Isolation and characterization of a syncytium-forming virus of raccoons

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Title:
Isolation and characterization of a syncytium-forming virus of raccoons
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xi, 92 leaves : ill. ; 29 cm.
Language:
English
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Laham, Stanley Nicolas, 1950-
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Raccoons   ( mesh )
Viruses   ( mesh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1978.
Bibliography:
Includes bibliographical references (leaves 88-91).
Statement of Responsibility:
by Stanley Nicolas Laham.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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oclc - 20298199
ocm20298199
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AA00011122:00001

Full Text












ISOLATION AND CHARACTERIZATION OF A
SYNCYTIUM-FORMING VIRUS
OF RACCOONS










By

STANLEY NICOLAS LAHAM


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA
1978




























To my mother, for her love, devotion and
unshakeable faith in my capabilities

and

To my father, for his patient understand-
ing and constant support.














ACKNOWLEDGMENTS


I am especially indebted to Dr. Jack Michael Gaskin,

my indefatigable mentor and friend without whom this thesis

would not have been realized, and Dr. Kenneth I. Berns who,

in his capacity as a member of my special committee and

department chairman, has always freely given the scientific

advice and general guidance so necessary during difficult

times. May they find here a sincere expression of my

gratitude.

I wish to thank the other members of my doctoral

committee: Dr. Philip Laipis who gave access to his

laboratory and valuable advice on reverse transcriptase

studies, Dr. Richard Crandall and Dr. Bill Hauswirth. I

also wish to thank Dr. Charles Simpson and Mr. J. W. Carlisle

of the College of Veterinary Medicine in whose laboratory

electron microscopic studies were performed. I would also

like to express my appreciation to Ms. Glenda Hall, whose

skills have always kept our laboratory in good operational

order, Mrs. Patricia Price and Mrs. Cecelia Turman, our

dedicated secretaries, who have helped on many occasions.

iii
















TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS . . iii

LIST OF TABLES .... . vii

LIST OF FIGURES . . .. viii

ABSTRACT. . . x

I. INTRODUCTION . ... .. 1

II. BACKGROUND REVIEW . .. 3

III. MATERIALS AND METHODS . 12

A. Laboratory Animals and Sera .. 12
B. Cells and Media . .. 13
C. Virus Isolation. . .. 14

1. Direct cell cultures .. 14
2. Tissue suspensions. .. 16
3. Inoculation of buffy coat cells into 16
established cell lines .
4. Swab samples . .. 17

D. Stock Virus Preparation .. 17
E. Viral Procedures . ... 18

1. Plaque assay .. .. 18
2. Immunofluorescent assay. 19

F. Serologic Methods . 19

1. Immunodiffusion 19










TABLE OF CONTENTS (Continued)





a. Antigen preparations. ... 19
b. Method . .. 21
c. Antisera. . .. 22

2. Inununofluorescence . 22

a. Preparation of FITC-labelled
conjugates . 22
b. Staining procedure. .. 23

3. Neutralization . .. 24

G. Sensitivity to Lipid Solvents .. 25
H. Viral Replication Studies . 25

1. Effect of BUDR .. . 25
2. Uridine uptake and effect of
Actinomycin D. . .. 26

I. Reverse Transcriptase Assay ... 27
J. Electron Microscopy . .. 28

IV. RESULTS . . 29

A. Virus Isolation. . 29
B. Antigen Preparation . 43
C. Experimental Infection. . 44
D. In Vitro Host Range . .. 46
E. Cytopathology and Assay of RSFV .. 52
F. Viral Neutralization. . .. 57
G. Sensitivity to Lipid Solvents 57
H. Viral Replication Studies .. 59

1. Effect of BUDR . 59
2. Uridine uptake and actinomycin D 61

I. Reverse Transcriptase Assays. 64
J. Electron Microscopy . .. 67
K. Serological Survey . 72










TABLE OF CONTENTS (Continued)


Page


V. DISCUSSION ..................

APPENDICES
I. FITC--CONJUGATION OF SERUM .
II. ERIOCHROME BLACK A COUNTERSTAIN
FOR IMMUNOFLUORESCENCE. . .

BIBLIOGRAPHY ....................

BIOGRAPHICAL SKETCH ................














LIST OF TABLES


Table Page

I Foamy Viruses .. .... 8

II Tissues from Which RSFV Was Recovered 43

III Host Range of RSFV. .... .. 47

IV Antibodies to RSFV as Determined by
Three Methods ...... .. 58

V Effect of BUDR on RSFV Replication; Also
Shows Inactivation of the Virus by Lipid
Solvents. .. ... .. . 60

VI Requirements for Manganese in R.T. Assay;
Also Shows Effect of Varying Concentration
of Triton-X 100 .. . 66

VII Recovery of Viral Reverse Transcriptase
Activity from a Sucrose Gradient. 68

VIII Survey of Raccoon Sera for Precipitating
Antibodies to RSFV; Geographical Distri-
bution by Counties. . ... 74


vii















LIST OF FIGURES


Figure Page

1 Uninfected BHK Cells Grown in MEM
Supplemented with 10% Calf Serum
(Magnification X100). .. 32

2 Second Passage BHK Cells Infected with
RSFV. ............. 32

3 Syncytial Structures Induced by RSFV
about to Become Detached from Culture
Flask . ... 34

4 BHK Cells Grown in Leighton Tube and
-Stained with 10% Giemsa .. 34

5 RSFV Infected BHK Cells Stained with
10% Giemsa ... .. 37

6 Typical Giant Cell Formation with Varying
Degree of Multinucleation .. 37

7 Precipitating Antibodies to RSFV Antigen
Detected in Infected Raccoon Sera by
Immunodiffusion . .. 40

8 Detection of Precipitating Antibodies to
RSFV Antigen in Serum of Rabbit Injected
with Infected SIRC Cells .. 40

9 Testing of Different Antigen Preparations
for Precipitation with Raccoon Anti-RSFV
Serum . . .42

10 Comparison of Precipitating Activity in
Serum of Experimentally Infected Rabbits
and Naturally Infected Raccoon Serum. 42


viii










FIGURES (Continued)


LIST OF

Figure

11



12


13


14



15


16



17


18





19


20


21


22


Immunodiffusion Showing Line of
Identity with Sera of Rabbits Infected
by Different Means. . .

Indirect Immunofluorescent Staining
of Uninfected BHK Cells .

Indirect Immunofluorescence on RSFV
Infected SIRC Cells . .

Indirect Immunofluorescent Staining
of RSFV Infected Primary Culture of
Rabbit Kidney Cells . .

Microscopic Plaque Induced in BHK
Cell Culture by RSFV. . .

-Immunofluorescence Showing Viral
Induced Syncytia with No Cell
Destruction . .

Immunofluorescence Showing Infected
Cells with No Apparent Cytopathology.

Incorporation of 3H-Uridine or
3H-Thymidine and Effect of
Actinomycin D on Uridine Incorpora-
tion. . .

RSFV-Associated Reverse Transcriptase
Activity. . .

Electron Microscopy Showing Preformed
Cytoplasmic Viral Cores .

E. M. showing Complete Virions in
Intracytoplasmic Vacuole. .

E. M. showing Extracellular Virons and
Nucleoids Budding Out of Cell or into
Intracytoplasmic Vacuole. .









Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ISOLATION AND CHARACTERIZATION OF A
SYNCYTIUM-FORMING VIRUS
OF RACCOONS

By

Stanley Nicolas Laham

June 1978

Chairman: Jack M. Gaskin
Major Department: Immunology and Medical Microbiology

A virus that shares all of the distinctive characteris-

tics of the syncytium-forming viruses of simian, bovine,

feline, hamster and human origin was isolated from the tissues

of raccoons. It was found to have a wide in vitro host range

with giant cell formation as its most characteristic cyto-

pathic effect. Viral antigen was detectable in the nuclei

of these cells by indirect immunofluorescence.

Persistent infection was experimentally induced in

rabbits and an antigen extracted from infected cells was

used to detect precipitating antibodies in infected animals.

A serological survey of Florida raccoons revealed a wide

geographical distribution of the virus.

An immunofluorescent assay was developed to quantitate

the agent. The virus was inactivated by the lipid solvents

ether and chloroform indicating it to be enveloped. It was

x










shown to incorporate 3H uridine but not 3H-thymidine and

this incorporation was inhibited by low levels of actinomycin

D. Furthermore, its replication was blocked by bromodeoxy-

uridine, indicating a DNA replicative stage which was con-

firmed by demonstration of virion associated reverse

transcriptase activity. The virus banded at a density of

1.18 g/cm3 in sucrose gradients.

Electron microscopic studies revealed an enveloped

spherical virus with a diameter of 85 to 105 nm. The viral

core (58 to 64 nm in diameter) was composed of an electron

opaque outer ring with an electron-lucent center. The

envelope was covered with small projections, and the virus

was seen to mature by budding of preformed nucleoids through

the plasma or intracytoplasmic membrane.















I. INTRODUCTION


When an organism or group of organisms is to be given

a functional appellation, a specific designation should be

used which conveys its uniqueness and separates it from

other related entities. If the name chosen does not accom-

plish this purpose, then further clarification becomes

necessary. That is the problem with the term "syncytium-

forming virus."

