_Archives
Vi rology
Arch Virol (1993) 128:389-394
© Springer-Verlag 1'993 Printed in Austria
In vitro selection of Junin virus antigenic variants
Brief R e p o r t Laura E. Alch~ and Celia E. Coto
Laboratory of Virology, Department of Biochemistry, School of Science, University of Buenos Aires, Buenos Aires, Argentine Accepted June 2, 1992
Summary. The establishment of an experimental persistent infection with Junin virus, the aetiological agent of argentine hemorrhagic fever, involves the emergence of antigenic variants in brain and blood of the cricetid Calomys musculinus. We demonstrate that antigenic variants can also be isolated in vitro under the selective pressure of polyclonal antibodies and from a long-term infected C. musculinus primary embryo fibroblast culture. The participation of neutralizing antibodies and host cells in the appearance of viral variants in vivo is discussed.
Genetic variation is a common feature of viral persistence since the ability of several viruses to undergo mutation during chronic infection has been amply documented [16-18]. Particularly, RNA viruses- including single isolates and clonal populations-consist of multiple variants collectively designated viral quasispecies [8]. However, the selective pressures involved in the emergence of these viral variants in vivo are not well understood. In the case of arenaviruses, perpetuation of the agents in nature depends on their efficiency to establish a long-term persistent infection in rodents, except for Tacaribe virus that was isolated from bats and never could be associated to other animal species. Both lymphocytic choriomeningitis virus (LCMV) and Junin virus (JV) have been studied in order to elucidate the mechanism(s) responsible for viral persistence in mice and cricetids, respectively. Ahmed et al. [2, 3-1 showed that most of the LCMV isolates obtained from the CNS of infected mice behaves similarly to the parental strain, since they induce potent virus-specific cytotoxic T lymphocyte (CTL) responses in adult mice. In contrast, LCMV clones derived from the lymphoid tissue of carrier mice cause persistent infections in adult mice associated with suppressed T cell responses. Both viral
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phenotypes only differed in a single or few mutations which determine the organ-specific selection of these mutants [1]. The infection of the cricetid Calomys musculinus from an outbred colony with the XJC13 strain of JV has been thoroughly studied in our laboratory. This strain is selectively neurotropic for cricetids since approximately 60% of neonatal animals infected by intraperitoneal route die after developing a clinical neurological disease. This acute stage of infection is also characterized by the detection of high viral titers in brain, an increased frequency of viremia and the onset of a humoral immune response from day 15 post-infection (p.i.) onwards [6, 13]. Among the survivors, some recover completely and others present persisting neurological symptoms associated with the simultaneous presence of neutralizing antibodies and high viral titers in brain [12]. The inability of circulating antibodies in clearing the virus from these animals could be explained by the emergence of antigenic variants in blood and brain, which are detected by cross neutralization assays against an antiserum raised to parental virus [4] and by kinetic neutralization tests against a serum sample from an infected C. musculinus, respectively [5]. The ease of deriving antigenically distinct mutant viruses in vivo enabled us to consider the occurrence of stable variants resistant to neutralization in vitro conditions. Thus, we first analyzed the role of polyclonal antibodies in the selection of antigenic variants during JV replication in Vero cells. For that purpose, approximately 200 PFU/0.2 ml of cloned JV (XJC13 strain) were mixed with two fold dilutions of heated (56 °C for 30 min) anti JV hyperimmune serum prepared in guinea pigs. Serum-virus mixtures were incubated at 37 °C during 60 min, and then titrated for remaining virus by plaque assay on Vero cells, by duplicate. One series was overlaid with a semisolid medium containing a 1 : 40 dilution of anti JV serum (A), while series B, which served as a control, received the same dilution of normal guinea pig serum. Plaques were stained with neutral red 7 days later. The dose-response curves of surviving activity versus antiserum dilution are shown in Fig. 1. Both curves exhibited a comparable pattern of neutralization although, in the case of the highest dilutions of antiserum, remaining infectivities of series A were considerably lower than those corresponding to series B. This effect can be adscribed to the additional JV antiserum present in the overlay. Therefore, well-separated plaques representing virus that had escaped neutralization and had been able to grow under the selective pressure of antibodies were picked-up and amplified in Vero cells. Due to a small plaque size, it was possible to recover infectivity in only two out of ten isolated clones, which were designated JVr and JVrI. When these viral isolates were assayed against the anti JV serum, both proved to be resistant to neutralization since no decrease in viral surviving fractions was detected, except for a slight loss of infectivity observed with the 1:40 antiserum dilution (Fig. 2). In consequence, external selective pressure displayed by polyclonal antibodies has led to the emergence of JV antigenic variants in vitro. According to these results, we can speculate
