Download Long-Term Persistent Vesicular Stomatitis Virus and Rabies Virus

J. gen. Virol. (I976), 33, I93-2II
I93
Printed in Great Britain
Long-Term Persistent Vesicular Stomatitis Virus and
Rabies Virus Infection of Cells In Vitro
By J. J. H O L L A N D , * L. P. V I L L A R R E A L , * I I R. M. W E L S H , t
M. B. A. O L D S T O N E , t D. K O H N E , * R. L A Z Z A R I N I ~ AND E. S C O L N I C K §
* Department of Biology, University of California-San Diego, and ~ Scripps Clinic and
Research Foundation, La Jolla, California 9zo37, ~ National Institute of Neurological and
Communicative Disorders and Stroke, NIH, Bethesda, Maryland 2ooi4, § National Cancer
Institute, N1H, Bethesda, Maryland zooM, U.S.A.
(Accepted
I2
July I976)
SUMMARY
BHK 2I carrier cells persistently infected with VSV Indiana for over 2 years
have been shedding generally very low levels of mature infectious virus or mature
T particles (averaging less than one-hundredth p.f.u./cell/day) yet most cells are
producing virus antigens and are resistant to homologous superinfection. However,
large amounts of biologically active T particle RNP can be recovered from cytoplasmic extracts of these carrier cells even at times when they are shedding no
detectable infectious virus. This recovered cytoplasmic RNP replicates (with helper
B virions) to produce mature T particles, interferes strongly after DEAE dextranfacilitated uptake and, together with B virions, allows the establishment of a persistent carrier state in exposed cells.
No 'provirus' DNA copies of the VSV RNA genome are detectable (less than
-1--4ocopy/cell or I copy per 40 cells) in carrier cells after more than 2 years of persistent
infection, and all transfection attempts have failed using DNA from these VSV
carriers or DNA from carrier cells persistently infected with some other negative
strand RNA viruses (measles, mumps, LCM, influenza, rabies).
Infectious viruses shed after more than I year from carrier cells originally infected
with wild-type B virions are small plaque mutants showing a slight temperature
sensitivity. Cured cell populations can be obtained from the long term VSV carrier
culture by cloning in the presence or absence of antiviral antibody.
INTRODUCTION
Holland & Villarreal (I974) showed that persistent non-cytocidal carrier infections of
B H K z1 cells in culture with vesicular stomatitis virus (VSV) required the presence of
defective interfering virus particles (DI) to modulate the virulence of the virus for these
highly susceptible cells. Doyle & Holland (1973) used completely purified DI to provide
prophylactic protection of mice against low virus challenge doses, and observed a slower,
altered disease process with DI in the presence of higher challenge doses of virus. Holland &
Villarreal (I975) found that DI can be generated and replicated entirely in vivo. These results
lend credence to the speculation of Huang & Baltimore (I97o) that DI generally might be
II Present address: Department of Biochemistry,Stanford University, Stanford, California, U.S.A.
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J. J. H O L L A N D
AND
OTHERS
involved in alteration of acute virus disease processes and in causing some persistent virus
diseases.
Kawai, Matsumoto & Tanabe 0975) recently demonstrated cyclic production of DI in
rabies carrier cultures. Palma & Huang 0974) had earlier shown that VSV DI and B virions
undergo cyclic replication if new susceptible cells are introduced regularly. Other factors,
such as interferon (Rodriguez & Henle, I965) and temperature-sensitive mutants of the virus,
undoubtedly may play a role in virus persistence. Preble & Youngner (I 973) with paramyxoviruses, Fields & Raine (t974) with reovirus, and Wagner (~974) and Stanners & Goldberg
0975) with VSV have all shown that ts mutants tend to give persistent infections in vitro and
slower disease processes in vivo.
The present study presents data concerning more than 2½ years of persistent infection of
BHK zI cells in vitro with VSV, and similar observations with rabies virus. It is shown that
DI play a major role in persistence and that temperature sensitivity and small plaque mutation
of B virions evolve during persistence and contribute to a more stable persistent virus-cell
relationship. It is also shown that biologically active DI RNP is present in persistently
infected cells and that virus yields are generally very low even though all carrier cells are
infected. It appears that DI and virion attenuation provide for RNA viruses the functional
equivalence of lysogeny in DNA viruses. Simpson & Iinuma 0975) and Zhdanov (I975)
have recently presented evidence that DNA provirus copies may be produced in carrier
cultures of enveloped RNA viruses and in human lupus erythematosis. We find no evidence
for DNA copies in our long-term carrier cultures of VSV.
METHODS
Cells and virus. Cells were grown at 37 °C in Eagle's minimum essential medium plus 7
heat-inactivated calf serum. BHK 2i baby hamster kidney cells were obtained originally
from Flow laboratories. The L cell mouse fibroblast line and HeLa human cervical carcinoma
cell line were originally obtained from the American Type Culture Collection. VSV, measles,
mumps and influenza virus plaque assays were carried out as previously described (Holland
& McLaren, I959). Rabies virus was plaque-assayed as described by Sokol et al. (I968), and
LCM virus as described by Welsh, O'Connell & Pfau (I972).
Except where otherwise stated the VSV carrier culture is the B H K 2~, VSV~nd CAR4
persistently infected carrier cell line described previously (Holland & Villarreal, I974). It
was established originally using B virions and T particles of the ts G3I mutant of Pringle
(I97o). This carrier was twice re-exposed to ts 31 B virions at m.o.i. = IOO to ensure infection
of every cell. (This was done twice within one week at 6 months after the carrier was established, and the present carrier represents the surviving cell population.) Carrier cells were
passaged by trypsin treatment at regular intervals (usually 2 to 3 days) when monolayers
became confluent. The generation time of these cells is less than 30 h except when cytopathic
episodes slow cell growth.
Biological assays for virus ribonucleoprotein activity. A modification of the DEAE dextranfacilitated uptake method of Cartwright, Smale & Brown 0970) was used in which cell
monolayers were pre-treated with 350 #g/ml of DEAE dextran, followed by addition of the
RNP in Hanks' balanced salt solution (BSS) for ~o to I5 min at 25 °C. The same regimen
was employed for assay of the replicating capacity and interfering ability of T particle
ribonucleoprotein cores from acutely infected cells or carrier cells. T particle RNP have been
shown elsewhere (Holland, Villarreal & Breindl, i976) to be biologically active in interference tests and in more sensitive amplification tests.
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Hybridization of carrier cell DNA to 3H-VSV virion probes. This was done as described
previously (Reichmann et al. I974) for characterization of VSV virion RNA hybridization
to in vitro mRNA. Virion RNA was labelled with iodine to a sp. act. of I-2 x Io 7 ct/min/#g
as described by Commerford (~97I), annealed at Cot 918 at a carrier cell DNA/probe ratio
of 2 × 106/1, and ribonuclease was used to determine the degree of hybridization. Because no
significant annealing was observed with carrier cell D N A or normal cell DNA, the intactness
of the probe after the incubation period was verified by annealing to VSV in vitro m R N A as
previously described (Reichmann et al. I974).