There are many other viruses that cause syncytium

formation in vitro including members of the orthomyxo-,

paramyxo-, corona-, oncorna-, and herpes virus group. The

syncytium-forming viruses (also referred to as foamy

agents) are RNA viruses having virion associated RNA-

dependent DNA polymerase. They are present in man and a

variety of animals, causing persistent infection with no

known associated disease (17, 25). The human and bovine

respiratory syncytial virus are not included in this group.

Although they share certain characteristics with the foamy

viruses (e.g.,cell association, lack of hemagglutinin

and syncytium-formation incell cultures), they do not






2


produce an antigen detectable in the nuclei of infected

cells, a principal feature of the syncytium-forming viruses.

In this text, the terms "syncytium-forming virus" and

"foamy virus" will be used interchangeably. Recently,

these agents have been recognized as a unique group and

classified as the subfamily Spumavirinae of the RNA virus

family Retroviridae.















II. BACKGROUND REVIEW


The first foamy viruses to be studied were of simian

origin. Enders and Peebles (7) in 1954 reported the spon-

taneous foamy degeneration of monkey kidney cell cultures.

However, apart from describing the cytopathic effect in

those cultures, no other data was given concerning their

agent.

A year later (during attempts to adapt dengue virus

to monkey kidney cell cultures), Rustigian et al. (28)

isolated an agent from kidneys of rhesus monkeys (Macaca

mulatta) and designated it Simian Foamy Virus (SFV) Type I.

They described the degenerative process in the kidney cell

cultures as follows: "There were areas in which individual

cells were no longer visible but appeared to be made up

of degenerated and perhaps fused cells. .clusters of

nuclei could be distinguished. The multiple nuclei, .

suggested large fused syncytial structure. There appeared,

at the same time, variable sized vacuoles which in some

instances were more prominent than the syncytial struc-

tures" (p. 10). This dilatation of vacuoles produced the

foamy appearance in their cell cultures.

3










They inoculated other cell types with filtered super-

natant fluids from their degenerated cultures. Similar

cytopathic effects were obtained in all instances except in

a mouse kidney cell culture which was unaffected by the

agent. With HeLa epithelial cells, they made the following

observations: "Of much greater contrast to reaction in

monkey cultures was the absence of vacuolation. Degenera-

tion seemed to consist mainly of a gradual increase in size

and number of lytic-like areas. .. .At the same time multi-

nucleated cellular masses representing either giant cells or

syncytial structures became more prominent" (p. 10). Thus,

syncytium-formation seemed to be a more universal manifesta-

tion of the virus than did vacuolation of infected cells.

Simian Foamy Virus Type II was isolated in 1961 from

cynomolgus monkeys (Macaca fascicularis) (20). Since that

time 7 other serotypes of SFV's have been recovered from

various species of monkeys and apes (21, 17, 18, 20, 31)

giving a total of 9 serotypes recognized to date. Cross-

neutralization studies have shown each serotype to be

antigenically distinct (8, 13, 18).

It was not until 1969 that a syncytium-forming virus

was isolated from a non-primate species. Malmquist et al.

(24) designated as bovine syncytial virus a foamy agent






5


which they recovered from both normal and lymphosarcomatous

cattle. It induced syncytia formation in bovine embryonic

spleen cells (BESP) and the rabbit cornea cell line SIRC.

Virus was isolated from direct cell cultures initiated from

lymph nodes and spleens of lymphosarcomatous cattle. It

was also recovered by co-cultivation of the cellular com-

ponent of milk and buffy coat cells of both normal and

lymphosarcomatous animals with BESP or SIRC cells.

The investigators developed an immunodiffusion test

to detect precipitating antibodies in infected cattle.

The antigen used in this test was prepared by sonically

disrupting heavy suspensions of cells from highly infected

cultures. They pointed out that "the presence of antibodies

demonstrable by the immunodiffusion test did not terminate

the carrier state" (p. 192) in infected cattle.

The bovine virus seemed to be very cell associated

and difficult to transmit with cell-free fluid. Nuclei of

infected cells exhibited viral antigens detectable by

immunofluorescence. Electron microscopy revealed a viral

core 35 to 45 nm in diameter enveloped by an outer membrane

covered with surface projections. The complete virion had

an overall diameter of 90-115 nm.










A feline syncytium-forming virus was isolated from

normal cats and cats with a variety of illnesses such as

feline infectious peritonitis, neoplasms and urolithiasis

(12, 13, 16, 19, 23, 27). Kasza et al. (23) isolated a

syncytium-forming agent from a sarcoma of a cat in an

established canine melanoma cell line. Importantly, they

observed that no cytopathology was evident in these cell

cultures in the first passage. After three passages by sub-

cultivation, 50 to 80% of their cells became involved in

syncytium-formation. The authors pointed out that the

cytopathic effect appeared most rapidly when cell monolayers

were infected before they were completely confluent.

Achong et al. (1) recovered a human foamy agent from

a nasopharyngeal carcinoma. The virus was shown to

share all the major characteristics of the syncytium-

forming viruses of the other species (8). It was not

neutralized by antisera to any of the known animal

foamy agents demonstrating that it is immunologically

distinct. Its cytopathogenicity was inhibited by addi-

tion of BUDR to culture fluids suggesting a DNA re-

plicative stage. By electron microscopy, the virus

could be seen to mature by entry of a preformed nucleoid

into a bud at the cell membrane. The completed virions










were covered with radiating spikes. The authors remark

that the morphology and morphogenesis of their virus was

very similar to that of the simian, bovine, and feline

agents. Since these viruses were unassignable to any

established morphological family, Epstein et al. (8)

suggested that they could represent members of a new group.

Parks and Todaro (25) have pointed that the syncytium-

forming viruses of feline, bovine and simian origin are

members of a unique group. Although all are able to cause

dilation of vacuoles producing a foamy appearance in the

cytoplasm of infected cells the authors state that "the

syncytium-forming ability of these viruses appear to be

a more characteristic manifestation of their cytopathology"

(p. 676). Table I lists the known established syncytium-

forming viruses. (A rabbit syncytial virus has been

described but not definitively characterized.) A common

group antigen has not been demonstrated.










Table I

Foamy Viruses (17)



Simian foamy viruses (9 serotypes)

Bovine syncytium-forming virus

Feline syncytium-forming virus

Hamster foamy virus (9)

Human foamy virus




The syncytium-forming viruses have a buoyant density

of 1.16 to 1.18 g/cm3 and have been shown to be sensitive

to chloroform and ether (8, 16, 17, 25). Low concentrations

of bromodeoxyuridine (BUDR) in culture fluids markedly

decrease virus yield (8, 25). Parks et al. (26) have shown

incorporation of tritiated uridine but not thymidine with

simian serotype 3. Furthermore, uridine incorporation is

inhibited by actinomycin D (26). This is a characteristic

of the retroviruses which go through a DNA replicative

intermediate stage via reverse transcriptase. The simian,

bovine and feline agents have been shown to have reverse

transcriptase activity (17, 26, 29), resulting in their

classification as a subfamily of the Retroviridae (3).










A plaque-forming assay was developed for simian foamy

viruses Types I and III but the same method proved un-

successful with the bovine agent (6). Generally syncytium-

forming viruses replicate to very low titers and require

several passages of infected cells before the cytopathic

effect becomes evident (17, 25). Park and Todaro (25) aptly

remark that biological studies are hindered by their low

level of infectivity, marked liability and lack of a quan-

titative in vitro assay procedure.

The foamy viruses seem to be ubiquitous in nature and

cause persistent infection in their hosts. For example,

Hackett et al. (16) reported isolation of feline syncytium-

forming virus (FeSFV) from 90% of cats in their laboratories

during the first four months of 1969. Lymphocytes have

been shown to be infected in the case of bovine agent (14,

24). A characteristic host response to infection by these

agents is formation of virus-specific precipitating anti-

bodies. Gaskin and Gillespie (12) working with FeSFV have

shown that the presence of precipitating antibodies is

indicative of chronic infection. Malmquist et al. (24)

have demonstrated the same with the bovine agent.

The syncytium-forming viruses infect a wide range of

cells in vitro. They propagate in epithelial and










fibroblastic cells of numerous mammals tested. Parks and

Todaro (25) and Hooks and Gibbs (17) list the characteristics

of the syncytium-forming (foamy) viruses as follows:

1. Wide in vitro host range for cell species and
cell types.

2. Syncytium formation as characteristic in vitro
cytopathic effect.

3. Inhibition of replication by BUDR and dactinomycin.

4. Necessity for actively replicating cells in order
to initiate infection.

5. Multiplication to low levels of infectivity
(102 to 105 TCID50/ml).

6. Virion--associated reverse transcriptase.

7. High resistance to inactivation by ultraviolet
light treatment.

8. Production of viral antigen in the nuclei of
infected cells detectable by immunofluorescence.

9. By electron microscopy, have nucleoids 30-35
nm in diameter which are usually seen in the
perinuclear cytoplasm. Are 100 to 140 nm in
diameter after budding through the plasma
membrane with no clearly observable symmetry.
The enveloped virions are covered with surface
projections or spikes.

