virus-like particle ELISA. Journal of Medical Virology 75:114-121. RUBIN, M. A., B. KLETER, M. ZHOU, G. AYALA, A. L. CUBI LLA, W. G. QUIT, AND E. C. PIROG. 2001. Detection and typing of human papillomavirus DNA in penile carcinoma: evidence for multiple independent pathways of penile carcinogenesis. American Jour nal of Pathology 159 (4):1211-1218. SAIKI, R. K., D. H. GELFAND S. STOFFE L, S. SCHARF, R. H. HIGUCHI, G. T. HORN, K. B. MULLIS, AND H. A. ERLICH 1988. Primer-directed enzymatic amplification of DNA with a thermost able DNA polymerase. Science 239:487491. SCHEFFNER, M., J. M. HUIBREGTSE, R. D. VIERSTRA, AND P. M. HOWLEY. 1993. The HPV-16 E6 and E6-AP complex f unctions as a ubiqui tin-protein ligase in the ubiquitination of p53. Cell 75: 495-505. SCHIFFMAN, M. H. 1992. Recent progress in defining the epidemiology of human papillomavirus infection a nd cervical neoplasia. Journa l of the National Cancer Institute 84: 394-398.

PAGE 115

103 SCHILLER, J. T., AND D. R.LOWY. 2000. Pa pillomavirus-like particle vaccines. Journal of the National Cancer Institute Monographs 28: 50-54. SCHWARZ, E., U. K. FREESE, L. GISSMANN, W. MAYER, B. ROGGENBUCK, A. STREMLAU, AND H. ZUR HAUSEN. 1985. Structure and transcription of human papillomavirus sequences in cerv ical carcinoma cells. Nature 314: 111-114. SCHWARZ, E., M. DURST, O. DEMANK WOSKI, R. LATTERMANN, E. ZECH, S. WOLFSBERGER, S. SUHAI, AND H. ZUR HAUSEN. 1983. DNA sequence and genome organization of human papillomavirus type 6b. European Molecular Biology Journal 2: 2341-2348. SEDLACEK, T. V., S. LINDHEIM, C. EDER, L. HASTY, M. WOODLAND A. LUDOMIRSKY, AND R. F. RANDO. 1989. Mechanism for human papillomavirus transmission at birth. American Journal of Obstetrics and Gynecology 161: 55-59. SEDMAN, J., AND A. STENLUND. 1995. Co -operative interaction between the initiator E1 and the transcriptional activator E2 is required for replicator specific DNA replication of bovine papilloma-viru s in vivo and in vitro. European Molecular Biology Journal 14: 6218-6228. SEEDORF, K., G. KRAMMER, M. DURST S. SUHAI, AND W. G. ROWEKAMP. 1985. Human papillomavirus type 16 DNA sequence. Virology 145:181-185. SHAMANIN, V., H. DELIUS, AND E. M. de VILLIERS. 1994. Development of a broad spectrum PCR assays for papillomav iruses and its application in screening lung cancer biopsies. Journal of General Virology 75: 1149-1156. SHOPE, R. E. 1933. Infectious papilloma tosis of rabbits; with a note on the histopathology. Journal of Expe rimental Medicine 58: 607-624. SHOPE, R. E., R. MANGOLD, L. G. MCNAMARA, AND K. R. DUMBELL. 1958. An infectious cutaneous fibroma of the Virginia white-tailed deer (Odocoileus virginianus) Journal of Experiment al Medicine 108: 797-802. SILVERBERG, N. B. 2004. Hu man papillomavirus infections in children. Current Opinion in Pediatrics 16: 402-409. SMITH, A. W., AND D. E. SKILLING. 1979. Viruses and virus di seases of marine mammals. Journal of the American Ve terinary Medical Association 175: 918-920. SMITS, H. L., L. M. TIEBEN, S. P. TJONG-A-HUNG, M. F. JEBBINK, R. P. MINAAR, C. L. JANSEN, AND J. TER SCHEGGET. 1992. Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spect rum of human papillomavirus types. Journal of General Virology 73:3263-3268.

PAGE 116

104 STAUFFER, Y., K. RAJ, K. MASTERNAK, AND P. BEARD. 1998. Infectious human papillomavirus type 18 pseudovirions. Journal of Molecular Biology 283: 529-536. STOCKFLETH, E., I. NINDL, W. STERRY C. ULRICH, T. SCHMOOK, AND T. MEYER. 2004. Human papill omaviruses in transplant-associated skin cancers. Dermatologic Surgery 30: 604-609. SUNDBERG, J. P. 1987. Papilloma virus infection in animals. In Papillomavirses and Human Disease, K. Syrjaenen, L. Gissm ann, and L.G. Koss (eds.), Springer Verlag, Berlin, pp. 40-103. SUNDBERG, J. P., A. A. RESZKA, E. S. WILLIAMS, AND M. E. REICHMANN. 1991. An oral papillomavirus that infected one coyote and three dogs. Veterinary Pathology 28: 87-88. SUNDBERG, J. P., M. VAN RANST, R. MO NTALI, B. L. HOMER, W. H. MILLER, P. H. ROWLAND D. W. SCOTT, J. J. ENGLAND R. W. DUNSTAN, I. MIKAELIAN, AND A. B. JENSON. 2000. Feline papillomas and papillomaviruses. Veterinary Pathology 37: 1-10. SYRJANEN, S. 2003. Human papillomavirus infections and oral tumors. Medical Microbiology and Immunology 192: 123-128. TACHEZY, R., A. RECTOR, M. HAVE LKOVA, P. F. WOLLNATS, G. OPDENAKKER, A. B. JENSON, J. P. SUNDBERG,. AND M. VAN RANST. 2002. Avian papillomavirus es: the parro t Psittacus erithacus papillomavirus (PePV) genome has a unique organizati on of the early protein region and is phylogenetically related to the ch affinch papillomavirus. BMC Microbiology 2: 1471-1478. TING, Y., AND M. M. MANOS. 1990. Detection and typing of genital human papillomaviruses, Chapter 42. In PCR Protocols: A Guide to Methods and Applications, M A. Innis, D. H. Gelfand a nd J. J. Sninsky (eds.), Academic Press, Inc., San Diego, California. pp. 356-367. TRIMBLE, C. L., A. HILDESHEIM, L. A. BRINTON, K. V. SHAH, AND R. J. KUMAN. 1996. Heterogeneous etiology of squamous carcinoma of the vulva. Obstetrics and Gynecology 87: 59-64. TROFATTER, K.F.J. 1997. Diagnosis of HPV genital tract infection. American Journal of Medicine, 102: 21-27. U. S. FISH AND WILDLIFE SERVICE. 2001. Florida manatee recovery Plan, (Trichechus manatus latirostris ), Third Revision, U. S. Fi sh and Wildlife Service. Atlanta, Georgia. 144 pp. + appendices.

PAGE 117

105 VAN BRESSEM, M.-F., K. VAN WAEREBEE K, G. E. PIERARD, AND C. DESAINTES. 1996. Genital and lingual wa rts in small cetaceans from coastal Peru. Diseases of Aquatic Organisms 26: 1-10. WALBOOMERS, J. M., M. V. JACOBS, M. M. MANOS, F. X. BOSCH, J. A. KUMMER, K. V. SHAH, P. J. SNIJDERS, J. PETO, C. J. MEIJER, AND N. MUNOZ. 1999. Human papilloma virus is a necessary cause of invasive cervical cancer worldwide. Journal of Pathology 189:12-19. WALSH, C. J., C. A. LUER, AND D. R. NOYES. 2005. Effects of environmental stressors on lymphocyte proliferation in Florida manatees, Trichechus manatus latirostris Veterinary Immunology and Immunopathology 103: 247-256. WOODRUFF, R. A., R. K. BONDE, J. A. BONILLA, AND C. H. ROMERO. Molecular identification of a papilloma viru s from cutaneous lesions of captive and free-ranging Florida manatees. Journa l of Wildlife Diseases, In Press. ZHOU, C., Y. YANG, AND A. Y. YONG. 1990. Mini-preps in ten minutes. BioTechniques 8: 172-173. ZUR HAUSEN, H. 1991. Viruses in human cancers. Science 254:1167-1173 ZUR HAUSEN, H. 1996. Papillomavirus inf ectionsa major cause of human cancers. Biochimica et Biophysica Acta (BBA) Reviews on Cancer 1288: F55-F78.

PAGE 118

106 BIOGRAPHICAL SKETCH Rebecca Ann Woodruff, daughter of Mike and Debbie Woodruff, was born and raised in Bradenton, Florida, along with her siblings, Jessica and Mike. Rebecca graduated from Bradenton Christian Hi gh School in 1997 and went on to Florida Southern College, Lakeland, Florida, where she majored in biology. Hoping to gain more opportunities and experiences in a larg e, public college, sh e transferred to the University of Florida after the completion of her sophomore year. Rebecca graduated from UF in May, 2001, with a bachelor of science in microbiology and cell sciences. After working for about a year at a plant viro logy research lab, she decided to go back to UF to obtain a masters degree. Upon meeti ng several professors, she decided to take the opportunity to become one of Dr. Carlos Ro meros students and conduct research in the exciting field of marine mammal viruses. After completing her masters defense, planning a wedding, and making a big move, sh e will be ready to relax in her new hometown of Fort Collins, Colorado.


Permanent Link: http://ufdc.ufl.edu/UFE0011885/00001

Material Information

Title: Detection and Molecular Characterization of Manatee Papillomavirus in Cutaneous Lesions of the Florida Manatee (Trichechus manatus latirostris)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011885:00001

Permanent Link: http://ufdc.ufl.edu/UFE0011885/00001

Material Information

Title: Detection and Molecular Characterization of Manatee Papillomavirus in Cutaneous Lesions of the Florida Manatee (Trichechus manatus latirostris)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0011885:00001


This item has the following downloads:


Full Text












DETECTION AND MOLECULAR CHARACTERIZATION OF MANATEE
PAPILLOMAVIRUS IN CUTANEOUS LESIONS OF THE FLORIDA
MANATEE (Trichechus manatus latirostris)













By

REBECCA ANN WOODRUFF


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Rebecca Ann Woodruff

































This document is dedicated to the graduate students of the University of Florida.















ACKNOWLEDGMENT

I would like to extend my greatest appreciation and thanks to my mentor, Dr.

Carlos Romero, for allowing me to join his laboratory and enjoy many opportunities that

I would not have been able to elsewhere. I would also like to thank my committee

members, Dr. Peter McGuire and Dr. Ayalew Mergia, for their time, assistance, and

suggestions. I would like to thank Dr. Ellis Greiner for first introducing me to the

pathobiology graduate program at the University of Florida. I extend great thanks to Bob

Bonde, not only for obtaining all of the manatee samples used in this study, but also for

providing his expertise and knowledge of manatees. This project was funded by the

Florida Fish and Wildlife Commission through the Marine Mammal Animal Health

Program of the College of Veterinary Medicine at the University of Florida, and would

not have been possible without the U. S. Geological Survey (USGS) Department of the

Interior, contributions of collaborators affiliated with numerous zoological parks, and

those involved with manatee capture release studies. I would also like to thank my fellow

lab-mates, Alexa Bracht, Kara Smolarek-Benson, Shasta McClenahan, and Rebecca

Grant, for their much valued friendships. I am greatly appreciative of my best friend,

David Kottke, for supporting me unconditionally and following me to pursue my dreams,

even if it meant putting his own aside. I would also like to thank my family for their

constant encouragement during these past two years.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T .......................................................................... .................... iv

LIST OF TABLES ...................... ................. ............... viii

LIST OF FIGURES ......... ........................................... ............ ix

ABSTRACT .............. .................. .......... .............. xi

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

C classification of Papillom viruses ................. ................................. .. ....................1
Papillomavirus Genome Organization and Characterization ......................................2
Papillom avirus R eplication......................................... ................................. 4
P apillom avirus T ransm ission....................................... .......................................5
Papillom avirus Pathogenesis ........................................ ................................. 6
D iagnosis of Papillom avirus Infection ...................................................... .............. 8
Treatment and Prevention of Papillomavirus Infection..............................................9
H um an Papillom viruses ...................................... ................................................ 10
N on-Hum an Papillom aviruses................................................... .........................
Marine Mammal Papillomaviruses.................. ................................. 13
The Florida Manatee (Trichechus manatus latirostris).............................................13

2 M ATERIALS AND M ETHOD S ........................................ ......................... 16

Clinical Data ................................................... ........ ........ ............... 16
Source of Samples ........................... ............ .............. 16
DNA Extraction from Papillomatous Lesions......... .......................................17
G general M ethods of P C R ...................................................................... ......... 18
T aq Polym erase R actions .............. ......................................... ..................... 18
AccuprimeTM Taq DNA Polymerase Reactions ...............................................19
Expand High Fidelity Plus Reactions..... .......... ........................................ 19
PCR Targeting the L 458-bp Fragm ent ....................................... ............... 20
PCR Targeting the AL1 TmlPV 458-bp Fragment................. ............................20
PCR Targeting the TmlPV L1-El Region....................................... ............... 20
PCR Targeting the Complete TmlPV E6 Gene ................................ ............... 21
PCR Targeting the Complete TmlPV E7 Gene ................................ ............... 21


v









PCR Targeting the TmlPV L1-L2 Region................................................. 21
PCR Targeting the Complete TmlPV L1 Gene ................................ ............... 22
PCR Targeting the Complete TmlPV L2 Gene .............................................. .22
G el Electrophoresis....................................................... ......... ... 22
General Cloning of PCR Products................. ............ ............. ............... 23
Cloning into pCR 2.1 TOPO T/A Vector ............................. .................. 23
Cloning into P-TargetTM Mammalian Expression Vector ...............................24
Cloning into pcDNA '"3.1 Directional TOPO Expression Vector ................24
A nalyzing R ecom binants ......... .. ............... ...........................................................25
Sub-cloning of Purified Recombinants.......... .... .......................................... 27
Sub-Cloning into pcDNA 3.1/Zeo+ Expression Vector................ .......... 27
L1 Gene Sub-Cloning into pFastBacTM1 Vector..............................................29
L1 Gene Sub-Cloning into pBlueBac 4.5................................. ............... 30
Sequencing of PCR-amplified and Cloned Products............................................... 30
Transfection of Insect Cell Cultures ..................................................................... 31
Culturing Insect C ells ................ .......... .......... .. ....... ...... 31
Transformation of MAX Efficiency DH1OBacTM Competent E. coli .............32
Transfection of Insect Cells with Bacmid DNA Recombinants........................33
Harvest of Recombinant Baculovirus Stocks .................................................34
Reverse Transcription PCR (RT-PCR) of Infected Cell Cultures....................34
Generation of Recombinant Baculovirus .......................................................35
Transfection of M am m alian Cells .......................................................................... 36
Culturing African Green Monkey Kidney (COS-7) Cells..................................36
Electroporation of COS-7 Cells with Recombinants for Immunofluorescence
A s sa y s ................................. .... .. .......................... .... .............. 3 7
Culturing Florida Manatee Respiratory Epithelial Cells..................................38
Transfection of TmlRE Cells with DNA Recombinants for
Im m unofluorescence A says ........................................ ........ ............... 39

3 R E S U L T S .......................................................................... 4 4

P C R R results .................................................... ................................. . 44
PCR Targeting the Papillomavirus L1 458-bp Fragment...............................44
PCR Targeting the AL1 TmlPV Fragment.........................................................45
PCR Targeting the TmlPV L1-El Region................................ ............... 45
PCR Targeting the Complete TmlPV E6 Gene............................................45
PCR Targeting the Complete TmlPV E7 Gene ............................................45
PCR Targeting the TmlPV L1-L2 Region.................. ................. 46
PCR Targeting the Complete TmlPV L1 Gene.......... .......... ............... 46
PCR Targeting the Complete TmlPV L2 Gene .............................................. 46
Sequencing Results and Genetic Analyses............................... ............. ........... 46
Tm lPV L 1 458-bp Fragm ents.................................................. ......... .......... .. 46
L 1-E 1 T m lP V R egion ............................................................... .....................4 8
Tm lPV E6 G ene ............................................................. 49
Tm lPV E7 G ene ............................................................. 49
Tm lPV L1 -L2 R egion............................................. ......... ......................... 50
Complete TmlPV L1 Gene from Captive Manatee Lorelei .............................51









Com plete Tm lPV L2 Gene.................................................... .. ..................51
Immunofluorescence and Gene Expression Assays ................................................52
M am m alian Expression System s...................................................................... 52
Bac-to-Bac Baculovirus Expression System .....................................................52
Bac-N -Blue Baculovirus Expression System ....................................................... 53
Phylogenetic A analysis ...................................... ................. .... ....... 53

4 D ISC U S SIO N ............................................................................... 78

LIST OF REFEREN CES ............................................................................. 95

BIOGRAPHICAL SKETCH ............................................................. ............... 106
















LIST OF TABLES


Table page

2-1 Samples obtained from skin lesions of captive Florida manatees.....................39

2-2 Samples obtained from skin lesions of free-ranging Florida manatees ..............40

2-3 PCR primers designed to target manatee papillomavirus sequences ..................40

2-4 Sequencing primers used to obtain the complete sequence of PCR amplified
T m lP V gene fragm ents .......................................................................... .... ... 4 1

3-1 PCR results of DNAs obtained from captive manatee skin lesions tested for
the presence of TmlPV infection. .............................................. ............... 55

3-2 PCR results of DNAs obtained from free-ranging manatee skin lesions tested
for the presence of TmlPV infection. ...................................... ............... 56

3-3 Sum m ary of PCR results. ................................................................................57

3-4 Accession numbers of manatee papillomavirus sequences deposited into the
GenBank tool of the NCBI website ............................................................58

3-5 Pair-wise comparisons of the amino acid sequences of the L1, L2, E6, and E7
gene fragments of manatee papillomavirus (TmlPV) with several human and
non-human papillomaviruses..................................................... 59

3-6 Pair-wise comparisons of the amino acid sequences of the L1, L2, E6, and E7
gene fragments of manatee papillomavirus (TmlPV) with several human and
non-human papillomaviruses..................................................... 60

3-7 Summary of the pair-wise comparisons of the amino acid sequences of
manatee papillomavirus gene fragments with several human and non-human
p ap illo m av iru ses..................................................................................... 6 1















LIST OF FIGURES


Figure page

1-1 Illustration demonstrating the genetic organization of a typical
papillom avirus genom e.. ............................. .... .....................................15

2-1 Linear representation of the open reading frames (ORFs) of the circular
manatee papillomavirus (TmlPV) genome with the relative positions of the
PCR primers used to amplify TmlPV DNA ........................................... ........... 16

3-1 Agarose gel electrophoresis of PCR amplified 458-bp fragments of the L1
capsid protein gene of manatee papillomavirus. ............................. ............... 62

3-2 Agarose gel electrophoresis of PCR amplified 2,772-bp fragment of the Ll-
El region of manatee papillomavirus........................................................ 62

3-3 Agarose gel electrophoresis of PCR amplified 587-bp fragments of the E6
gene of manatee papillomavirus. ............. ................................. .................63

3-4 Agarose gel electrophoresis of PCR amplified 489-bp fragments of the E7
gene of manatee papillomavirus. ............ .. .................................. .................63

3-5 Agarose gel electrophoresis of PCR amplified 3,208-bp fragments of the L1
gene plus the L2 gene of manatee papillomavirus ............................................64

3-6 Agarose gel electrophoresis of PCR amplified 1,712-bp fragments of the
complete L1 gene of manatee papillomavirus.............................................64

3-7 Agarose gel electrophoresis of PCR amplified 1,660-bp fragments of the
complete L2 gene of manatee papillomavirus............................... ..............65

3-8 Multiple alignment of the amino acid sequences deduced from the nucleotide
sequences of the L1 gene fragment of manatee papillomavirus identified in
cutaneous lesions of captive and free-ranging Florida manatees ......................66

3-9 Electron micrograph of Sf21 insect cell cultures transfected with
pFastBacl/TmlPV L1 gene recombinant................................................67

3-10 Agarose gel electrophoresis demonstrating the PCR amplification of the
manatee papillomavirus complete L1 gene fragment from cDNAs obtained
from infected Sf21 cell cultures. .............................................. ............... 68









3-11 Agarose gel electrophoresis demonstrating restriction enzyme digests of the
manatee papillomavirus E6 gene and E7 gene recombinants. ..........................69

3-12 Agarose gel electrophoresis demonstrating restriction enzyme digests of the
manatee papillomavirus L1 complete gene recombinant...................................69

3-13 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of
the complete L1 gene of several human and non-human papillomaviruses........70

3-14 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of
the complete L2 gene of several human and non-human papillomaviruses........72

3-15 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of
the complete E6 gene of several human and non-human papillomaviruses ......74

3-16 Neighbor-Joining phylogenetic tree of the deduced amino acid sequences of
the complete E7 gene of several human and non-human papillomaviruses ........76

4-1 Papillomatous skin lesions on the flipper of a captive Florida manatee
(Trichechus manatus latirostris) housed at Homosassa Springs State Wildlife
Park, H om osassa, Florida .................................. ............... ............... 93

4-2 Typical papillomatous skin lesions of free-ranging Florida manatees ..............44















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Master of Science

DETECTION AND MOLECULAR CHARACTERIZATION OF MANATEE
PAPILLOMAVIRUS IN CUTANEOUS LESIONS OF THE FLORIDA MANATEE
(Trichechus manatus latirostris)

By

Rebecca Ann Woodruff

August 2005

Chair: Carlos H. Romero
Major Department: Veterinary Medicine

Papillomaviruses are widespread and successful pathogens associated with the

development of benign warts and malignant neoplasia in humans and domestic and wild

animals. Phylogenetic comparisons have classified human and non-human

papillomaviruses into 16 genera within the Papillomaviridae family. With the advent of

PCR and molecular cloning, significant advances have been made in understanding

human papillomaviruses; however, little is known about marine mammal

papillomaviruses. DNA extracted from skin lesions of captive and free-ranging manatees

was assayed by polymerase chain reaction (PCR) to detect papillomavirus infection.

