ISOLATION AND CHARACTERIZATION OF A
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
To my mother, for her love, devotion and
unshakeable faith in my capabilities
To my father, for his patient understand-
ing and constant support.
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
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.
TABLE OF CONTENTS
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)
V. DISCUSSION ..................
I. FITC--CONJUGATION OF SERUM .
II. ERIOCHROME BLACK A COUNTERSTAIN
FOR IMMUNOFLUORESCENCE. . .
BIOGRAPHICAL SKETCH ................
LIST OF TABLES
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
LIST OF FIGURES
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
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
Stanley Nicolas Laham
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
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.
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-
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
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
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
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.
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
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.
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
2. Syncytium formation as characteristic in vitro
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
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
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
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
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
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
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
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
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
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
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.
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
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
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.
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.
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
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-
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-
Fig. 4. BHK cells grown in a Leighton tube. The cover
slip was removed, fixed and stained with 10%
(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
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
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.
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
3--1 treated with chloroform
4--Concentrated virus preparation from viral stock
preparation treated with chloroform prior to
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.
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.
Tissues from Which RSFV Was Recovered
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
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.
Host Range of RSFV
Cell Types Indirect CPE
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.
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-
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
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)
Fig. 15. Microscopic plaque surrounded by large multi-
nucleated cells as seen after Giemsa staining
Fig. 16. Indirect immunofluorescent staining of in-
fected cover slip showing many syncytia with
no area of cell lysis.
... ... ". ::
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
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 + +
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
Effect of BUDR on RSFV Replication; Also Shows
Inactivation of the Virus by Lipid Solvents
Grown in BUDR
3.2 x 103 FFU*
.8 x 103 FFU
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
- 4- )
in ,-1 co n o 1!
' CM H H o C -i
N CN H O r-
r-l r t Ir
-n m CN
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
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.
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
Recovery of Viral Reverse Transcriptase
Activity from a Sucrose Gradient
Fraction Counts/min % Sucrose Buoyant
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
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-
"A L bl
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
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
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
Survey of Raccoon Sera for Precipitating Antibodies to
RSFV; Geographical Distribution by Counties
Total 120 56
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
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-
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
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
1. Similar buoyant densities.
2. Virion-associated reverse transcriptase.
3. Inhibition of replication by BUDR and dactinomycin.
4. High resistance to inactivation by ultraviolet
They differ with respect to:
1. Formation of syncytia as characteristic CPE.
2. Production of an intranuclear antigen detectable
3. Unique morphogenesis as determined by electron
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
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
5. Centrifuge 1400 g for 20 minutes.
6. Dissolve pellet in distilled water to 1/2 the original
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
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).
ERIOCHROME BLACK: A COUNTERSTAIN
ERIOCHROME BLACK A COUNTERSTAIN FOR
(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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2. Andrewes, C. H. and D. M. Horstmann. The susceptibility
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