10. No cellular transformation in vitro or tumor
production in vivo yet observed.

Work with the foamy viruses of various species has

shown that they are common in nature, cause persistent in-

fections and persist in the leukocytic cells of their host.










Scott (30) concluded that FeSFV was a nuisance to virologists,

oncologists, and vaccine producers working with feline cells.

Hackett and Manning (15) point out that since this agent

is strongly cell-associated and replicates to such low

titers it can easily be overlooked. It is now feared that

some of the early poliovirus vaccines were contaminated with

simian foamy viruses since neutralizing antibodies to SFV

type III were detected in one out of 20 sera of human

vaccinees checked (22). There should be a concerted effort

to uncover and characterize these viruses so that virolo-

gists, cancer researchers, and vaccine producers can be

made aware of them.

This dissertation is concerned with the characteriza-

tion of a previously undescribed syncytium-forming virus

of raccoons (RSFV) and establishing that it belongs to the

newly recognized subfamily Spumavirinae (foamy agents) of

the RNA virus family Retroviridae. A serological survey

of Florida raccoons was carried out to determine the-fre-

quency of occurrence and geographical distribution of the

virus.















III. MATERIALS AND METHODS


A. Laboratory Animals and Sera


Raccoons used in this study were obtained from the

Florida Game and Fresh Water Fish Commission and the

National Wildlife Refuge Service and from a local supplier.

They were caged individually.

Serum samples were obtained from anesthetized animals

by jugular venipuncture using sterile disposable syringes

or Vacutainer needles and tubes. The blood was then trans-

ferred to plastic centrifuge tubes and allowed to clot.

The clot was then reamed from the walls of the tube with a

glass rod and left to recede overnight at 40C. The serum

was collected after centrifugation at 2000 rpm for 20

minutes in a refrigerated centrifuge.

Raccoons which were to be euthanatized for micro-

biologic examination of their tissues were anesthetized

with an appropriate dose of ketamine hydrochloride. Death

by exsanguination was then achieved by cardiac puncture,

generally by use of a sterile 30 ml syringe and 18 gauge

1 inch sterile disposable needle. After careful and










asceptic opening of the carcass, various organ and tissue

samples were placed in sterile disposable petri dishes for

transport to the tissue culture facility. Swab samples of

various body orifices were also taken for virus isolation

attempts.

Rabbits were obtained through the facilities of the

College of Veterinary Medicine. Three animals were experi-

mentally infected with the raccoon virus. Pre-inoculation

and subsequent blood samples were obtained by stylet

puncture of a marginal ear vein. Serum was obtained as

described above. Rabbit anti-RSFV serum was used in

indirect immunofluorescence tests with fluorescein-labeled

goat anti-rabbit globulin serum.

Raccoon sera collected from throughout the state of

Florida were made available by Dr. William Bigler of the

Florida Department of Health and Rehabilitative Services

in Tallahassee, Florida.


B. Cells and Media


Cell cultures used in this study include the estab-

lished cell lines Statens Seruminstitut Rabbit Cornea

(SIRC), Baby Hamster Kidney (BHK) and Crandell Feline Kidney

(CrFK). In addition, primary cultures of rabbit kidney










and kitten lung cells were prepared in the laboratory using

standard trypsin digestion methods. For storage, heavy

suspensions of these cells in growth medium with 10%

dimethyl sulfoxide were dispensed into individual 1 ml

glass ampoules and flame-sealed. The ampoules were then

placed in alcohol bath and cooled to 4C for twenty minutes.

They were transferred to a -20 C freezer for thirty minutes

and then to a -700C freezer for one hour. After this time,

the ampoules were removed from the alcohol bath and stored

in liquid nitrogen for subsequent use.

Eagle's Minimal Essential Medium supplemented with

10% fetal calf serum and 0.5% lactalbumin hydrolysate

was used for growing cell cultures. Penicillin (100

units/ml) and streptomycin (100 ug/ml) were also added

to the medium.


C. Virus Isolation


1. Direct cell cultures: Aseptically collected

tissue samples were finely minced, washed with isotonic

phosphate buffered saline and subjected to .25% trypsin

digestion for 45 minutes while being magnetically

stirred. The tissue digests were filtered through

gauze-wrapped funnels to get rid of larger pieces and










centrifuged for 15 minutes at 900 gs. The supernatants

were discarded and the pellets resuspended in growth media.

The cell suspensions were dispensed into culture flasks.

Cultures initiated in that manner were serially

transferred at least three times as soon as they would

become confluent. Passage was accomplished by trypsin-

versene dispersion of the cells, resuspension into fresh

culture media and seeding into new culture flasks.

With each transfer, companion Leighton tube cultures

were initiated. After the cells had grown-in the cover

slips were removed and subjected either to Giemsa staining

or indirect immunofluorescent staining. For Giemsa staining,

the cover slips were placed on racks, rinsed with isotonic

phosphate buffered saline, fixed for 10 minutes in methyl

alcohol, stained with 10% Giemsa for 20 minutes, dehydrated

in acetone and xylene, and mounted on glass slides for

microscopic examination. Indirect immunofluorescence

techniques are detailed in a later section (III, F, 2).

Cover slip preparations were found to be the best way

to monitor infection and cytopathic effect of the

virus.










2. Tissue suspensions: Portions of each tissue

collected were minced and ground with Ten Broeck tissue

grinders. The supernates were decanted, centrifuged to get

rid of debris and stored at -70 C. For assay of viral

content, 1 milliliter of the preparations was inoculated

into growing cultures of SIRC or BHK cells. Such cultures

were passed at least three times and accompanying Leighton

tube cultures were prepared. After the cells grew to

confluency, the cover slips were removed and stained as

described above.


3. -Inoculation of buffy coat cells into established

cell lines: Blood collected aseptically into 10 ml

syringes was quickly ejected into flasks and swirled to

promote defibrination and prevent clotting. The defi-

brinated blood was centrifuged at 2000 rpm for 10 minutes.

Pasteur pipettes were used to aspirate buffy coat cells

from the top of the pellet. These were immediately

inoculated into growing cultures of SIRC or BHK cells.

After 24 hours at 370C, the growth media were decanted and

the monolayers washed. Fresh growth fluid was then added.

After reaching confluency, cultures were passed with com-

panion Leighton tubes and monitored for infection as

described above.










4. Swab samples: Two milliliters of growth medium

were dispensed into sterile screw-cap tubes. Cotton swabs

on wooden applicator sticks were used. Prior to obtaining

a sample, a sterile swab was moistened with medium and

then used to vigorously swab the appropriate area: throat,

nasal, rectal or vaginal area. The swab was then placed

in one of the tubes containing the growth medium, and the

applicator stick broken off the wall of the tube. The

samples were stored at -70C until virus recovery was

attempted.

To check for viral content, each sample was filtered

through a .3 micron filter and a .2 ml volume inoculated

into growing cultures of BHK or SIRC cells. The cultures

were passed at least three times with companion Leighton

tube. The presence of virus was checked by Giemsa or

indirect immunofluorescence as previously described.


D. Stock Virus Preparation


Two virus stocks were used, one grown in BHK cells

and one grown in SIRC cells. The stocks were prepared by

inoculating .5 ml of virus isolate into cultures in which

the cells had not reached confluency. After the cells had

grown in, blind passages were performed by subcultivation










of the cultures. Companion Leighton tube cultures were

initiated with each transfer. When the cells were grown

in, the cover slips were removed and stained by immuno-

fluorescence. When 60% or more of the cells became infected,

usually after 2 or 3 transfers, the cultures were frozen,

thawed and centrifuged at 2000 rpm for twenty minutes.

Freeze-thawing was done to release some of the cell-

associated viruses. The supernatant growth medium con-

taining cell-free virus constituted the viral stock pre-

paration and was conserved at -70 C.


E. Viral Procedures


1. Plaque assay: The method of Parks and Todaro

(25) was followed. BHK cells were seeded into 50 mm petri

dishes. After 24-48 hours, before the cells reached con-

fluency, the drained monolayers were inoculated with the

appropriate viral dilutions. Each dilution was done in

quadruplicates. The virus was allowed to adsorb for a

2 hour period at 370C; fluid medium was then replaced.

After a further 24 to 48 hours incubation, 1% agar in

growth medium was applied as an overlay. Seven days

later, a second overlay with a 1 to 10,000 dilution of

neutral red was added and after another 24 hours in the










dark, plaques were counted using indirect lighting to

enhance visualization.


2. Immunofluorescent assay: BHK cells were seeded

into Leighton tubes and inoculated with doubling dilutions

of virus preparation. The first dilution was a ten-fold

dilution which was serially doubled, usually up to a

dilution of one in 2560. Four Leighton tube cultures were

used for each dilution. After a five-day incubation period

at 370C, the cover slips were removed and subjected to the

indirect immunofluorescence procedure using rabbit anti-

serum to the virus and fluorescein isothiocyanate (FITC)

labeled goat anti-rabbit immunoglobulin serum. The last

dilution at which specific fluorescent staining was observed

was considered the end-point titer. The titers were cal-

culated according to the appropriate dilution and expressed

as fluorescent Focus-Forming Units (FFU) (10).