Initially, primers were based on the widely used MY11 and MY09 human papillomavirus

(HPV) primer set, which target a highly conserved region of the L1 capsid protein. The

MY11 and MY09 primers directed the amplification of a 458-bp fragment from DNA

extracted from captive and free-ranging Florida manatee (Trichechus manatus latirostris)

skin lesions. Sequences of this fragment led to the development of AL1 PCR primers,









which specifically target the L1 capsid protein of Florida manatee papilloma virus

(TmlPV). The L1 and AL1 DNA fragment sequences were 100% identical, suggesting

that there is only one genotype of manatee PV present in captive and free-ranging

manatees around Homosassa Springs State Wildlife Park. PCR primers were also

designed to specifically target the complete TmlPV E6, E7, L1, and L2 genes.

Amplification of the TmlPV E6 gene product yielded a 589-bp fragment, which included

the complete TmlPV E6 gene, and amplification of the TmlPV E7 gene product yielded a

487-bp fragment, which included the complete TmlPV E7 gene. Targeting the TmlPV

L1 capsid protein gene yielded amplification of a 1,712-bp fragment, which included the

complete TmlPV L1 gene, and targeting the TmlPV L2 capsid protein yielded

amplification of a 1,660-bp fragment, which included the complete TmlPV L2 gene. The

complete TmlPV L1, E6, and E7 genes were successfully cloned into mammalian and

insect expression vectors, allowing for the future production of recombinant genes and

the development of serologic assays, such as ELISA. The described assays based on

PCR and direct sequencing of amplicons have allowed for the molecular detection of

TmlPV in captive and free-ranging Florida manatees and have allowed for the

phylogenetic comparisons of TmlPV to several human and non-human papillomaviruses.

The TmlPV is a unique papillomavirus based on phylogenetic analyses of the deduced

amino acid sequences of the L1, L2, E6, and E7 proteins, quite distinct from another

marine papillomavirus of Burmeister's porpoises, most closely related to HPVs that cause

common skin warts, and least related to high-risk HPVs involved in malignancy.














CHAPTER 1
INTRODUCTION

The papillomaviruses (PVs) are a group of small DNA viruses that cause benign

warts and malignant neoplasia in humans (Walboomers et al., 1999) and domestic and

wild animals (Sundberg, 1987). To date, more than a hundred human PV (HPV) types

have been partially identified and a wide variety of PV types have also been detected in

mammals and birds (de Villiers et al., 2004). Papillomavirus infection has also been

detected in genital and cutaneous lesions of several species of marine mammals (Bossart

et al., 2002; Kennedy-Stoskopf, 2001; Van Bressem et al., 1996) and studies have shown

that the healthy skin of humans and animal species can harbor sub-clinical PV infections

in the absence of overt lesions (Antonsson and Hansson, 2002, and Antonsson et al.,

2000).

Classification of Papillomaviruses

Papillomaviruses were originally grouped together with the polyomaviruses in the

family Papovaviridae based on their small size, non-enveloped capsids, and circular,

double-stranded genomes. However, a lack of overall homology of particle size, genome

organization, and sequence similarities between the two viral genomes led to the

recognition of two separate families, Polvomaviridae and Papillomaviridae (Howley and

Lowy, 2001). Viruses within the Papillomaviridae family are defined by genomic

properties, rather than serology, and are therefore described as PV genotypes, not

serotypes. Papillomavirus genotypes are classified by analysis of only part of the viral

genome that encompasses the combined nucleotide sequences of the E6, E7, and L1 open









reading frames (ORFs) (Chan et al., 1995). Recent phylogenetic comparisons of the L1

ORF nucleotide sequences of 96 HPVs and 22 animal PVs have further classified PV

types into genera and species. Higher-order phylogenetic clusters (major branches) of

PV types have now been classified into genera, sharing less than 60% identity in the L1

ORF nucleotide sequences. Each genus within the 16 genera identified is defined by its

biological properties and genome organization. Lower-order clusters (minor branches) of

PV types, or PV subtypes, have now been termed species. Species within each genus

share 60-70% identity at the nucleotide level (de Villiers et al., 2004). Nucleotide

sequence analyses of the L1 ORF of various HPV types have been used to construct

phylogenetic trees that display clusters of PV types with similar tissue tropisms and

oncogenic potentials (Chan et al., 1995).

Papillomavirus Genome Organization and Characterization

Papillomaviruses have double-stranded, circular DNA genomes of about 8,000 base

pairs (bp) in size. The papillomavirus particles (52 to 55 nm in diameter) contain the

viral genome within a spherical capsid composed of 72 capsomers. All ORFs are located

on one strand, indicating that transcription occurs on only one strand. Transcription is

regulated by the differentiation state of the infected cells and is complex, due to the

presence of multiple promoters, alternate and multiple splice patterns, and differential

production of mRNA in different cells (Howley and Lowy, 2001).

A PV genome usually contains seven major ORFs that code for five early (E)

proteins and two late (L) capsid proteins, plus an upstream regulatory region (URR), or

long coding region (LCR) (Tachezy et al., 2002). The early region of the PV genome

encodes the viral regulatory proteins El, E2, E4, E6, and E7, which are necessary for

initiating viral DNA replication. The late region encapsulates the genome and encodes









the L1 and L2 capsid proteins (Howley and Lowy, 2001). The LCR does not contain any

ORFs, but does contain the origin of viral DNA replication. Elements present in the LCR

regulate viral DNA replication and transcription (Desaintes and Demeret, 1996).

Unusual ORFs have been described in several types of PVs (Tachezy et al., 2002). The

genomes of European elk (Odocoileus alces) PV, white-tail deer (Odocoileus virginianus)

PV, and reindeer (Odocoileus hemionus) PV contain a transforming E9 gene (Eriksson et

al., 1994). The African grey parrot (Psittacus erithacus timneh) papillomavirus (PePV)

genome does not contain the typical E6 or E7 ORFs (Tachezy et al., 2002) and the

Burmeister's porpoise (Phoecena spinipinnis) papillomavirus (PsPV-1) genome also

lacks the E7 ORF (Van Bressem et al., 1996). Two ORFs with unknown functions, E3

and E8, may also be present in a PV genome (Lowy and Howley, 2001). An illustration

of a typical PV genome is shown in Figure 1-1.

The PV genome is contained within the capsid region which consists of the major

and minor structural proteins, L1 and L2, respectively. The L1 ORF is the most highly

conserved ORF within the papillomavirus genome (de Villiers et al., 2004) and represents

80% of the total viral protein (Howley and Lowy, 2001). The L1 protein can self-

assemble into virus-like particles (VLPs) or in combination with L2, although the L2

protein is not required (Hagensee et al., 1993). The L2 protein may enhance packaging

(Stauffer et al., 1998) and infectivity (Roden et al., 2001).

The early genes E6 and E7, and in some PV types, E5, contain oncogenic

properties that can modulate the transformation process (Baker and Howley, 1987). The

E6 protein of bovine papilloma virus type 1 (BPV-1) and of the oncogenic HPVs is

believed to function through binding cellular targets (Howley and Lowy, 2001), such as









binding and degrading the p53 tumor suppressor protein (Scheffner et al., 1993). The E7

protein of the oncogenic HPVs binds a number of important cellular regulatory proteins,

such as the retinoblastoma tumor suppressor protein (pRB) and the related pocket

proteins, p107 and p130 (Dyson et al., 1989). The papillomaviruses may activate cellular

genes necessary for the replication of their own DNA through the binding of the viral E6

and E7 proteins to cellular factors (Howley and Lowy, 2001).

The early genes El and E2 are regulatory proteins that modulate transcription and

replication (Baker and Howley, 1987). The El and E2 proteins are essential for viral

DNA replication (Chiang et al., 1992). The El protein has been shown to bind a number

of cellular proteins, including the cellular DNA polymerase a-primase, thus recruiting the

cellular DNA replication initiation machinery to the viral origin of replication (Park et al.,

1994). The multifunctional E2 protein can activate or repress viral promoters, has critical

roles in viral DNA replication (Ham et al., 1991), and targets the El protein to the

replication origin (Sedman and Stenlund, 1995). Although the E4 ORF is located in the

early region of the PV genome, it is expressed as a late gene (Howley and Lowy, 2001).

The precise role of the E4 protein is unclear, but studies have shown E4 expression

coincides with the onset of viral genome amplification (Peh et al., 2002).

Papillomavirus Replication

Key life cycle events seem to be similarly regulated in both human and non-human

PVs (Peh et al., 2002). The PV life cycle is strictly dependent on differentiation of the

epithelial tissue (Barksdale and Baker, 1993) and PV replication can be divided into three

stages (McBride et al., 2000). First, the PV virion must bind to a basal keratinocyte,

although studies have shown that the PV virions can bind a wide variety of cell types

(Muller et al., 1995). During this stage, the viral genome is maintained as an episome









within the nucleus (McBride et al., 2000). The viral genome is then amplified and the

viral copy number is increased up to 1,000 per haploid cell genome (Lepik et al., 1998).

As the basal cells differentiate, the viral DNA is maintained as a stable plasmid (Howley

and Lowy, 2001). During this second, maintenance stage, the viral genome replicates in

synchrony with the host cell chromosome (Gilbert and Cohen, 1987). The PVs rely on

cellular replication factors and enzymes (Muller et al., 1994), such as replication protein

A (RPA) (Mannik et al., 2002), in order to replicate their genomes from a single origin of

replication (Melendy et al., 1995). The earliest PV DNA synthesis is within the fragment

containing the PV replication origin and synthesis proceeds in both directions from the

replication origin (Melendy et al., 1995). The third replication stage takes place in the

terminally differentiated epithelial cells of the papilloma (Howley and Lowy, 2001). In

this next layer of stratified epithelium, the stratum granulosum, late viral gene expression,

synthesis of capsid proteins, vegetative viral DNA synthesis, and assembly of virions

occur (McBride et al., 2000). The PV DNA is thought to remain in the basal epithelial

cells and to be reactivated when levels of immune system monitoring decline (Doorbar, J.

2005).

Papillomavirus Transmission

Papillomavirus enters infected skin via skin surface abrasions, allowing the virus

access to the proliferative cells of the skin (Egawa, 2003), where it promotes cellular

proliferation and accelerated epithelial growth (Silverberg, 2004). Infection with HPV is

primarily found on the extremities, face, and body, and moist skin is more likely to allow

viral transmission (Silverberg, 2004). Transmission of high-risk mucosal HPVs occurs

predominately through sexual contact, but horizontal and vertical routes have also been

identified. Vertical transmission of mucosal HPVs may be acquired from the mother in









utero, across the placenta, intrapartum, during birth through an infected birth canal, or

post partum (Cason and Mant, 2005). In 1989, Sedlacek et al. described the first

confirmed vertical transmission of HPV infection in nasopharyngeal samples of infants

born to HPV positive mothers.

Papillomavirus Pathogenesis

Papillomas (warts) are induced in the skin and mucosal epithelia at specific sites

(de Villiers et al., 2004) and differ in their tissue specificity and the associated disease

(McMurray et al., 2001). The highly tissue-specific papillomaviruses can be divided into

two groups: one group is primarily found in cutaneous epithelia (skin), in which there is

thickening of the epidermis, and the other group is predominantly present in mucosal

epithelia (Smits et al., 1992), involving the oral pharynx, esophagus, or genital tract

(Howley and Lowy, 2001). Warts are diagnosed by physical examination and are defined

by morphology, location, and host immune response. The three main types of lesions

observed are: common warts (rough plaques of skin), mosaic warts (groups of common

warts), and flat warts (smooth, flesh-colored papules). Mucosal warts may appear as

single plaques or as a group with a grapelike appearance (Silverberg, 2004).

The genital mucosal HPV types are defined as either high-risk or low-risk based on

their involvement with lower genital tract cancers (McMurray et al., 2001). In the low-

risk types, such as HPV-6 or HPV-11, benign warts proliferate. In the high-risk types,

such as HPV-16 or HPV-18, the virus deregulates checkpoints that normally monitor the

fidelity of chromosome replication and segregation, leading to the development of cancer

(Galloway, 2003). Anogenital carcinomas caused by HPV infection include penile

(Rubin et al., 2001), vaginal (Daling et al., 2002), vulvar (Trimble et al., 1996), and anal

cancers (Krzyzek et al., 1980). The high-risk HPV types are also associated with cervical









dysplasia (Kurman et al., 1982), uterine cancer (Parkin et al., 1999, and zur Hausen,

1996), and cervical cancer, one of the most common cancers of women world-wide

(Schiffman, 1992, and zur Hausen, 1991). The E6 and E7 genes of the high-risk types

are expressed in cervical cancer (Schwarz et al., 1985). The roles of E6 and E7 of the

low-risk types are unclear, but studies suggest that they may act in a similar manner as

observed in the high-risk E6 and E7 proteins (Oh et al., 2004). Risk factors associated

with cervical cancer and other anogenital tumors include cigarette smoking, sexual

factors, and, possibly, genetic susceptibility (Daling et al., 2002).

Both the low-risk and high-risk HPV types cause low-grade squamous

intraepithelial lesions (LSIL) of the cervix, but the high-risk HPV types cause high-grade

SIL (HSIL), carcinoma in situ, or invasive cancer. The steps leading to cervical

carcinogenesis include infection with an oncogenic (high-risk) HPV, development of

HSIL, progression of HSIL to carcinoma in situ, and, then, invasive cancer (Baseman and

Koutsky, 2005). Frequent integration of the HPV genome into the host genome usually

occurs in the case of high-risk PVs (Mannik et al., 2002).

At least 25 types of HPVs have also been detected in oral lesions on the lips, hard

palate, and gingiva (Syrjanen, 2003). The high risk HPV types 16 and 18 are highly

associated with oropharyngeal and laryngeal squamous cell carcinomas (Kreimer et al.,

2005) while the low risk HPV types 6 and 11 are predominant in benign lesions of the

oral mucosa (Syrjanen, 2003). Infection with HPV is a significant independent risk

factor for oral squamous cell carcinoma (Miller and Johnstone, 2001).

The pathogenesis of HPVs differs for viruses that are considered high risk or low

risk group members. In the case of epidermodysplasia verruciformis (EV), the disease









behaves like a genetic cancer of viral origin, which may result from an abnormal

recessive gene (Jablonska, 1991). Clinical forms of EV may be benign or malignant.

The benign form is associated with HPV-3 and/or HPV-10 and induces flat, wart-like

lesions over the trunk and limbs. The malignant form is associated with EV-HPV and

induces reddish, polymorphous lesions, flat wart-like lesions, and pre-malignant lesions

disseminated over the body. Epidermodysplasia verruciformis is the first human genetic

condition in which cutaneous cancer is associated with HPV infection (Majewski and

Jablonska, 1995).

There is an increased risk of developing cutaneous HPV-associated disease if the

virus is not cleared from the skin (Silverberg, 2004). Cutaneous HPV types, such as

HPV-5 and HPV-8, may contribute to skin cancer development in immunosuppressed

individuals (Stockfleth et al., 2004). In renal transplant studies, 90% of recipients

developed HPV-induced warts (Blessing et al., 1989) and up to 40% of recipients

developed nonmelanoma skin cancer within 15 years after transplantation (Birkeland et

al., 1995). HPV-5 and HPV-8 were also predominately detected in squamous cell

carcinoma of patients diagnosed with EV (Orth, 1987). Immunosuppression may

increase the activity of HPV, which may lead to the development of cancer (Stockfleth et

al., 2004).

Diagnosis of Papillomavirus Infection

Testing for the presence of HPV viral DNA includes methods such as Southern

blots, dot blots, in situ hybridization, polymerase chain reaction (PCR), and solution

hybridization (hybrid capture assay) (Trofatter, 1997). Detection of HPV by PCR is

more sensitive than the other methods, enables the detection of a single genome copy per

cell for HPV DNA that has integrated (Shamanin et al., 1994), and allows for the









detection of a broad spectrum of genital HPVs (Ting and Manos, 1990). The widely used

consensus PCR primers, MY09 and MY11, are based on sequences obtained from the

highly conserved PV L1 capsid protein gene (Manos et al., 1989). The primers were

designed from homologous regions 20 to 25 base pairs (bp) in length that were identified

in genital HPV types 6, 11, 16, 18, and 33 (Ting and Manos, 1990). These primers are

known to amplify a 458 base pair (bp) fragment when used with DNA from most types of

genital HPVs (Bernard et al., 1994). Human papillomavirus DNA can be detected by

method of PCR in fresh or frozen cervical biopsies (Li et al., 1988; Manos et al., 1989),

condylomata acuminata (genital warts) tissues (Brown et al., 1999), cutaneous wart

tissues (Harwood et al., 1999), and in swab samples taken from the top of lesions

(Forslund et al., 2004). Harwood et al. (1999) have described a degenerate nested PCR

that is capable of detecting cutaneous, mucosal, and EV HPV types. A new method of

HPV detection using high-density DNA microarrays is able to detect single and multiple

mucosal HPV infections (Klaassen et al., 2003).

Treatment and Prevention of Papillomavirus Infection

The host response to HPV infection is a complex process of skin barrier protection,

innate immunity, and acquired immunity (Silverberg, 2004). Warts generally will regress

over time and after six months of infection, 30 percent of warts will clear on their own

(Messing and Epstein, 1963). Following immune regression, PV DNA persists in a

latent state, with only a few cells, if any, capable of supporting the productive cycle that

occurs during epithelial cell differentiation (Doorbar, 2005). A possible approach to

controlling the level of HPV-associated disease is to prevent HPV infection (McMurray

et al., 2001); however, the PV life cycle requires a differentiated stratified epithelium to

replicate and this has been difficult to generate in cell culture (McBride et al., 2000).









Due to the fact that PVs could not previously be propagated in cell cultures, the

development of a capsid-directed vaccine was hindered for a long time (Biemelt et al.,

2003). A recently described raft system now allows for the genetic analysis of the

complete viral life cycle of BPV-1. Using a combination of organotypic raft cultures and

xenografts on nude mice, BPV-1 DNA can be amplified and capsid antigens and

infectious BPV-1 virus particles can be produced (McBride et al., 2000).