Hybridization of' 3H-VSV cDNA with unlabelled DNA and RNA from normal and carrier
cells. D N A was prepared from the VSV carrier cells after 25 months of persistent infection
by the urea-phosphate technique of Britten, Graham & Neufeld 0974). Carrier cell
R N A was isolated from the same cells by dialysing the urea-phosphate fraction not binding
to hydroxyapatite against o'25 M-NaCI and precipitating RNA with ethanol. 3H-VSV
cDNA was prepared with purified reverse transcriptase and 3H-labelled thymidine triphosphate. A positive strand 3H-cDNA was selected for these experiments by reacting
total 3H-cDNA with excess VSV virion RNA, absorbing the R N A - D N A duplex to
hydroxyapatite in o'~4 M-sodium phosphate, pH 6.8, o.2 ~ SDS at 6o °C and eluting with
o'3 M-sodium phosphate. This positive strand D N A was freed of RNA by alkali digestion.
The sp. act. of the 3H-cDNA was 2 × IO7 ct/min/#g.
Annealing reaction mixtures contained o'48 M-phosphate buffer, pH 6.8, Io -4 M-EDTA
and IO3 ct/min. 3H-cDNA and unlabelled denatured cell D N A in o.2 to o.4 ml total volumes.
Incubation was at 68 °C, with samples being withdrawn at appropriate times and assayed
by binding to hydroxyapatite (Kohne & Britten, I97I) to determine ct/min 3H reannealed.
Optical density at 260 nm was used to follow unlabelled bulk cell D N A reassociation.
Transfection experiments with DNA from persistently infected carrier cells. Monolayers of
2 x io 7 target cells were pre-treated for 2 min with 350 #g/ml DEAE dextran in Hanks's
balanced salt solution (BSS), then immediately exposed to the total undenatnred D N A from
between 2 × IO7 to I x io 8 carrier cells in I ml o'I5 M-NaC1. D N A was extracted from carrier
cells by proteinase K treatment in o'I5 M-NaC1, o.I ~ sarksosyl, followed by vigorous
shaking with equal volumes of phenol-chloroform, phase separation and repeated extraction
with ether. In some experiments additional shearing was introduced by long-term chloroformoctanol shaking or by ultrasonic shearing for various times. After a Io to I5 rain uptake
period at 25 °C the monolayers were rinsed with BSS and incubated for 7 days or more at
37 °C (32 °C for rabies virus), with periodic assays of the medium on other sensitive cells
in attempts to detect infectious virus. Target cells were also transferred to glass coverslips
or suspended in medium at intervals and examined for expression of specific virus antigens
on the cell surface or in the cytoplasm by fluorescent antibody techniques described below.
Fluorescent antibody techniques; ceil staining and fluorescence microscopy. Two x Io 5
washed cells were incubated with 2o #1 of fluorescein-conjugated rabbit IgG antibody to
VSV. After 3o rain incubation at room temperature, cells were washed three times in M E M
and examined for expression of virus antigens on the cell surface using a Zeiss RA fluorescent
microscope with an HBO-2oo (Osram) light source. An FITC filter system (Optisk Laboratorium Lyngly, Denmark) was used for most analyses. To determine cytoplasmic VSV
antigens in carrier cells, the cells were grown on coverslips, fixed, handled, and viewed as
described by Joseph & Oldstone, I974, and Oldstone & Dixon, I97I).
Radiolabel assay; measurements of VSV antigens at cell surfaces. We determined virus
antigens on the surfaces of infected cells using the radiolabelled antibody technique (Joseph,
Perrin & Oldstone, I976). Briefly, IgG isolated from rabbit serum containing antibodies to
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J. J. HOLLAND AND OTHERS
VSV was labelled with 1~51by the chloramine T method to asp. act. in the range o'7/zCi/#g.
Normal rabbit IgG was labelled with 131I by the chloramine T method to about the same
level. Approx. o.8 ~ of the l~sI-IgG antiserum preparation contained specific antibody to
VSV. Radiolabelled preparations were deaggregated by centrifuging at I oo ooo g for 3o rain;
unlabelled IgG isolates were individually deaggregated in a similar manner. Cells of each
type (i.e. acute VSV, persistent VSV and uninfected B H K cells) were placed in individual
glass tubes at concentrations of 5 × Io 4, I × Io 5 and 5 × Io5 cells per tube. Unlabelled IgG
was added to radiolabelled IgG at a ratio of I o : I , and a total of 2oo #g was added to each
tube (a25I-labelled IgG plus unlabelled antibody IgG to VSV was added to one set, and lzaIlabelled IgG plus unlabelled IgG devoid of antibody to VSV were added to another).
These amounts had been previously determined to saturate virus antigens expressed on
surfaces of B H K cells acutely infected with VSV (i2 h post infection). IgG cell mixtures were
incubated for 3o min at room temperature, washed 6 times with 5 ml cold M E M containing
IO ~ FCS, pelleted and counted in a Baird Atomic ~25I/~31Ipaired labelled gamma counter.
Counts were corrected for background and ~3~I crossover in the a25I window. The number
of IgG molecules of antibody to VSV binding specifically per cell in each sample was
determined by the following formula:
(6"023 x Io ~a) x (#g125I-IgG- #g 13'I-IgG fixed on the cells) x io -6
Number of cells x mol. wt. of IgG
RESULTS
VSV T particles produced during long-term persistence
Wagner et aL 0963) reported that L cells infected with a small plaque mutant of VSV
evolved into a persistently infected carrier state with frequent cytopathic 'crises'. The basis
for the persistence was not explored, but interferon was suggested as a possible factor.
Holland & Villarreal (1974) showed that DI was a necessary component along with infectious
virus for the establishment of persistence, and it was shown that a long T particle produced
continuously by the carrier B H K 2i cells could lead to the establishment of persistence
whenever it infected B H K aI cells along with B virions.
We have now monitored the B H K 21 vsgind CAR4 carrier cells during more than 2½
years of persistent infection and have always found these cells to be producing T particles.
The long T particle previously described is sometimes the sole T particle present, but other
sizes are often present in addition to, or in place of, the very long T particles. Fig. I shows
an example of some of the patterns of T particles seen after co-cultivation of the B H K
2i, VSV~nd CARd carrier with uninfected B H K zI cells during the period from I½ to 2½
years after establishment of the carrier state. A variety of different sizes of T particles
was produced at different times. Apparently, no single T particle can achieve continuous
dominance over other types as the persistently infected cells are maintained for long periods
at 37 °C. Similar unpredictable changes in the T particle population were seen in the carrier
initiated with wild-type B virions plus long T particles. J. Perrault (manuscript in preparation) has characterized a variety of these different carrier T particIes with regard to the
secondary structure of the DI virion RNA. Some exhibit extensive or moderate levels of
'snap back' self-annealing (Lazzarini et al. 1975; Perrault, I976); others exhibit timedependent, concentration-dependent self-annealing; and yet others show little or no self
annealing. Thus far no characteristic has been found which distinguishes carrier cell T
particle varieties from a wide variety of T particles produced during acute infection.
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I97
W
N
i
~---- B
I
!
,J
I
Fig. i. Diagrammatic representation of the variety of different sized T particles being shed from
cells persistently infected with VSV during different time intervals in the period from I½ to 2½years
after establishment of the carrier state. Each tube shows the position in a sucrose gradient of VSV
B virions and the T particle size classes produced at each time by co-cultivation of carrier cells
with normal BHK 2~ cells. The longest T particles (s2o,w= 470) are usually, but not always, present.