F. Serologic Methods


1. Immunodiffusion


a. Antigen preparations: BHK cells were grown

in one liter bottle in a roller apparatus. One hundred

milliliters of medium were sufficient to grow the cells to










confluency when the bottles were rotated at .5 revolutions

per minute. With the roller method, greater quantities of

cells could be grown more economically than with culture

flasks. The bottles were inoculated at an early stage with

2 milliliters of viral stock. When 60% or more of the

monolayer consisted of syncytia (usually after two passages

by subcultivation) the cells were scraped from the bottle

wall with a rubber policeman and centrifuged (900 gs for 20

minutes) in the medium in which they were grown. The super-

natant fluid was decanted, the centrifuge tubes drained and

the cells resuspended in the small volume of medium still

remaining. The concentrated suspension of infected cells

was frozen and thawed several times and then centrifuged

again at 900 gs for 20 minutes. The opalescent supernatant

material constituted the precipitating antigen. It was

stored at -20C until needed. Each liter bottle gave

about .4 ml of antigen.

Antigen was also prepared by direct viral concentra-

tion. One hundred milliliters of viral stock preparation

were centrifuged at 25,000 rpm for 3 hours to pellet the

virus. The viral pellet was then resuspended in .2

milliliters of TNE buffer (.01 M Tris-HCl, .1 M NaC1,

.005 M EDTA). Some viral preparations were treated with









chloroform prior to concentration in order to eliminate

envelope structures from the antigen preparation.


b. Method: Immunodiffusion tests were carried

out in a micro-diffusion system in order to conserve

antigen. The method developed by Crowle (6) using plexi-

glass templates to hold the reagents was used.

One per cent Noble agar dissolved in borate buffer

(pH 8.5) with 1:10,000 merthiolate was used to fill the

space between a plexiglass block and a glass slide. The

space was created by supporting the block on each side by

three layers of electrical tape. The layer of agar thus

formed was about 1 mm thick. After allowing the agar to

solidify, the block was removed and replaced by a template.

The wells were filled (.05 ml) using 1 ml syringes with

22 gauge needles.

The slides were then held at room temperature for

36-72 hours in a humidified atmosphere created by putting

them in glass petri dishes containing wet filter paper.

At the end of the time, the templates were carefully

removed and the precipitin lines read. The preparations

were then soaked in PBS for 24 hours and then in distilled

water for another 24 hours to remove unreacted reagents.










They could then be photographed by indirect lighting or

dried and stained with Buffalo black solution for permanent

records.


c. Antisera: The antisera used for precipi-

tation were those of raccoons and experimentally infected

rabbits. Usually the antigen was placed in the center well

and the sera to be checked in the surrounding wells.

Templates with one central well and six or four surrounding

wells were used.


2. Immunofluorescence


a. Preparation of FITC-labelled conjugates:

Goat anti-rabbit globulin serum was already available in

the laboratory. Rabbit anti-raccoon globulin serum was

prepared specifically for this study. Thirty milliliters

of raccoon serum was precipitated at 50% and 33%

saturation with ammonium sulfate and dialysed against

PBS (pH 7.5).

A spectrophotometer was used to evaluate the protein

content and the preparation was diluted to 1 mg/ml with PBS.

A rabbit was immunized with the preparation. One

milliliter emulsified in an equal volume of Freund's










complete adjuvant was injected I.M. in the "hamstring"

muscles. Three weeks later it was bled and its serum

checked for precipitating activity with raccoon globulin.

One week later 1 mg of raccoon globulin preparation was

injected I.V., and after another week the rabbit was

anesthetized and exsanguinated by cardiac puncture.

Goat anti-rabbit and rabbit anti-raccoon serums were

conjugated with FITC in the manner described in Appendix I.

The goat-rabbit indirect system was used to detect and quan-

titate infection in cells grown in Leighton tubes. The

rabbit anti-raccoon conjugate was used with known infected

cover slips to detect the presence of antibody to RSFV in

raccoon serums.


b. Staining procedure: Infected cover slips

from Leighton tubes were removed and placed in staining

racks. The racks were then rinsed in phosphate-buffered

saline and fixed in acetone. The cover slips were allowed

to dry and placed on glass slides. With a Pasteur pipette

a thin layer of the anti-viral serum was applied on the

cover slips. They were then incubated for one hour at 370C

in humidified glass petri dishes. The slips were then

removed from the glass slides, placed back in the racks










and washed with 2 changes of stirred PBS. The racks were

rinsed in distilled water after which the cover slips were

allowed to dry and placed again on glass slides. The FITC

labeled antiserum was layered on them. The preparations

were again incubated for an hour at 37C in humidified petri

dishes. After this time the cover slips were removed,

reinserted in racks and the washing process with PBS and

distilled water repeated. They were then dipped 3 times

in eriochrome black solution as a counter-stain and rinsed

in two changes of distilled water. The backs of the cover

slips were dried with tissue paper and they were mounted,

cell side down, with 10% buffered saline in glycerine on'

glass slides. The preparations were then examined for

specific fluorescence using dark-field, ultraviolet light

microscopy.


3. Neutralization: Sera from raccoons and from

experimentally infected rabbits were tested for neutralizing

activity. All sera checked for neutralizing antibodies were

inactivated for 30 minutes at 560C. Doubling dilutions of

the serum to be tested were made. Each dilution (1 ml) was

incubated with an equal amount of viral stock containing

200 FFUs. The mixtures were incubated for 1 hour at 370C.









Then .2 milliliter of each dilution was added to a set of

four Leighton tubes containing 1.8 ml of BHK cell suspension

at a concentration of 105 cells/mi. The tubes were placed

in racks and incubated 5 days at 370C. At the end of this

period, the cover slips were removed and stained by indirect

immunofluorescence using the goat-rabbit reagents. The

reciprocal of the last dilution at which no specific stain-

ing could be seen was considered the titer.


G. Sensitivity to Lipid Solvents


The method of Andrewes and Horstmann (2) was followed.

A mixture of 13 parts virus in MEM and 1 part anhydrous

ether was kept at 40C for 16 hours with periodic vigorous

shaking. The ether was then decanted, residuum evaporated,

and titrations done by immunofluorescence.

The effect of chloroform was tested using 1 part chloro-

form and 19 parts viral culture (4). The preparation was

also incubated for 16 hours at 40C with intermittent shaking.

The virus culture was then decanted and titrated.


H. Viral Replication Studies


1. Effect of BUDR: The method of Parks and Todaro (25)

was used. Two growing cultures of BHK cells were inoculated

with 2 milliliters of viral stock preparation. After a 1-2








hour adsorption, medium alone or medium with BUDR (1. ug/ml)

was added and incubated in the dark. Twenty-four hours later,

the culture fluids were removed, the monolayers washed 3 times

with media and fresh medium added. At 48 hours post-

inoculation, virus was harvested by freezing and thawing

the infected cultures. They were then centrifuged at 900 gs

for 20 minutes to pellet cell debris. The supernatants were

then assayed by the indirect immunofluorescence technique.


2. Uridine uptake and effect of Actinomycin D: The

effect of actinomycin D on tritiated uridine uptake was stu-

died as described by Parks et al. (26). BHK cells at ad-

vanced stages of infection were incubated for 24 hours with

30 uCi/ml of 3H-thymidine, 3H-uridine alone, and 3H-uridine

plus actinomycin D (2 ug/ml). The actinomycin D was added 4

hours prior to the label. The cultures were freeze-thawed;

the supernatant fluids were clarified by centrifugation

(900 gs, 20 minutes) and precipitated with saturated ammonium

sulfate. The precipitate was redissolved 1.5 ml of TNE buffer

and .2 ml of this preparation was layered on top a 15-55%

sucrose gradient and centrifuged at 200,000 g for 2 hours.

Collected fractions were counted in scintillation fluid in

a liquid scintillation counter.








I. Reverse Transcriptase Assay


Reverse transcriptase assays were performed on virus

preparations concentrated one hundred fold. Two hundred milli-

liters of virus stock were centrifuged at 75,000 g for 3 hours.

The pellet was then resuspended in 2 milliliters of TNE

buffer. The assays were performed under conditions described

by Parks et al. (26). The viral concentrate was filtered

through a .3 micron filter and varying amounts (10, 20, 30

and 50 lambdas) were incubated for 2 hours with 50 lambdas of

a reaction mixture containing 40 mM tris HCl, 60 mM KC1, 2 mM

MnC12, 2 mM of dithiothreitol, 20 uM of 3H-TTP, 5 mM of each

dATP, dCTP, dGTP, and .2% triton X-100. To each reaction,

1.25 x 10-3 mg of the synthetic nucleic acid template rA:dT

was added. After incubation, .1 ml of "carrier" salmon sperm

DNA (2.5 mg/ml), .1 ml of .1 M PPi and .5 ml of cold 3.5%

perchloric acid-0.35o uranyl acetate were added per .1 ml

of reaction mixture. After 10 minutes at 00, the resulting

precipitate was collected on a Whatman glass filter (GF/C

2.4 cm) and washed 3 times (10 ml portions) with cold 1 N

HC1, 3 times with 1 N HCl-0.1 M PPi and once with absolute

ethanol. The filter was dried and its radioactivity de-

termined. The absolute requirement for manganese was checked

by carrying out the reactions with magnesium ion instead.









