Vesicular Stomatitis Virus Can Establish Persistent ...

J. gen. Virol. (1982), 63, 493-497. Printed in Great Britain

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Key words: vesicular stomatitis virus/persistent infection/pathogenesis

Vesicular Stomatitis Virus Can Establish Persistent Infections in Syrian Hamsters (Accepted 4 July 1982)

SUMMARY Persistent infections by vesicular stomatitis virus (VSV) of the Indiana serotype were readily established in adult Syrian hamsters following intraperitoneal injection of the virus. Plaque-forming virus, identified as VSV by serological and physical criteria, was isolated from brain homogenates of five hamsters that were tested 3 to 8 months after infection. Four of these animals had exhibited either transient or permanent paralysis, whereas the fifth appeared healthy, during the period of observation. At the time of sacrifice all hamsters had high titres of anti-VSV-neutralizing antibodies in their sera. Vesicular stomatitis virus (VSV), a negative-strand RNA virus, has been used by several investigators to study mechanisms of virus persistence in cultured cells of hamster and mouse origin (Holland & Villarreal, 1974; Holland et al., 1976; Ramseur & Friedman, 1977, 1978; Sekellick & Marcus, 1978, 1979; Youngner et al., 1976). Depending on the system used, temperature-sensitive (ts) mutants, defective-interfering (DI) particles, and interferon have been shown to be important in either the establishment or maintenance of persistent infections by this highly cytopathic virus. Whether these in vitro studies are relevant to mechanisms of persistent infections in living animals is not known since long-term in vivo infections by VSV have not been demonstrated. Perhaps the closest model was provided by Jones et al. (1980) who recovered virus from nude mice after injection of VSV-carrier BHK-21 cells. We now report that persistent infections by VSV Indiana can be achieved regularly in immunocompetent adult Syrian hamsters. Virus, identified as VSV by serological and physical criteria, has been recovered, from brain, spleen and liver up to 8-5 months (the longest time period tested) after intraperitoneal (i.p.) inoculation of hamsters with VSV. We have recovered VSV Indiana following prolonged persistent infections of hamsters that were infected for unrelated experiments; these hamsters had received different doses of VSV (Table 1). In one experiment, LSH and M H A strain hamsters that normally die of an acute systemic disease within 3 days of i.p. injection of low doses of VSV (Toronto strain of the Indiana serotype) (Fultz et al., 1981 a), were protected from death by the simultaneous injection of VSV and homologous DI particles (Fultz et al., 1981 b, 1982). The inocula contained 104 p.f.u. of VSV and either 107 or 108 DI-LT particles (a DI particle from the 3' end of the VSV genome) (Petric & Prevec, 1970). VSV and DI particles were co-injected i.p. into sixteen (LSH and MHA) hamsters. Seven of the infected hamsters died and of the nine survivors, three developed transient or permanent paralysis of one or more limbs during the first few weeks post-infection. No obvious changes in clinical status occurred subsequently and the animals appeared in good health over the next several months. Paralysed animals were considered to be more likely to harbour virus for extended periods, so survivors that exhibited no signs of paralysis were not kept in these initial experiments. At 6 or 8 months after initial infection with wild-type VSV and DI particles, the three hamsters (Table 1 ; LSH-I, LSH-2 and MHA) were sacrificed. Selected tissues were analysed for plaque-forming VSV by titration of 10 % (w/v) homogenates on monolayers of confluent L cells using an agar overlay but no virus plaques were seen. When 0.5 ml samples of tissue homogenates were incubated overnight at 37 °C on monolayers of confluent rat cells of the R(B77) line, virus eventually was detected in brain tissues of all three animals and from spleen and liver tissues of the M H A hamster. Four serial, undiluted passages of medium from R(B77) monolayers infected with LSH-1 or LSH-2 brain tissue were required before virus was detected, 0022-1317/82/0000-5208 $02.00© 1982 SGM

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Table 1. Description of hamsters pers&tently infected with vesicular stomatitis virus

Animal LSH-I LSH-2 LSH-3 MHA UTI

Inoculum* 107 DI + 10~ VSV 107 DI + l04 VSV Poly(I).poly(C) + 104 VSV 108 DI + 10~ VSV 106 VSV