Production of virus by carrier cultures
Between 25 months and 3o months following establishment of the carrier state, these cells
have become stabilized and generally show few microscopic signs of infection. Only at rare
intervals of several days during months of culture does cytopathology appear briefly. At
z5 months, it was observed that no virus could be recovered in repeated attempts over
several days. Thereafter, we have sampled the supernatant medium from the carrier cultures
every 14 h and determined the yield of p.f.u./cell]day. Fig. z shows that the virus yields
fluctuate in a cyclic manner. They are nearly always less than I p.f.u.[cell/day and average
less than o-oI p.f.u./celI/day and at times the yield is below one millionth p.f.u./cell/day
average (i.e. not detectable). During only one z-day period (during a cytopathic crisis
indicated by the arrow in Fig. 2) was virus produced at levels averaging greater than I p.f.u./
cell/day. Clearly this carrier has evolved toward a non-producing state similar to that of
certain L C M (Welsh et al. I97z; Staneck et al. 197z) or measles (Rustigian, I966) carriers
or SSPE brain cells (Payne, Baublis & Itabashi, 1969). Shifting this long term stabilized
carrier culture to 3z °C did not increase levels of virus shed over a 3-day period.
Most carrier cells produce VSV antigens
Fluorescent antibody staining of unfixed B H K 2I VSV~nd CAR4 cells for surface virus
antigens between 2 and 2½ years of persistent infection always showed that over 95 ~ of the
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J. J. H O L L A N D AND OTHERS
I
I
[
I
2t
1
0
p
I
20
I
I
[
40
60
8O
Days
Fig. 2. Yields/cell/day of infectious B virion p.f.u, from cells persistently infected with VSV for
more than 2 years. At 25 months after establishment of the VSV carrier state in BHK 21 cells it
was noticed that infectious virus could not be recovered from the carrier cell medium at certain
times. Thereafter, the medium of carrier cells was sampled each day, plaque assayed at 32 °C, and the
yield/cell/day plotted for the period from z5 months to 28 months after the establishment of
persistent infection. In order to lessen the problems of interference by T particles shed into the
medium, o'3 ml samples of the medium (and dilutions of the medium) were plated on to very large
monolayers of BHK 2I cells for plaque formation. Monolayers of z x IO7 BHK 2I cells were used
instead of monolayers of 2 x lO6 cells. This lessens the probability of a B virion and a T particle
co-infecting the same cell or cell grouping. Plaques were developed after 50 h at 3z °C under MEM
with o'4 ~ agarose. The arrow indicates an episode of severe cytopathic crisis.
cells were antigen positive. Fig. 3 shows a typical field with a patchy p a t t e r n of surface
staining seen o n some cells, and a c o n t i n u o u s p a t t e r n on others. F u r t h e r m o r e , a quantitative
i m m u n o g l o b u l i n b i n d i n g assay (Table I) demonstrated that these carrier cells were expressing
virus surface antigens at levels a b o u t 5o to 8o ~ of the level observed d u r i n g acute infection.
This a n t i b o d y - b i n d i n g assay was performed at a time when the cells were producing less
t h a n IO-4 p.f.u./cell/day. Clearly, virus antigens were being p r o d u c e d at significant levels in
most cells, despite the paucity of infectious virus p r o d u c t i o n after 2 years in the carrier state.
Carrier cells can be 'cured' by cloning
Walker (I964) has reviewed evidence for persistent carrier infections with enveloped R N A
viruses in which prolonged a n t i b o d y t r e a t m e n t or cloning can lead to recovery of cured
cells. The B H K 2~ VSV~d CAR4 carrier studied here appears somewhat different from
either of the types outlined by W a l k e r (~964). Table 2 shows that a n t i b o d y t r e a t m e n t of
large n u m b e r s of cells for I m o n t h did n o t cure the cultures, b u t identical t r e a t m e n t of very
lightly seeded m o n o l a y e r cultures did cure them. Likewise, Table 2 shows that cloning in
the absence of a n t i b o d y did n o t lead to recovery of cured clones if there were more t h a n a
few clones growing in each culture plate (unless a n t i b o d y was present), b u t it generally did
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Fig. 3. P h o t o m i c r o g r a p h of B H K 21 cells persistently infected for over 2 years with VSV and
incubated with monospecific fluorescenated rabbit I g G antibody to VSV. Virus antigens are distributed over cell surfaces. These cells were not releasing detectable infectious virus at the time o f
immunofluorescent assay.
Table I. 1~5Iantibody binding assay for VSV antigens expressed on the surface of BHK 2~
cells acutely infected or persistently infected f o r over 2 years with V S V
B K H 21 cells employed
Uninfected (control)
Acute infections 7 h
Acute infection i2 h
Persistent carrier cells
Persistent carrier cells
Measles carrier (control)
Counts b o u n d per lO5 cells
~
~
1311
125][
3177
33IO
3 I9I
3 252
4 oi o
3 821
5317
I526I
22223
16170
19323
5121
Approximate n u m b e r
of antibody molecules
bound/cell*
-P3 x io 6
2"4 × IOn
I. 6 x I o ~
2- I × 106
(No significant binding)
* D a t a in this table were derived f r o m duplicate samples over a range of cell concentrations (5 x io 4,
I x io 5, 5 x io5), and are corrected for background and crosstalk. Linear response was seen at the various
cell dilutions. Comparable data were obtained in two additional experiments similar to the one presented.
Sp. act. o f the I g G was 4"5 x io 7 ct//~g/3o s. Acutely infected cells were infected at m.o.i. = 3.
lead to cured clones whenever only one or a few clones grew in each culture plate. We have
observed similar curing of carrier established with wild-type VSV B virions. Note that the
cloning efficiency of the VSV carrier culture is rather low, but has improved from o'o3
to better than ~ %0-The above curing effect of antibody and of cloning suggests strongly that
at least occasional cell to cell spread of virus is necessary to maintain the carrier state, It
appears that infected cells can eliminate or outgrow infectious B virions by cell division
because of the strong interfering effect of D I on virus replication. In mass monolayer cultures,
cured cells are presumably soon re-infected, but in sparse cultures with antibody, or in
isolated clones the cure can become permanent. As indicated in Table 2, cured cells show
no virus antigen and exhibit normal plating efficiency and give normal virus yields with
VSV Indiana even after 2½ years in the carrier state, during which they were resistant to
homologous virus. Thus, there is no cellular genetic basis for resistance in the carrier state.
14
vIR 33
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J . J . H O L L A N D A N D OTHERS
T a b l e z. Curing of long-term VSV carrier cells by cloning or by antibody treatment of
sparsely seeded monolayers
Treatment of carrier cells*
Cloned in absence of antibody
Cloned in absence of antibody
Cloned in absence of antibody
Cloned in presence of antibody
2 × 106 cells plus antibody (30 days)*
5 x io 4 ceils plus antibody (30 days)
5 x io 4 cells, no antibody (3o days)
Clones/dish
Cloning efficiency
~
Cured cells
recovered ?