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Fig. 1. Dose-responsecurves of JV against an anti JV serum. Neutralization assays were done with a hyperimmune serum prepared against JV in guinea pigs. O Normal guinea pig serum (series B) and • anti JV serum (series A) were added to the overlay medium (dilution 1:40) Fig. 2. Dose-responsecurves of JV and viral variants JVr and JVrI isolated in vitro against the anti JV serum. Neutralization assays were done with the anti JV serum against JV (A), JVr (Q), and JVrI ( • )
that the infection of C. musculinus with JV would constitute an appropriate experimental model for the selection of serological variants achieved by circulating antibodies, although non immunological forces can also be involved. In fact, it was demonstrated that LCMV variants are likely to emerge when they have a growth advantage in certain cell types [11]. In order to analyze the importance of host cell in the occurrence of variant JV populations, primary C. museulinus embryo fibroblasts (CEF) cultures were established from 15-18 days old C. musculinus embryos. Fetuses were rinsed in PBS and afterbirths and heads were removed. Cell suspension was obtained by trypsin dissociation and then seeded. Secondary cultures were used to be infected with a multiplicity of infection of 0.I of JV. With the purpose of following the yield of viral progeny from infected stationary CEF cultures, samples of culture fluids were collected at 17, 27 and 53 days p.i. (named JV/ 17, JV/27 and JV/53), and titrated by plaque assay on Vero cells. After an initial peak of virus production of 1 06/ P F U ml at 10 days p.i., the amount of infectious virus particles progressively diminished till day 53 p.i., reaching less than 102 PFU/ml (Fig. 3). This replicative curve is comparable to the pattern of multiplication of JV in persistently infected Vero cells [7], although no cytopathic effect was detected in the case of infected CEF cultures.
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Fig. 3. Virus release from CEF cultures infected with JV (m.o.i. = 0.1). Supernatants were harvested at days 17, 27, and 53 p.i. and were titrated by plaque formation on Vero cell monolayers. Each point represents the mean value of two CEF cultures analyzed Fig. 4. Dose-response curves of JV and viral variants isolated from infected CEF cultures. Neutralization assays were done with an anti JV serum (which belongs to a lot of antisera exhibiting a higher homologous titer than that observed in Figs. 1 and 2), against JV ( l ) , JV/17 (A), JV/27 (O) and JV/53 (A) When viral stocks obtained were tested in a neutralization assay against the anti JV serum, the dose-response curves of JV/17 and JV/27 were similar to that observed for the parental strain (Fig.4). However, JV/53 escaped the neutralizing activity of antibodies, indicating the emergence of a novel population of viral mutants after a long-term infection in CEF cultures. The experiments above described demonstrate that antigenic variants of JV can be obtained in vitro under selective immunological pressure and, besides, by means of a CEF-specific selection during chronic infection. It is important to remark that the appearance of viral mutants through the incorporation of polyvalent antibodies in cell cultures was primarily used with lentiviruses [14, 15], since the selection of different stable variants from other viral families were only reported in the presence of monoclonal antibodies [9, 10]. Nevertheless, the role of circulating antibodies in the selection of serological variants in vivo still remains obscure, especially if we consider that the earliest mutant viral isolation from blood occurred at day 8 p.i., before the onset of the humoral immune response (unpubl. results). On the other hand, the influence of host cells in the emergence of JV variants was also analyzed. Thus, the detection of antigenic variants in the supernatants of long-term infected CEF cultures suggests the involvement of a host-dependent mechanism of variation independent of the immunological pressures which
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provokes antigenic drift. This feature may be supported by preliminary results, which indicate that viruses yielded by peritoneal macrophages obtained from infected cricetids at 20 days p.i. behaved as antigenic variants (unpubl. data). In consequence, it is likely that better replication in fibroblasts and/or macrophages may be one of the underlaying cause of chronic infection, as it was demonstrated in the case of L C M V [i 1]. On the basis of the results presented in this study, we suggest that viral stock would contain or generate a phenotypically mixed population even after plaque purification. In any case, those JV variants which exhibited a greater fitness during the course of infection will succeed, either as a consequence of the exposure to the immune system and/or due to the multiple replication cycles in the various organs and cell types of the host. In conclusion, antigenic variation of JV would be the result of the interaction of different biological strategies essential for the establishment and/or maintenance of the persistent state i n vivo. The high tendency of virus variability observed combined with an appropriate environment provided by the host may play a crucial role in the survival of the virus in nature.