Several animal models of PV infection have shown that neutralizing antibodies can

block new infection (Galloway, 2003). Vaccination against PV infections using virus-

like particles (VLPs) based on the L1 capsid protein or the L1 plus the L2 protein is

currently being developed (Leder et al., 2001). Vaccines based on VLPs are desirable

because they retain repetitive, highly immunogenic epitopes found on the surface of

infectious virions, but lack the potentially harmful PV genomes. Three types of HPV

VLP-based vaccines are currently being developed. The first, most basic type is designed

to prevent genital HPVs by inducing virus-neutralizing antibodies against the L1 major

capsid protein. The second type of vaccine is based on chimeric VLPs which incorporate

polypeptides of other viral and cellular proteins into the VLPs. These vaccines induce

cell-mediated responses to nonstructural viral proteins, such as the HPV E7 protein. The

third type of vaccine incorporates self-peptides into the outer surface of the VLPs and is

designed to induce antibodies against central self-antigens (Schiller and Lowy, 2000).

Vaccination with HPV VLPs has been well tolerated, induces high titers of antibodies,

and shows evidence of T-cell responses (Galloway, 2003).

Human Papillomaviruses

Papillomaviruses are one of the most important viruses associated with benign and

malignant neoplasia in humans (Chan et al., 1995). Papillomaviruses were first isolated









almost 30 years ago (Orth et al., 1977) and the first HPVs fully sequenced were HPV-1

(Danos et al., 1982), HPV-6 (Schwarz et al., 1983), and HPV-16 (Seedorfet al., 1985).

To date, more than 100 types of human papilloma viruses (HPVs) have been identified,

of which 96 have been cloned and characterized (deVilliers et al., 2004). More than 50

types of HPVs have been found to infect the genital tract (Galloway, 2003). Low-risk

(benign) genital HPVs include: types 6, 11, 40, 42-45, 53-55, 57, 67, 69, 71, and 74.

High-risk (oncogenic) genital HPVs include: types 16, 18, 31-35, 51, 52, 56, 58, 66, 68,

70, and 73 (deVilliers et al., 2004 and McMurray et al., 2001), with types 26, 53, and 66

being probably oncogenic (Munoz et al., 2003). The HPV types 59, 61, and 82 may be

associated with benign or malignant lesions (deVilliers et al., 2004). Infection with a

single HPV type or infection with multiple HPV types can occur (Munoz et al., 2003).

Non-Human Papillomaviruses

Warts in animals have been recognized for centuries. Equine papillomas were

described as early as in the 9th century A. D. and the first experimental transmission of

animal papillomas occurred in 1898 (Lancaster and Olson, 1982). Warts in wild

cottontail rabbits were the first animal papillomas thoroughly examined for properties of

transmissibility, etiology, and histology. The activities and characteristics of the

papilloma-producing agent in cottontail rabbits classified it as a virus (Shope, 1933).

Additional non-human PVs initially characterized include: BPV (Lancaster and Olson,

1978), equine PV (Fulton et al., 1970), canine oral PV (Chambers and Evans, 1959), deer

fibromavirus (Shope et al., 1958), and chaffinch PV (Lina et al., 1973). Presently, 22

animal PVs have been fully characterized and classified into genera and species based on

the L1 ORF sequences (de Villiers et al., 2004). As many as 53 putative new animal PV

types have been identified by polymerase chain reaction (PCR) in 7 animal species,









including chimpanzees, gorillas, spider monkeys, long-tailed macaques, domestic cattle,

aurochs, and European elk (Antonsson and Hansson, 2002).

Animal papillomas can be divided into four groups based on tissue tropism and

histology of lesions. These groups comprise animal PVs that can induce neoplasia of

cutaneous stratified epithelium, fibromas with a minimally hyperplastic cutaneous

epithelium, cutaneous papillomas and fibropapillomas (an underlying fibroma of

connective tissue), and hyperplasia of either normal non-stratified squamous epithelium

or metaplastic squamous epithelium. Infection with non-human PVs is generally

contained to the skin or mucous membranes of the host species (Lancaster and Olson,

1982); however, canine oral PV can also infect the eyelid, conjunctival epithelium, and

skin around the nose and mouth (Chambers and Evans, 1959). Some animal PV types,

such as BPV, cottontail rabbit PV, and European harvest mice PV, have been implicated

in cancers (Antonsson and Hansson, 2002), with BPV being the most oncogenic of the

PVs (Lancaster et al., 1977). The E5 protein of BPV type 1 (BPV-1) transforms cells and

functions by altering the activity of cellular membrane proteins that are involved in

proliferation (DiMaio et al., 1986).

Most PVs are species-specific or may infect closely related animals within the

same genus (Sundberg et al., 2000), although BPV-1 and BPV-2 can induce fibroblastic

tumors in a strain of inbred mice (Boiron et al., 1964), hamsters (Cheville, 1966), and

rabbits (Breitburd et al., 1981). The presence ofBPV-specific DNA has also been

detected in both naturally occurring tumors and BPV-induced tumors of horses

(Lancaster et al., 1977). An oral PV that infected one coyote and three dogs has also

been described (Sundberg et al., 1991). Studies have shown that many domestic and









wild species of mammals and birds can be infected by one or more PVs (Sundberg et al.,

2000).

Marine Mammal Papillomaviruses

Viruses and viral diseases have long been identified in marine mammals (Smith

and Skilling, 1979) and new PVs in marine mammals have recently been described (Van

Bressem et al., 1996, and Bossart et al., 2002). Papillomavirus-like particles have been

observed in association with genital lesions of male sperm whales (Physeter catodon)

(Lambertsen et al., 1987), dusky dolphins (Lagenorhynchus obscurus), and Burmeister's

porpoises (Phocoena spinipinnis) (Van Bressem et al., 1996). The high prevalence of

papillomatous lesions in several small cetaceans (L. obscurus, P. spinipinnis, Delphinus

capensis, Tursiops truncatus) indicates a possible venereal transmission of the disease

(Van Bressem et al., 1996). Squamous papillomas and fibropapillomas have also been

identified on the skin, the surface of the penis, and the tongue of mysticetes and

odontocetes (Geraci et al., 1987) and gastric papillomas containing PV-like particles have

been observed in a significant amount of beluga (Delphinapterus leucas) inhabiting the

St. Lawrence River (Martineau et al., 2002).

The Florida Manatee (Trichechus manatus latirostris)

The Florida manatee is a marine mammal that is primarily found in the

southeastern United States waters and the Gulf of Mexico and is listed as endangered at

both the state and federal levels (U. S. Fish and Wildlife Service, 2001). The manatee

immune system appears to be highly developed and it has been hypothesized that natural

disease in manatees is uncommon (Bossart et al., 2002). Environmental diseases that

may represent emerging problems for the Florida manatee include brevetoxicosis and

cold stress syndrome (Bossart, 2001). Recently, it has been shown that exposure to









multiple stressors, such as cold weather and harmful algal blooms (Karenia brevis), may

have synergistic effects on the immune function of manatees (Walsh et al., 2005).

Papillomas in Florida manatees were initially identified in 1997 in a captive

population maintained at Homosassa Springs State Wildlife Park (HSSWP), Homosassa,

Florida. These seven captive manatees developed multiple, cutaneous, pedunculated

papillomas located on the pectoral flippers, upper lips, external nares, and periorbital

regions. Approximately three years later, four of the manatees developed papillomas that

were clinically distinct from the previously observed lesions. These lesions were sessile,

firm, and more diffuse and numerous than those biopsied in 1997. Based on histological,

ultrastructural, and immunohistochemical findings, PV was considered to be a theoretical

causative agent in both outbreaks. Electron microscopy evaluation of the lesions

revealed the presence of round to hexagonal 45- to 50-nm virions that were

ultrastructurally identical to those of known PVs. Positive immunohistochemical staining

was demonstrated with polyclonal antibodies against BPV-1. This was the first viral

infection described in Florida manatees (Bossart et al., 2002). Similar papillomatous skin

lesions have since been observed in free-ranging Florida manatees inhabiting two

locations in Florida waters (Woodruff et al., in press).

The first molecular detection of PV infection in Florida manatees was performed

by amplifying PV DNA from papillomatous lesions of captive and free-ranging manatees

using the degenerative HPV primers MY09 and MY11. These primers amplified a 458-

bp DNA fragment of the highly conserved L1 capsid protein gene of manatee

papillomavirus (TmlPV) (Woodruff et al., in press). Recently, the entire TmlPV genome

has been completely sequenced and characterized. The complete TmlPV genome









(TmPV-1) contains 7,722 bp and consists of seven major ORFs that encode five early

proteins and two late capsid proteins (Rector et al., 2004). Sequences of nine L1

fragments previously described by Woodruff et al. (2005) were 100% identical to the

corresponding L1 region of the published manatee papillomavirus, TmPV-1, suggesting

that there is only one type of manatee PV that causes skin lesions in manatees (Rector et

al., 2004). Although the existence of a manatee papillomavirus has been well

documented, further work is required in order to better understand the overall impact and

possible oncogenic potential of papillomavirus infection in the already endangered

Florida manatee. The goals of this study were to determine if more than one TmlPV is

associated with papillomatous lesions in captive and free-ranging manatees, to

genetically characterize the TmlPV genome(s), and to develop serological assays with the

potentially oncogenic E6 and E7 proteins and with the L1 capsid protein.

LCR

.9 5 E6 7




.00oo HPV 16 2.oo El



4.00



E5
Figure 1-1. Illustration demonstrating the genetic organization of a typical
papillomavirus genome. The HPV-16 genome is divided into early (E) and
late (L) regions depending on the timing of protein expression.














CHAPTER 2
MATERIALS AND METHODS

Clinical Data

Nine adult female Florida manatees (Trichechus manatus latirostris) comprised the

captive population dwelling at Homosassa Springs State Wildlife Park (HSSWP) located

in Homosassa, Florida. Manatees at HSSWP were housed in Homosassa Springs, a

natural freshwater spring at the headwaters of the Homosassa River, Citrus County,

Florida. An underwater fence placed at the junction of the spring and the river confined

the manatees to an area of approximately 2 acres (Bossart et al., 2002). Free-ranging

manatees were occasionally observed at the outer perimeter of the underwater fence. The

free-ranging manatees found in this area are winter residents that use the springs for

thermoregulation (Woodruff et al., in press).

Source of Samples

Between January, 2003, and February, 2005, skin lesions from captive and free-

ranging manatees were brought to our laboratory fresh, on ice, or fixed in either 10%

non-buffered formalin (NBF) or Dimethyl Sulfoxide (DMSO). Papillomatous skin

lesions from captive Florida manatees were received from various parks, including

HSSWP, Florida, and other marine parks in Florida and California. Papillomatous skin

lesions were also obtained from free-ranging Florida manatees inhabiting several bodies

of Florida waters, including Crystal River, Homosassa River, Port of Isles, and Tampa

Bay, and from free-ranging Antillean manatees (Trichechus manatus manatus) inhabiting

the offshore waters of the Drowned Keys, Belize. One papillomatous penile lesion









preserved in 10% NBF was obtained from a free-ranging manatee carcass examined at

the Fish and Wildlife Research Institute, St. Petersburg, Florida, and an uninfected,

normal manatee liver was obtained from the Marine Mammal Pathology Laboratory, St.

Petersburg, Florida. Samples biopsied from HSSWP captive manatees between July,

1998, and January, 2000, were preserved in 10% NBF or DMSO and samples biopsied

from free-ranging manatees in April, 2004, and August, 2004, were preserved in 10%

NBF. All other manatee tissue samples (captive and free-ranging) arrived fresh or on ice.

Sample description and information are located in Table 2-1 and Table 2-2.

Blood serum from captive manatees housed at HSSWP was brought to our

laboratory on dry ice, courtesy of Bob Bonde, U. S. Geological Survey, Gainesville,

Florida. Serum samples were obtained from the following captive manatees (date

obtained): Holly (July 9, 1998), Betsy (February, 1999), Oakley (Feb 25, 1999; June 20,

2002), Willoughby (January 13, 2000), Amanda (Jan 13, 2000), and Lorelei (Jun 20,

2002). Purified anti-manatee IgG monoclonal antibody (1.2 mg/ml) was obtained

courtesy of Dr. Peter McGuire, Department of Biochemistry, University of Florida, and

Ms. Linda Green, Hybridoma Core Laboratory, University of Florida, Gainesville,

Florida.

DNA Extraction from Papillomatous Lesions

Total DNA was extracted from all tissue samples using the DNeasy tissue kit

(Qiagen Inc, Valencia, California, USA) according to the protocol recommended by the

manufacturer. Working in a laminar flow cabinet equipped with an HEPA filter,

approximately 25 mg of each papillomatous skin lesion was minced with a sterile

surgical blade and placed and ground in a sterile 1.5-ml micro centrifuge tube. The

tissues were incubated overnight at 550C in a mixture containing 1801l of digestion









buffer ATL and 20 [tl proteinase K (20 mg/ml) until lysis was complete. Then, 200 ptl of

buffer AL and 200 [il of 100% molecular grade ethanol were added to precipitate the

DNA. The solution was centrifuged in a DNeasy Spin Column to bind the DNA to the

membrane and the membrane was washed with 500 ptl of buffers AW1 and AW2 for 1

minute each time. A final centrifugation step was performed to eliminate residual ethanol

remaining in the membrane. The DNA was eluted in 200 ptl of buffer AE and evaluated

for yield and purity by spectrophotometry using the Ultrospec 3000 (Amersham

Biosciences Corp., Piscataway, New Jersey, USA). A negative tissue sample from

uninfected manatee skin or uninfected manatee liver was extracted along with each set of

tissue samples for use as a negative control in PCR analyses. The eluted DNA samples

were stored in 1.5-ml screw-cap tubes at -800C.

General Methods of PCR

Taq Polymerase Reactions

The PCR reaction in a 0.2 ml tube contained: 200 nM of each primer (IDT,

Coralville, IA, USA), 2 mM MgSO4, 100 [IM of each deoxynucleoside triphosphate

(dNTP), 20 mM Tris-HCl (pH 8.4), 10 mM KC1, 0.1 % Triton X-100 (pH 8.8), 10 mM

(NH4)2S04, 1 unit of Taq DNA polymerase (New England BioLabs, Beverly,

Massachusetts, USA), 0.5-1.0 tg of template DNA, and ultrapure H20 in a final volume

of 50 l. A total of 40 PCR cycles were performed in a PTC-100 thermal cycler (MJ

Research, Inc., Waltham, Massachusetts, USA) for the amplification of the manatee

papillomavirus (TmlPV) L1 fragments using the modified MY11/MY09 primers

(CR333/CR332) and the AL1 TmlPV-specific primers (CR490/CR491). Following an

initial denaturation step at 940C for 1 min, reactions were subjected to 39 cycles of: a

denaturation step at 940C for 1 min, an annealing step at 480C for 1 min, and an









elongation step at 720C for 2 min. An elongation step at 720C for 10 min was

incorporated in the final cycle.

AccuprimeTM Taq DNA Polymerase Reactions

The PCR reaction in a 0.2 ml tube contained: 400 nM of each primer, 200 mM

Tris-HCl (pH 8.0), 500 mM KC1, 15 mM MgCl2, 2 mM of each dNTP, 2 units of

Accuprime Taq DNA polymerase (Invitrogen, Carlsbad, California, USA), 0.2-0.5 pg of

template DNA, and ultrapure H20 in a final volume of 50 il. Cycling conditions for the

amplification of the complete TmlPV E6 gene fragment included: an initial denaturation

step at 940C for 2 min, then, 39 cycles of: a denaturation step at 940C for 30 sec, an

annealing step at 530C for 30 sec, and an extension step at 680C for 1 min. Cycling

conditions for amplification of the L1 fragments and the complete E7 complete gene

were similar, except that the annealing temperatures were set at 590C and 510C,

respectively.

Expand High Fidelity Plus Reactions

The PCR reaction in a 0.2 ml tube contained: 400 nM of each primer, 200 iM of

each dNTP, 1.5 mM MgCl2, Expand High Fidelity Plus Reaction Buffer diluted to 1.5

mM MgCl2 (Roche Applied Science, Mannheim, Germany), 2.5 units of Expand High

Fidelity Plus Enzyme Blend (Roche Applied Science), 0.5 pg of template DNA, and

ultrapure H20 in a final volume of 50 [il. Cycling conditions for amplification of the

TmlPV L1-El region were: an initial denaturation at 940C for 2 min, followed by 39

cycles of 940C for 30 sec, 500C for 30 sec, and 720C for 3 min, with a final extension

step at 720C for 7 minutes. Cycling conditions for amplification of the TmlPV L1-L2

capsid region were similar, except that the annealing temperature was set at 590C and the

extension step was performed at 680C for 3.5 min. Cycling conditions for amplification









of the TmlPV complete L1 capsid gene were similar, but the extension steps were

performed at 720C for 1.5 min. For amplification of the TmlPV complete L2 capsid

gene, cycling conditions were similar and the annealing temperature was set at 530C and

the extension step performed at 720C for 2 min.

PCR Targeting the L1 458-bp Fragment

The MY11 and MY09 L1 consensus primers that amplify a 458-bp fragment of the

L1 capsid protein gene of several human papillomaviruses (HPVs) (Manos et al., 1989)

were modified in our laboratory to contain deoxyinosines at positions of nucleotide

degeneracy [forward primer (FP) CR333 and reverse primer (RP) CR332] (Table 2-3).

DNA obtained from a lesion from captive manatee Oakley was used as the positive

control. A blank sample (water) and DNA extracted from a liver of an uninfected

manatee were used as negative controls.

PCR Targeting the AL1 TmlPV 458-bp Fragment

Based on newly generated nucleotide sequences of the amplified TmlPV L1

fragment obtained in our laboratory, the MY11/MY09 HPV L1 primers (Manos et al.,

1989) were modified to contain TmlPV L1-specific nucleotides at positions of nucleotide

degeneracy. The AL1TmlPV primers, FP CR490 and RP CR491 (Table 2-3), target a

458-bp sequence contained within the L1 capsid gene of TmlPV. DNA obtained from a

lesion from captive manatee Oakley was used as the positive control. A blank sample

(water) and DNA from a negative tissue (manatee liver) were used as negative controls.

PCR Targeting the TmlPV L1-E1 Region

Using a PCR method described by Forslund and Hansson (1996), oligonucleotide

primers were designed to target a region of the TmlPV genome that spans an area from

within the 3' end of the TmlPV L1 ORF to an area within the 5' end of the TmlPV El









ORF. A plus-strand oligonucleotide primer designed from the TmlPV L1 458-bp

fragment nucleotide sequence was used in a PCR reaction together with a minus-strand

HPV consensus primer from the El ORF (Smits et al., 1992). The TmlPV L1 FP CR442

and the HPV El RP CR441 (Table 2-3) direct the amplification of an approximately

3,000-bp fragment. DNA obtained from a lesion from captive manatee Oakley was used

as the positive control. A blank sample (water) and DNA from a negative tissue

(manatee liver) were used as negative controls.

PCR Targeting the Complete TmlPV E6 Gene

Based on the nucleotide sequences of the TmlPV L1-El region obtained by us,

oligonucleotide primers FP CR498 and RP CR499 (Table 2-3) targeting the complete

TmlPV E6 gene were designed for cloning and expression. DNA obtained from a lesion

from captive manatee Oakley was used as the positive control. A blank sample (water)

and DNA from a negative tissue (manatee liver) were used as negative controls.

PCR Targeting the Complete TmlPV E7 Gene

Based on the nucleotide sequences of the TmlPV L1-El region obtained by us,

oligonucleotide primers FP CR500 and RP CR501 (Table 2-3) targeting the complete

TmlPV E7 gene were designed for cloning and expression. DNA obtained from a lesion

from captive manatee Oakley was used as the positive control. A blank sample (water)

and DNA from a negative tissue (manatee liver) were used as negative controls.

PCR Targeting the TmlPV L1-L2 Region

Based on nucleotide sequences of the complete TmlPV genome (AY609301)

(Rector et al., 2004), a plus strand oligonucleotide primer FP CR572 (Table 2-3) was

designed from an area upstream of the L2 ORF. Used together with an L1 minus strand

primer RP CR574 (Table 2-3), designed by us, these primers target a region of the









TmlPV genome that spans an area upstream of the 5'end of the TmlPV L2 ORF to an

area downstream of the 3'end of the TmlPV L1 ORF. DNA obtained from a lesion from

captive manatee Oakley was used as the positive control. A blank sample (water) and

DNA from a negative tissue (manatee liver) were used as negative controls.