Paralysis Permanent Transient None Permanent Permanent

Time of sacrificer (months) 6 6 3 8 8.5

Age at death (months) 18 18 8 13 14

Brain yielding virus + + + +:~ +

* Animals were infected i.p. with VSV (diluted in balanced salt solution) by injecting a volume of 0.1 ml; VSV and the homologousDI-LT particles were co-injected in a volumeof 0-2 ml; 100[xgof poly(I), poly(C) (dissolved in PBS) were injected i.p. in a volume of 0.1 ml immediately prior to injection of VSV. t At various times after infection, the animals were placed under ether anaesthesia and were bled by cardiac puncture. The blood was allowed to clot and serum was prepared. Following exposure of the animals to a lethal dose of ether, various organs were removed and 10~ homogenates of all tissues were made with a ground-glass homogenizer in Dulbecco's modified Eagle's medium. Tissues and sera were stored at - 70 °C until used. :~Spleen, liver, and lymph node homogenates from this animal were also tested: VSV was recovered from spleen (second passage) and liver (third passage), but not from lymph node (four consecutive passages).

while the M H A brain tissue yielded detectable virus after only one passage. The additional passages required for the LSH tissues may have been due either to the presence of DI particles or to anti-VSV-neutralizing antibodies in tissue homogenates (see below). In the former case, eventual detection of virus in the presence of DI particles could result from the cyclic nature of DI interference (Palma & Huang, 1974) or from altered interactions of virus and DI particles, as has been described for VSV isolated from persistently infected cells in vitro (Horodyski & Holland, 1980). Another possibility is that the LSH tissues contained only a small proportion of viruses capable of growing at 37 °C and, thus, several passages were required before a sufficient amount for detection of a virus band in sucrose gradients was possible. Virus was detected in supernatants of the overnight R(B77) cultures by applying pelleted material, obtained after centrifugation (90 min at 25000 rev/min in an SW27 rotor) of supernatants, to linear 5 to 3 0 ~ sucrose gradients. Visible bands of virus particles that sedimented in the same position as wildtype VSV were collected and used as stocks for subsequent tests. These recovered viruses were able to form plaques on L cells. To ensure that the recovered viruses were VSV, parallel plaque-reduction assays were done with viruses from brain tissue and the original stock virus using hyperimmune rabbit anti-VSV serum. The dilutions of serum that reduced the n u m b e r of plaques by 5 0 ~ were: LSH-1, 1/15000; LSH-2, 1/15500; MHA, 1/13000; original VSV, 1/17000. These results show that the viruses recovered from hamsters as many as 8 months after infection were serologically identical to VSV. A fourth hamster that exhibited paralysis after i.p. injection of VSV was of the UT1 strain which is genetically resistant to lethal VSV disease (Fultz et al., 1981 a). This animal had been inoculated i.p. with l06 p.f.u, of VSV and, in this one experimental group, was the only animal out of ten survivors that became paralysed. This hamster died 8.5 months after infection, possibly of causes unrelated to the virus infection. Virus was recovered from brain tissue of this UT1 hamster as shown by a band in a sucrose gradient after one overnight incubation of 0.5 ml of brain homogenate with R(B77) cells. When tested with rabbit anti-VSV serum, 5 0 ~ reductions in plaques of the UTl-recovered virus and of VSV occurred with the same dilution of antiserum, showing that this virus also was VSV Indiana. Since VSV was recovered many months after infection from hamsters that exhibited some form of paralysis, we subsequently tested a survivor of VSV infection that had shown no evidence of paralysis or sickness following infection (Table 1 ; LSH-3). This animal was one of ten survivors out of a group of twelve hamsters that had received an i.p injection of 100 ~tg poly(I), poly(C) at the same time that they received 104 p.f.u, of VSV; 3 months after infection one animal was chosen at random and was sacrificed. As with the paralysed animals, virus was not detected by direct plaque assay of tissue homogenates, but was identified as a band in a


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Table 2. VSV-neutralizing titres o f sera from persistently infected hamsters Animal LSH-I LSH-2 LSH-3 MHA Control1"

Time post-infection 6 months 6 months 3 months 8 months 2-6 weeks

Titre* 1700 14000 7000 2500 7200:~ (2900-16000)

* Values given are the reciprocals of the dilution of sera from infected hamsters required to reduce the number of VSV plaques by 50%. Equal volumes of virus and serum were incubated 30 min at 37 °C, after which 0.1 ml samples were added to L cell monolayers. Virus was allowed to adsorb for 45 min at 37 °C, and then the cell monolayers were overlaid with agar. The plates were incubated for 2 days before plaques were counted. t Controls were LSH and UT1 hamsters that survived VSV infection and that were bled by cardiac puncture at the indicated times post-infection. :~Titre given is the average from thirteen hamsters. The values in parentheses are the ranges of titres in sera from the thirteen control hamsters.