The effect of decreasing Triton X-100 concentration to .02%

was also determined. The absolute requirement for man-

ganese was checked by carrying out the reactions with

magnesium ion instead. The effect of decreasing Triton

X-100 concentration to .02% was also determined.

Viral concentrate was layered on top of a 15-60%

sucrose gradient and centrifuged at 200,000 gs for 2 hours.

Fractions were collected by bottom puncture and assayed

directly for polymerase activity. The per cent sucrose of

each fraction was read in a refractometer and the relative

buoyant densities determined.


J. Electron Microscopy


Infected SIRC cells were examined. The cells were

fixed with 3% buffered glutaraldehyde and 1% osmium tetroxide.

The preparations were then dehydrated stepwise in 50, 70,

80, 95 and 100% ethanol and finally with absolute propylene

oxide. The resulting tissues were embedded in epoxy resin

in gelatin capsules which were cured for 3 days at 450C.
0
Sections of tissue 200 to 500 A thick were cut from

the resin using a Porter Blum ultramicrotome manufactured

by Ivan Sorvall, Inc. The sections were placed on grids,

stained with uranyl acetate and lead citrate, and viewed.















IV. RESULTS


A. Virus Isolation


The raccoon syncytium-forming virus (RSFV) was first

isolated from various organs of raccoons captured on Marco

Island, Collier County. Four animals were received from

the Florida Game and Fresh Water Fish Commission. They

were euthanatized by exsanguination via cardiac puncture

and their sera collected. Primary cell cultures were de-

rived from spleen, kidney, lung and greater momentum. The

cells grew normally to confluency in the tissue culture

flasks without any noticeable abnormalities.

After 2 to 3 passages by subcultivation definite

cytopathic effect manifested by syncytia formation became

evident in the organ cultures of 3 of the 4 raccoons.

Affected cells became rounded and more refractile in

appearance by light microscopy. Simultaneously with these

changes, vacuoles developed in the cytoplasms of these

cells giving a foamy appearance under low powered magnifi-

cation. In affected areas, cells lost their regular

orientation in the monolayer. These changes were presumed










to be due to the presence of a latent virus. Its cytopathic

effect closely paralleled that of the syncytium-forming

viruses of simian, bovine and feline origins.

To confirm this hypothesis, cells from cultures which

exhibited these manifestations were co-cultured with the

established cell lines SIRC and BHK. After one subculti-

vation, syncytia formation appeared. Upon observation over

a period of time, progressive involvement of cells in

syncytia formation could be seen. Further passage resulted

in a sharp increase of fused and vacuolated areas (see

Figs. 1, 2 and 3). More nuclei could be seen accumulating

into syncytia through a process of incorporation of adjacent

cells. With longer incubation, some of the large multi-

nucleated cells would become detached from the bottom of

the culture flask leaving irregularly shaped microscopic

plaques in the monolayer. Infected cultures could not be

transferred beyond third or fourth passage since this

resulted in total cell destruction.

Companion Leighton tube cultures were initiated at

the time of each transfer. After the cells were grown in,

the cover slips were removed and stained with 10% Giemsa

(see Figs. 4 and 5). Syncytia with varying degree of

multinucleation could be seen in these stained preparations
















Fig. 1. Uninfected BHK cells grown in MEM supple-
mented with 10% calf serum (Magnification
X100).


























Fig. 2. Second passage BHK cells inoculated with RSFV.
Large refractile giant cells can be seen
along with an area of cell lysis. Cells have
lost their regular orientation in the mono-
layer.






32



































































i,
r;-r~ 4 ` jy;Jy-~ ~ \

















Fig. 3. RSFV-infected BHK cells. Irregularly shaped
microscopic plaque surrounded by large syncytial
structures that have rounded up and are about
to become detached from the bottom of the cul-
ture dish.


























Fig. 4. BHK cells grown in a Leighton tube. The cover
slip was removed, fixed and stained with 10%
Giemsa.













(see Fig. 6). The nuclei involved could be seen evenly

distributed in the cytoplasm, clustered in certain areas,

or in ring formation around the peripheral cytoplasm.

Since a characteristic host response to infection by

syncytium-forming viruses is the production of precipitating

antibodies, viral antigen was extracted from infected cells

to check various sera for precipitating activity. A con-

centrated lysate of infected cells constituted the pre-

cipitating antigen. In order to conserve antigen tests

were performed employing a microsystem developed by Crowle

(6). The sera of animals from which the virus could be

isolated had precipitating activity with the antigen. In

contrast no virus could be isolated from blood or any of the

organs of raccoons whose sera showed no line of precipitation

(see Fig. 7). Thus, a procedure was developed by which

raccoons could be quickly screened for infection with

RSFV.

Persistent infection of a rabbit with RSFV was pro-

duced by administering infected SIRC cells intravenously.

A preinoculation bleed showed that the animal's serum

contained no activity against the viral antigen. A month

later a second bleed was performed and precipitating

antibodies were demonstrated in the serum. Precipitating




















Fig. 5. RSFV infected BHK cells after second passage
(Magnification X100). Cells were grown in a
Leighton tube and the cover slip was stained
with 10% Giemsa.






















Fig. 6. RSFV-induced syncytia in infected BHK cells.
Varying degrees of multinucleation can be
seen in giant cells (Magnification X200).













activity persisted for over a year (see Fig. 8). Peri-

pheral blood leukocytes (buffy coat) of the rabbit were

inoculated into growing SIRC cell cultures. The latter

developed characteristic CPE after subcultivation and

totally degenerated upon further passage. Furthermore,

antigen extracted from SIRC cells infected with the rabbit

buffy coat precipitated with positive raccoon sera, demon-

strating the animal to be persistently infected with RSFV.

Two raccoons were received from the National Wildlife

Refuge on Merritt Island. Both were bled and their sera

checked for antibodies to RSFV by immunodiffusion and

immunofluorescence and both were negative. Throat, nasal,

rectal and vaginal swabs and buffy coat cells from each

raccoon were inoculated into growing cultures of SIRC cells.

After 3 passages, the cover slips from companion Leighton

tubes were stained by immunofluorescence. All were negative.

The raccoons were killed by exsanguination and primary

cultures were initiated from spleen, momentum, kidney and

lungs. After 3 passages no CPE was observable and immuno-

fluorescence performed on companion cover slips proved

negative. Also, suspensions from each of the organs were

inoculated into SIRC cells with no effect after 3 passages.

These experiments reinforced prior observation that the



















Fig. 7. Immunodiffusion using antigen extracted from
infected cells. The antigen was placed in the
center well and various raccoon sera in the
six surrounding wells.
Sera 1, 2 and 4 gave 2 lines of precipitation
Serum 5 produced one band
Sera 3 and 6 did not react


Fig. 8.


Detection of precipitating antibodies to RSFV
antigen in rabbit infected by injection of
infected SIRC cells intravenously. The rabbit
serum was placed in well 1 and shows a line
of identity with positive raccoon sera in
wells 2 and 6. The preparation was stained
with Buffalo black.








40
















Fig. 9. Immunodiffusion with untreated and chloroform
treated antigen. Positive raccoon serum (75-1)
was placed in the center well.
1--Antigen prepared from infected BHK cells
2--Concentrated virus
3--1 treated with chloroform
4--Concentrated virus preparation from viral stock
preparation treated with chloroform prior to
concentration.



















Fig. 10. Immunodiffusion with antigen prepared from
infected BHK cells.
1--Rabbit injected with infected SIRC cells
2--Rabbit receiving 2 ml of cell-free virus
stock prepared from infected BHK cells
3--Raccoon serum 75-1
4--Same as 2
5--Same as 3
6--Rabbit infected by injection of 5 ml of whole
blood from rabbit 1.








42










presence of precipitating antibodies in serum could be

used as a presumptive test for viral infection.

Table II shows tissues from which virus was recovered.



Table II

Tissues from Which RSFV Was Recovered



Spleen

Greater momentum

Kidney

Lungs

Peripheral blood leukocytes




B. Antigen Preparation


As mentioned above, RSFV antigen for immunodiffusion

was prepared by freeze-thawing very heavy suspensions of

infected cells and collecting the lysate. This is the

method described by Malmquist et al. (24) with the bovine

agent and Gaskin and Gillespie (12) with FeSFV. Treating

the RSFV antigen with chloroform did not destroy its

precipitating property (see Fig. 9). This suggests that

there is no lipid in the antigenic sites involved.









Attempts were made to prepare RSFV antigen by concen-

trating viral stock preparations. Five hundred-fold concen-

trations were made by ultracentrifugation. Viral core con-

centrates were also prepared by treating virus stock with

chloroform prior to ultracentrifugation. Neither viral

nor chloroform treated viral concentrates precipitated with

RSFV antisera (see Fig. 9).

The number of precipitation lines seen in gel diffusion

depended both on the serum and the antigen preparation.

Some sera produced two precipitin lines and others only one

with the same antigen. When two bands were seen, one was

usually quite pronounced and the other weak and diffuse.