PCR Targeting the Complete TmlPV L1 Gene

Based on nucleotide sequences of the L1-L2 fragment obtained in our laboratory, a

plus-strand oligonucleotide primer FP CR573 (Table 2-3) was designed upstream of the

L1 start codon to be used together with the L1 FP CR574 for amplification of the

complete L1 gene for cloning and expression. DNA obtained from a lesion from captive

manatee Oakley was used as the positive control. A blank sample (water) and DNA from

a negative tissue (manatee liver) were used as negative controls.

PCR Targeting the Complete TmlPV L2 Gene

Based on nucleotide sequences of the L1-L2 fragment obtained in our laboratory, a

minus-strand oligonucleotide primer RP CR622 (Table 2-3) was designed downstream of

the L2 stop codon to be used together with the L2 FP CR572 for amplification of the

complete L2 gene. DNA obtained from a lesion from captive manatee Oakley was used

as the positive control. A blank sample (water) and DNA from a negative tissue

(manatee liver) were used as negative controls. A linear representation of the circular

TmlPV genome with the relative positions of the PCR primers used in this study is shown

in Figure 2-1.

Gel Electrophoresis

Between 20-30 [tl of PCR products were resolved by horizontal electrophoresis in

1.0% agarose gels containing ethidium bromide (0.5 [g/ml). Amplified DNA fragments









were visualized under ultraviolet light and photographed using a gel documentation

system (Bio-Rad Laboratories, Inc., Hercules, California, USA).

General Cloning of PCR Products

As described under gel electrophoresis, PCR products containing amplified

fragments of only the expected size were purified for use in cloning reactions. PCR

products containing additional amplified fragments were resolved in 1.2% low-melting

point (LMP) agarose and the band of the appropriate size was excised and purified for

use in cloning reactions. PCR products and excised gel pieces were purified using the

Wizard SV Gel and PCR Clean-Up System (Promega Corporation, Madison, Wisconsin,

USA) according to the protocol provided by the manufacturer. Briefly, an equal volume

of membrane binding solution was added to the PCR product, the prepared PCR product

was added to the SV minicolumn assembly, and the minicolumn was washed twice with

the membrane wash solution. The purified DNA was eluted in 50 ptl of nuclease-free

water and stored at -800C until further use in cloning reactions.

Cloning into pCR 2.1 TOPO T/A Vector

Purified PCR products were cloned into the pCR 2.1 TOPO vector (Invitrogen

Life Technologies, Carlsbad, California, USA) for sequencing analysis. In a 0.2 ml tube,

ligation reactions contained: 1 itl of salt solution (1.2 M NaC1, 0.06 M MgC12), 1 Ol of the

TOPO T/A vector (Invitrogen), 50 ng of purified PCR or gel product, and ultrapure H20

in a final volume of 6 l. Reactions were incubated at room temperature for 1 hour; then,

3 l of the reaction were added to one vial (50tl) of DH5a or TOP-10 chemically

competent Escherichia coli cells (Invitrogen). Tubes were placed on ice for 1 hour, heat

shocked for 30 sec at 420C in a water bath, and returned to ice. After adding 250 pl of

S.O.C. medium (Invitrogen), the tubes were shaken horizontally at 220 rpm at 370C for 1









hour. Reactions (100-200 .il) were spread onto bacterial agar plates containing ampicillin

(100 pg/ml) (Roche Applied Science) and blue/white selection medium [75 pl 2XYT:

16g Bacto-tryptone, 10 g Bacto-Yeast Extract, 15 g Bacto-Agar (Beckton Dickinson,

Franklin Lakes, New Jersey, USA), 5g enzyme grade NaCl (Fisher Scientific

International Inc., Hampton, New Hampshire, USA) in 1 L of ultrapure H20, 20 pl IPTG

(100mg/ml) (Invitrogen), and 5 .l UltraPure Bluo-gal (100mg/ml) (Invitrogen)] spread

on the agar plates surface one hour before the plates were inoculated with cloning

reactions. Plates were incubated overnight at 370C.

Cloning into P-TargetTM Mammalian Expression Vector

Purified PCR products were directly cloned into the P-TargetTM mammalian

expression vector (Promega) to test for protein expression after transfection and

immunofluorescence. In a 0.5 ml tube, the ligation reaction contained: 1 p1 of T4 DNA

ligase constituents, 1 il of T4 DNA ligase, 1 il of pTargetTM cloning vector (ProMega),

51p of purified PCR product, and ultrapure H20 in a final volume of 10 il. The ligation

reaction was incubated at 40C overnight in a refrigerated block. Five il of the ligation

reaction were added to one vial (501l) of JM109 high efficiency competent cells

(Promega) and incubated on ice for 20 min. The cells were heat shocked for 45 sec at

420C in a water bath and returned to ice for 2 min. After adding 450p1 of S.O.C.

medium, the tubes were shaken at 150 rpm at 370C for 1.5 hrs. The transformation

reaction was spread in 100p1 volumes onto bacterial agar plates containing ampicillin

(100 lg/ml) and blue/white selection medium.

Cloning into pcDNA TM3.1 Directional TOPO Expression Vector

In order to pair with the -GTGG- overhang of the pcDNATM 3.1 Directional

TOPO Expression Vector, the TmlPV E7 FP CR 500 was modified at the 5'end to









contain a corresponding 4-bp CACC sequence. To amplify the complete TmlPV E7 gene

with the TOPO overhang, the modified E7 FP CR 530 (Table 2-3) was used in a PCR

reaction with the TmlPV E7 RP CR501. The PCR reaction in a 0.2 ml tube contained:

200 nM of each primer, 300 mM Tris-SO4 (pH 9.1), 90 mM (NH4)2SO4, 10 mM MgSO4,

200KM of each dNTP, 2 units of Elongase enzyme mix (Invitrogen), 0.5-1.0 [g of

template DNA, and ultrapure H20 to a final volume of 50 [il. Cycling conditions for the

amplification of the E7 TOPO PCR products were: an initial denaturation step at 940C

for 2 min, then 39 cycles of a denaturation step at 940C for 30 sec, an annealing step at

510C for 1 min, and an extension step at 680C for 1 min. Purified E7 TOPO PCR

products were directly cloned into the pcDNATM 3.1 Directional TOPO expression

vector for use in immunofluorescence assays. In a 0.2 ml tube, the ligation reaction

contained: 1 ll of salt solution (50 mM NaC1, 2.5 mM MgC12), 1 l1 of pcDNATM 3.1

TOPO vector (Invitrogen), 50 ng of purified E7 TOPO PCR product, and ultrapure

H20 in a final volume of 6[l. Ligation reactions were incubated at room temperature for

1 hour; then, 3 l of the reaction were added to one vial (50 l) of One-shot TOP10

chemically competent E. coli cells (Invitrogen). Tubes were placed on ice for 1 hour,

heat shocked for 30 sec at 420C in a water bath, and returned to ice. After adding 250 il

of S.O.C. medium (Invitrogen), the tubes were shaken horizontally at 220 rpm at 370C

for 1 hour. Transformations (100-200 pl) were spread onto bacterial agar plates

containing ampicillin (100 .g/ml) and the plates were incubated overnight at 370C.

Analyzing Recombinants

Using sterile toothpicks, bacterial colonies were selected from agar plates and

added to 10 ml sterile glass tubes containing 3 mL 2XYT and 3ptl ampicillin (100lg/ml)

and the tubes were shaken overnight at 275 rpm at 370C. DNA was extracted from









approximately 1 ml of overnight culture using the 10-minute Mini-Prep Protocol (Zhou et

al., 1990). Briefly, overnight cultures were centrifuged for 10 sec in a 1.5 ml capped

tube. The supernatant was discarded, 300.l of TENS was added to the cell pellet, and the

tube was vortexed for 2-5 sec. Then, 150 pl of 3.0 M sodium acetate (pH 5.2) (Gibco

Life Technologies, Carlsbad, California, USA) were added and the tube was vortexed and

centrifuged. The supernatant was transferred to a fresh 1.5 ml capped tube and mixed

thoroughly with 0.9 ml of 100% molecular grade ethanol which had been pre-cooled to -

200C. After centrifugation, the plasmid DNA pellet was washed twice with 1 ml of 70%

ethanol, allowed to dry, and resuspended in 50 [l of TE buffer (pH 8.0). Recombinants

were analyzed by restriction enzyme digestion using endonucleases HindIII, Apal,

BamHI, EcoRI, and the combination of Apal and BamHI (Invitrogen). Restriction digest

reactions contained: 0.3 [il of restriction enzyme, 3.0 [il of the appropriate enzyme buffer,

1.0 pl of mini-prep DNA, and ultrapure H20 in a final volume of 30 il. Reactions were

incubated at 370C for 1 hour in a dry block and analyzed by gel electrophoresis.

Recombinants containing inserts of the appropriate size were further propagated in

competent E. coli cells and purified with the AuruTM Plasmid Mini Kit (Bio-Rad

Laboratories, Inc., Hercules, CA, USA) for sub-cloning and sequencing. Following the

protocol provided by the manufacturer, 1 ml of overnight culture was added to a 2.0 ml

tube, centrifuged, and the supernatant decanted. Then, 250 [l of resuspension solution

and 2501l of lysis solution were added, the tube was inverted 6-8 times, and the mixture

was allowed to incubate at room temperature for 5 min to ensure lysis was complete.

After adding 350 [l of neutralization solution, the tube was inverted 6-8 times and

centrifuged. The cleared lysate was transferred to a mini spin column and the column









was washed once with 750 [l of wash solution. The DNA was eluted in 50 il of elution

solution and evaluated for yield and purity by spectrophotometry.

Sub-cloning of Purified Recombinants

In order to obtain a high yield and purity of vector and recombinant DNAs for sub-

cloning reactions, midi-prep DNAs were prepared with the Qiagen plasmid purification

kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol.

Transformation reactions containing 1 pl of DNA were propagated overnight in 100 ml

of 2XYT medium (100.l ampicillin), the overnight cultures were centrifuged, and the

supernatants were removed. The bacterial pellets were resuspended and vortexed in 4 ml

of buffer P1 and mixed gently in 4 ml of buffer P2. The lysis reactions were allowed to

proceed at room temperature for 5 min and 4 ml of chilled buffer P3 were added. The

reactions were mixed gently, incubated on ice for 15 min, and centrifuged for 1 hour until

the supernatant was clear. Each supernatant was added to an equilibrated Qiagen-tip 100

and allowed to enter the resin by gravity flow. The Qiagen-tip was washed twice with

buffer QC and the DNA was eluted in 5 ml of buffer QF. The DNA was precipitated in 5

ml of isopropanol, washed with 2 ml of 70% ethanol, and allowed to dry for 15-20 min.

The DNA pellet was redissolved in 200 pl of buffer EB and evaluated for yield and

purity by spectrophotometry.

Sub-Cloning into pcDNA 3.1/Zeo+ Expression Vector

The complete TmlPV E6 and L1 capsid genes were sub-cloned from pCR 2.1

TOPO T/A into pcDNA 3.1/Zeo+ expression vector at positions of compatible restriction

sites present in the vector multiple cloning sites. The TmlPV E6 recombinants were sub-

cloned into the expression vector at the EcoRI site, and the TmlPV complete L1 gene

recombinants were sub-cloned into the expression vector at the HindIII and Xbal sites.









Purified recombinant DNAs and purified pcDNA 3.1/Zeo DNA were cut with identical

restriction enzyme(s) in separate digest reactions. Restriction digests of pcDNA 3.1/Zeo

contained: 3.0 .il of restriction enzyme, 6.0 .il of the corresponding buffer (Invitrogen),

3.0 pl of bovine serum albumin (Invitrogen), 1 pg of purified pcDNA 3.1/Zeo vector

DNA, and ultrapure H20 in a final volume of 601. Restriction digests of TOPO T/A

recombinants were similar, except that they contained 3 pg of purified recombinant DNA.

Digest reactions were incubated at 370C in a dry block for 2 hours and resolved by

horizontal electrophoresis in 1.2% low-melting point (LMP) agarose gels containing

ethidium bromide (0.5 ig/ml). The bands of the appropriate size for the digested pcDNA

3.1/Zeo+ vector and for the digested recombinants were excised from the LMP gel and

purified using the Wizard SV gel and PCR clean-up system. DNA from the purified gel

products was used in the ligation reaction that contained: 4 [il of 5X T4 ligase buffer, 1.5

pl of T4 ligase (Invitrogen), a 1 [1 of gel purified recombinant DNA, 3 pl of gel purified

pcDNA 3.1/Zeo and ultrapure H20 in a final volume of 20[l. Ligation reactions were

incubated overnight at 140C in a refrigeration block. Ten [il of the ligation reaction were

added to one vial (50 [l)ofDH5a competent E. coli cells, incubated on ice for 1 hour,

heat shocked at 420C for 30 sec, and returned to ice. Then, 600 pl of 2XYT containing

50 mM glucose were added and the vial was shaken horizontally at 220 rpm at 370C for 1

hour. Reactions (200p1) were spread onto agar plates containing ampicillin, and plates

were incubated overnight at 370C. Bacterial colonies were selected from the plates and

propagated in 2XYT medium containing ampicillin. Recombinant DNA was purified

using the 10 minute mini-prep protocol and inserts of the appropriate size cloned into the

pcDNA 3.1+ vector were confirmed by restriction digest using 6-base cutter enzymes









Nsil, known to cut the TmlPV E6 fragment once, and Spel, known to cut the TmlPV L1

fragment sequence once.

L1 Gene Sub-Cloning into pFastBacTM1 Vector

The TmlPV complete L1 gene was sub-cloned from pCR 2.1 TOPO T/A into the

pFastBacTM1 vector (Invitrogen) at positions of compatible restriction sites present in the

vector multiple cloning site for use in the Bac-to-Bac Baculovirus expression system

(Invitrogen). Purified L1 recombinant DNA and purified pFastBacTMI vector DNA were

cut with identical restriction enzyme(s) in separate digest reactions. Restriction digests of

pFastBacTMI contained: 3.0 .il of restriction enzyme EcoRI (Invitrogen), 6.0 .il of the

corresponding 10X buffer III (Invitrogen), 3.0 pl of bovine serum albumin (Invitrogen), 1

tg of purified pFastBacTM1 maxi-prep DNA, and ultrapure H20 in a final volume of

60p l. Restriction digests of the TOPO T/A TmlPV L1 complete gene recombinant were

similar, except that they contained 3 tg of purified maxi-prep recombinant DNA. Digest

reactions were incubated at 370C in a dry block for 2 hours and resolved by horizontal

electrophoresis in 1.2% low-melting point (LMP) agarose gels containing ethidium

bromide (0.5 [tg/ml). Bands of the appropriate size for the digested pFastBacTMI vector

and the digested TOPO T/A TmlPV L1 complete gene recombinant were excised from

the LMP gel and purified using the Wizard SV gel and PCR clean-up system. The

ligation and transformation reactions of the L1 complete gene into pFastBacTM1 were set

up similar to the reactions previously described for sub-cloning into the pcDNA 3.1+/Zeo

expression vector, except that transformations were performed in TOP-10 competent E.

coli cells (Invitrogen). Individual bacterial colonies were selected from the plates and

propagated in 2XYT medium containing ampicillin (100lg/ml). DNA was extracted

from the overnight cultures using the 10 minute mini-prep protocol (Zhou et al., 1990)









and pFastBacTM1 L1 recombinants were identified by restriction digest with enzymes

EcoRI, Hind III, and BamHI, a 6-base cutter enzyme known to cut the TmlPV L1

sequence at one site.

L1 Gene Sub-Cloning into pBlueBac 4.5

The TmlPV complete L1 gene was sub-cloned from pCR 2.1 TOPO T/A after

digesting with enzymes Xbal and SstI into the pBlueBac 4.5 vector (Invitrogen), which

was digested with the same enzymes. The ligation and transformation reaction of the

TmlPV L1 gene into the pBlueBac vector was set up similar to the described sub-cloning

reaction into the pFastBacI vector. Individual bacterial colonies were selected from agar

plates and propagated in 2XYT medium containing ampicillin (100lg/ml). DNA was

extracted from overnight cultures using the 10 minute mini-prep protocol (Zhou et al.,

1990) and pBlueBac L1 recombinants were identified by restriction digest with Xbal and

SstI enzymes.

Sequencing of PCR-amplified and Cloned Products

As described under gel electrophoresis, amplified PCR products of the expected

size were purified and sequenced directly using the PCR primers diluted 1:5 in sterile

H20. Cloned PCR products were sequenced with the corresponding vector sequencing

primers diluted 1:10 in sterile H20. Additional sequencing primers (1:5) were used to

obtain the complete sequence of cloned DNA fragments that were greater than 600

nucleotides in length. A list of sequencing primers is contained in Table 2-4. Between

50-100 fmol of purified PCR products or purified recombinants were sequenced in

duplicate using specific forward and reverse primers in the Beckman-Coulter CEQ

2000XL sequencing instrument (Beckman-Coulter Inc., Fullerton, California, USA).

Chromatograms were checked manually for errors in nucleotide sequences using the









Chromas 2.3 software (Technelysium Pty Ltd., Tewantin, Queensland, Australia), and the

assembled sequences were analyzed using the seqed, gap, translate, and multiple

alignment functions of the University of Wisconsin Package Version 10.2 (Genetics

Computer Group [GCG], University of Wisconsin, Madison, Wisconsin, USA). The

Basic Local Alignment Search Tool (BLAST) function of the National Center for

Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) was used to

identify papillomavirus sequences most closely related to sequences obtained from

TmlPV fragments. Neighbor-Joining phylogenetic trees were generated by PAUP 4.0

(Sinauer Associates, Sunderland, Massachusetts, USA) software, using Clustal W slow

and accurate function using Gonnet residue weight table, gap penalty of 35 and gap

extension penalty of 0.75 for pairwise alignment parameters, and gap penalty of 15 and

gap extension penalty of 0.3 for multiple alignment parameters. Phylogenetic trees were

constructed with the deduced amino acid sequences of the TmlPV L1 ORF, L2 ORF, E6

ORF, and E7 ORF DNA fragments. Trees were based on the amino acid sequences of

human and non-human PVs obtained from the GenBank database through the NCBI

website and from the HPV database of the Los Alamos National Laboratory Theoretical

Biology and Biophysics website (http://hpv-web.lanl.gov/stdgen/virus/hpv/).

Transfection of Insect Cell Cultures

Culturing Insect Cells

Sf21 (Spodoptera frugiperda) insect cells (Invitrogen) were used as indicator cell

cultures to generate recombinant baculoviruses. Sf21 cells were cultured in 75 cm2 flasks

(TPP, Switzerland) in serum free Sf-900 II medium (Invitrogen) containing: 200 pg/ml

of antibiotic/antimycotic [penicillin (1X104 units/ml)], streptomycin sulfate (10 mg/ml),

amphotericin B (25 pg/ml)] (Gibco), 200 pg/ml of gentamicin (100 pg/ml) (Gibco), and









10% fetal bovine serum (Gibco). Insect cells were incubated at 270C in a humidified

incubator. After one week, the cell monolayer was scraped with a sterile, disposable cell

scraper (Greiner Bio-One, Kremsmuenster, Australia), resuspended in 10 ml of Sf-900

11/10% FBS, and counted with a hemocytometer (Reichert Scientific Instruments,

Buffalo, NY). Approximately 1 X 106 Sf21 cells in 2 ml of Sf-900 I1/10% FBS medium

were added to 35 mm dishes and incubated at 270C. All steps were performed in a

laminar-flow cabinet under sterile conditions.