sucrose gradient following two passages of medium from cells incubated with brain homogenate. This virus, also, was identified as VSV I n d i a n a by plaque-reduction assay. A common feature of the above hamsters was that antibodies capable o f neutralizing VSV were present in their sera. The levels of neutralizing antibodies found in sera from these hamsters up to 8 months after infection were similar to levels routinely found in animals 2 to 6 weeks after infection with VSV (Table 2). The only tissue available from the UT1 hamster was brain. W e presume that, because antibodies to VSV were detected (by radioimmunoassay; d a t a not shown) in brain tissue of the UT1 hamster, high systemic titres of VSV-neutralizing antibodies also were present in this animal. Our data demonstrate that persistent infections can be established in Syrian hamsters that are genetically susceptible (LSH and M H A ) to VSV as well as in genetically resistant (UT1) hamsters. Since virus was recovered from spleen and liver tissues o f the M H A hamster, it would appear that persistent infection by VSV is not limited to the central nervous system (CNS). Also, the fact that virus was recovered from one hamster (LSH-3) that showed no signs o f paralysis suggests that injury to the C N S resulting in motor i m p a i r m e n t is neither a prerequisite nor a consequence of persistent infection. However, the non-paralysed animal had been injected with poly(I), poly(C) and VSV while the others had received only virus particles; therefore, persistence in the different animals may have involved different mechanisms. F o r example, none of the ten survivors in the poly(I), poly(C) experiment (that included LSH-3) became paralysed. Since all of the recovered viruses were neutralized by rabbit anti-VSV serum, it would a p p e a r that mutations that alter antigenic determinants in a major way had not accumulated. The sensitivity of the plaque-reduction assay is such that subtle alterations in antigenicity m a y not be detected. Preliminary characterization of virus isolated from the above hamsters indicates that many of the recovered viruses are small-plaque mutants and that approximately 90~o of the plaque-purified clones are ts. It will be of interest to see if during in vivo persistent infections, multiple genome mutations occur in VSV in a manner analogous to that found by Holland et al. (1979) after long-term in vitro persistence of VSV in BHK-21 cells. In their system the accumulated mutations appeared to be directed by D I particles. Various factors that may operate in allowing viruses to persist for extended periods of time in apparently immunocompetent hosts have been suggested by in vivo and in vitro experiments. Virus-specific antibodies have been shown to participate in the persistence of visna virus in sheep (Clements et al., 1980; N a r a y a n et al., 1981) and of measles virus in cultured cells (Oldstone, 1977). In the former case, antibodies appear to exert selective pressure for antigenic variants of visna virus that are no longer neutralized by antibodies to the virus originally inoculated. For measles virus, antibodies modulate virus antigen expression on cell surfaces so that infected cells are no longer susceptible to destruction by immune cytolysis. Presently, we are monitoring anti-VSV antibody levels in susceptible and resistant hamster strains that have survived primary VSV infections, and we find that apparently normal as well

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as paralysed hamsters have similar levels of serum anti-VSV antibodies up to 1 year postinfection. Also, there appears to be no correlation between the degree of paralysis and antibody levels due to persistent VSV infection. The mechanism of RNA virus persistence in whole animals is likely to be complex, and the exact roles of antibodies, DI particles, or virus mutants are unclear at present. Our hamsterVSV system, however, provides an excellent model for in vivo persistence and continued study may provide a better understanding of the roles of these various factors in the establishment or maintenance of RNA virus persistence. P.N.F. gives special thanks to Dr John Holland whose comments were responsible for the initiation of this work. We also thank Drs Brad Ozanne, David Hart and Stuart Nichol for helpful discussions, and Ms Diane Proia for technical assistance. This work was supported by U S P H S grants RR01133 and CA-09082.

Departments o f ~Cell Biology and 3Microbiology University o f Texas Health Science Center at Dallas Dallas, Texas 75235, U.S.A. and 2Department o f Veterinary Pathobiology University o f Illinois, Urbana-Champaign Urbana, Illinois 61801, U.S.A.