2
3
0'03
I "5
Yest
Yes
li
o-8
No
28
1-2
Yes
No
Yes
No
* Cloning was carried out in glass culture flasks for 3 weeks at 37 °C. Carrier cells were trypsin-dispersed,
counted and plated at various dilutions.
t Curing was verified by return of cured cell populations to full susceptibility to VSV Indiana (i.e. normal
plaquing efficiency and normal virus yields); by lack of B virion or T particle shedding in cured cell cultures;
and (in two cases) by failure to detect VSV antigens with the fluorescent antibody assay.
Antibody treatment of carrier cell monolayers was carried out by placing 150 5o ~ plaque reduction
units of rabbit antiviral antibody in the medium over sparse (5 x io 4 cells/bottle) or confluent (2 x io ~ cells/
bottle) monolayers of carrier cells for 30 days at 37 °C (with passage of the monolayers as needed to maintain
confluent or sparse monolayers after passage).
Infectious centre assays
W e previously r e p o r t e d ( H o l l a n d & Villarreal, 1974) that all or nearly all carrier cells
d u r i n g the first m o n t h s after establishment o f persistent infection registered as infectious
centres. This is no longer true in the stabilized carrier state after 212 years. I n two instances,
no c o n c e n t r a t i o n o f carrier cells p l a t e d on B H K 2I cell m o n o t a y e r s yielded a n y plaques
(i.e. less t h a n I cell in Io 6 p r o d u c e d a plaque), a n d in a third infectious centre assay only 93
cells o u t o f lO 5 registered as infectious centres. This is n o t unexpected in view o f the low
levels o f virus being p r o d u c e d each d a y (Fig. 2).
Reduced production of mature T particles
W e r e p o r t e d previously ( H o l l a n d & Villarreal, I974) that carrier cells were over 99 ~o
resistant t o h o m o l o g o u s VSV I n d i a n a B virion infection a n d this is still true o f the stabilized
carrier after 21 years. (The yield p e r cell is reduced over 99"9 ~ as c o m p a r e d to c o n t r o l
n o r m a l B H K 2I cells when e a c h are challenged with m.o.i. = IOO I n d i a n a B virions.) The
related N e w Jersey yield is reduced only a b o u t 9o ~o, however, a n d u n r e l a t e d N W S influenza
virus yield are n o t reduced. Influenza ( N W S ) virus forms n o r m a l p l a q u e s on m o n o l a y e r s o f
the l o n g - t e r m VSV carrier cells. This strongly implicates D I a n d n o t interferon as the interfering agent. As r e p o r t e d above, co-cultivation o f carrier cells with n o r m a l B H K 2I cells
leads to p r o d u c t i o n o f large a m o u n t s o f B virions a n d T particles o f v a r y i n g sizes. A l l
a t t e m p t s to p u r i f y visible a m o u n t s o f B virions o r T particles directly f r o m the m e d i u m o f
large n u m b e r s o f carrier cells have failed. Interference tests on the m e d i u m o f carrier cultures
were negative except d u r i n g crisis (Table 3) indicating t h a t very few T particles are shed
into the m e d i u m at m o s t times d u r i n g the carrier state. Even a sensitive one cycle amplification test ( H o l l a n d & Villarreal, I975) was negative in 2 o u t o f 3 tests done on different days.
Therefore, there are generally less t h a n I o 7 T particles/ml o f carrier culture m e d i u m (Table
3). However, T a b l e 3 shows t h a t T particles are present in small n u m b e r s even when no B
virions are shed, since extremely sensitive two-cycle amplification tests were positive. I t is
obvious t h a t these carrier cultures generally shed only very small n u m b e r s o f m a t u r e B
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T a b l e 3. Interference and amplification tests for the presence of T particles
in the medium of long-term carrier cell cultures
B virions shed
inte carrier cell
medium
(p.f.u./cell)
Interference tests for
the presence of
T particles in the
medium
( ~ interference)
4× lO-8
0"15
None
Amplification tests for the presence
of T particles in the medium of
carrier cellst
~
~- ,
One cycle
Two cycles
-(<30 ~)
+(71 ~oo)*
-- ( < 30 ~o)
-+
--
+
+
+
* This medium showing a positive interference test and one cycle amplification test was from the carrier
cells at a time when they were undergoing a rare episode of cytopathetogy (slight c.p.e.).
Amplification tests were as described elsewhere (Holland & Villerreal, 1975; Holland et al. 1976). The
two-cycle amplification test is lo4-fold more sensitive than a single-cycle test. These assays involve infection
of BHK 2I cells by B virions, and exposure to a source of T particles. This allows measurement of the T
particles because each T particle is replicated about ]o4-fold at each cycle (as long as the T particle m.o.i.
is less than I.O).
T a b l e 4. Purification of T particles from yields of long-term carrier cells
superinfected with related heterologous B virions
Virus particles purified
from yields of cells infected
with VSV Indiana B virions
Virus particles purified from
yields of cells infected with
VSV New Jersey B virions
Cured carrier cells
No virus bands visible after
purification1"
B virion band only
B virion band plus 2 T particle
bands
B virion band only
Normal BHK 21 cells
B virion band only
B virion band only
Cells challenged with B virions
alone (m.o.i. = 1oo)*
BHK 21 VSVIndCAR~ carrier cells
* The challenge virus pools employed were first passage clonal pools prepared by low m.o.i, passage of
wild-type virus picked directly from a plaque. They will not generate visible T particle bands on normal cells
until the fourth passage (three additional undiluted passages (Holland et aL 1976).
t Following challenge of ]o 8 cells of each type, the cells were incubated at 37 °C for 2o h and the virus
yields purified and displayed on a sucrose gradient to visualize bands of B virions and T particles. Homologous
interference was so great that no virus band was seen after challenge of the carrier cells with VSV Indiana.
v i r i o n s o r m a t u r e T particles, yet n e a r l y every cell m u s t have B v i r i o n R N P in its c y t o p l a s m
(in o r d e r to p r o d u c e virus antigens) a n d T particle R N P (in o r d e r to resist h o m o l o g o u s
challenge). T h e r e f o r e , we a t t e m p t e d to ' rescue' T particle R N P f r o m these cells b y B v i r i o n
challenge.
VSV New Jersey B virion challenge elicits production of mature T particles
C h a l l e n g e i n f e c t i o n o f the stabilized carrier cells with m.o.i. = IOO h o m o l o g o u s I n d i a n a
s t r a i n B v i r i o n s led to cell d e s t r u c t i o n b u t n o visible B v i r i o n s o r T particles c o u l d b e purified
f r o m the m e d i u m f r o m 2 × IO7 cells ( T a b l e 4)- S t r o n g h o m o l o g o u s i n t e r f e r e n c e p r e v e n t e d
significant B o r T v i r i o n p r o d u c t i o n . H o w e v e r , T a b l e 4 shows that s i m i l a r challenge o f these
l o n g t e r m carrier cells w i t h m.o.i. = ]oo V S V s t r a i n N e w Jersey led to release o f B v i r i o n s
a n d T particles i n t o the m e d i u m in a m o u n t s sufficient to be directly purified a n d visualized.