Acknowledgements We thank S. Coronato for technical assistance. This research was supported by grants from Secretaria de Ciencia y Tecnologia, Argentina.
References 1. Ahmed R, Hahn CS, Somasundaram T, Villarete L, Matloubian M, Strauss JH (1991) Molecular basis of organ-specific selection of viral variants during chronic infection. J Virol 65:4242-4247 2. Ahmed R, Oldstone MBA (1988) Organ-specific selection of viral variants during chronic infection. J Exp Med 167:1719-1724 3. Ahmed R, Salmi A, Butler LD, Chiller J, Oldstone MBA (1984) Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice: role in suppression of cytotoxic T lymphocyte response and viral persistence. J Exp Med 60:521-540 4. Alch6 LE, Coto CE (1986) Antigenic variants of Junin virus isolated from infected Calomys musculinus. Arch Virol 90:343-348 5. Alch6 LE, Coto CE (1988) Differentiation of Junin virus and antigenic variants isolated in vivo by kinetic neutralization assays. J Gen Virol 69:2123-2127 6. Alch~ LE, Coulombi6 FC, Coto CE (1985) Aislamiento de virus Junin a partir de sangre y linfo-monocitos perif~ricos de Calomys muscutinus infectados.. Rev Arg Microbiol 17:17%181 7. Candurra NA, Damonte EB (1985) Influence of cellular functions on the evolution of persistent infections with Junin virus. Arch Virol 86:275-282 8. Domingo E, Martinez-Salas E, Sobrino F, de la Torre JC, Portela A, Ortin J, L6pezGalindez C, Prrez-Brena P, Villanueva N, Nfijera R, VandePol S, Steinhauer D, de Polo N, Holland JJ (1985) The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance-a review. Gene 40:1-8 9. Howard CR, Lewicki H, Allison L, Salter M, Buchrneier MJ (1985) Properties and characterization of monoclonal antibodies to Tacaribe virus. J Gen Virol 66: 13831395
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10. Iorio RM, Bratt MA (1985) Selection of unique variants of Newcastle disease virus with neutralizing monoclonal antibodies and anti-immunoglobulin. Proc Natl Acad Sci USA 82:7106-7110 11. King C, de Fries R, Kolhekar SR, Ahmed R (1990) In vivo selection of lymphocytetropic and macrophage-tropic variants of lymphocytic choriomeningitis virus during persistent infection. J Virol 64:5611-5616 12. Laguens RM, Lampuri JS, Coto CE, Laguens RP (1982) Patologia de la infeccidn persistente del Calomys musculinus con una cepa atenuada de virus Junin. Medicina (Buenos Aires) 42:273-278 13. Lampuri JS, Vidal M del C, Coto CE (1982) Respuesta del Calomys musculinus a la infeccidn experimental con virus Junin. Medicina (Buenos Aires) 42:61-66 14. Narayan O, Clements JE, Griffin DE, Wolinsky JS (1981) Neutralizing antibody spectrum determines the antigenic profiles of emerging mutants of visna virus. Infect Immun 32:1045-1050 15. Reitz MS, Wilson C, Naugle CH, Gallo RC, Robert-Guroff M (1988) Generation of neutralization-resistant variant of HIV is due to a selection for a point mutation in the env gene. Cell 54:57-63 16. Smith DB, Inglis SC (1987) The mutation rate and variability of eukaryotic viruses: an analytical review. J Gen Virol 68:2729-2740 17. Steinhauer DA, Holland JJ (1987) Rapid evolution of RNA viruses. Annu Rev Microbiol 41:409-433 18. Strauss JH, Strauss EG (1988) Evolution of RNA viruses. Annu Rev Microbiol 42: 657-683 Authors' address: Laura E. Alch6, Laboratorio de Virologia, Depto. Quimica Biol6gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabell6n II, 40 piso, Cdad. Universitaria, 1428 Buenos Aires, Argentina. Received March 17, 1992