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Journal of General Virology (1994), 75, 1173 1176. Printed in Great Britain 1173 Difference in neutralization between lactate dehydrogenase-elevating virus isolated from acutely and chronically infected mice Philippe M o n t e y n e * and J e a n - P a u l Coutelier Unit of Experimental Medicine, International Institute of Cellular and Molecular Pathology and Universitd CathoIique de Louvain, Avenue Hippocrate 74, B-1200 Brussels, Belgium Mouse infection with lactate dehydrogenase-elevating virus (LDV) leads to lifelong viraemia, despite the production of neutralizing antiviral antibodies. To test whether viral persistence correlated with the development of resistance to these antibodies, we compared the neutralization of viral particles derived from acutely and chronically infected animals, using polyclonal and monoclonal anti-LDV antibodies. Whereas virus isolated during acute infection was efficiently neutralized, titres of LDV from chronically infected mice were only slightly reduced by antiviral antibodies. In addition, LDV from animals acutely infected with such poorly neutralizable virus from chronically infected mice was resistant to anti-LDV antibodies like their parental viral particles. These results suggest that LDV variants capable of escaping neutralization by antiviral antibodies can emerge in chronically infected animals. Lactate dehydrogenase-elevating virus (LDV), a mouse virus related to equine arteritis virus and simian haemorrhagic fever virus (de Vries et al., 1992; Godeny et al., 1993; Kuo et al., 1992) and to Lelystad virus (Meulenberg et al., 1993), induces in adult mice a lifelong viraemia, despite the production of antibodies that have been shown to neutralize virus particles efficiently in vitro (Cafruny et al., 1986; Coutelier et al., 1986; Coutelier & Van Snick, 1988; Notkins et al., 1966; Plagemann et al., 1992). Whereas anti-LDV antibodies do not prevent in vivo virus replication in macrophages and subsequent chronic infection, they can protect susceptible mouse strains against virus-induced polioencephalomyelitis (Harty & Plagemann, 1990). In addition to direct neutralization, binding of anti-LDV antibodies to virus particles may lead to the formation of infectious complexes that can be further neutralized with anti-mouse immunoglobulin reagents (Notkins et al., 1968; Coutelier & Van Snick, 1988). Such a disparity in the results of antibody-virus interactions suggested heterogeneity in the viral population, a hypothesis also supported by the isolation of neutralization escape variants after serial incubations of viral particles with anti-LDV monoclonal antibodies (Harty & Plagemann, 1988). If such variants were naturally selected in vivo during chronic infection, this could explain, at least partly, why viraemia does not disappear in the presence of a well established neutralizing anti-LDV response. To test this hypothesis, we isolated LDV during the acute [1 day post-infection (p.i.)] and chronic (2 months p.i.) phases of infection from five animals in a pool of 30 female 129/Sv mice (bred by G. Warnier at the Ludwig Institute for Cancer Research, Brussels, Belgium) that had been infected by intraperitoneal injection of 2 x 107 IDs0 of LDV (Riley strain; from the ATCC). To prepare polyclonal antiviral antibodies, we collected the sera of the 30 mice 2 months p.i., pooled the samples and adjusted the pH to 8 with Tris-HCl-buffered saline. The immunoglobulins were then extracted by anion exchange on DEAE-Sephadex before precipitation with 40% ammonium sulphate. Samples of LDV were purified by centrifugation through a 2.8 ml 10 % sucrose cushion for 1 h at 40500 r.p.m, in a SW60 rotor (Beckman) and adjusted to a concentration of approximately 105 IDs0/ml. Of these samples 25 lal was then incubated in 100 lal PBS, alone or with 10 lag anti-LDV antibodies for 15 min at 37 °C, followed by 1 h at 4 °C. Viral titres were subsequently measured after injection of serially diluted samples into groups of four N M R I mice and assay of plasma lactate dehydrogenase 4 to 5 days later (Rowson & Mahy, 1975). Whereas anti-LDV antibodies reduced the titre of virus from acute infection by more than 104fold, they displayed only a marginal effect on the virus derived from the same animals when chronically infected (Fig. 1). After that, the virus derived from chronically infected mice was transmitted into a new group of four N M R I mice by injecting 1 lal of plasma (diluted in 500 lal isotonic solution) into each mouse. One day later, a new LDV sample (called here 'chronic-derived', with a titre similar to that of the original acute virus) was prepared from plasma from these animals and incubated with antiLDV antibodies as already described. This virus prep- 0001-1845 © 1994 SGM 1174 Short communication iI -F 6 >6 4 O O +a ..a .