N o r m a l B H K 2t c o n t r o l cells infected w i t h the same B v i r i o n s o f N e w Jersey s t r a i n yielded
o n l y B v i r i o n s (visible after purification). O b v i o u s l y , T particle g e n o m e s p r e s e n t i n the c a r r i e r
cells replicated ( a n d interfered a b o u t 9o ~ ) d u r i n g r e p l i c a t i o n o f the challenge N e w Jersey
strain. (This is a b o u t the s a m e degree o f h e t e r o l o g o u s interference seen i n acute i n f e c t i o n o f
o u r B H K 2I cells w i t h o u r N e w Jersey s t r a i n B v i r i o n s a n d I n d i a n a T particles at high m.o.i.)
I4-2
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J . J . H O L L A N D AND OTHERS
T a b l e 5. The presence in carrier cell cytoplasmic extracts of T particle ribonucleoproteins able
to replicate, interfere and re-establish a new carrier state (in cells exposed to them along with
homologous V S V Indiana B virions)
Mature B
virions in
medium,
p.f.u./cell
None
o'o3
One-cycle amplificaOne-cycle amplification test for mature Interference
tion test for T
Interference test
Ability of
T particles in cell test for mature particle RNP in
for T particle
cytoplasmic
culture medium
T particles in
cytoplasmic
RNP in cyto- RNP to estab(highest positive
medium ( ~
extracts (highest plasmic extracts
lish new
dilution)
interference) positive dilution)* ( ~ interference)t carrier state~
- §
+ (undiluted)
Long-term carrier cells
-- (<30%)
+ (Io -3)
+ (>99"5~)
+
-
(<3O~o)
+
(96%)
+
-
(<30%)
-
(<30%)
-
+
(Io -~)
Cured carrier cells
None
-
Control normal cells
None
* Amplification tests for cytoplasmic particle RNP were as described (Holland et al. I976) for cells
acutely infected with B virions plus T particles. 2 × Io 7 cells were disrupted after i min swelling in the o-oI MNaC1 by five strokes of a glass tissue grinder at o °C, and the mixture was made o. 15 M with respect to NaC1
by addition of 2 M-NaCI. Nuclei were sedimented at t ooo g for 2 min and various dilutions of the cytoplasmic
supernatant were absorbed to 2 × IOv BHK 21 cells after DEAE dextran treatment (see Methods and Cartwright et aL 197o).
~ Cytoplasmic T particle RNP were tested for interference with B virions (m.o.i. ~ Ioo) after DEAE
dextran-facilitated uptake of undiluted cytoplasmic extracts. In the absence of DEAE dextran, amplification
tests and interference tests were negative.
Following interference tests, surviving cells were maintained at 37 °C and medium replenished at regular
intervals for 3o days. Persistently infected carrier cells, when they appeared, were verified to be shedding
both B virions and T particles.
§ Although this carrier culture medium was negative, a Ion-fold more sensitive two-cycle amplification
test showed the presence of small numbers of T particles in the medium.
Biologically active T particle R N P in the cytoplasm of long term carrier cells
C a r t w r i g h t et aL (r97o) showed t h a t subviral r i b o n u c l e o p r o t e i n ( R N P ) structures from
VSV are infectious, r a t h e r efficiently so, when u p t a k e is facilitated b y D E A E dextran. W e
have recently extended this to show that T particle R N P (but n o t T particle R N A ) is also
biologically active after facilitated u p t a k e with D E A E dextran ( H o l l a n d et al. I976).
T particle R N P f r o m d i s r u p t e d virions or f r o m infected cell c y t o p l a s m caused the p r o d u c t i o n
o f large a m o u n t s o f T particles in a one-cycle amplification test in which helper B virions
were a d d e d to B H K 2I cells after D E A E d e x t r a n facilitated u p t a k e o f the T particle R N P .
T a b l e 5 shows t h a t d i s r u p t i o n o f carrier cells releases biologically active T particle R N P
which replicated (and interfered with helper h o m o l o g o u s VSV I n d i a n a B virions) to p r o d u c e
large a m o u n t s o f m a t u r e T particles visible after purification as m a j o r b a n d s in sucrose
gradients. C y t o p l a s m i c extracts f r o m carrier cells n o t p r o d u c i n g detectable infectious virus
yielded replicable T particle R N P . Extracts o f cured carrier cells did not. The a m o u n t s o f
replicable R N P recoverable varied, b u t carrier cells often c o n t a i n e d nearly as m u c h biologically active T particle R N P as cells late d u r i n g acute infection with B virions plus T particle
(~ ]5ooo dilutions o f the latter gave positive amplification tests). These c y t o p l a s m i c T particle
r i b o n u c l e o p r o t e i n s f r o m carrier cells were also able directly to facilitate the e s t a b l i s h m e n t
o f persistent infections o f B H K 2t cells when a d s o r b e d before B virion infection. The surviving cells o f these B virion-carrier cell R N P d o u b l e infections quickly established new
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VS V persistence
A
B
C
D
20 3
E
F
Fig. 4. Summary of various attempts at recovery of B virions and T particles from long-term carrier
cultures. Each tube illustrates the results of a sucrose gradient analysis for the presence of virus.
(A) Direct purification of the medium from 5 x i& carrier cells failed to reveal visible bands of B
virions or T particles in a number of attempts. (B) Co-cultivation of carrier cells with normal BHK 2 I
cells always produced visible bands of T particles and B virions. (C) Super-infection of VSVlnd
carrier cells with VSVIna B virions (m.o.i. = Ioo) failed to produce visible bands of virus from
2 x io 8 cells. (D) Superinfection with VSV New Jersey serotype B virions (m.o.i. = Ioo) led to
production of a B virion band plus two bands of T particles rescued from the carrier cells. (E) Onecycle amplification tests for the presence of mature T particles shed into the medium of carrier ceils
were negative. (F) Amplification tests for T particle RNP in cytoplasmic extracts of long-term
carrier cells always showed the presence of biologically active T particle RNP (if DEAE dextran
was used to facilitate RNP uptake into assay cells). Since all of these procedures were carried out
on the carrier within t week, the same two T particles sizes were rescued in each case. Others were
seen at other times in this carrier culture.
carrier cell p o p u l a t i o n s which themselves shed B virions and T particles (Table 5). These
results with c y t o p l a s m i c extracts p r o v i d e direct evidence t h a t carrier cells c o n t a i n T particle
R N P even when they are n o t shedding significant a m o u n t s o f m a t u r e B virions o r T particles,
a n d can explain resistance to h o m o l o g o u s superinfection in the absence o f large n u m b e r s
o f m a t u r e T particles.
Fig. 4 graphically shows the T particles p r o d u c e d f r o m carrier R N P on one occasion,
a n d summarizes the situation with r e g a r d to T particles in the stabilized carrier cells after
2-{ years in culture. It shows sucrose g r a d i e n t analysis results in which no visible a m o u n t o f
B virions or T particles can be purified directly f r o m the culture m e d i u m o f even very large
n u m b e r s o f carrier cells (Tube A), b u t co-cultivation o f carrier cells a n d n o r m a l B H K 21
cells yielded a visible b a n d o f B virions a n d two T particle b a n d s (Tube B). I n d i a n a challenge
yielded no visible virions or D I b a n d s (Tube C) b u t N e w Jersey challenge caused p r o d u c t i o n
o f large n u m b e r s o f the same two T particles f r o m the carrier cells (Tube D). Finally, amplification tests (one cycle) on the m e d i u m o f carrier cells generally failed to show significant
levels o f shed T particles (Tube E), except when B virion shedding was elevated, b u t amplification tests on c y t o p l a s m i c R N P were positive (Tube F). Again, the same two T particles
were p r o d u c e d as with co-cultivation a n d N e w Jersey challenge tests done at this time. A t
o t h e r times in the carriers' evolution these three techniques revealed different sizes o f T
particles than those seen here, b u t at any given time the same classes o f T particle are recovered
f r o m the carrier regardless o f the technique used to detect their presence. W e conclude t h a t
i m m a t u r e , biologically active T particle R N P are present in the c y t o p l a s m o f m o s t or all o f
these persistently infected cells.