The major band was in identity with that of sera produc-

ing only one line of precipitation. With some antigen

preparations, however, only the major band was produced no

matter what antisera were used. The same phenomenon was

observed by Malmquist et al. (24) who developed the system

for the bovine agent. This was presumed to be due to a

concentration effect.


C. Experimental Infection


As mentioned in section A, chronic infection of a

rabbit was achieved by administering infected SIRC cells










intravenously. Serum precipitating antibodies have per-

sisted for over a year and virus could be routinely re-

covered from its peripheral blood leukocytes.

When 5 milliliters of whole blood from the infected

rabbit was injected into a marginal ear vein of a second

rabbit, circulating antibodies to RSFV antigen could be

detected five weeks later. By immunodiffusion, lines of

identity with the donor rabbit serum and positive raccoon

serum could be seen (see Fig. 10).

A third rabbit was infected with 2 milliliters of

cell-free viral stock preparation (grown in BHK cells) which

was injected intravenously. A month later, its serum showed

a very weak line of precipitation against RSFV antigen.

This line was in identity with precipitin lines formed by

other anti-RSFV sera (see Fig. 10). Another precipitation

band was also observed. Since the virus stock was prepared

by freeze-thawing BHK cultures to release intracellular

virus, the preparation also contained cellular and medium

components. Therefore, the rabbit's serum was tested

against antigen prepared with uninfected cells and formed

a precipitin band indicative that the rabbit had responded

to at least one antigen of non-viral origin.










Twenty-five days after the first bleed, a second

serum sample was taken. This sample no longer had pre-

cipitating activity against BHK antigen but gave a strong

line of identity against RSFV antigen when tested with

other positive sera (see Fig. 11). The persistence of

such antibodies is indicative of chronic infection. There-

fore, it was found that infection could be initiated with

infected cells, whole blood from an infected donor and

with cell-free virus.


D. In Vitro Host Range


The syncytium-forming viruses replicate and induce CPE

in numerous cell lines from a variety of mammalian hosts.

The viruses productively infect epithelial and fibroblastic

cells. The raccoon isolate was shown to infect primary

cell cultures of rabbit kidney and kitten lung and the

established cell lines SIRC, BHK and CrFK.

In each cell culture system, it was necessary to

inoculate growing cultures in order to observe infection.

No CPE could be readily detected in the monolayers until

they were subcultivated. Kitten lung and BHK cells proved

to be more susceptible to viral induced CPE than the others

since syncytia and areas of cellular destruction appeared










more rapidly. The infected kitten lung cells rarely sur-

vived a second transfer. None of the infected cells tested

could be transferred beyond 4 passages.

With each subcultivation, companion Leighton tube

cultures were initiated. When the cells were grown in, the

cover slips were removed and stained by indirect immuno-

fluorescence using rabbit immune serum and FITC labeled

goat anti-rabbit globulin serum. Large syncytia with intense

nuclear staining could be seen (see Figs. 13 and 14). The

reddish appearance of control cells is due to the erio-

chrome black counterstain (see Fig. 12). There was a ten-

dency of nuclei involved in giant cell formation to arrange

themselves uniformly around the periphery of the cytoplasm.

Table III shows that all cells showing foamy degeneration

exhibited viral antigens detectable by immunofluorescence.


Table III

Host Range of RSFV


Cell Types Indirect CPE
Tested Immunofluorescence

SIRC + +
BHK + ++
RK + +
CrFK + +
KL + +++


















Fig. 11. Immunodifussion showing line of identity
produced by the sera of three rabbits, each
infected by a different method as indicated
in Fig. 10.
























Fig. 12. Indirect immunofluorescence on uninfected
BHK cells. The red appearance is due to
the eriochrome black counterstain.













49











































- -















Fig. 13.


Indirect immunofluorescence on infected SIRC
cells. Rabbit antiserum to RSFV and FITC-
labeled goat anti-rabbit globulin serum were
used to stain Leighton-tube cover slips.
Syncytia with varying degree of multinucleation
can be seen. Note the intense nuclear stain-
ing.


Fig. 14. Indirect immunofluorescent staining of RSFV
infected primary rabbit kidney cell culture.
Nuclei in ring formation around the periphery
of the cytoplasm can be seen.





























~~










E. Cytopathology and Assay of RSFV


Parks and Todaro (25) have pointed out that the lack

of adequate quantitative assay procedures has hindered the

study of the foamy agents. They described a plaque assay

system which proved successful with simian foamy virus

Types I and III but not with the bovine agent. This method

consisted of the application of a 1% agar overlay 24 to 48

hours after inoculation of the virus into 50-mm petri

dishes. Seven days later a second overlay with a 1 to

10,000 dilution of neutral red was added and plaques

counted 24 hours later. This method did not succeed with

RSFV. A.3% ion-agar overlay, followed by removal of the

agar 7 days later and staining with crystal violet was

also unsuccessful.

The problem was similar to that encountered by Parks

and Todaro (25) with the bovine agent in that most of the

plaques that did form were not large enough to be counted

microscopically and not all foci of infection developed

plaques. By performing titrations in Leighton tubes and

then staining the cover slips with Giemsa or by indirect

immunofluorescence, an appraisal was made of the cytopathic

properties of RSFV. The number of macroscopically visible










plaques was less than 50% of the plaques that could be

detected microscopically (see Fig. 15). In addition, the

total number of plaques did not accurately reflect the

number of infectious units inoculated into the system.

Giemsa staining demonstrated many syncytia where no plaque

was apparent. That these syncytia were of viral origin

was confirmed by immunofluorescent staining (see Fig. 16).

Immunofluorescence also showed another characteristic of

RSFV: cells containing viral antigens were not always

involved in syncytia formation or any other manifestations

of CPE (see Fig. 17).

Some simian foamy viruses have been reported to in-

duce a carrier state in certain cell lines (5). This was

attempted with RSFV, but after repeated passages (usually 2

to 4) all cells tested were ultimately destroyed.

It was found that the most accurate way to quantitate

the virus was by an immunofluorescent end-point assay.

Different preparations of viral stocks (prepared by freeze-

thawing infected cultures to release cell-associated virus

and collecting the supernatants) were found to contain

from .8 to 3.2 x 103 fluorescent Focus-Forming Units (FFU)

per ml.


















Fig. 15. Microscopic plaque surrounded by large multi-
nucleated cells as seen after Giemsa staining
of BHK.
























Fig. 16. Indirect immunofluorescent staining of in-
fected cover slip showing many syncytia with
no area of cell lysis.









55















.
... ... ". ::






















































Fig. 17. Indirect immunofluorescence revealing
infected cells without any apparent
cytopathic effect. In this preparation
the infected BHK cells show nuclear
and cytoplasmic staining.










F. Viral Neutralization


Neutralizing antibodies have been found in sera of

hosts infected by the foamy agents. Malmquist et al. (24)

and Gaskin and Gillespie (12) have shown that precipitating

antibodies were indicative of chronic infection with the

bovine and feline agents. Gaskin (11) has also shown pre-

cipitating antibodies could be demonstrated in the absence

of neutralizing antibodies.

Neutralization tests were carried out with five raccoon

sera that were checked for precipitating activity. Indirect

immunofluorescence on infected cover slips using rabbit

anti-raccoon globulin conjugate was also carried out to

detect the presence of anti-RSFV antibodies. Table IV

shows the result of these experiments. They confirm that

the precipitin test is a very reliable method to screen

sera for infection with RSFV.


G. Sensitivity to Lipid Solvents


As with all other enveloped viruses the syncytium-

forming viruses are sensitive to lipid solvents such as

ether and chloroform. As indicated in Table V, treatment

of the raccoon syncytium-forming virus with ether and

















Table IV


Antibodies to RSFV as Determined
by Three Methods


Raccoon Neutraliza- Immuno- Indirect
Number tion Titer diffusion Immunofluorescence

G-75-1 32 + +

G-431 128 + +

G-556 16 + +

70F-2 + +

LR-5-77-1 -

DC-4 (Rabbit) 64 + +










chloroform resulted in total inactivation, indicating

the destruction of essential lipids in the viral envelope.

Since the integrity of the envelope seemed to be essential

for initiating infection, it can be assumed that special

attachment sites on the envelope are necessary for viral

entry into target cells.


H. Viral Replication Studies


1. Effect of BUDR: Low concentrations of bromo-

deoxyuridine in culture fluids have been shown to markedly

inhibit replication of the foamy agents (25). In contrast,

iododeoxyuridine has been shown to have much less of an

effect. Parks and Todaro (25) have suggested that since the

brominated derivative of uridine structurally resembles thymi-

dine more closely than the iodonated derivative, it could

be that the reverse transcriptase of the syncytium-forming

viruses is able to discriminate between the two halogenated

derivatives of thymidine.

The incorporation of 10 ug/ml of BUDR in growth media

completely inhibited the replication of the RSFV. Controls

grown under identical conditions without BUDR gave sub-

stantial yields. Table V shows the results of the

experiment.















