Two neutralizing monoclonal antibody (MAb)-resistant variants selected from an isolate of foot-and-mouth disease virus (FMDV) type A5 were repeatedly passaged in cell culture and monitored for susceptibility to neutralization by the selecting MAb. A variant isolated with a MAb to a conformational epitope (1-OG2) lost resistance in 20 passages, while a variant isolated with a MAb to a linear epitope (1-HA6) persisted for 30 passages. In both cases, the virus population emerging after passage was

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... antigenicaily and genetically indistinguishable from the original wild-type parental virus (FMDV A5 . Coinfection assays with the wild type and each variant, and between the variants, showed rapid conversion to a homogeneous population. Wild-type virus prevailed over the variants and for coinfection between the variants, the linear epitope variant 1-HA6. While both variants arose from a single nucleotide substitution and reversion to wild type occurred for each, it appears that the variant based on the continuous epitope (1-HA6) was more stable. We discuss the implications of these results for the antigenic diversity of FMDV and its relationship to virus evolution. Foot-and-mouth disease virus (FMDV), a member of the family Picornaviridae, is the cause of an economically devastating disease of cloven-hoofed animals. The virus possesses a single-stranded positive RNA molecule greater than 8,000 nucleotides (2) encapsidated in an icosahedral capsid made of 60 copies each of four proteins: VP1, VP2, VP3, and VP4 (30). The genomic heterogeneity of RNA viruses is now clearly established (for reviews, see references 16 and 18). The presence of mixtures of very similar genomes coexisting in a single population constitutes experimental support for the quasispecies model (21, 22) . This model implies that molecules that replicate with limited fidelity will be organized as distributions of related but nonidentical genomes that include one (or more) master sequences. These may represent a small proportion of molecules in a dynamic equilibrium with a mutant spectrum consisting of a distribution of singleand multiple-residue mutants. The proportion of each mutant depends not only on the frequency with which it appears but also on its competitive behavior with all the variants present at that moment in the population (4, 16, 20) . The emergence of (genetic) variants during the replication of FMDV has been extensively characterized in cell culture (8, 11, 13, 15, 32, 33) , even after extensive plaque purification (41), and in field isolates (27-29, 34, 35). Genetic and antigenic heterogeneity has also been described in virus populations recovered from infected swine (10) and in persistently infected cattle (24). This antigenic diversity of FMDV and emergence of new strains are significant impediments for the design of more broadly protective vaccines (for a review, see reference 9). A recent approach to study of the diversity of FMDV populations is to locate and characterize antigenic sites through the isolation of monoclonal antibody (MAb)-resistant variants (for a review, ence 19). This has allowed the definition of multiple antigenic sites on FMDV, depending on the virus serotype analyzed. Most of these antigenic sites have been located on VP1, but recently neutralizing epitopes located outside (or at least partially outside) VP1 have been described for serotypes 0 (3, 26) and A (7, 8, 31, 38) . We have examined the behavior of two FMDV type A5 antigenic variants when passaged in tissue culture, both alone and in competition with the parental wild type or the other variant, but in the absence of neutralizing antibodies. The data presented show the appearance of antigenic strains similar or identical to the parental virus after serial passages in vitro of plaque-purified MAb-resistant variants. In addition, we found a quick domination of the parental type over the variants in coinfection experiments and the imposition of one variant virus strain over the other when the variants coinfected cell cultures. We discuss these results in relation to the antigenic diversity of RNA viruses and the implication in viral evolution. Virus strains, MAbs, and cells. FMDV type A5 Spain-86 (A5 Sp-86) was obtained from an isolate of a recent outbreak in Spain (1986) and three times plaque purified. Production and characterization of the MAbs used have been described elsewhere (32). MAb-resistant variants 1-HA6 and 1-OG2 were obtained from the parental virus population (A5 Sp-86) as previously described (31). Serial passages of the variant strains in the absence of neutralizing MAbs were made on monolayers of a continuous bovine kidney cell line (LFBK; 37) in 25-cm2 tissue culture flasks, using a multiplicity of infection of 1 to 3 PFU per cell. A 10-fold dilution of the recovered material was used to infect a new flask, and this procedure was repeated 20 to 30 times, depending on the virus studied. Cultures were also coinfected with equal mixtures of the parental A5 virus and each variant or with a mixture of the two variants, and serial passages were made in a similar fashion. The virus passage stocks are identified by strain designation and a postscript passage designation; i.e., 1-HA6P30 indicates the population recovered from virus variant 1-HA6 after 30 serial passages; A5:1-OG2C6 indicates the viral population recovered after coinfection and 3949 Vol. 65, No. 7 on May 5, 2020 by guest