Transformation of MAX Efficiency DH1OBacTM Competent E. coli

In order to transfect insect cell cultures and generate a recombinant baculovirus, the

pFastBacTM1 L1 constructs were first transformed into MAX Efficiency DH10BacTM

competent E. coli (Invitrogen) that contain a baculovirus shuttle vector (bacmid) and a

helper plasmid. Briefly, 1 [l of purified pFastBacTM1 recombinant (Ing/il) was added to

50pl of DH10BacTM E. coli, incubated on ice 30 min, heat shocked at 420C for 45 sec,

and returned to ice for 2 min. The reaction was transferred to a 15 ml screw-cap conical

tube (Sarstedt Inc., Newton, North Carolina, USA) and 900 pl of S.O.C. medium were

added. The reactions were shaken at 225 rpm at 370C for 4 hours and transformations

were spread onto bacterial agar plates containing kanamycin (50.g/ml), tetracycline

(10g/ml), gentamicin (7 pg/ml) (Gibco), and blue/white selection medium. Plates were

incubated overnight at 370C. Individual bacterial colonies were selected and propagated

in 3ml 2XYT medium containing kanamycin, tetracycline, and gentamicin and shaken

overnight at 275 rpm at 370C. Overnight cultures were purified using the 10 minute

mini-prep protocol and recombinant bacmids were confirmed by PCR analysis. The Bac-

to-Bac M13 primers, forward primer CR 613: 5'-CCC AGT CAC GAC GTT GTA

AAA CG-3' and reverse primer CR 614: 5'-AGC GGA TAA CAA TTT CAC ACA GG-









3', were used together in a PCR reaction that contained: 200 nM of each primer, 2 mM

MgSO4, 100 pM of each dNTP, 20 mM Tris-HCl (pH 8.4), 10 mM KC1, 0.1 % Triton X-

100 (pH 8.8), 10 mM (NH4)2S04, 1 unit of Taq DNA polymerase (New England

BioLabs), 1 [1 of mini-prep DNA, and ultrapure H20 in a final volume of 50il. For

amplification ofbacmid DNAs, cycling conditions were: an initial denaturation step at

930C for 3 min, followed by 29 cycles of denaturation at 940C for 45 sec, annealing at

510C for 45 sec, and extension at 720C for 5 min. An elongation step at 720C for 7 min

was incorporated into the final cycle. Bacmid DNA recombinants containing the L1

complete gene were expected to be approximately 4,000-bp in size (2,300 bp bacmid

DNA plus 1,712 bp L1 complete gene) and bacmid DNA amplicons that were not

recombinants were expected to be approximately 300-bp in size. PCR products were

resolved by horizontal electrophoresis in 1.0% agarose gels containing ethidium bromide

(0.5 [g/ml) and visualized under UV light. Recombinant bacmids were further

confirmed to contain the pFastBacTM1 L1 gene by PCR analysis targeting the TmlPV L1

complete gene.

Transfection of Insect Cells with Bacmid DNA Recombinants

In a 10 ml glass bacteriological tube (Fisher Scientific), 1 [tg of L1 bacmid DNA

was added to 100[l of unsupplemented Grace's insect cell culture medium (Gibco). In a

separate glass tube, 6 [il of Cellfectin Reagent (Img/ml) (Invitrogen) were added to

100p1 of unsupplemented Grace's insect cell culture medium. The mixtures were

combined and the tube was incubated at room temperature for 45 min. Prior to

transfecting Sf21 insect cells, the cell monolayer was washed with 2 ml of Grace's

medium and incubated for 5 min at room temperature. The medium was removed and

800 [l of Grace's medium were added gently and directly to the cells. The









Bacmid/Cellfectin transfection mix was then added dropwise onto the cells and the

cultures were incubated at 270C for 5 hours. At this time, the transfection mixture was

removed and 2 ml of unsupplemented Grace's medium was added to the transfected cells.

Cultures were incubated at 270C in a humidified incubator. Sf21 cells fed with

unsupplemented Grace's medium (without baculovirus DNA) and Sf21 cells transfected

with purified baculovirus DNA (without insert) were used as negative controls. All steps

were performed in a laminar-flow cabinet under sterile conditions.

Harvest of Recombinant Baculovirus Stocks

After the transfected cell cultures were incubated for approximately 96 hrs, the cell

monolayer and supernatant (-2 ml) were collected with a sterile pipette and inoculated

onto Sf21 cultures in 60 mm tissue culture dishes. One ml of the cell supernatant plus 1

ml of Sf-900 11/5% FBS medium were added to fresh 60 mm dishes containing

approximately 2 X 106 Sf21 cells. The cells were incubated at 270C for 1.5 hrs, the

inoculum was removed, and the cultures were transfected with 3 ml of unsupplemented

Grace's medium. Infected cultures were incubated at 270C for approximately 96 hrs and

sub-cultured again into fresh cultures. The infected cultures were sub-cultured a total of

5 times and the final harvest of cells and supernatants were obtained from 150 mm

dishes. All steps were performed in a laminar-flow cabinet under sterile conditions.

Harvests from infected cell cultures were analyzed by electron microscopy, by Mr.

Woody Frazer, from the Florida Animal Disease Diagnostic Laboratory, Florida

Department of Agriculture and Consumer Services, Kissimmee, Florida.

Reverse Transcription PCR (RT-PCR) of Infected Cell Cultures

RNA was extracted from infected Sf21 cell cultures transfected with the pBlueBac

plasmid containing the L1 capsid gene to determine whether messenger RNA (mRNA)









encoding the TmlPV L1 capsid protein was present. To use as negative controls, RNA

was extracted from untransfected Sf21 cell cultures and from Sf21 cells transfected with

purified parental pBlueBac vector. Cells and supernatants were harvested 96 hrs after

transfection, centrifuged at 4,000 rpm at 100C for 10 min, the supernatant was removed,

and RNA was extracted from the cell pellets using the Aurum total RNA mini kit (Bio-

Rad). The RNA samples were treated with twice the recommended amount (160p1) of

DNase I solution for 30 min, eluted from a mini column with 100l1 of elution solution,

and treated again with 160[l of DNase I solution. The alcohol precipitated RNA pellet

was resuspended in 601l of RNase-free H20 and analyzed for yield and purity by

spectrophotometry. Total RNA was used in reverse transcription reactions in order to

obtain a first-strand cDNA product for use in PCR analysis. Synthesis of cDNA was

performed with SuperScript II (Invitrogen) according to the manufacturer's protocol.

Reactions contained random hexamer primers (Invitrogen) or a TmlPV L1 gene-specific

primer (RP CR574) designed by us. The reverse transcription assays were performed in

duplicate with or without the incorporation of SuperScript II reverse transcriptase

enzyme (Invitrogen) in order to ensure that the DNA had been completely degraded by

the DNase I treatment during the RNA extraction process. The cDNAs were used as

templates for amplification in TmlPV L1 PCR assays according to the PCR protocol

targeting the TmlPV complete L1 gene.

Generation of Recombinant Baculovirus

Recombinant vector pBlueBac 4.5 containing the complete TmlPV L1 gene under

the control of the polyhedron promoter and the Bac-N-Blue baculovirus DNA

(Invitrogen) (0.5kg DNA in 10pl volume) were incubated at room temperature for 10

min. Then, 1 ml of unsupplemented Grace's insect medium (Invitrogen) was added to









the tube, followed by 20Cl of Cellfectin reagent (Invitrogen). The reaction was gently

mixed for 10 sec and allowed to incubate at room temperature for 15 minutes. Prior to

transfecting the Sf21 insect cultures (3X106) seeded in 60 mm dishes, the medium was

removed and the cell monolayer was rinsed gently twice with 2 ml of fresh,

unsupplemented Grace's insect medium without FBS. The transfection mixture was

added dropwise onto the cells and incubated at 270C for six hours in a humidified

incubator. After the incubation period, 2 ml of complete TNM-FH medium (Invitrogen)

containing gentamycin (10g/ml) and FBS (10%) were added to each dish. The dishes

were incubated at 270C for 10 days, at which time cells and medium were harvested and

stored at 40C prior to screening and purification of the recombinant viruses. A second

dish containing Sf21 cell cultures was transfected with the transfer vector pBlueBac 4.5

containing the L1 capsid gene, but no baculovirus DNA, and treated similarly. A third

culture ofuntransfected Sf21 cells served as a negative control. After 72 hrs, 500[l of

the medium was harvested from each dish and transferred to a sterile 15 ml screw-cap

tube to which 2[l ofBluo-gal substrate (200[g/pl in DMSO) was added. The tubes were

incubated at 270 and monitored for the development of a blue color.

Transfection of Mammalian Cells

Culturing African Green Monkey Kidney (COS-7) Cells

African green monkey kidney (COS-7) cells were propagated in 75 cm2 flasks in

Dulbecco's modified eagle medium (DMEM) (Gibco) containing gentamycin,

antibiotic/antimycotic, and 10% FBS. Cells were incubated at 370C in a humidified

incubator.









Electroporation of COS-7 Cells with Recombinants for Immunofluorescence Assays

COS-7 cells were dispersed with trypsin/EDTA (0.25% Trypsin/lmM EDTA)

(Gibco), resuspended in 10 ml DMEM/5% FBS in a 15 ml screw cap tube, and counted

with a hemocytometer. The cells were centrifuged at 1,500 rpm for 10 min at 100C in a

refrigerated centrifuge (Jouan, Unterhaching, Germany). The medium was aspirated

from the cells and the cell pellet was resuspended in cold, sterile IX PBS (Gibco). Then,

0.4 ml of cell suspension (1 X 107 cells) and 5 g of purified recombinant plasmid DNA

were added to a 0.4 cm cuvette placed inside the Gene Pulser II unit shocking chamber

(Bio-Rad Laboratories). COS-7 cell electroporation assays were performed using either

the E6 complete gene in pcDNA 3.1+/Zeo or the L1 complete gene in pcDNA 3.1+/Zeo

vector. Cells were electroporated in the Gene Pulser II Electroporation System (Bio-Rad

Laboratories) for 0.64 msec with the low capacitor set at 25[iF and the voltage set at 0.6

KVolts. Then, 200 [il of electroporated cells were added to each chamber of a four

chamber glass slide (Lab-Tek, Nalge Nunc Intl., Naperville, Illinois, USA), each

containing 1 ml ofDMEM/10 % FBS and zeocin, and the chambered coverglass sides

were incubated at 370C. Recombinant plasmid DNAs with the genes cloned in the wrong

orientation and parental plasmid DNA (without insert) were used as negative controls.

Cells in each chamber of the slide were fixed at four different times; 12 hrs, 24 hrs, 36

hrs, and 48 hrs. Briefly, the medium was aspirated from the chamber and the cells were

rinsed with lml of 1X Hanks balanced salt solution (Gibco). The cells were then fixed

for 1 min using 1 ml of cold acetone: methanol (1:1) solution, the fixative was removed,

and the chamber slide was stored at -200C until use. Immediately before use, to

equilibrate the cells, 1 ml of PBS/ 3% BSA was added to each slide chamber, allowed to

soak the monolayer for 5 minutes at room temperature, and removed. Serum obtained









from captive manatees (Holly, Betsy, Oakley, Willoughby, Amanda, and Lorelei) with a

history of papillomavirus infection was diluted 1:20 in PBS/3% BSA solution and 500 [l

of diluted serum were added to the appropriate chamber. The cells were incubated at

370C for 1 hr, the serum was removed, and the cells were washed three times with 1 ml

PBS/0.025% Tween 20 (Fisher Scientific, Fairland, New Jersey, USA) solution. Purified

anti-manatee IgG monoclonal antibody (1.2mg/ml) was diluted 1:20 in PBS/3% BSA

solution and 500 pl of diluted monoclonal antibody were added to each chamber. The

chambers were incubated at 370C for 1 hr, the monoclonal antibody was removed, and

the cell monolayer was washed three times with 1 ml PBS/0.025% Tween 20 solution.

Fluorescein-labeled protein G conjugate (250[g/ml) (Sigma-Aldrich, Steinheim,

Germany), was diluted 1:40 in PBS/3% BSA, 200 pl were added to each slide chamber,

and the chamber slides were incubated at 370C for 1 hr. The monolayers were gently

rinsed three times with 1 ml PBS/0.025% Tween 20 solution, the cover and lining of the

chamber were removed, and ProLong gold anti-fade reagent (Molecular Probes,

Carlsbad, California, USA) was added dropwise to the monolayer. Cover slips were

placed on the anti-fade reagent and the obtained monolayers were evaluated for specific

immunofluorescence using a fluorescent microscope (Zeiss Axiovert 25). Similar

immunofluorescence assays were also performed using fluorescein-labeled Protein A

(Sigma-Aldrich), instead of Protein G, according to the methods described.

Culturing Florida Manatee Respiratory Epithelial Cells

Florida manatee respiratory epithelial (TmlRE) cell cultures were obtained from

Dr. Mark Sweat (Fish and Wildlife Research Institute, St. Petersburg, Florida) and

propagated in 75 cm2 flasks in Dulbecco's modified eagle medium (DMEM) (Gibco)









containing antibiotic/antimycotic, gentamycin, and 10% FBS. Cells were incubated at

370C in a humidified incubator.

Transfection of TmlRE Cells with DNA Recombinants for Immunofluorescence
Assays

Methods for transfecting TmlRE cells were the same as the methods described for

transfecting COS-7 cells. TmlRE cell transfection assays were performed with E7

pcDNA 3.1+ TOPO and the L1 complete gene pcDNA 3.1 /Zeo recombinant plasmids.


Table 2-1. Samples obtained from skin lesions of captive Florida manatees. DNA
extracted from lesions was tested by PCR for the presence of papillomavirus
infection. HSSWP: Homosassa Springs State Wildlife Park, FL: Florida, SD:


San Diego, CA:California.
Sample I.D. Manatee I.D.
CR130 Oakley
V368 Betsy
V369 Holly
V370 Betsy
V371 Amanda
V372 Amanda
V373 Willowby
V374 Willowby
V375 Lorelei
V376 Lorelei
V377 Lorelei
V684 Rosie
V685 Lorelei
V686 Betsy
V687 Amanda
V909 SW04031
V910 SW04031
V989 TM0334
V991 Stubby
V995 TM0341
V996 TM0341


Location
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
HSSWP, FL
SD, CA
SD, CA
Orlando, FL
Orlando, FL
Orlando, FL
Orlando, FL


Date Obtained
July, 2002
July, 1998
July, 1998
July, 1998
January, 2000
January, 2000
January, 2000
January, 2000
January, 2000
January, 2000
January, 2000
October, 2003
October, 2003
October, 2003
October, 2003
January, 2004
January, 2004
January, 2004
January, 2004
January, 2004
January, 2004









Table 2-2. Samples obtained from skin lesions of free-ranging Florida manatees. DNA
extracted from lesions was tested by PCR for the presence of TmlPV
infection. CR: Crystal River, FL: Florida, HR: Homosassa River, DK:
Drowned Keys, BZ: Belize, POI: Port of Isles (Everglades City),
TB=Tampa Bay,*: manatee penile lesion, **: uninfected manatee liver.
Sample I. D. Manatee I. D. Location of Manatee Date Obtained
V378 RKB-1029-17 CR, FL January, 2003
V389 RKB-1035-31 CR, FL February, 2003
V390 RKB-1036-19 HR, FL February, 2003
V396 RKB-1040-23 HR, FL February, 2003
V397 RKB-1039-13 HR, FL February, 2003
V408 RKB-10474 CR, FL February, 2003
V556 BZ01M16 DK,BZ May, 2003
V1329 TNP-29 POI, FL April, 2004
V1330 TNP-29 POI, FL April, 2004
V1331 TNP-29 POI, FL April, 2004
V1332 TNP-31 POI, FL April, 2004
V1333 TNP-32 POI, FL April, 2004
V1334 Unidentified POI, FL April, 2004
V1335 Unidentified POI, FL April, 2004
V1343 BZ04M64 DK, BZ May, 2004
V1437* MEC0449 TB, FL August, 2004
V1351 BZ04M58 DK, BZ May, 2004
V1774 THR-02 HR, FL April, 2005
V1776 THR-03 HR, FL April, 2005
V1777 THR-04 HR, FL April, 2005
V1855** MEC-0515 TB, FL February, 2005

Table 2-3. PCR primers designed to target manatee papillomavirus sequences


Target
L1 458-bp
Fragment



L1--El
Region

Complete E6
Gene


E6 TOPO
Fragment


PCR Primer
FP CR333
RP CR332
FP CR490
RP CR491

FP CR442
RP CR441

FP CR498
RP CR499

FP CR529
RP CR499


PCR Primer Sequence
5'- GCI CAG GGI CAT AAI AAT GG-3'
5'- CGT CCI AII GGA IAC TGA TC-3'
5'- CAG GGG CAT AAG AAT GGT ATT G -3'
5'- GAG GGG AGA CTG ATC GAG TTC TG-3'

5'- CCT GCT GAA AAT GAT GAT CC -3'
5'- TTA TCA IAT GCC CAI TGT ACC AT -3'

5'- CAA CCA TCT TCT ACA TGC TTA GT-3'
5'- CGT ATT CTT GGA TAT GTG GTG -3'

5'- CAC CCA ACC ATC TTC TAC ATG CTT AGT-3'
5'- CGT ATT CTT GGA TAT GTG GTG -3'









Table 2-3. Continued


Target
E7 TOPO
Fragment

L1-L2
Region
Complete L1


Gene


Complete L2
Gene


PCR Primer
FP CR530
RP CR501

FP CR572
RP CR574
FP CR573

RP CR574

FP CR572
RP CR622


PCR Primer Sequence
5'-CAC CTT AGA AGAC ACA GCA CGT ATC-3'
5'- ATC TGT TGT ATC CGA GTC AC -3'

5'- TAA CCG CAT TTA ATG GGC AAT TTG -3'
5'- AAT AAA ATG ATG CAC AGT GCC AG -3'
5'- CAC CTA CAA TCC TTA TTG ATT TTC AAT C
-3'
5'- AAT AAA ATG ATG CAC AGT GCC AG -3'

5'- TAA CCG CAT TTA ATG GGC AAT TTG -3'
5'-TTC GGT ATT GAG GAT GCG GG-3'


Table 2-4. Sequencing primers used to obtain the complete sequence of PCR amplified
TmlPV gene fragments.
Target Sequencing Primer Primer Sequence
L1 458-bp FP CR333 5'- GCI CAG GGI CAT AAI AAT GG -3'
Fragment RP CR332 5'- CGT CCI All GGA IAC TGA TC -3'
FP M13 5'- GTA AAA CGA CGG CCA G -3'
RP M13 5'- CAG GAA ACA GCT ATG AC -3'


L1-El
Region


Complete E6
Gene



Complete E7
Gene


L1-L2
Region


T7 Promoter
RP CR216
FP CR579
RP CR478
FP CR492
RP CR493

FP M13
RPM13
T7 Promoter
RP CR532

FP M13
RPM13
T7 Promoter
RP CR532

FP M13
RPM13
FP CR583
RP CR580
FP CR588
RP CR601
FP CR 592


5'- TAA TAC GAC TCA CTA TAG GG -3'
5'- TAC AAG ACA GGT TTA AGG AGA C -3'
5'- TGC GCA TAG TTA CTT CTG AG -3'
5'- CAG TGT ACC ATT GAA GAT AAG TC -3'
5'- ATG TAT GAA GTA TAA ATA GCA C -3'
5'- CAA CTC TAC CTG TAC GTT CC -3'

5'- GTA AAA CGA CGG CCA G -3'
5'- CAG GAA ACA GCT ATG AC -3'
5'- TAA TAC GAC TCA CTA TAG GG -3'
5'-TAG AAG GCA CAG TCG AGG-3'

5'- GTA AAA CGA CGG CCA G -3'
5'- CAG GAA ACA GCT ATG AC -3'
5'- TAA TAC GAC TCA CTA TAG GG -3'
5'-TAG AAG GCA CAG TCG AGG-3'

5'- GTA AAA CGA CGG CCA G -3'
5'- CAG GAA ACA GCT ATG AC -3'
5'- AGA TTA CAC CAG AGG CTC C -3'
5'- ATT CAT TGT ATG TAT GTG GG -3'
5'- CCC TAT CTT TGA CAA TTC TG -3'
5'- GAG CGT CTG CTT TCG TGT GT -3'
5'-GGG AGA CTC CAC TGA TAC CA-3'






42


Table 2-4. Continued
Target Sequencing Primer Primer Sequence


Complete L1 FPM13
Gene RP M13
FP CR579
RP CR580


5'- GTA AAA CGA CGG CCA G -3'
5'- CAG GAA ACA GCT ATG AC -3'
5'- TGC GCA TAG TTA CTT CTG AG -3'
5'- ATT CAT TGT ATG TAT GTG GG -3'






















L2ORF FP CR572
-- I0


L1ORF FP CR573
-- 0


L1 FP CR333/
L1FP CI90
E6 FP CR498
L1FP CR442 -


S2 ORF L1 ORF NCR I ORF 7 ORF NCR E ORF


L20RF RP CR622
4-


L1ORF RP CR574
4-


E6 RP CR499
4-


E7 RP CR500
4-


E1 RP CR441
4-


L1 RP CR332/
L RPCR491

Figure 2-1. Linear representation of the open reading frames (ORFs) of the circular manatee papillomavirus (TmlPV) genome with
the relative positions of the PCR primers used to amplify TmlPV DNA. FP=Forward primer, RP=Reverse primer.