~ 3 0 ~z Acute Chronic Chronicderived Virus isolate Fig. l. LDV neutralization with polyclonal antiviral antibodies. LDV samples obtained during acute (24 h p.i.) or chronic (2 months p.i.) phases of infection of a group of 129/Sv mice, or 24 h after secondary passage (chronic-derived) were adjusted to a concentration of approximately 105 IDs0/ml before in vitro neutralization with polyclonal antiviral antibodies prepared from the pool of infected mice. Titres of virus incubated with (hatched columns) or without (black columns) antibody, ±s.o., are indicated. aration was found to resist neutralization in a way similar to its parental LDV isolate (Fig. 1). This chronicderived virus was again purified by centrifugation on a sucrose cushion and transmitted to another group of mice by injecting each animal with 500 lal of a 12 x l0 % fold dilution of this sample. Again, we observed a significantly lower neutralization of the chronic-derived virus (10179-fold reduction of LDV titre) in comparison with the neutralization of the acute virus sample identically transmitted to another group of mice (more than 10~-fold reduction of LDV titre). To confirm this result, LDV samples were also incubated with 50 gg of a pool of three different purified monoclonal antibodies recognizing the VP3 viral envelope protein. This mixture of antibodies, at this dose, had been previously found to be the most efficient for 0 Acute Chronic Chronicderived Virus isolate Fig. 2. LDV neutralization with monoclonal antibodies. LDV samples, as described in Fig. 1, were neutralized with a pool of purified monoclonal anti-VP3 IgG3 antibodies (C3904H12, C3901B3M and C7506B12M, from BALB/c mice infected with LDV-Riley strain). Titres of virus incubated with (hatched columns) or without (black columns) antibody, + s.D., are indicated. neutralization of the virus in vitro (Coutelier & Van Snick, 1988). Again, a strong reduction in the titre of virus isolated from acutely infected animals was observed (Fig. 2). In contrast, both LDV derived from the same mice at a chronic stage of infection and chronic-derived virus obtained after secondary passage were only poorly neutralized by this pool of monoclonal antibodies. Since experiments with pooled reagents obtained from groups of animals might not exactly reflect what happens naturally in one infected host, LDV samples were derived from individual NMRI mice 1 day and 2 months p.i., or 24 h after secondary passage of the chronic virus into groups of four animals. Antiviral antibodies were also prepared from the serum of the same individual mice that were repeatedly bled after isolation of the chronic virus. Because of the limited amount of anti-LDV antibodies that we could obtain from these individual animals, virus neutralization could only be compared for Short communication 1175 Table 1. Neutralization of LD V isolated from individual N M R I mice LDV titre (logl0)t Mouse Anti-LDV antibody 1 Polyclonal:~ 2 Polyclonal 2 Monoclonal§ 3 Monoclonal 4 Monoclonal LDV isolate* Without antibody Acute Chronic Acute Chronic-derived Acute Chronic-derived Acute Chronic-derived Acute Chronic-derived 4.38 ± 0-35 4.33 ±0.36 6.50±0.41 5'00 ± 0.53 6.50±0.31 6"33 ± 0.36 4.50±0.41 4.67 ± 0.36 4.67 ± 0-36 5.00 + 0.44 With antibody 2.33 ± 0-36 4.00±0-53 4-00±0.57 4.33 ± 0.36 < 2-50 3.67 ± 0.36 2.33±0.36 3.33 ± 0-36 2.33 ± 0.36 4.50 _+0.41 Reduction of LDV titre (logx0) 2-05 0.33 250 0.67 > 4.00 2.66 2.17 1.34 2,34 0-50 * LDV isolated during acute (24 h p.i.) or chronic (2 months p.i.) phases of infection, or after secondary passage. t LDV infectivity titre (ID~0/ml) ± s.o. :~ Prepared from pooled bleedings from the individually analysed mice. § Pool of three anti-VP3 monoclonal antibodies, as described in Fig. 2. LDV obtained during acute infection and either virus from chronically infected mice or chronic-derived LDV. The results obtained with virus isolated from individual mice were similar to those observed with pools of animals (Table 1). LDV derived from acutely infected mice was efficiently neutralized by antibodies produced later by the corresponding animal, whereas virus particles obtained when the ant±viral response was fully developed, or the progeny of this chronic virus, escaped neutralization by the same antibody preparation. This difference between acute virus and LDV particles obtained after secondary infection with a chronic virus was also observed when neutralization experiments were performed with the pool of anti-VP3 monoclonal antibodies, however with some variations from one mouse to another (Table 1). Our results confirm the heterogeneity of the viral population suggested by differences in sensitivity towards a given preparation of antibodies (Coutelier & Van Snick, 1988). Heterogeneity between viral isolates, including variants that were selected after passages in particular strains of mice, has also been demonstrated by neutralization with rabbit anti-LDV serum (Cafruny & Plagemann, 1982), by RNA oligonucleotide fingerprint analysis (Brinton et