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J. J. H O L L A N D
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OTHERS
Table 6. Recovery of infectious R N P from the cytoplasm of long-term persistently
infected carrier cells
Infectious intact B virions
Infectious R.NP recovered
releasedinto medium by
from carrier cell cytoplasm carriercells during previous
(p.f.u./cell)*
24 h (p.f.u./cell)
2 × IO -3
2 " 5 × IO -5
o (none detected)
o (none detected)
2 X IO -2
O'I
1.6× 10 -3
3 × I0-2
O'I
7 × to -4
* Infectious RNP levels were determined after DEAE dextran-facilitated uptake of RNP in dilutions of
cytoplasmic extracts from carrier cells at various times between 25 and z9 months after establishment of
the persistent carrier state. The subviral nature of the infectious p.f.u, was confirmed by omitting the DEAE
dextran facilitation step, in which case no p.f.u. (or > thousand-fold fewer) were obtained. (See Cartwright
et al. 0970); however, we cannot rule out the possibility that some of the p.f.u, measured in the RNP assays
were due to mature infectious virions.)
Infectious B virion RNP
We next attempted to detect infectious p.f.u, in the cytoplasm of carrier cells which are
producing very little or no virus, since this might be a useful way to recover virus from
persistent infections such as SSPE in which virus recovery is difficult despite extensive virus
antigen production (Payne, et al. I969; Horta-Barbosa et al. I969). Table 6 shows that
infectious p.f.u, of B virion R N P were recoverable from carrier cell cytoplasmic extracts.
Sometimes, more p.f.u, of infectious R N P were recoverable from these extracts of carrier
cells than could be recovered from the medium of the same carrier cells. But at one time
when the carrier cultures were producing no infectious mature B virions, no infectious p.f.u.
of B virion R N P could be recovered from cytoplasmic extracts. The recoveries of infectious
R N P in Table 6 represent significant amounts of B virion R N P in the cytoplasm of these
cells since Cartwright et aL (I97o) showed that VSV R N P are over io4-fold less infectious
than intact virus from which they were prepared (even with D E A E dextran facilitation).
Failure to detect DNA 'provirus' copies of VSV RNA in long-term carrier cells
Simpson & Iinuma (i975) recently reported the recovery of infectious provirus D N A
from cells persistently infected with respiratory syncytial virus. The cells yielded infectious
D N A capable of initiating the synthesis of complete virus in exposed cells.
Zhdanov (I975) also reported D N A provirus copies of measles virus and of other R N A
viruses in cells persistently infected by these viruses. In repeated attempts, we were unable
to transfect cells with D N A from the long-term VSV carrier. No induction of infectious
virus occurred in recipient B H K zr cells, HeLa cells or L cells and no induction of virusspecific antigens was detectable by immune fluorescent microscopy of recipient cell populations. Similar attempts employing D N A from cells persistently infected with lymphocytic
choriomeningitis virus, measles virus, mumps virus, influenza virus, or rabies virus gave
negative results. Several short term L cell carriers were included as D N A donors in the
hope that the abundant endogenous retrovirus (L cell virions) in these cells might increase
the likelihood of obtaining reverse copies, but these were also negative.
Table 7 shows the results of attempts to detect D N A copies of the VSV genome (or part of
the genome) in the B H K zI VSV~ndCAR~ carrier cells after z years of persistent infection.
A labelled D N A reverse transcript of the VSV genome was used as a probe. This probe
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V S V persistence
205
Table 7. Hybridization of aH-VSV cDNA with unlabelled DNA and RNA from
long-term VSV carrier culture
Source and type of
unlabelled nucleic acid
VSV-BHK 21 carrier
cell DNA*
Ratio of
unlabelled
nucleic acid to
3H-cDNA
Cot
aH-cDNA
reacted
annealing
of 3H-cDNA
with added
~ unlabelled VSV RNA at
DNA reacted end of reaction
(2 X 107)/1
74~
5'5
74"9
z457
7
86.2
74.z
6825
6"3
86.6
Control normal BHK 2t
(I'67 x toT)/I
424
5"4
66.8
DNA
14o4
6'3
7z'7
72-8
39o3
8"7
77
BHK 21 VSV carrier cell
(3"2 x IO7)/i
186o
39
RNA*
4o92
34
* DNA and RNA were extracted from VSV carrier cells after 25 months of persistent VSV infection.
The preparation of the ~H-cDNA probe and annealing techniques are described in Methods. A similar
failure to detect DNA provirus copies was encountered with a 3H-labelled VSV virion probe as described
in Methods.
was rendered solely of positive strand polarity by prior selection by annealing to virion
R N A and hydroxyapatite chromatography. At a Cot of 6825 and a ratio of carrier cell D N A
to VSV probe D N A of 2 x io 7 to I, no significant hybridization occurred even though 86.6
of the carrier-cell D N A had reannealed as would be expected at these Cot levels. We verified
that the probe was intact and still capable of specific annealing at the end of the incubation
period (75 ~ annealed back to VSV virion RNA). We conclude that there is less than onefortieth of a D N A provirus copy of the VSV genome per cell after 2 years of persistent
infection (or less than I cell in 4o with a complete copy). Since we cannot verify that a D N A
probe of this very high sp. act. is representative of the entire genome of VSV, we also prepared a high sp. act. - uSI-labelled VSV virion R N A probe by the method of Commerford
(I97I). Again, no annealing was detected at a ratio of carrier cell D N A to R N A probe of
z x Io 6 to I, and at a Cot of 918. Again the probe was able to anneal specifically (to VSV
virion R N A ) after termination of the incubation and this also rules out the presence of
D N A provirus copies (at a level of one-sixth of a genome per cell).
These negative results show that although D N A proviruses need not be present in cells
in order to achieve long-term stable, persistent infection by R N A viruses, they do not negate
the possibility that D N A provirus copies may occur under certain conditions. The ability
of this positive-strand D N A probe to anneal to carrier cell R N A (over one-third protected)
indicates the presence in carrier cells of significant levels of virion polarity minus strand
RNA. This could be B virion R N A or T particle R N A (probably mostly the latter).