P A T R I C I A N . F U L T Z 1*IJOHN A . SHADDUCK 2 C . YONG K A N G 3 J. WAYNE STREILEIN 1

t Present address: Department of Biology C-016, University of California, San Diego, La Jolla, California 92093, U.S.A. REFERENCES CLEMENTS, J. E., PEDERSEN, F. S., NARAYAN,O. & HASELTINE, W. A. (1980). G e n o m i c changes associated with antigenic variation of visna virus during persistent infection. Proceedings of the National Academy of Sciences of the United States of America 77, 4454-4458. FULTZ, P. N., SHADDUCK, J. A., KANG, C. Y. & STRE1LEIN, J. W. (1981 a). Genetic analysis of resistance to lethal infections of vesicular stomatitis virus in Syrian hamsters. Infection and Immunity 32, 1007-1013. FULTZ, P. N., SHADDUCK,J. A., KANG, C. Y. & STREILEIN, J. W. (1981 b). On the m e c h a n i s m of DI particle protection against lethal VSV infections in hamsters. In The Replication of Negative Strand Viruses, pp. 893-899. Edited by D. H. L. Bishop & R. W. Compans. New York: Elsevier/North-Holland. FULTZ, P. N., SHADDUCK,J. A., KANG, C. Y. & STREILEIN, J. W. (1982). Mediators of protection against lethal systemic vesicular stomatitis virus infections in hamsters: defective interfering particles, polyinosinate-polycytidylate, and interferon. Infection and Immunity 37, 679-686. HOLLAND, J. J. & VILLARREAL, t. P. (1974). Persistent noncytocidal vesicular stomatitis infections mediated by defective T particles that suppress virion transcriptase. Proceedings of the National Academy of Sciences of the United States of America 71, 2956-2960. HOLLAND,J. J., VILLARREAL,L. P., WELSH,R. M., OLDSTONE,M. B. A., KOHNE, D., LAZZARINI,R. & SCOLNICK,E. (1976). Long-term persistent vesicular stomatitis virus and rabies virus infection of cells in vitro. Journal of General Virology" 33, 193-211. HOLLAND, J. J., GRABAU, E. A., JONES, C. L. & SEMLER, B. L. (1979). Evolution of multiple genome mutations during long-term persistent infection by vesicular stomatitis virus. Cell 16, 495-504. HORODYSKI,F. M. & HOLLAND,J. J. (1980). Viruses isolated from ceils persistently infected with vesicular stomatitis virus show altered interactions with defective interfering particles. Journal of Virology 36, 627-631. JONES, C. L., SPINDLER, K. R. & HOLLAND,J. J. (1980). Studies on tumorigenicity of cells persistently infected with vesicular stomatitis virus in nude mice. Virology 103, 158-166. NARAYAN,O., CLEMENTS,J. E., GRIFFIN, D. E. & WOLINSKY,J. S. (1981). Neutralizing antibody spectrum determines the antigenic profiles of emerging mutants of visna virus. Infection and Immunity 32, 1045-1050. OLDSTONE,M. B. A. (1977). Role of antibody in regulating virus persistence: modulation of viral antigens expressed on the cell's plasma m e m b r a n e and analyses of cell lysis. In Developmentof Host Defenses, pp. 223-232. Edited by M. D. Cooper & D. H. Dayton. New York: Raven Press. PALMA, E. L. & HUANG, A. (1974). Cyclic production of vesicular stomatitis virus caused by defective interfering particles. Journal of lnJectious Diseases 129, 402~,10. PETRIC, M. & PREVEC, L. (1970). Vesicular stomatitis virus-a new interfering particle, intracellular structures, and specific RNA. Virology 31, 615-630. RAMSEUR, J. M. & FRIEDMAN, R. M. (1977). Prolonged infection of interferon-treated cells by vesicular stomatitis virus : possible role of temperature-sensitive m u t a n t s and interferon. Journal of General Virology37, 523-533. RAMSEUR, J. M. & FRIEDMAN, R. M. (1978). Prolonged infection of L cells with vesicular stomatitis virus. Defective interfering forms and temperature-sensitive mutants as factors in the infection. Virology 85, 253-261.


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SEKELLICK, M. J. & MARCUS,P. I. (1978). Persistent infection. I. Interferon-inducing defective-interfering particles as mediators of cell sparing: possible role in persistent infection by vesicular stomatitis virus. Virology85, 175-186. SEKELLICK,M. I. & MARCUS,P. I. (1979). Persistent infection. II. Interferon-inducing temperature-sensitive m u t a n t s as mediators of cell sparing: possible role in persistent infection by vesicular stomatitis virus. Virology95, 3(w 47 YOUNGNER, J. S., DUBOVI, E. J., QUAGLIANA,D. O., KELLY, M. & PREBLE, O. T. (1976). Role of temperature-sensitive mutants in persistent infection initiated with vesicular stomatitis virus. Journal of Virology 19, 90-101.

(Received 22 April 1982)