al., 1986) and by nucleotide and amino acid sequence analysis (Kuo et al., 1992). In our study, LDV isolated early after inoculation, but not the virus obtained later from the same animal, was efficiently neutralized by antibodies that were secreted in response to infection. The presence of non-neutralizing anti-LDV antibodies in chronically infected mice leading to the formation of infectious virus-antibody complexes (Notkins et aI., 1966) could easily explain the difference in neutralization between acute and chronic virus. However, the observation of the persistent resistance after passage of the poorly neutralizable chronic virus in a new set of mice favours the hypothesis that variants of LDV resisting neutralization are selected during the infectious process. This hypothesis is further supported by the observation that virus isolated after a secondary passage remained poorly neutralizable. Indeed, it is quite unlikely that significant amounts of ant±viral antibodies have been transferred to the animals that received the equivalent of 4x 10 5 lal of plasma from the initial chronically infected mice and therefore that reduction in neutralization of the virus purified from these animals is related to passive immunization with non-neutralizing anti-LDV antibodies. The emergence of variants that we report was not strain-specific since it was observed with 129/Sv mice (Fig. 1 and 2) as well as with NMRI mice (Table l). Therefore, our results indicate that the selection of neutralization escape mutants, which has been demonstrated by others after incubation with monoclonal antibodies (Harty & Plagemann, 1988), may happen also naturally in the course of infection in response to ant±viral immunity. Hence this phenomenon could contribute to some variations that have been reported between LDV isolates in addition to other possible mechanisms such as selection by cellular immune responses. Antigenic variants escaping neutralization by ant±viral antibodies have been isolated for other viruses. Lent±viruses, like LDV, persist indefinitely in their hosts, replicate continuously during the lifelong infection and undergo antigenic variations in the presence of neutralizing antibodies. The development of an animal model in which a standard strain of equine infectious anaemia virus induces a chronic disease allowed the characterization of viral antigenic variations and the study of genomic alterations that were associated with this 1176 Short communication persistent infection (Payne et al., 1984). The analysis of visna virus mutants appearing during persistent infection of sheep showed a small number of changes in their oligonucleotide pattern (Clements et al., 1980). Variations were also detected in the genome of human immunodeficiency virus isolates derived from persistently infected individuals (Hahn et al., 1986). A role, if any, of anti-LDV antibodies in controlling virus replication is still to be demonstrated. It has been shown that LDV titres fluctuate because of limited availability of permissive cells rather than because of the immune response. For example, immunodeficient nude mice develop a viraemia comparable to that of their normal counterpart (Onyekaba et al., 1989). The selection of antibody-resisting LDV variants in immunocompetent animals is compatible with this similarity in viral titres. Indeed, it can be postulated that in the absence of escape mechanisms anti-LDV antibodies would be capable of eliminating the virus. The role of naturally occurring neutralization escape variants in persistent viraemia, as well as the mechanisms by which these viruses resist neutralization remain to be determined. Such an analysis would be greatly facilitated by the development of a cell line allowing LDV cloning and amplification and by sequence analysis of isolated variants. We are indebted to J. Van Snick and to P. L. Masson for critical reading of the manuscript. This work was supported by the Fonds National de la Recherche Scientifique (FNRS), Fonds de la Recherche Scientifique M6dicale (FRSM), Loterie Nationale, Op6ration T616vie, the State Prime Minister's Office Science Policy Programming (interuniversity attracting poles, grant 44), and the French Community (concerted actions, grant 88/93-122), Belgium. P. Monteyne is research assistant and J.-P. Coutelier is research associate with the FNRS, Belgium. References BRINTON, M. A., GAVIN, E. I. & FERNANDEZ,A. V. (1986). Genotypic variation among six isolates of lactate dehydrogenase-elevating virus. Journal of General Virology 67, 2673-2684. CAFRUNY,W. A. & PLAGEMANN,P. G. W. (1982). Immune response to lactate dehydrogenase-elevating virus: serologically specific rabbit neutralizing antibody to the virus. Infection and Immunity 37, 1007-1012. CAFRLINY, W.A., CHAN, S.P.K., HARTY, J.Z., YOUSEFI, S., KOWALCHYK, K., McDONALD, D., FOREMAN,B., BUDWEG, G. & PLAGEMANN,P. G. W. (1986). 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