Table V

Effect of BUDR on RSFV Replication; Also Shows
Inactivation of the Virus by Lipid Solvents


Virus


Titer


RSFV
Control

RSFV
Grown in BUDR

RSFV treated
with Chloroform


3.2 x 103 FFU*


None detectable


None detectable


None detectable


RSFV treated
with Ether


.8 x 103 FFU


RSFV maintained**
16 hours at 40C


*Titer expressed as fluorescent Focus-Forming Units

**Since the protocol for chloroform and ether treatment
required 16 hours at 40C, the effect of such a treatment
alone was determined.


_ _










2. Uridine uptake and actinomycin D: The foamy

viruses incorporate uridine and not thymidine like all

other RNA viruses. But the incorporation of uridine is

inhibited by actinomycin D which is a characteristic of

the retroviruses. This inhibition led to the belief that

this group of virus go through a DNA replicative inter-

mediate stage and to the postulation of the existence of

the reverse transcriptase.

Parks et al. (26) have shown that the simian foamy

agents incorporate tritiated uridine but not thymidine and

band on a sucrose gradient at a buoyant density of 1.16

g/cm This band did not form if the virus was grown in the

presence of low levels of actinomycin D.

BHK cells infected with RSFV were incubated with

3H-thymidine, 3H-uridine and 3H-uridine plus actinomycin D.

Supernatant fluids containing the labeled virus were con-

centrated and layered on top a 15-55% sucrose gradient and

spun at 200,000 gs for 2 hours. Fractions were then collected

and counted. The virus incorporated the 3H-uridine but not

the 3H-thymidine (see Fig. 18). The peak activity was at

a buoyant density of 1.18 g/cm3 similar to that reported

for the feline agent. The band did not form when virus was

grown in the presence of low levels of actinomycin D

(2 ug/ml).
























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(C-OT) Wd3










I. Reverse Transcriptase Assays


The bovine, feline and some of the simian foamy viruses

have been shown to have RNA-dependent DNA polymerase acti-

vity (17). Supernatant fluids from cultures infected with

RSFV were concentrated one hundred-fold and filtered through

.3 micron filters. Reverse transcriptase assays were

carried out on the viral concentrate under conditions

described by Parks et al. (26). The raccoon agent was

shown to have virion-associated RNA-dependent DNA poly-

merase (see Fig. 19).

Parks et al. (26) also reported that optimal concen-

tration of triton X-100 for demonstration of simian foamy

virus type 3 reverse transcriptase activity was .1 to .2%

instead of .014% as has been reported for murine leukemia

viruses. Therefore the effect of varying the triton

X-100 from .02 to .2% was studied, but was found to be

minimal (Table VI). More characteristic of the virion-

associated reverse transcriptase activity is the require-

ment for manganese ion which cannot be substituted for by

magnesium ion (29). Table VI shows that the reverse

transcriptase of RSFV had an absolute requirement for Mn++
transcriptase of RSFV had an absolute requirement for Mn





















RSFV
15






0
S10






5




Control


10 20 30 50

Amount of Virus preparation (Lambdas)






Fig. 19. Reverse Transcriptase Assay with concen-
trated raccoon sycyntium-forming virus--
Graph shows effect of increasing virus
concentration. Control shows results
obtained with uninfected BIHK cells treated
in the same manner as the infected cells.















Table VI

Requirement for Manganese in R.T. Assay; Also Shows
Effect of Varying Concentration of Triton-X 100



Substrate Ion T.X.100 CPM


Control Mn++ .2% 163

RSFV Mn++ .2% 3640

RSFV Mn++ .02% 3188

RSFV Mg++ .2% 244

RSFV Mg++ .02% 249

Blank Mn++ .2% 140









Attempts to recover reverse transcriptase activity

after sucrose gradient centrifugation with simian foamy

virus type III showed clearly three separate peaks of acti-

vity (26). The enzymatic action was recovered at sucrose

densities of 1.22 g/cm3 (thought to be viral cores),

1.16 g/cm3 (the viral fraction), and of 1.08 g/cm3 which

the authors termed the soluble fraction.

The RSFV concentrate was layered on top a 15 to 60%

sucrose gradient and spun at 200,000 gs for 2 hours.

Fractions were collected and assayed for reverse transcrip-

tase (Table VII). Peaks of activity were detected at

1.29 g/cm3, at 1.18 g/cm3 and at 1.07 g/cm3.


J. Electron Microscopy


The foamy viruses are enveloped spherical viruses

differing slightly in size. Intracellular particles are 35

to 50 nm in diameter and consist of an electron dense ring

surrounding an electron-lucent center. Completed virions

are seen extracellularly or within cytoplasmic vacuoles

and have a diameter of 100 to 140 nm. The envelopes are

covered with surface projections or spikes.

Electron microscopic studies on SIRC cells infected

with RSFV revealed preformed cytoplasmic particles (different











Table VII

Recovery of Viral Reverse Transcriptase
Activity from a Sucrose Gradient


Relative
Fraction Counts/min % Sucrose Buoyant
Number Densities

1 679 59 1.28
2 1362 57.7 1.29
3 717 54.9 1.25
4 600 52.5 1.24
5 679 50.9 1.23
6 856 49 1.22
7 901 47 1.21
8 830 45 1.20
9 650 43 1.19
10 1123 40 1.18
11 726 38 1.17
12 849 36 1.16
13 923 32 1.14
14 850 29 1.12
15 890 25.5 1.10
16 761 22 1.09
17 1234 18 1.07

Blank 850


Viral concentrate was layered on top a 15-60% sucrose
gradient and spun at 200,000 gs for 2 hours using a Beckman
S W 50.1 rotor. Fractions were collected and 50 lambda of
each assayed for R.T. activity. The % sucrose and buoyant
density of the fractions were determined on a refractometer.
Peak activities are underscored.
















Fig. 20. Electron microscopy showing preformed intra-
cytoplasmic viral cores composed of an
electronlucent center surrounded by electron-
opaque shell. Their dismeters were estimated
at 58 to 64 nm by comparison with beads that
were 188 nm in diameter (Magnification X40,000).





















Fig. 21. E. M. showing complete virions in intra-
cytoplasmic vacuole. Diameters estimated at
85 to 105 nm. Surface projections or spikes
can be seen on the outer envelopes (Magnifi-
cation X50,000).









70















"A L bl


















3.~E7'


cell or into intracytoplasmic vacuole
and acquiring their envelope in the
process (Magnification X35,000).










from C type) of 58 to 64 nm in diameter (see Fig. 20), as

estimated by comparison with beads that were 188 nm in

diameter. Completed enveloped RSFV could be seen inside

cytoplasmic vacuole (see Fig. 21). Extra cellular virions

and virions observed in cytoplasmic vacuoles were 85-105

nm in diameter (see Fig. 22). Preformed viral cores

could be seen to mature acquiring their outer envelope by

budding out of the cell or into cytoplasmic vacuoles

(see Fig. 22). The envelope was observed to be covered with

small projections.

The RSFV has all of the morphological characters

ascribed to the members of the Spumavirinae. The morpho-

genesis of RSFV as observed by E.M. was more like that of

the myxoviruses than of the C-type oncornaviruses.


K. Serological Survey


The foamy viruses are ubiquitous in nature causing

persistent infection in their respective hosts. As an

example, Hackett et al. (16) reported isolation of feline

syncytium-forming virus from 90% of the cats studied in

their laboratory during the first four months of 1969.

Since the foamy viruses are usually widespread in their

hosts, a serological survey of Florida raccoons was made






73



to study the frequency of occurrence and geographical

distribution of RSFV. Sera from numerous counties were

provided by Dr. William Bigler of the Florida Department

of Health and Rehabilitative Services in Tallahassee,

Florida. The survey (Table VIII) indicated that infection

with RSFV is widespread in the raccoon population of

Florida.















Table VIII

Survey of Raccoon Sera for Precipitating Antibodies to
RSFV; Geographical Distribution by Counties


Counties

Alachua

Clay

Collier

Columbia

Dade

Duval

Levy

Monroe

Polk

Sarasota

Suwannee


Number Tested

8

2

68

4

4

5

3

3

2

16

5


Number
Positives

3

1

39

0

1

1

0

0

2

9

3


Total 120 56















V. DISCUSSION


The raccoon syncytium-forming virus described herein

has all of the distinctive characteristics of the other

members of the Spumavirinae. The cythopathology produced

in infected cells was manifested principally in formation

of syncytia. Intense nuclear staining by indirect immuno-

fluorescence showed production of viral antigen in the

nuclei of these cells, a main difference with the other

members of the Retroviridae and the human and bovine

respiratory syncytial viruses. Electron microscopy revealed

a morphology and morphogenesis distinct from the C-type

particles.

RSFV nucleocapsids are first observed in the cytoplasm

and migrate to the cell membrane or intracytoplasmic mem-

branes, acquiring an envelope by budding. In contrast, the

nucleocapsids of the C-type oncornaviruses form at the plasma

membrane as the viruses bud through. Electron microscopic

studies of the feline and bovine agents have indicated that

nucleocapsids are assembled in the nuclei of infected cells

(17). Immunofluorescent studies of all of the foamy agents










thus far examined demonstrate that viral antigens are

initially detected in the cell nuclei (17). In contrast,

cells infected with C-type particles show membrane and

cytoplasmic fluorescent staining.