Temperature-sensitive and small p&que characteristics of carrier B virions
These long-term carrier cells were originally established with the ts G3I mutant, which is
leaky and revertable (Pringle, I97O). Since its establishment and maintenance at 37 °C this
carrier has produced only leaky ts B virions which do not revert to wild type. Presumably
this non-reverting characteristic is due to additional mutations. Furthermore, these carrier
B virions have mutated into small plaque variants which produce smaller plaques than the
original ts G3I mutant at all temperatures from 25 to 33 °C (at 32 °C, I to 3 m m diam.
plaques are formed in 48 h; this is about half the size of ts G3r plaques under these
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J.J. HOLLAND AND OTHERS
conditions). Apparently the carrier state selects for multiple B virion mutations (ts and
otherwise) which attenuate virus virulence (since any cell in which more virulent B virions
are present is more likely to die or be outgrown by other cells). Another carrier cell line established with a virulent wild-type non-ts B virion (plus long T particles) illustrates this effect
more clearly. The original wild-type virus averages 2 m m plaques in 27 h at 37 °C, and B
virions recovered from the carrier after z months gave identical plaque numbers and plaque
size as the input wild type at 37 and 39 °C. But after I to a years of persistent infection the
virions shed by this carrier are all slightly ts, and give smaller plaques at all temperatures
than did the wild-type input B virions (after I year the plaque size in 4o h at 37 °C averages
2 m m and these shed B virions give 3 m m plaques at 33 °C in 4o h). Clonal pools from these
carrier B virion plaques still give Ioo ~o yields (_+ 2o ~o) at 32 and 38 °C, but give essentially
no yields at 4 ° °C after m.o.i. = 5o infection. This represents more than a lOOOOO-fold
yield reduction whereas wild-type yield is reduced only 97 ~ at 4o °C and B virions shed
after 2 months were reduced 98"5 ~ at 4o °C. Clearly the wild-type B virions are slowly
evolving into ts and small plaque mutants (even though they still give normal yields at
37 °C - the temperature at which the carrier is maintained). It should be noted that this
carrier established with wild-type virus shows frequent severe episodes of cytopathic crises
(unlike the carrier established with the ts G31 mutant) so temperature sensitivity has an
obvious stabilizing role in persistent carrier infections. Nevertheless cloned B virions from
these carrier cultures have never been able to establish new carriers without DI present.
They always destroy all B H K 2t cells exposed at 37 °C regardless of the ts nature of the
carrier virus. In one case cloned carrier B virions did establish a new carrier but the B virion
pool was found to have picked up carrier T particles during cloning and the new carrier was
shedding these T particles. In their original report o f a VSV carrier cell Wagner et al. (I963)
showed that small plaque mutation was important. The above results confirm that small
plaque and ts mutants are selected in carrier cells.
Persistent carrier state in less permissive cell lines
We have shown elsewhere (Holland et al. I976) that our line of HeLa cells is unable to
replicate VSV T particles. It was therefore of interest to determine whether they are capable
of becoming persistently infected by VSV. We attempted to establish a persistent carrier
state by exposure of these H e L a cells to a mixture of B virions and T particles from the
long term B H K 2I carrier (at m.o.i. = lo of B virions and Iooo of T particles). Although
this B virion T particle mixture readily established a carrier state in B H K 2I cells, it failed
in three separate attempts to establish persistence in the H e L a cells. In two cases all cells
died and in one case several clones grew up from the survivors of Io 7 infected cells, but
these clones were free of virus. In a fourth attempt, clones of surviving cells grew up only
after 2 months to form a carrier population. Since then (for 25 days) these persistently
infected cells have continuously shed B virions and T particles (so cells able to replicate D I
have been selected in establishing this persistent infection).
Since these H e L a cells are severalfold to tenfold less efficient in replicating VSV B virions
we examined several other less permissive cell lines. L cells did establish a persistent carrier
state with VSV for several months, but two independent L cell VSV carrier cell lines ' cured'
spontaneously after 3 months of shedding B virions and T particles, and no further virus
could be recovered thereafter. RKI~ rabbit cells, which are very non-permissive for VSV
Indiana, exhibited an initial cytopathology followed by outgrowth of normal cells which
shed no virus.
C. Blifeld & J. Holland (unpublished data) have failed to find mature T particles in a
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207
Table 8. Ability of clonal virus pools and high passage rabies virus pools to establish
persistent carrier infections of BHK 2I cells in culture
Virus inoculum pool
Clonal~
Clonalt
3rd undiluted passaget
3rd undiluted passaget
m.o.i.
too
o.oi
Ioo
o-oi
Degree of c.p.e.
at days p.i.
r - - ~ ' - - ~
Day 4
Day 8
2+
4+
2+
4+
0
2+
i+
3+
T particles present
in yield, at
c- ~ ' - - - - ,
Day 4
Day 8
+
+
+
+
+
+
Persistent carrier
state established
by surviving
cells*
Yes
Yes
Yes
Yes
* Following extensive c.p.e. (4 + ) nearly all cells degenerated and only a small percentage of cells survived
to repopulate the culture when they were maintained at 37 °C and medium was replenished regularly
beginning on day 8 p.i. at 32 °C. By 3o days all cultures had resumed vigorous growth and were shedding B
virions and T particles.
The clonal virus pool was the first passage pool of rabies HEP Flury virus prepared by low (m.o.i. =
o.oot) multiplicity passage of virus recovered directly from a clone and harvested at day 6. The undiluted
3rd passage pool was the same clonal pool passaged z more times on BHK 2t cells at m.o.i. = ioo and
harvested each time on day 7.
Drosophila melanogaster cell line persistently shedding small amounts o f virus after infection
with VSV B virions alone or B virions plus T particles. These insect cells do not exhibit
cytopathology when infected with VSV at high m.o.i., and they generate multiple m u t a n t
p r o g e n y o f input B virions ( M u d d et al. I973). Thus, it appears that certain cells are not able
to establish persistent infection with, or without, DI, whereas at least some insect cells are
able to do so without generating detectable levels o f mature T particles. E a t o n (I975)
recently showed that Semliki Forest virus D I do not interfere or replicate well in mosquito
cells.
Generation of carrier cultures by cloned rabies virus
W i k t o r & Clark 0 9 7 2 ) showed that rabies virus establishes persistent infection o f cells in
culture. Crick & Brown (I974) demonstrated that rabies virus generates T particles and
Kawai et al. (3975) and Holland & Villarreal (1975) have recovered D I from chronically
infected rabies carrier B H K 23 cells. We have attempted to determine whether rabies T
particles are required to establish persistence by infecting cells with cloned B virion pools
alone (or with the addition o f purified rabies T particles). Table 8 shows that clonal pools
o f rabies virus generated a persistently infected carrier state in B H K 23 cells whether or not
T particles were added. In the presence of added T particles, cytopathology was lessened
and surviving cells resumed growth within Io days. In the absence o f added T particles the
clonal rabies B virion pool caused extensive cytopathology, and most o f the cell degenerated.
However, the surviving cells eventually resumed growth and established a persistent carrier
state in which both B virions and T particles were continuously shed into the medium. This
ability o f clonal pools o f rabies virus to generate carrier cultures alone seemed to differ
f r o m the results seen with VSV in which T particles are required. However, this apparent
discrepancy m a y be explained (Table 8) by the finding that even clonal pools o f our H E P
Flury rabies virus contain (or quickly generate) T particles. Perhaps rabies virus generated
D1 more readily than VSV, but in any case the 7-day period o f plaque formation and the
additional 5 days required to prepare a pool from cloned rabies virus allows ample time
for D I generation. Regardless o f the reasons, our clonal pools of rabies virus show only
B virions visible during the first 4 days following infection of B H K 23 cells, but in the days
thereafter a variety of T particles appear (Holland et al. 1976). Differing abilities to generate
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J. J. H O L L A N D
AND OTHERS
DI may help explain the varying capacities of different RNA viruses (or different strains) to
establish persistent infection. It has been found that even clonal pools of VSV have already
generated T particles even though it takes several additional high multiplicity passages to
visualize them directly (Holland et al. 1976); so merely cloning does not assure freedom from
DI or their biological effects. Crick & Brown (1974) also reported that their rabies virus
pools showed interfering activity after passages at m.o.i, less than I-o.