E7FP CR500













CHAPTER 3
RESULTS

PCR Results

Total DNA extracted from 13 skin lesions of captive Florida manatees (Trichechus

manatus latirostris) and six skin lesions of free-ranging Florida manatees from the

vicinity of Homosassa Springs State Wildlife Park (HSSWP) amplified DNA fragments

of the expected size in PCR assays for the detection of manatee papillomavirus (TmlPV).

DNA fragments of the expected size could not be amplified from DNA extracted from

three skin lesions of free-ranging Antillean manatees (T. manatus manatus) in PCR

assays and, therefore, served as negative controls in subsequent PCR assays. DNA

extracted from one manatee liver was also incorporated into the PCR in order to serve as

a negative control and to validate the results in subsequent PCR assays. PCR results of

manatee skin lesions assayed for the presence of TmlPV DNA are shown in Tables 3-1,

3-2, and 3-3 (summary).

PCR Targeting the Papillomavirus L1 458-bp Fragment

Oligonucleotide primers MY11 and MY09 (Manos et al., 1989), known to amplify

a 458-bp fragment within the L1 capsid gene of several human papillomaviruses and

modified by us to contain deoxyinosines at positions of nucleotide degeneracy (primers

CR333 and CR332), amplified DNA fragments of identical size from five captive

manatee skin lesions, out of 11 tested, and from four free-ranging manatee skin lesions

(near HSSWP), out of seven tested.









PCR Targeting the AL1 TmlPV Fragment

The AL1 TmlPV oligonucleotide primers CR490 and CR491, designed to amplify a

458-bp fragment within the L1 capsid gene of TmlPV, amplified DNA fragments of

identical size from 11 captive manatee skin lesions, out of 15 assayed, and from five free-

ranging manatee skin lesions, out of 15 tested for the presence of papillomavirus

infection. Total DNA from four skin lesions from which no amplification of the 458-bp

DNA fragments could be achieved using the MY11 and MY09 HPV L1 primer set

amplified DNA fragments of the expected size (458-bp) using the TmlPV-specific

CR490 and CR491 primer set (Figure 3-1).

PCR Targeting the TmlPV L1-E1 Region

Oligonucleotide primers CR442 and CR441, known to amplify an approximately

3,000 bp fragment of the L1-El region of the HPV-70 genome (Forslund and Hansson,

1996), amplified a fragment of similar size from one captive manatee skin lesion that

was tested with the CR442/CR441 primers (Figure 3-2).

PCR Targeting the Complete TmlPV E6 Gene

Oligonucleotide primers CR498 and CR499, designed to amplify a 587-bp

fragment of the TmlPV genome that contains the complete TmlPV E6 gene ORF,

amplified DNA fragments of the expected size from five captive manatee skin lesions

(Figure 3-3), out of eight tested for the presence of papillomavirus infection.

Amplification was not obtained from 12 free-ranging manatee skin lesions tested.

PCR Targeting the Complete TmlPV E7 Gene

Oligonucleotide primers CR500 and CR501, designed to amplify a 489-bp

fragment of the TmlPV genome that contains the complete TmlPV E7 gene ORF,

amplified DNA fragments of the expected size from five captive manatee skin lesions









(Figure 3-4), out of five tested for the presence of papillomavirus. Similar fragments

were not amplified from any of the seven free-ranging manatee skin lesions tested.

PCR Targeting the TmlPV L1-L2 Region

Oligonucleotide primers CR572 and CR574, designed to amplify a 3,208-bp

fragment of the TmlPV genome that contains the complete L1 gene ORF plus the

complete L2 gene ORF, amplified DNA fragments of the expected size from two skin

captive manatee skin lesions (Figure 3-5), out of two tested for the presence of

papillomavirus infection.

PCR Targeting the Complete TmlPV L1 Gene

Oligonucleotide primers CR573 and CR574, designed to amplify a 1,712-bp

fragment of the TmlPV genome that contains the complete L1 capsid gene ORF,

amplified DNA fragments of the expected size from three captive manatee skin lesions

(Figure 3-6), out of three tested for the presence of papillomavirus infection.

PCR Targeting the Complete TmlPV L2 Gene

Oligonucleotide primers CR572 and CR622, designed to amplify a 1,611-bp

fragment of the TmlPV genome that contains the complete L2 capsid gene ORF,

amplified DNA fragments of the expected size from two captive manatee skin lesions

(Figure 3-7), out of two tested for the presence of papillomavirus infection.

Sequencing Results and Genetic Analyses

TmlPV L1 458-bp Fragments

Sequencing of papillomavirus L1 capsid gene fragments amplified with the

modified HPV primers MY11 and MY09 (CR333/CR332) revealed that the fragments

were 458-bp in length, supporting the universality of the MY11 and MY09 primers,

which are known to amplify a 458-bp DNA fragment from most types of genital HPVs









(Bernard et al., 1994). Sequencing of papillomavirus L1 capsid gene fragments amplified

with the AL1 TmlPV-specific primers revealed that the fragments were also 458-bp in

length. Nucleotide sequences of the 458-bp TmlPV L1 gene fragments were entered into

the Basic Local Alignment Search Tool (BLAST) of the National Center for

Biotechnology Information Website (NCBI, Bethesda, Maryland) to identify

papillomavirus homologues that had the highest similarity and identity to TmlPV L1.

This demonstrated that the amplified TmlPV fragments were contained within a highly

conserved domain of the L1 capsid protein gene of papillomaviruses. Nine TmlPV L1

fragment sequences, obtained from the DNA of three captive manatee lesions and six

free-ranging manatee lesions, were submitted to the GenBank database of the NCBI

website (Table 3-4).

The TmlPV L1 gene fragments translated correctly from the first nucleotide of

forward primer (FP) MY11 and FPALITmlPV (CR 490) into protein fragments

consisting of 152 amino acids. The GAP function of the GCG Genetic Package (GCG,

University of Wisconsin, Madison, Wisconsin, USA) showed that the nine TmlPV L1

fragment sequences were 100% identical at the nucleotide and the amino acid level. The

multiple alignment of the deduced amino acid sequences of the TmlPV L1 fragments

demonstrated the identity of the TmlPV L1 sequences (Figure 3-8). Comparisons of the

TmlPV L1 amino acid sequence with homologues from human and non-human

papillomaviruses demonstrated identities that ranged between 36% and 57% (Table 3-5)

and similarities that ranged between 57% and 67% (Table 3-6). After removal of the L1

primer sequences, the L1 fragment sequence was 100% identical to the corresponding L1









region of the TmPV-1 sequence obtained by another research group and recently

deposited in the GenBank database (AY609301) and published (Rector et al., 2004).

L1-E1 TmlPV Region

Sequencing of the approximately 3,000-bp papillomavirus DNA fragment

amplified from one lesion of a captive manatee (V369, Holly) revealed that the fragment

was 2,772 nucleotides in length. The ORF(Open Reading Frame) Finder tool of the

NCBI website revealed that the 2,772-bp sequence contained partial sequences of the L1

ORF, the complete E6 ORF, the complete E7 ORF, and partial sequences of the El ORF.

Further genetic analysis of the 2,772-bp sequence showed that it contained: partial

sequences of the 3' end of the TmlPV L1 ORF (261-bp, including the L1 stop codon),

followed by a large non-coding region (816-bp), the TmlPV E6 ORF (414-bp, including

the E6 stop codon), then, separated by two nucleotides, the TmlPV E7 ORF (348-bp,

including the E7 stop codon), a small non-coding region (334-bp), and partial sequences

of the 5' end of the TmlPV El ORF (599-bp). The 2,772-bp sequence that spans the L1-

El region of the TmlPV genome was submitted to the GenBank database of the NCBI

website (Table 3-4). Nucleotide sequences of the TmlPV E6 ORF translated into a

protein consisting of 137 amino acid residues. Comparisons of the TmlPV E6 137 amino

acid sequence with homologues from human and non-human papillomaviruses

demonstrated identities that ranged between 22% and 32% (Table 3-5) and similarities

that ranged between 28% and 42% (Table 3-6). The TmlPV E6 ORF sequence obtained

from sequencing the 2,772-bp fragment was submitted to the GenBank database of the

NCBI website (Table 3-4). Nucleotide sequences of the TmlPV E7 ORF translated into a

protein consisting of 115 amino acid residues. Comparisons of the TmlPV E7 115 amino

acid sequence with homologues from human and non-human papillomaviruses









demonstrated identities that ranged between 23% and 41% (Table 3-5) and similarities

that ranged between 31% and 51% (Table 3-6). Pair-wise comparisons were not

performed with Phocoena spinipinnis PV (PsPV-1) because the PsPV-1 genome does not

encode an E7 ORF. The complete TmlPV E7 ORF sequence obtained from sequencing

the 2,772-bp fragment was submitted to the GenBank database of the NCBI website

(Table 3-4).

TmlPV E6 Gene

Sequencing of amplified DNA fragments containing the complete TmlPV E6 gene

from four lesions of captive manatees revealed that the fragments were 587 nucleotides in

length. Genetic analysis of the 587-bp TmlPV E6 gene fragment revealed that the

sequence contained the complete TmlPV E6 ORF (414-bp, including the E6 stop codon)

that translated into a protein consisting of 137 amino acid residues. Comparisons of the

four obtained TmlPV E6 sequences showed that the nucleotide identity ranged from 99.5-

100.0% and the amino acid identity was 100.0% (not shown). The TmlPV E6 gene

sequence obtained from the positive control manatee DNA (CR130) demonstrated 100%

identity and similarity to the corresponding region of the TmPV-1 genome described by

another research group (Rector et al., 2004). The TmlPV E6 ORF sequences were

submitted to the GenBank database of the NCBI website (Table 3-4).

TmlPV E7 Gene

Sequencing of amplified DNA fragments containing the complete TmlPV E7 gene

from four lesions of captive manatees revealed that the fragments were 489 nucleotides in

length. Genetic analysis of the 489-bp TmlPV E7 gene fragments revealed that the

sequences contained the complete TmlPV E7 ORF (348-bp, including the E7 stop codon)

that translated into a protein consisting of 115 amino acid residues. Comparisons of the









four obtained TmlPV E7 ORF sequences showed that the nucleotide identity ranged from

99.4% tol00.0 % and the amino acid identity ranged from 99.1% to 100.0% (not shown).

The TmlPV E7 sequence obtained from positive control manatee DNA (CR130) had

100% identity and similarity to the corresponding region of the recently published

TmPV1 genome (Rector et al., 2004). The TmlPV E7 ORF sequences were submitted to

the GenBank database of the NCBI website (Table 3-4).

TmlPV L1-L2 Region

Sequencing of the amplified TmlPV L1-L2 gene fragments from two papillomatous

lesions of captive manatees Oakley and Holly revealed that the fragments were 3,208

nucleotides in length. Genetic analysis revealed that the fragments contained the

complete TmlPV L1 ORF (1,518-bp, including the L1 stop codon) plus the complete

TmlPV L2 ORF (1,536-bp, including the L2 stop codon). The start codon of the

TmlPVL 1 ORF sequence was contained within the TmlPV L2 ORF sequence, and the

TmlPV L1 and TmlPVL2 ORFs had an overlapping region of 20 nucleotides. Nucleotide

sequences of the TmlPV L1 ORF translated into a protein of 505 amino acid residues.

Comparisons of the TmlPV L1 505 amino acid sequence with homologues from human

and non-human papillomaviruses demonstrated identities that ranged between 31% and

57% (Table 3-5) and similarities that ranged between 41% and 68% (Table 3-6). The

complete TmlPV L1 sequence obtained from a lesion of manatee Oakley (CR130)

demonstrated 100% identity and similarity to the corresponding region of the TmPV1

genome deposited in the GenBank database by another research group (AY609301). The

two TmlPV L1 ORF sequences obtained from lesions of captive manatees Oakley and

Holly shared 99.7% nucleotide identity and 99.8% amino acid identity (not shown). The

TmlPV L1 ORF sequences were submitted to the GenBank database of the NCBI website









(Table 3-4). Nucleotide sequences of the TmlPV L2 ORF translated into a protein of 511

amino acid residues. Comparisons of the TmlPV L2 511 amino acid sequence with

homologues from human and non-human papillomaviruses showed identities that ranged

between 35% and 42% (Table 3-5) and similarities that ranged between 43% and 51%

(Table 3-6). The complete TmlPV L2 sequence obtained from positive control manatee

DNA (CR130) demonstrated 100% identity and similarity to the corresponding region of

the TmPV1 genome (AY609301). The two TmlPV L2 ORF sequences obtained from

lesions of captive manatees Holly and Oakley shared 99.9% nucleotide identity and

99.6% amino acid identity (not shown). The TmlPV L2 ORF sequences were submitted

to the GenBank database of the NCBI website (Table 3-4).

Complete TmlPV L1 Gene from Captive Manatee Lorelei

Sequencing of the amplified fragment containing the complete TmlPV L1 gene

fragment from one lesion of captive manatee Lorelei (V685) revealed that the fragment

was 1,712 nucleotides in length. Genetic analysis revealed that the fragment contained

the complete TmlPV L1 ORF sequence that translated into a protein of 505 amino acid

residues. The complete TmlPV L1 ORF sequence was shown to be 99.7% and 99.4%-

99.7% identical to the previously obtained TmlPV L1 ORF sequences at the nucleotide

and amino acid levels, respectively (not shown). The TmlPV L1 ORF sequence (Lorelei)

was submitted to the GenBank database of the NCBI website (Table 3-4).

Complete TmlPV L2 Gene

Total DNA extracted from two lesions of captive manatees Oakley and Holly from

which the complete TmlPV L2 gene (1,611-bp) was amplified was also positive for

amplification of the TmlPV L1-L2 fragment (3,208-bp). This fragment had previously









been sequenced; therefore, sequencing of the complete TmlPV L2 genes from these

DNAs (V369 and CR130) was not repeated.

Immunofluorescence and Gene Expression Assays

Mammalian Expression Systems

Fluorescence was not detected in immunologic assays utilizing monkey kidney

(COS-7) cells transfected with TmlPV L1 or TmlPV E6 genes cloned in eukaryotic

vectors under the control of the CMV promoter. Likewise, specific fluorescence was not

observed in assays using Florida manatee respiratory epithelial (TmlRE) cells transfected

with the eukaryotic vectors containing the TmlPV L1 gene under control of the CMV

promoter. A few TmlRE cells transfected with the E7 gene displayed a faint halo of

fluorescence; however, fluorescence was considered subjective and ambiguous and did

not provide conclusive evidence of protein expression. Restriction digests of the TmlPV

E6 and E7 pcDNA3.1+/Zeo recombinants are shown in Figure 3-11.

Bac-to-Bac Baculovirus Expression System

Supernatant and cell harvests from Sf21 insect cell cultures transfected with TmlPV

L1 capsid protein bacmid DNA were analyzed by electron microscopy by Mr. Woody

Frazer, Florida Animal Disease Diagnostic Laboratory, Florida Department of

Agriculture and Consumer Services, Kissimmee, Florida for the presence of virus-like

particles (VLPs). Amorphous clumps were observed after negative staining that

resembled capsid protein particles; however, papillomavirus-like particles of the expected

size (50-55 nm) were not observed (Figure 3-9). The cDNAs obtained by RT-PCR of

RNAs extracted from infected cell cultures showed that mRNA expressing the TmlPV L1

gene was being produced in the cell cultures (Figure 3-10). Restriction digests of the









TmlPV L1 pFastBacl recombinant used in the transfection experiment are shown in

Figure 3-12.

Bac-N-Blue Baculovirus Expression System

After ninety-six hours of incubation at 270C, supernatants were harvested from

Sf21 cell cultures transfected with the recombinant pBlueBac 4.5 L1 vector DNA. The

baculovirus DNA developed blue color after 48 hrs of incubation (270C) in the presence

of the P-galactosidase substrate Bluo-gal (Invitrogen). This finding indicated that

recombination had occurred between the homologous sequences in the baculovirus DNA

(Bac-N-Blue DNA) and the transfer vector (pBlueBac 4.5 L1 plasmid), reconstituting the

essential sequences necessary for replication of the newly generated recombinant virus.

Supernatants corresponding to the cultures transfected with the pBlueBac 4.5 L1 vector

(no baculovirus DNA) and those from the untransfected cultures did not express the P-

galactosidase enzyme, as judged by the lack of development of blue color. Although no

direct proof has yet been obtained on the expression of the L1 capsid protein by the

generated baculoviruses, it is speculated at this point that unless the L1 capsid gene has

been inadvertently mutagenized, protein expression after the isolation and purification of

recombinant baculovirus will be demonstrated by Western blot analysis.

Phylogenetic Analysis

Multiple sequence alignments and the construction of phylogenetic trees

demonstrated the genetic relatedness of the TmlPV amino acid sequences to the amino

acid sequences of several human and non-human papillomaviruses. Trees were

constructed using the following PVs (with their GenBank accession numbers): Human

papillomavirus type la HPV-la (V01116), HPV-2a (X55964), HPV-3 (X74462), HPV-4

(X70827), HPV-5 (M17463), HPV-6 (AF092932), HPV-7 (X74463), HPV-9 (X74464),









HPV-11 (M14119), HPV-13 (X62843), HPV-15 (X74468), HPV-16 (K02718), HPV-18

(X05015), HPV-20 (U31778), HPV-21 (U31779), HPV-26 (X74472), HPV-27

(X74473), HPV-30 (X74474), HPV-32 (X74475), HPV-33 (M12732), HPV-34

(X74476), HPV-41 (X56147), HPV-51 (M62877), HPV-63 (X70828), HPV-65

(X70829), HPV-92 (NC_004500), HPV-95 (AJ62010), Bovine PV type 1 BPV-1

(X02346), BPV-2 (M20219), Canine oral PV COPV (L22695), Cottontail rabbit PV

CRPV (AJ243287), White-tail deer PV DEERPV (M11910), Equus caballus PV ECPV

(NC_003748), European elk PV EEPV (M15953), Ovine PV type 1 OPV-1 (U83594),

OPV-2 (U83595), Rhesus monkey PV RhMPV (M60184), Phocoena spinipinnis PV

PsPV1(AJ238373), and Manatee papillomavirus type 1 TmPV1 (AY609301). Florida

manatee PV (TmlPV) sequences obtained from positive control manatee DNA (CR130)

were used in all phylogenetic analyses. The L1 capsid protein gene phylograms indicated

that the TmlPV sequence formed a unique branch, distinct from known human and

animal papillomavirus L1 sequences (Figure 13). The TmlPV complete L2 capsid

protein gene also formed a separate branch in the L2 capsid protein phylograms (Figure

14), indicating the uniqueness of this virus. The E6 protein gene based phylograms

indicated that TmlPV was the sole member of a unique branch (Figure 3-15). Similarly,

the E7 protein gene claded by itself to form a branch that is closely rooted to the

papillomaviruses of hoofed animals (Figure 3-16).












Table 3-1. PCR results of DNAs obtained from captive manatee skin lesions tested for the presence of
TmlPV infection. POS: positive, DNA fragments of the expected size were amplified;
NEG: negative, DNA fragments of the expected size were not amplified; X: assay not


ORF: open reading frame.