A precipitating antigen preparation extracted from

infected cells was used to screen raccoons for infection with

RSFV. This antigen may be a viral structural protein or a

protein produced in the course of viral infection that is not

incorporated into the virus. Since viral antigens are pre-

dominantly observed in the nuclei of infected cells by

immunofluorescence and because electron microscopic studies

indicate that nucleocapsids are formed in the nucleus or

perinuclear cytoplasm, it is likely that it is this antigen

which is responsible for the precipitating activity since

immunodiffusion is less sensitive than immunofluorescence.

Attempts to demonstrate that the antigen is a viral struc-

tural protein did not succeed. Ultracentrifuged concentrates

of cell-free virus did not react by immunodiffusion. It is

possible, because RSFV replicates to low titers, that the

ultracentrifuged virus preparations were not sufficiently

concentrated to give a visible precipitin band. Another

possibility, that the antigen is a non-structural virus

protein, could also be true. A third hypothesis, that the










antigen is a structural protein subunit of which the reactive

site is hidden when the nucleocapsid is fully assembled,

is also possible. The nature of this antigen preparation

is not addressed in the literature on the foamy viruses and

needs more study.

As can be observed in Figures 7 and 8, either one or

two precipitation bands were produced by immunodiffusion with

the antigen extract. Raccoon and rabbit antisera gave

similar reactions and, when sera from each species were

placed in adjacent wells, the lines which formed upon re-

action with the antigen were observed to fuse, indicating

identity. When two lines formed, one was always more pro-

nounced than the other. The major line was sometimes

observed to be nearer the antigen well, sometimes nearer

the antiserum well. The relative location or distribution

of the two lines appeared to vary with the antigen prepara-

tion used.

The raccoon agent incorporated tritiated uridine but

not thymidine and this incorporation was inhibited by

actinomycin D. This indicated that RSFV is an RNA virus

requiring a DNA replicative intermediate. The high back-

ground of tritiated thymidine recovered throughout the

gradient (see Fig. 18) probably reflects cellular DNA










synthesis. Raccoon syncytium-forming virus was shown to

possess virion-associated RNA dependent DNA polymerase.

Only weak bands of activity were recovered from fractions

collected after sucrose density gradient centrifugation.

This activity, however, was found at the same densities in

several repetitions of the experiment, and was distributed

in the same fashion as the reverse transcriptase activity of

the simian foamy viruses as described by Parks et al. (26).

Work with the foamy viruses of various species has

shown that they are common in nature, cause persistent

infections, and persist in the leukocytic cells of their

respective animal hosts. Scott (30) concluded that FeSFV

was a nuisance to virologists, oncologists, and vaccine

producers working with feline cells. Hackett and Manning

(15) point out that since this agent is strongly cell-

associated and replicate to such low titers it can easily

be overlooked.

Much significance has been placed on the discovery of

reverse transcriptase in cell of humans and other animals.

Until recently reverse transcriptase was considered to be an

exclusive property of the tumor producing oncorna viruses.

The foamy viruses share the following properties with that

group (25):










1. Similar buoyant densities.

2. Virion-associated reverse transcriptase.

3. Inhibition of replication by BUDR and dactinomycin.

4. High resistance to inactivation by ultraviolet
irradiation.

They differ with respect to:

1. Formation of syncytia as characteristic CPE.

2. Production of an intranuclear antigen detectable
by immunofluorescence.

3. Unique morphogenesis as determined by electron
microscopy.

4. No cellular transformation in vitro or tumor
production yet observed.

As Parksand Todaro (25) so aptly point out this last

difference may not be absolute. All cells thus far tested

have supported viral replication. A non-permissive system

has not been studied. Such systems may be subject to

transformation by the foamy viruses. Their ubiquitous

nature and persistence in their hosts makes it important

to find ways to readily detect these viruses. As pointed

out earlier, one such virus (simian foamy virus Type I) has

been shown to induce a carrier state in certain cells.

These viruses can contaminate cell cultures and be respon-

sible for enzyme activities associated with oncogenesis.










Since the syncytium-forming viruses of different

species have been shown to have similar biological proper-

ties, such studies may very well help in understanding the

newly isolated human agent. Lately some researchers have

been studying reverse transcriptase in normal human

lymphocytes. From experience with other species, a syncy-

tium-forming virus could be responsible for this activity.

There should be a concerted effort to uncover and charac-

terize these viruses so that virologists, cancer re-

searchers, and vaccine producers can be made more aware

of them.


































APPENDICES



































APPENDIX I

FITC--CONJUGATION OF SERUM












FITC--CONJU:AT2ION OF SERUM

(As cited in Gaskin (1973)(11))


1. To a two-fold dilution of serum in phosphate buffered

saline (.01 M phosphate, .15 M NaCI, pH 7.2), add an

equal volume of saturated ammonium sulfate, stirring

constantly with a magnetic stirrer. Continue stirring

for 1 hour.

2. Centrifuge 1400 g for 20 minutes.

3. Dissolve pellet in PBS to the original volume.

4. Add dropwise with continuous stirring 1/2 volume of

saturated ammonium sulfate. Continue stirring for

1 hour.

5. Centrifuge 1400 g for 20 minutes.

6. Dissolve pellet in distilled water to 1/2 the original

serum volume.

7. Dialyse, with stirring, against PBS at 4C using at least

5 changes of at least 50 times volume per change.

Clarify dialysate by centrifugation.

8. Read absorbance of a 1:100 dilution of dialysate at a

wavelength of 276 nm in a spectrophotometer.

Reading X 100 t 1.37 = protein concentration in

mg/ml.










10. Make .5 M carbonate buffer, pH 9.5 as follows: 3.7

gin NaHCO3, 0.6 gm anhydrous Na2CO3, made up to 100 ml

with distilled water.

To a volume of globulin solution (20 mg/ml),

add 1/2 volume of carbonate buffer containing 0.013 mg

FITC for each mg of protein to be conjugated.

11. Stir at 40C for 6-10 hours.

12. Pass immediately through medium or coarse Sephadex

G-25 equilibrated with PBS; one cm of a 2.5 X 40 cm

column for each ml of conjugate. Elute with PBS and

collect the first yellow band.

13. Dialyse the conjugate against .1 M Tris-HCl buffer,

pH 7.8 until equilibrated (4C). Pass through DEAE-

Sephadex (2 cm of a 2.5 X 40 cm column per ml of

conjugate) equilibrated with the same buffer.

Elute with .1 M Tris-HCl, pH 7.8

.1 M Tris-HCl + .1 M NaC1, pH 7.8

.1 M Tris-HCl + .2 M NaC1, pH 7.8

.1 M Tris-HCl +t .3 M NaC1, pH 7.8

Collect each fraction separately, monitoring the

collection with a black light as necessary.










14. Concentrate the fractions by dialysis against poly-

vinylpyrrolidone to a volume equivalent to the

original serum volume or less.

15. Test the fractions, if possible, by setting up an

immunodiffusion test against the antigen concerned.

Formed precipitin lines should fluoresce under black

light after the test has been washed overnight in PBS

to elute most unreacting conjugated proteins.

Determine the molecular fluorescein:protein ratio

by reading a 1:50 or other suitable dilution in a

spectrophotometer at 280 and 495 nm, and employing the

nomograph of The and Feltcamp.* A molecular F:P ratio

of 1-4 is supposed to be optimal, giving good specific

fluorescence while minimizing non-specific fluorescence.

16. Add 1:10,000 merthiolate if desired. Centrifuge the

fractions at high speed (e.g., 10,000 g) for clarifica-

tion. Store at -200 to -70C in small aliquots.

Test dilutions of conjugate on acetone-fixed

infected cover slip preparations to determine the effi-

cacy of the conjugate and the optimally effective dilu-

tion for use.



*The, T. H. and T. E. W. Feltcamp. Conjugation of
fluorescein isothiocyanate to antibodies. II. A reproducible
method. Immunol. 18:875-881 (1970).


































APPENDIX II

ERIOCHROME BLACK: A COUNTERSTAIN
FOR IMMUNOFLUORESCENCE












ERIOCHROME BLACK A COUNTERSTAIN FOR
IMVMUINOF LUORESCENCE
(As cited by Gaskin, 1973 (11))

I. Chelating Agent

A. 1. N, N-dimethylformamide 200 ml

2. Distilled H20 80 ml

3. 0.1 M Aluminum Chloride 40 ml
(2.4 gm/100 ml)

4. 1 M Acetic Acid 40 ml
(1 M = 5.7 glacial acetic
acid/100 ml H20)

Total 360 ml

B. Adjust to pH 5.2 with 1 M sodium hydroxide
(1 M = 4 gm NaOH/100 ml H20 need approx.
10 ml (?)

C. Make up to total of 400 ml with distilled H20

II. Preparation of Dye:

1. Weigh .628 gm Eriochrome Black A

2. Add dye to 80 ml N, N-dimethylformamide

3. Add 400 ml chelating agent slowly while swirling
the dye solution

III. Dilute 1:5 or more with distilled water as necessary

Stain tissue culture 1-3 seconds

Stain tissue sections 10-30 seconds














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