DISCUSSION
The above results further implicate DI as a major factor in long-term, persistent infections
by VSV. Amplification assays for DI RNP now allow DI detection in persistent infections
where DI and even infectious virus may otherwise be difficult to recover and where their
presence may hitherto have been overlooked.
The role of ts mutants in resistance is more difficult to evaluate. Our results show that
wild-type non-ts VSV will establish carriers in the presence of DI but not in their absence.
Even the slightly ts small plaque mutants (from the wild-type carrier cells) and frank ts
mutants require T particles to establish persistent infection. We have not as yet achieved
a persistent carrier state with rhabdoviruses in which the carrier cells were not producing
DI. On the other hand, the carrier persistently infected with the ts G31 mutant (plus DI) is
much more stable and undergoes fewer and less severe episodes of cytopathology than wild
type carriers, so temperature sensitivity is a stabilizing factor in this balanced host-virus
relationship. This is reinforced by the fact that the virus carrier originally established with
wild-type virus has shed only virus that is slightly ts after more than a year of persistent
infection. Temperature-sensitive mutations are not the only ones which occurred during
persistence, since the shed virus is a small plaque mutant even at low temperatures. It would
be surprising if a variety of mutations did not occur during persistent infection by an R N A
virus. Some of these must be selected for the attenuating effect they have on virus virulence
(since any carrier cell in which virulent virus is present or arises by mutation is more likely
to die or be outgrown).
It should be emphasized that the B H K 2t-VSV system is one which is normally very
virulent, with up to 2oooo p.f.u./cell being produced in acutely infected cells in the absence
of DI. Less susceptible cells may show different modes of persistence or failure to achieve
long-term persistence (as reported for Drosophila cells or RKla cells alone). Since insects,
for example, are natural vectors for some rhabdoviruses, their cellular resistance to VSV
cytopathology (Mudd et al. I973) may allow limited replication and persistent shedding of
(mainly mutant) virus.
The ability of cloning (or antiviral antibody on light monolayers) to 'cure' cells from
carrier cultures in which all or nearly all cells were producing large quantities of virus
antigen suggests that DI RNP must be able to completely out-replicate B virion RNP at
times in carrier cells. This could lead directly t o ' curing', or indirectly by cell division diluting
out all B virion RNP in some daughter cells. In any case the 'cured' cells behave as fully
susceptible normal B H K 21 cells so the carrier state cannot be attributed to cellular mutations
which lead to virus resistance. This tendency for DI to overwhelm B virions is well illustrated
by the data of Table 5 in which large numbers of biologically active DI were recovered at
one time period during which no infectious B virions or infectious RNP could be recovered.
This can explain why curing might occur with fairly high probability. In the absence of
antibody,' cured' cells should form a clone of cells which are highly susceptible to re-infection
by virus from neighbouring cells once all of their DI RNP are degraded or diluted. When
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V S V persistence
209
such cured clones represent only a small percentage of the total cartier monolayer, their reinfection would lead to only low virus yields and minor or undetectable cytopathology,
especially if some DI RNP remain in some cells of the clone (or arrive in the clone at about
the same time as infectious virions). A larger percentage o f ' c u r e d ' cells with no DI RNP
could give high yields of virus sufficient to trigger overt cytopathology. It is obvious from
the very low virus levels in the carrier medium at any time (Fig. 3) that only a tiny proportion
of cells in the carrier culture is ever cured of B virion RNP and of T particle RNP. If only
o.1% of the cells in the carrier culture were fully susceptible and became infected simultaneously, the amount of infectious virus released into the medium from these cells alone
would approximate Io p.f.u./cell (since cured cells and normal B H K 2I cells yield IOOOOto
2oooo p.f.u./cell). Because this amount of virus is very rare in the carrier cell medium this
situation must occur only infrequently, probably because DI spread from cell to cell at
least as frequently as B virions.
All of the above discussion is based on the supposition that our plaque assay can detect
most infectious B virions released by the carrier culture. We cannot rule out the possibility
that non-cytopathic mutant virions are released from the carrier culture, or even that they
are a predominant component of the carrier virus population. In any event, the ability of
isolated cells or 'antibody-protected' cells to cure spontaneously helps to explain why persistent infection in vivo is not a commonplace event at least at a level of tissue involvement
that leads to overt disease. Such 'curing' may also be important in recovery from acute
virus disease. The ability of antibody to cause capping and modulation of virus antigens
under normal conditions at the cell surface (Joseph & Oldstone, I974, 1975) could increase
the probability of cell ' curing' in vivo by preventing T cell destruction or complement lysis
until 'curing' is achieved. However, cells which divide infrequently in vivo or not at all (e.g.
neurons) may be much more resistant to 'curing', and tend to provide foci of persistent
infection more readily than would dividing cell populations in which cured cells could
out-compete the infected population.
The accompanying paper (Villarreal & Holland, 1976)shows that VSV virion R N A replication is usually undetectable in carrier cells and VSV m R N A synthesis is very low. This helps
to explain how curing can occur at a detectable frequency in carrier cells. We conclude
that the VSV carrier state studied here is due to constantly changing levels of B virions and
T particles (and their intracellular RNP). Although the yields of virus and DI are much
lower than in the rabies carrier cells of Kawai et al. 0975) the mechanisms seem to be very
similar. In both cases there are cyclic variations in virus and DI yields. These are probably
explainable by the cycling model of DI replication (Palma & Huang, I974), with the added
complexity of carrier cell survival and even curing, so that carrier cell cycling may involve
RNP cycles within single cells as well as at the cell population - mature virion - mature DI
level. Thus, DI (and evolution of attenuated B virion mutants) provide for RNA viruses
the functional equivalent of lysogeny in D N A viruses.
We thank Estelle Ashcraft for excellent technical assistance. This work was supported
by a grant from the National Cancer Institute USPHS no. CAio8o2 to J. J. H. and USPHS
grants no. Aio9484 and Aio7oo7 to M. B. A.O. and NSI2428 to R.W. This is contribution
no. 1IOI from Scripps Clinic and Research Foundation, La Jolla, California.
Note added at p r o o f
Youngner & Quagliana (1976) recently interpreted VSV persistence in L cells as being
due to ts mutant B virion interference with wild-type VSV. Their interference tests are
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210
J.J. HOLLAND
AND OTHERS
difficult t o e v a l u a t e s i n c e t h e y u s e d w i l d - t y p e v i r u s a t m . o . i . = o. I a n d t - m u t a n t a t m . o . i .
= ~.o. H o w e v e r , c l o n e d B v i r i o n s f r o m o u r c a r r i e r cells a f t e r 21 y e a r s s h o w e d n o i n t e r f e r e n c e w i t h w i l d - t y p e V S V w h e n b o t h w e r e i n f e c t e d a t e q u a l m . o . i . ( I o r ~oo) a n d w h e n
verified t o b e free o f D I .
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