Sample
I. D. No.
CR130
V368
V369
V370
V371
V372
V373
V374
V375
V376
V377
V684
V685
V686
V687
V909
V910
V989
V991
V995
V996


performed,
L1 458-bp
Fragment
POS
NEG
POS
NEG
NEG
NEG
NEG
POS
POS
POS
NEG
X
X
X
X
X
X
X
X
X
X


TmlPV AL1
Fragment
POS
X
X
POS
POS
X
X
X
POS
POS
X
POS
POS
POS
POS
NEG
POS
NEG
NEG
POS
NEG


L1-L2
Region
POS
X
POS
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X


L1-El
Region
X
X
POS
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X


E6 ORF
POS
X
POS
X
NEG
X
X
X
POS
X
X
X
POS
POS
X
X
NEG
X
X
NEG
X


E7 ORF
POS
X
POS
POS
X
X
X
X
X
X
X
X
POS
POS
X
X
X
X
X
X
X


L1 ORF
POS
X
POS
X
X
X
X
X
X
X
X
X
POS
X
X
X
X
X
X
X
X


L2 ORF
POS
X
POS
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X












Table 3-2. PCR results of DNAs obtained from free-ranging manatee skin lesions tested for the presence of TmlPV infection. POS:
positive, DNA fragments of the expected size were amplified; NEG: negative, DNA fragments of the expected size were
not amplified; X: assay not performed, ORF: open reading frame, *= penile skin lesion, **= normal manatee liver DNA


Location of
Animal
CR, FL
CR, FL
HR, FL
HR, FL
HR, FL
CR, FL
DK, BZ
POI, FL
POI, FL
POI, FL
POI, FL
POI, FL
POI, FL
POI, FL
DK, BZ
DK, BZ
TB, FL
HR, FL
HR, FL
HR, FL
TB, FL


Animal L1 458-bp
I. D. Fragment


V378
V389
V390
V396
V397
V408
V556
V1329
V1330
V1331
V1332
V1333
V1334
V1335
V1343
V1351
V1437*
V1774
V1776
V1777
V1855**


POS
NEG
NEG
POS
POS
POS
NEG
X
X
X
X
X
X
X
X
X
X
X
X
X
X


TmlPV AL1
Fragment
X
POS
POS
POS
POS
POS
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG
NEG


L1-L2
Region
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
NEG
NEG
NEG
X


L1
ORF
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
NEG
NEG
NEG
X


L2
ORF
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
NEG
NEG
NEG
X


L1-E1
Region
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X


E6
ORF
X
NEG
NEG
NEG
NEG
NEG
X
NEG
NEG
NEG
NEG
NEG
NEG
NEG
X
X
X
X
X
X
X


E7
ORF
X
X
X
X
X
X
X
NEG
NEG
NEG
NEG
NEG
NEG
NEG
X
X
X
X
X
X
X













Table 3-3. Summary of PCR results. DNAs obtained from skin lesions of captive and free-ranging manatees were tested for the
presence of manatee papillomavirus infection.


CAPTIVE
MANATEES


FREE-RANGING
MANATEES


Tissues
No. +ve

No. tested

No. +ve
No. tested


CR332/CR333
Fragment
5
11


AL1
Fragment
11
15


L1-L2
Region
2


L1-El
L1 ORF L2 ORF Region


E6 ORF E7 ORF
5 5
8 5









Table 3-4. Accession numbers of manatee papillomavims
GenBank tool of the NCBI website.
Animal
Amplicon I.D. No.
TmlPV L1 458-bp Fragment V369
V396
V389
V390
V397
V408
V375
V378
CR130


TmlPV L1-El Region

TmlPV Complete E6 Gene





TmlPV Complete E7 Gene





TmlPV L -L2 Region


TmlPV Complete L1 Gene



TmlPV Complete L2 Gene


V369

CR130
V369
V685
V686

CR130
V369
V685
V686

CR130
V685

CR130
V369
V686

CR130
V369


sequences deposited into the

GenBank
Accession No.
AY455940
AY455941
AY496568
AY496569
AY496570
AY496571
AY496572
AY496574
AY496575


DQ099425

DQ099425
AY830703
AY830704
AY830705

DQ099427
AY830706
AY830707
AY830708

DQ099423
DQ099424

AY994164
AY994166
DQ099422

AY994165
AY994167









Table 3-5. Pair-wise comparisons of the amino acid sequences of the L1, L2, E6, and E7
gene fragments of manatee papillomavirus (TmlPV) with several human and
non-human papillomaviruses. Numbers represent percent identity to
corresponding TmlPV sequences obtained from Oakley's DNA. X=no
sequence available.

PV Type L1 Fragment L1 ORF L2 ORF E6 ORF E7 ORF
HPVla 55.0 53.0 37.0 32.0 40.0
HPV2a 51.0 52.0 37.0 30.0 36.0
HPV3 57.0 53.0 37.0 29.0 39.0
HPV4 57.0 56.0 40.0 28.0 32.0
HPV5 53.0 56.0 41.0 26.0 35.0
HPV6 55.0 54.0 37.0 25.0 40.0
HPV7 50.0 54.0 37.0 27.0 27.0
HPV9 54.0 56.0 40.0 29.0 40.0
HPV11 56.0 54.0 36.0 23.0 38.0
HPV13 56.0 55.0 37.0 22.0 39.0
HPV15 50.0 54.0 41.0 26.0 41.0
HPV16 51.0 52.0 37.0 30.0 35.0
HPV18 43.0 52.0 38.0 22.0 31.0
HPV20 57.0 58.0 39.0 31.0 35.0
HPV21 57.0 57.0 40.0 30.0 38.0
HPV26 55.0 54.0 38.0 26.0 28.0
HPV27 51.0 51.0 39.0 28.0 35.0
HPV30 56.0 55.0 39.0 23.0 30.0
HPV32 52.0 52.0 40.0 31.0 31.0
HPV33 54.0 31.0 38.0 30.0 28.0
HPV34 53.0 52.0 36.0 25.0 32.0
HPV41 55.0 51.0 36.0 31.0 28.0
HPV51 55.0 54.0 37.0 28.0 32.0
HPV63 55.0 51.0 36.0 31.0 27.0
HPV65 57.0 57.0 39.0 28.0 28.0
HPV92 51.0 55.0 40.0 30.0 38.0
HPV95 57.0 57.0 40.0 32.0 24.0
BPV1 49.0 50.0 36.0 28.0 28.0
BPV2 50.0 50.0 35.0 28.0 27.0
OPV1 49.0 50.0 36.0 25.0 31.0
OPV2 51.0 50.0 35.0 22.0 28.0
CRPV 53.0 57.0 37.0 32.0 25.0
DeerPV 47.0 46.0 35.0 22.0 23.0
ECPV 55.0 51.0 40.0 29.0 32.0
EEPV 50..0 51.0 37.0 25.0 21.0
RhMPV 54.0 53.0 38.0 27.0 39.0
COPV 53.0 54.0 41.0 27.0 34.0
PsPV1 53.0 51.0 42.0 26.0 X
TmPV1 100.0 100.0 100.0 100.0 100.0









Table 3-6. Pair-wise comparisons of the amino acid sequences of the L1, L2, E6, and E7
gene fragments of manatee papillomavirus (TmlPV) with several human and
non-human papillomaviruses. Numbers represent percent similarity to
corresponding TmlPV sequence obtained from Oakley's DNA. X=no sequence
available
PV Type L1 Fragment L1 ORF L2 ORF E6 ORF E7 ORF
HPVla 65.0 63.0 47.0 42.0 51.0
HPV2a 60.0 62.0 47.0 36.0 40.0
HPV3 66.0 63.0 45.0 36.0 44.0
HPV4 66.0 65.0 49.0 36.0 43.0
HPV5 64.0 66.0 51.0 40.0 44.0
HPV6 64.0 63.0 46.0 34.0 50.0
HPV7 61.0 63.0 47.0 35.0 36.0
HPV9 65.0 66.0 49.0 39.0 50.0
HPV11 63.0 62.0 47.0 33.0 48.0
HPV13 64.0 64.0 45.0 32.0 45.0
HPV15 63.0 64.0 51.0 39.0 49.0
HPV16 60.0 61.0 48.0 36.0 43.0
HPV18 66.0 62.0 47.0 32.0 41.0
HPV20 66.0 62.0 49.0 42.0 44.0
HPV21 67.0 68.0 50.0 41.0 45.0
HPV26 66.0 64.0 47.0 33.0 40.0
HPV27 61.0 62.0 48.0 37.0 40.0
HPV30 64.0 64.0 46.0 30.0 38.0
HPV32 63.0 63.0 48.0 42.0 40.0
HPV33 64.0 41.0 48.0 37.0 36.0
HPV34 62.0 62.0 47.0 32.0 42.0
HPV41 65.0 62.0 46.0 41.0 36.0
HPV51 65.0 64.0 46.0 37.0 43.0
HPV63 65.0 62.0 46.0 41.0 36.0
HPV65 66.0 65.0 48.0 37.0 40.0
HPV92 64.0 65.0 50.0 40.0 45.0
HPV95 67.0 67.0 50.0 38.0 36.0
BPV1 63.0 61.0 44.0 36.0 31.0
BPV2 64.0 61.0 44.0 34.0 31.0
OPV1 60.0 61.0 43.0 33.0 34.0
OPV2 61.0 60.0 44.0 31.0 31.0
CRPV 65.0 66.0 46.0 36.0 34.0
DeerPV 57.0 56.0 46.0 28.0 31.0
ECPV 62.0 60.0 51.0 36.0 37.0
EEPV 60.0 61.0 46.0 32.0 32.0
RhMPV 63.0 62.0 46.0 38.0 51.0
COPV 62.0 64.0 51.0 40.0 41.0
PsPV1 62.0 61.0 50.0 36.0 X
TmPV1 100.0 100.0 100.0 100.0 100.0









Table 3-7. Summary of the pair-wise comparisons of the amino acid sequences of
manatee papillomavirus gene fragments with several human and non-human
papillomaviruses.
TmlPV Fragment Most Similar PV Type (%) Least Similar PV type (%)
L1 458-bp HPV-21 (67.0%) DeerPV (57.0%)


HPV-21 (67.0%)
HPV-20 (66.0%)
HPV-65 (66.0%)


OPV-1 (60.0%)
EEPV (60.0%)
HPV-2a (60.0%)


Complete L1 ORF





Complete L2 ORF





Complete E6 ORF





Complete E7 ORF


HPV-21 (68.0%)
HPV-95 (67.0%)
HPV-5 (66.0%)
HPV-9 (66.0%)

HPV-5 (51.0%)
HPV-15 (51.0%)
COPV (51.0%)
ECPV (51.0%)

HPV-la (42.0%)
HPV-20 (42.0%)
HPV-32 (42.0%)
HPV-63 (41.0%)

HPV-la (51.0%)
RhMPV (51.0%)
HPV-15 (49.0%)
HPV-11 (48.0%)


Fragment


HPV-33
DeerPV
OPV-2
ECPV

OPV-1
OPV-2
BPV-1
BPV-2

DeerPV
HPV-30
OPV-2
HPV-18

BPV-1
BPV-2
OPV-2
DeerPV


(41.0%)
(56.0%)
(60.0%)
(60.0%)

(43.0%)
(44.0%)
(44.0%)
(44.0%)

(28.0 %)
(30.0%)
(31.0%)
(32.0%)

(31.0%)
(31.0%)
(31.0%)
(31.0%)
























MM 1 2 3 4 5 6 7 8 9 10 11 12
Figure 3-1. Agarose gel electrophoresis of PCR amplified 458-bp fragments of the L1
capsid protein gene of manatee papillomavirus. MM: 1 Kb Molecular marker,
Lane 1: V375, Lane 2: V376, Lane 3: V389, Lane 4: V390, Lane 5: V396,
Lane 6: V397, Lane 7: V408, Lane 8:V370, Lane 9:V371, Lane 10:V556,
negative tissue; Lane 11: negative tube, water; Lane 12: Positive control
captive manatee Oakley (CR130). C: Captive manatee, FR: Free-ranging
manatee


MM 1 2 3
Figure 3-2. Agarose gel electrophoresis of PCR amplified 2,772-bp fragment of the Ll-
El region of manatee papillomavirus. MM: 1 Kb Molecular marker, Lane 1:
Captive manatee Holly (V369), Lane 2: negative tube, no DNA; Lane 3:
negative tissue control (V1855).


C, C, P- IP- IP- IP- IP- C, C






















MM 1 2 3 4 5 6
Figure 3-3. Agarose gel electrophoresis of PCR amplified 587-bp fragments of the E6
gene of manatee papillomavirus. MM: 1 Kb Molecular marker, Lane 1:
Captive manatee Holly (V369), Lane 2: Captive manatee Betsy (V686), Lane
3: Captive manatee Lorelei (V685), Lane 4: Positive control captive manatee
Oakley (CR130), Lane 5: negative tube, no DNA; Lane 6: negative tissue
control (V1855).


.. ... ... .











MM 1 2 3 4 5 6
Figure 3-4. Agarose gel electrophoresis of PCR amplified 489-bp fragments of the E7
gene of manatee papillomavirus. MM: 1 Kb Molecular marker, Lane 1:
Captive manatee Holly (V369), Lane 2: Captive manatee Betsy (V686), Lane
3: Captive manatee Lorelei (V685), Lane 4: Positive control captive manatee
Oakley (CR130), Lane 5: negative tube, no DNA; Lane 6: negative tissue
control (V1855).






















MM 1 2 3 4
Figure 3-5. Agarose gel electrophoresis of PCR amplified 3,208-bp fragments of the L1
gene plus the L2 gene of manatee papillomavirus. MM: 1 Kb Molecular
marker, Lane 1: Captive manatee Holly (V369), Lane 2: Positive control
captive manatee Oakley (CR130), Lane 3: negative tube, no DNA; Lane 4:
negative tissue control (V1855).


MM 1 2 3 4 5
Figure 3-6. Agarose gel electrophoresis of PCR amplified 1,712-bp fragments of the
complete L1 gene of manatee papillomavirus. MM: 1 Kb Molecular marker,
Lane 1: Captive manatee Holly (V369), Lane 2: Captive manatee Lorelei
(V685), Lane 3: Positive control captive manatee Oakley (CR130), Lane 4:
negative tube, no DNA; Lane 5: negative tissue control, (V1855).


























1 2 3 4
SAgarose gel electrophoresis of PCR amplified 1,660-bp fragments of the
complete L2 gene of manatee papillomavirus. MM: 1 Kb Molecular marker,
Lane 1: Captive manatee Lorelei (V685), Lane 2: Positive control captive
manatee Oakley (CR130), Lane 3: negative tube, no DNA; Lane 4: negative
tissue control (V1855).


MM
Figure 3-7






















































V408



V397



V396



V390



V389



V378



V375



V369



P31


Consensus AQGHKNGIAW QNQLFVTILD NTRGTNMTVS VSTQNALVVD HYDDNDYAQY LRHAEEFELS FVFQLCKVQL TTEALAHIHT MNPKILEDWH IGLRPPPSAS


V408



V397



V396



V390



V389



V378



V375



V369



P31


Consensus VEDQYRYIQS LATRCPPKEV PAENDDPYKT KKFWVVDLST RFSTELDQSP LG












Figure 3-8. Multiple alignment of the amino acid sequences deduced from the nucleotide sequences of the L1 gene fragment of




manatee papillomavirus identified in cutaneous lesions of captive and free-ranging Florida manatees.


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........


..........



..........



..........



..........



..........



..........



..........



..........



..........











Rod-shaped
Baculovirus particles


Amorphous
viral clumps











Figure 3-9. Electron micrograph of Sf21 insect cell cultures transfected with pFastBacl
TmlPV L1 gene recombinant. Amorphous viral clumps that resemble capsid
protein particles and rod-shaped Baculovirus (non-recombinant) particles
were found present. Photo supplied by Mr. Woody Fraser.









MM 1R 2S 3R 4S 5R 6S 7R 8S 9 10 MM











Figure 3-10. Agarose gel electrophoresis demonstrating the PCR amplification of the
manatee papillomavirus complete L1 gene fragment from cDNAs obtained
from infected Sf21 cell cultures. R= Random hexamer primers used in RT-
PCR reactions, S= TmlPV L1 gene-specific primers used in RT-PCR
reactions; MM=Molecular marker, 1 KB ladder; Lanes 1 and 2=Sf21 cell
cultures (negative control); Lanes 3 and 4=Sf21 cell cultures infected with
bacmid DNA (no recombinant); Lanes 5 and 6=Sf21 cell cultures infected
with TmlPV L1 gene recombinant; Lanes 7 and 8= Sf21 cell cultures infected
with TmlPV L1 gene recombinant (no SuperScript); Lane 9=blank sample
(H20), negative PCR control; Lane 10=TmlPV L1 plasmid DNA, positive
PCR control. All of the samples were treated with Dnasel solution, but the
samples in Lanes 7 and 8 were not treated with the SuperScript enzyme and
were not transcribed to cDNA. This demonstrates that the Dnase I solution
effectively degraded the DNA in these samples and validated the results of
this assay. Samples in lanes 5 and 6 were treated with the Dnasel solution and
also with SuperScript enzyme, therefore the TmlPV L1 DNA fragment was
able to be amplified from the cDNA.























MM 1 2 3 4
Figure 3-11. Agarose gel electrophoresis demonstrating restriction enzyme digests of the
manatee papillomavirus E6 gene and E7 gene recombinants. MM: 1 Kb
Molecular marker, Lane 1: pcDNA 3. l+/Zeo E6 recombinant, Hind III; Lane
2: pcDNA 3.1+/Zeo E6 recombinant, EcoRI; Lane 3: pcDNA 3.1+/Zeo E7
recombinant, Hind III; Lane 4: pcDNA 3. 1+/Zeo E7 recombinant, EcoRI
















MM 1 2 3 4
Figure 3-12. Agarose gel electrophoresis demonstrating restriction enzyme digests of the
manatee papillomavirus L1 complete gene recombinant. MM: 1 Kb
Molecular marker, Lane 1: pcDNA 3. 1+/Zeo L1 recombinant, Hind III; Lane
2: pcDNA 3.1+/Zeo L1 recombinant, HindIII plus Xhol; Lane 3: pFastBacl
L1 recombinant, Hind III; Lane 4: pFastBacl L1 recombinant, EcoRI.











HPVla
HPV63

CFPV
PsPV1

I TPV-1
M-.0- E HP"2a,
-- HFV27
HPV2
s HPV3
HPV13
HPV30
HPV26
~~~____~~______________--- HPkr41
HPVr51
HPV7
HPV13
HPW1
LE HPV B
&&-7Joao PV11
HPV 32
RIiMPV
t 7-- HPV34
S HPV16
.4 HPV33
HPVB95
HPV4
HPBkr 55
Rs HPV5

HPV21





ECPV
BPV 1
--4 BPV2
SOPV1
p opyl

A DEERPV
EEPV

Figure 3-13. A. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences
of the complete LI gene of several human and non-human papillomaviruses.
The tree was generated by Clustal W slow and accurate function using Gonnet
250 residue weight table, gap penalty of 35 and gap extension penalty of 0.75
(pairwise alignment parameters), gap penalty of 15 and gap extension penalty
of 0.3 (multiple alignment parameters). In the rectangular cladogram format,
numbers represent the percent confidence of 1000 bootstrap replications. B. In
the radial format, the 0.1 divergence scale represents 0.1 substitutions per site











DEERPV



EEPV



TmPV1
TmlPV

SCRPV
COPV

HPV63
SHPV1a






HPV3

HPV2a
HPV27


B


0.1


Figure 3-13. Continued


S I HPV13
HPV11 HPV6
HPV7

















































Figure 3-14. A. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences
of the complete L2 gene of several human and non-human papillomaviruses.
The tree was generated by Clustal W slow and accurate function using Gonnet
250 residue weight table, gap penalty of 35 and gap extension penalty of 0.75
(pairwise alignment parameters), gap penalty of 15 and gap extension penalty
of 0.3 (multiple alignment parameters). In the rectangular cladogram format,
numbers represent the percent confidence of 1000 bootstrap replications. B. In
the radial format, the 0.1 divergence scale represents 0.1 substitutions per site.













HPV4
HPV95 \


HPV20
HPV65 HPV5 I


HPV41

ECPV

PsPV1





OPV2
OPV1


DEERPV


BPV2 '0
BPV1


HPV11 / I
HPV6 /
HPV13 HPV32
HPV7 RhMPV


B 0.1


HPV21
HPV9
HPV15 TmPV1
/ TmlPV

CRPV


HPV63
HPVla


HPV27
HPV2a
HPV18
HPV26


HPV3
116


Figure 3-14. Continued












HPVZ'

HPV2T
H PY*
F8PV1
H FY'ZT


H PVI ?



H PY1F

H PV51
H P'~4








Tm1--lPV
Tl7 hM PV

H PY25

HFr41



T00 PV

H PAI

HPV1



1.4H P92
HPVW

HPV
---------- ( BP1
W S------------- T H P19 3








9.A HEVE2P
H P.
HPV15
ECPV
_BPW

Ii- [DEH RPV

A E EPV
O PV1




Figure 3-15. A. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences
of the complete E6 gene of several human and non-human papillomaviruses.
The tree was generated by Clustal W slow and accurate function using Gonnet
250 residue weight table, gap penalty of 35 and gap extension penalty of 0.75
(pairwise alignment parameters), gap penalty of 15 and gap extension penalty
of 0.3 (multiple alignment parameters). In the rectangular cladogram format,
numbers represent the percent confidence of 1000 bootstrap replications. B. In
the radial format, the 0.1 divergence scale represents 0.1 substitutions per site.











HPV30 HPV51 PsPV1
HPV26I HPV32 HPV7
/ / HPV13
hMIHPV6
SI / HPV11


EEPV
DEERPV


HPV41

-- CRPV

TmPV1
TmlPV


0.1


Figure 3-15. Continued











COPV
H PV7
H PV13
H PV3
H PV32
H PV1a
RhHPV
H PV34
H PVl1
HPM3,
H PW S
H PV20
.0 H PV2T
HP V1
HPV11
HP V3D
HPV2B
SHPV51
HPV1B
HPV33
EC PV
BPV1
SBPV2
s;B I DEERPV
73A1
EE PV



I TniI _

CPV'
HP V41
H PV5
H PV20
H PuV21
H PV92
H PV'15
H PV9
SH PV95
H PV4
1 .0
H PLWS5


Figure 3-16. A. Neighbor-Joining phylogenetic tree of the deduced amino acid sequences
of the complete E7 gene of several human and non-human papillomaviruses.
The tree was generated by Clustal W slow and accurate function using Gonnet
250 residue weight table, gap penalty of 35 and gap extension penalty of 0.75
(pairwise alignment parameters), gap penalty of 15 and gap extension penalty
of 0.3 (multiple alignment parameters). In the rectangular cladogram, numbers
represent the percent confidence of 1000 bootstrap replications. B. In the
radial format, the 0.1 divergence scale represents 0.1 substitutions per site.













HPV26 HPV18


HPVla
S.HPV63


ECPV


% BPV2
BPV1

DEERPV


0.1 TmlPV
TmPV1


Figure 3-16. Continued














CHAPTER 4
DISCUSSION

Papillomatous lesions harvested from the skin of captive and free-ranging manatees

contained manatee papillomavirus (TmlPV) DNA and were morphologically similar to

the previously described papillomatous lesions of captive Florida manatees (Trichechus

manatus latirostris) housed at Homosassa Springs State Wildlife Park (HSSWP),

Homosassa, Florida (Bossart et al., 2002). Lesions were either flat and sessile or

pedunculated and distributed over the manatee body, including the flippers, nares, and

contact regions of the anterior body (Figure 4-1 and Figure 4-2). Conclusions could not

be made about the stage of the infection based on the appearance of the lesions; however,

the TmlPV L1 fragment sequence (GenBank Accession number AY496572) isolated

from a pedunculated skin lesion in the present study was 100% identical to the manatee

papillomavirus type 1 (TmPV-1) sequence that had been deposited in the GenBank

(AY609301) from another research group and had been isolated from a sessile skin

lesion. These findings strongly suggest that the same virus may cause both types of

papillomatous skin lesions in manatees, and is also suggested by those authors (Rector et

al., 2004).

Until recently, papillomavirus studies dealt mainly with transmission experiments,

virus ultrastructure and chemical composition, and pathological description of lesions.

The main impediment for the analysis of these viruses has been the lack of a reproducible

cell culture system permissive for their replication (Lancaster and Olson, 1982). The

study of PVs has been limiting, as only a few HPV types have been purified in quantities









sufficient for structural analysis, due to the low virus load of many lesions and the

inability to develop tissue culture systems for large scale propagation of the virus

(Rommel et al., 2005). However, the papillomavirus field has advanced recently,

especially in the areas of molecular biology techniques and molecular cloning, which

have made these viruses more amenable to study (Lancaster and Olson, 1982). Despite

the absence of advanced molecular tools, immunohistochemical data from previous

studies of papillomatous lesions in manatees indicated the presence of a species-specific

PV. The immunologic data also suggested that manatee PV (TmlPV) infection might be

latent and, possibly, that it might have been activated after immunosuppression (Bossart

et al., 2002). While electron microscopy evaluation and immunohistochemistry staining

of lesions showed the presence of a PV, these assays did not provide genetic information

about the type of PV involved. The primary objectives of this study were: 1.) To develop

a molecular diagnostic assay for the detection of TmlPV infection in skin lesions, 2.) to

molecularly characterize the TmlPV genome and define some of its organization, and 3.)

to develop a serological assay based on virus-like particles (VLPs) absorbed to an ELISA

plate for the detection and quantitation of antibodies against TmlPV.

The steps for developing a molecular diagnostic assay included: extracting DNA

from papillomatous lesions; designing PCR primers and PCR protocols; and sequencing

amplified products to confirm the identity of the virus involved. Due to the lack of

sequence data available on marine mammal papillomaviruses, specifically, of the

manatee, PCR assays for the molecular detection of TmlPV DNA were initially based on

the MY11 and MY09 primer set that targets a highly conserved region of the human PV

(HPV) L1 ORF. Our PCR protocol utilized annealing temperatures 100 below the









primers melting temperatures, in order to maximize the chances of amplifying the TmlPV

L1 DNA fragment, and incorporated deoxyinosines at positions of nucleotide degeneracy

in the primer sequences. After testing the modified MY11 and MY09 primers

(CR333/CR332) with positive TmlPV DNA, the annealing temperatures of the primers

were gradually increased until amplification of a single band of the appropriate size was

obtained. Using these primers in PCR assays, five TmlPV positive lesions were

identified from captive manatees and four positive skin lesions from free-ranging

manatees. These results confirmed the usefulness of the widely used MY11 and MY09

consensus primers and provided an improved method for the detection of TmlPV in

cutaneous lesions. Based on the sequences generated from the TmlPV L1 fragment using

the MY11 and MY09 primers, these primers were modified to contain TmlPV L1-

specific sequences at positions of nucleotide degeneracy. These TmlPV L1 specific

primers effectively amplified DNA fragments of the expected size from DNA samples

extracted from lesions of 11 captive and five free ranging manatees. Four manatee DNA

samples that had been negative for L1 fragment amplification of fragments of the

expected size using the modified MY11 and MY09 primers were shown to contain

TmlPV DNA fragments of the expected size using the more TmlPV L1-specific primers

(CR490/CR491). These results demonstrated the robustness of the TmlPV L1-specific

primers and improved the use of PCR as a tool for the detection of TmlPV in skin lesions.

Nucleotide sequences and deduced amino acid sequences of L1 fragments amplified with

the MY11/MY09 primer set and with the TmlPV Li-specific primer set were 100%

identical (Figure 3-8), indicating that only one genotype of TmlPV was detected in the

captive manatees of HSSWP and a few of the free-ranging animals that swim around









HSSWP. Three skin lesions (V372, V373, V377) obtained from captive manatees

preserved in DMSO were negative for amplification of the TmlPV L1 fragment;

however, DMSO is known to crosslink DNA, making it difficult to amplify DNA

fragments from these samples. These manatees may have been infected with TmlPV, but

due to the method of sample preservation, the TmlPV DNA fragments were not able to be

amplified from these samples. This demonstrates the need for fresh tissue samples in the

diagnosis of TmlPV infection and has implications on the methods of sample

preservation.

Positive TmlPV skin lesions, identified by PCR amplification of DNA fragments of

the appropriate size, were purified and sequenced directly or purified, cloned, and

sequenced. Nucleotide sequences of cloned products and of directly sequenced products

were compared to confirm the nucleotide identity of the sequences. In a study by Saiki et

al. (1988), an overall error frequency of 0.25% was observed in the sequences of 239-bp

amplified products after 30 cycles of amplification of the fragment. In our PCR assays,

whenever long DNA fragments (>1 Kb) were amplified, high fidelity enzymes that are

endowed with proofreading activity were used in order to minimize possible nucleotide

incorporation errors made by the polymerase during DNA amplification. Furthermore, at

least two clones from each recombinant DNA were completely sequenced and analyzed

and, often, both the purified mini-prep recombinant DNAs and the purified maxi-prep

recombinant DNAs of cloned products were sequenced, as the bacteria could have

possibly introduced nucleotide copying errors during transformations. Nucleotide

sequence differences were observed between the TmlPV E6 ORF sequences (99.5-

100.0% identity), E7 ORF sequences (99.4-100.0 % identity), L1 ORF sequences (99.7%









identity), and L2 ORF sequences (99.9% identity). These minor sequence variations may

have been due to nucleotide errors made either by the polymerase in PCR assays or by

the bacteria after transformation with plasmid DNA. Comparisons of the E6 ORF, E7

ORF, L1 ORF, and L2 ORF sequences obtained from the TmlPV DNA (CR130), used as

positive control throughout this study, with the corresponding sequences of the TmPV-1

genome recently published by another group (Rector et al., 2004) revealed that the

sequences were almost 100% identical at the nucleotide and amino acid levels. These

results indicated the truthfulness of the sequences obtained from the positive control

TmlPV DNA (CR130) and also validated the use of this DNA (CR130) for all future

genetic and phylogenetic analyses. Additionally, the identities shared by the manatee PV

sequences obtained from two different manatee papilloma isolates supports the presence

of only one type of manatee PV in skin lesions from captive and free-ranging manatees

around HSSWP.

Our results confirmed the presence of TmlPV in skin lesions of captive manatees

and extended the knowledge to encompass skin lesions from a few free-ranging manatees

inhabiting Crystal River and Homosassa River, Florida, the nearby waters of HSSWP.

Free-ranging manatees are often seen at the perimeter of the underwater fence that

separates the known TmlPV-positive captive HSSWP manatees from the free-ranging

manatees in this area. DNA samples obtained from skin lesions of free-ranging manatees

inhabiting more distant bodies of water in Florida (Port of the Isles, Tampa Bay) and

Belize (Drowned Keys) were always negative for TmlPV DNA. Therefore, it is thought

that a few of the free-ranging manatees that swim in the vicinity of the infected

population at HSSWP may have acquired the infection by direct contact through this









underwater fence. Manatee papillomavirus DNA was detected in skin lesions that were

harvested from free-ranging manatees during winter months (Table 2-2). Low water

temperatures have been suspected as a potential factor in immunologic suppression in the

known TmlPV-infected HSSWP manatees (Bossart et al., 2002), and exposure to cold

water has been directly correlated with impairment of the immune function in free-

ranging Florida manatees (Walsh et al., 2005). Cold stress syndrome (CSS), induced by

prolonged exposure to cold water, followed by nutritional, metabolic, and immunologic

disturbances culminating in multi-systemic, life-threatening opportunistic infectious

disease (Bossart et al., 2003), has been documented as a frequent (18%) cause of death in

Florida manatees (Bossart et al., 2004). It is likely, then, that when the manatee immune

system is compromised by ill-defined immunosuppressive factors, such as CSS, the

papilloma virus may become activated, invasive, and produce cutaneous lesions.

Exposure to harmful algal blooms (Karenia brevis) can also impair immune function in

manatees (Walsh et al., 2005) and may be involved in TmlPV activation and production

of lesions.

In an attempt to obtain the entire nucleotide sequence of the TmlPV genome, PCR

primers were designed to amplify overlapping PCR products that, if obtained, would

together span the complete TmlPV genome. PCR primers effectively amplified the first

amplimer that spans the L1-El region of the TmlPV genome; however, additional PCR

primers that were based on the overlapping PCR product method (Forslund and Hansson,

1996) did not amplify the subsequent amplimers. Problems encountered in amplifying

the overlapping PCR products included: the lack of sequence data of marine mammal

PVs available for primer design, low quantities and/or poor quality of starting extracted









DNA, and, leastly, significant sequence variation between the targeted regions of TmlPV

and homologous regions in the HPVs. Sequences obtained from the first amplimer

allowed for the partial characterization of the TmlPV genome, which included partial

sequences of the L1 ORF, the complete sequence of the E6 ORF, the complete sequence

of the E7 ORF, and partial sequence of the El ORF. Sequences obtained from the first

amplimer also allowed for the design of PCR primers that specifically targeted the

TmlPV complete E6 and E7 ORFs, widening the targets for the diagnosis of papillomas

of manatees. Primers targeting the TmlPV E6 ORF and E7 ORF effectively amplified

DNA fragments of the expected size from five DNA samples from HSSWP captive

manatees. These results expanded the use of molecular tools for the detection of TmlPV

infection and provided a method for complete gene amplification for gene expression

assays. However, the TmlPV E6 and E7 ORF primers did not effectively amplify

fragments of the expected size from five free-ranging manatee DNA samples and two

captive manatee DNA samples that had previously been positive for amplification of the

L1 458-bp fragment. These captive manatees were housed at marine parks (other than

HSSWP) in Florida and California and the TmlPV E6 and E7 ORF primers were specific

for TmlPV sequences obtained from the HSSWP captive manatees. The E6 and E7 ORFs

are less conserved among PV types than the L1 ORF, the most highly conserved region

of the PV genome (deVilliers et al., 2004), and sequence variation within the E6 and E7

ORFs may have lowered the efficiency of the E6 and E7 primers. Also, the primers that

targeted the E6 and E7 ORFs were located upstream and downstream of the ORFs, within

the non-coding regions of the TmlPV genome; unlike the TmlPV L1 fragment primers

that targeted a very highly conserved region within the L1 ORF. The quality and quantity









of the manatee DNA samples may have adversely affected the efficiency with which the

TmlPV E6 and E7 primers amplified DNA fragments of the expected size. The DNA

samples were frozen and thawed several times for the development of PCR assays, which

may have caused the DNAs to degrade. These results demonstrate the importance of

sequencing more than one genome of TmlPV. A reverse primer based on our own

sequences and a forward primer based on the sequence revealed in a recent publication of

the TmPV-1 genome (Rector et al., 2004) effectively amplified the complete L1 and L2

capsid protein genes from three captive manatee DNA samples and from two captive

manatee DNA samples, respectively. These results also expanded the use of molecular

tools for the detection of TmlPV infection and provided a method for complete gene

amplification for gene expression assays. Sequences obtained from the L1-E1 amplimer

plus the sequences obtained from the complete L1 and complete L2 amplimers allowed

for further characterization of the TmlPV genome.

Pair-wise comparisons of the TmlPV sequences with the corresponding sequences

of several human and non-human PVs revealed amino acid identities (Table 3-5) and

similarities (Table 3-6) of these viruses. Identities refer to the extent to which two amino

acid sequences are invariant and similarities refer to changes at a specific position of an

amino acid sequence that preserve the physico-chemical properties of the original

residue. Sequence identities and similarities also give an idea of the overall similarity

homologyy) of the TmlPV sequence to each virus to which it was compared, but do not

provide phylogenetic data on the genetic relatedness of the viruses. Comparisons of the

TmlPV L1 fragment with several human and non-human PV types suggested that the

TmlPV L1 fragment sequence was more similar to the sequences of cutaneous HPV types









(HPV-3, HPV-4, HPV-20, HPV-21, HPV-65, HPV-95) than to sequences of the high risk

genital mucosal HPV types (HPV-16, -18) and ungulate PV types that induce

fibropapillomas (Deer PV, OPV-1, EEPV). Comparisons of the TmlPV complete L1

ORF with several human and non-human PV types suggested that the TmlPV L1 ORF

sequence was more similar to the sequences of the cutaneous PV types (HPV-20, HPV-

65, HPV-95) than to the high-risk genital mucosal HPV-33 sequence and the

fibropapilloma-inducing Deer PV sequence. These L1 ORF sequence comparison results

were as expected, as TmlPV infection has so far only been associated with the presence

of benign skin lesions and not with genital mucosal lesions or aggressive fibropapillomas.

Since the L1 ORF is the most highly conserved region among all members of the PV

family (deVilliers et al., 2004), results of the L1 ORF comparisons are, possibly, more

reliable than results of comparisons made with less conserved, more variable regions of

the PV genome.

Comparisons of the TmlPV L2 ORF with several human and non-human PV types

shows that the TmlPV L2 ORF was more similar to another genetically characterized

marine mammal PV sequence (PsPV-1) isolated from a Burmeister's porpoise (Phocoena

spinipinnis) and to sequences of cutaneous HPV types (HPV-5, HPV-15), than to the

sequences of the fibropapilloma PV types (BPV, OPV, Deer PV). The TmlPV L2 ORF

sequence would be expected to be more similar to cutaneous PV types than to mucosal or

malignant papillomas, as TmlPV infection has so far only been associated with the

presence of skin lesions. The TmlPV L2 ORF sequence showed the greatest similarity to

the Burmeister's porpoise (Phocoena spinipinnis) papillomavirus (PsPV-1) (Van Bressem

et al., 1996), the only other papillomavirus of marine mammals that has been molecularly









characterized. Although marine in nature, these viruses do not seem to share a common

ancestor, as PsPV-1 and TmlPV clade independently of each other in phylogenetic trees.

Also, PsPV-1 is known to have caused genital warts in cetaceans (Van Bressem et al.,

1996), whereas the TmlPV has so far only been associated with cutaneous lesions. These

results demonstrated the overall similarity of the TmlPV L2 ORF sequence to that of the

L2 ORF sequences of several PV types, but did not provide an accurate phylogenetic

relationship of the viruses.

Comparisons of the TmlPV E6 ORF with several human and non-human PV types

suggested that the TmlPV E6 ORF sequence was similar to sequences of a wide variety

of PV types, including a rabbit PV, a high risk mucosal genital PV type (HPV-32), and

cutaneous PV types (HPV-la, HPV-20, HPV-95). Inferences could not be made from

these results, as they suggested that the TmlPV E6 ORF sequence is similar to both non-

oncogenic (HPV-la, HPV-20, HPV-95) and oncogenic (HPV-32) PV types. However,

the oncogenic potential of TmlPV has not yet been determined, and it is possible that the

oncogenic determination of the TmlPV may be a consequence of a fine balance between

the E6 and E7 regions and unidentified sequences in a different region of the TmlPV

genome. This is partially the case in other types of terrestrial PVs, such as deer PV,

European elk PV, and reindeer PV, which contain a novel, transforming E9 gene in their

genomes (Erikkson et. al, 1994). The results of the E6 ORF sequence comparisons

showed more variability than the comparisons of the highly conserved L1 and L2 regions,

as the E6 ORF is a segment with little sequence conservation among PV viruses (de

Villiers et al., 2004).









Comparisons of the TmlPV E7 ORF with several human and non-human PV types

suggested that this sequence is most similar to sequences of cutaneous PV types (HPV-9,

HPV-15) and a benign mucosal PV type (HPV-6). Results from these comparisons were

not surprising, as TmlPV, so far, has only been associated with benign cutaneous lesions.

Cladistic phylogenetic diagrams reflect hypotheses about the evolutionary

relationships of organisms. A clade is formed by all species which share derived

ancestral characters and a most common ancestor (Chan et al., 1995). The bootstrapped

cladograms and the radial divergence trees representing the L1 ORF, L2 ORF, and E6

ORF sequences demonstrated that TmlPV forms a single, distinct branch, or constitutes

its own clade. Since no PVs have been characterized from species closely related to the

manatee, it is not surprising that TmlPV clades by itself in these phylograms (Figure 3-

13, -14, -15, -16). These results indicated that TmlPV is a unique virus, distinct from the

known human and non-human PVs. The single branch formed by the TmlPV in the L1

ORF phylograms may be associated with the type of cell surface receptors used by the

virus. The PVs enter a wide range of cells and the tropism of infection by the different

PVs is controlled by events downstream of the initial binding and uptake (Muller et al.,

1995). Studies have shown that the L1 major capsid proteins of low-risk HPV-11 binds

to the Kap alpha-2 adapter and the Kap beta-2 import receptor and also interacts with the

Kap beta-3 import receptor (Nelson et al., 2003), while the L1 capsid protein of high-risk

HPV-45 interacts with Kap alpha-2 beta-1 heterodimers (Nelson et al., 2000). It has not

yet been determined what the specific receptor of the TmlPV L1 capsid protein is;

however, if these receptors differ from those described in other PV types, it may explain

why the TmlPV L1 protein is phylogenetically characterized as a single, distinct branch.