Viral fitness can influence the repertoire of virus variants selected by antibodies.

Minority genomes in the mutant spectra of viral quasispecies may differ in relative fitness. Here, we report experiments designed to evaluate the contribution of relative fitness to selection by a neutralizing monoclonal antibody (mAb). We have reconstructed a foot-and-mouth disease virus (FMDV) quasispecies, with two matched pairs of distinguishable mAb-escape mutants as minority genomes of the mutant spectrum. Each mutant of a pair differs from the other by 11-fold or 33-fold in relative fitness. Analysis of the mutant spectra of virus populations selected with different concentrations of antibody in infections in liquid culture medium has documented a dominance of the high fitness counterpart in the selected population. Plaque development as a function of increasing concentration of the antibody has shown that each mutant of a matched pair yielded the same number of plaques, although the high fitness mutant required less time for plaque formation, and attained a larger plaque size at any given time-point. This result documents equal intrinsic resistance to the antibody of each mutant of a matched pair, confirming previous biochemical, structural, and genetic studies, which indicated that the epitopes of each mutant pair were indistinguishable regarding reactivity with the monoclonal antibody. Thus, relative viral fitness can influence in a significant way the repertoire of viral mutants selected from a viral quasispecies by a neutralizing antibody. We discuss the significance of these results in relation to antibody selection, and to other selective forces likely encountered by viral quasispecies in vivo.

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes.

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.

Expansion of host-cell tropism of foot-and-mouth disease virus despite replication in a constant environment.

Foot-and-mouth disease virus (FMDV) variants adapted to BHK-21 cells showed an expanded host-cell tropism that extended to primate and human cell lines. Virus replication in human HeLa and Jurkat cells has been documented by titration of virus infectivity, quantification of virus RNA, expression of a virus-specific non-structural antigen, and serial passage of virus in the cells. Parallel serial infections of human Jurkat cells with the same variant FMDVs indicates a strong stochastic component in the progression of infection. Chimeric viruses identified the capsid as a genomic region involved in tropism expansion. These results indicate that, contrary to theoretical predictions, replication of an RNA virus in a constant cellular environment may lead to expansion of cellular tropism, rather than to a more specialized infection of the cellular type to which the virus has been adapted.

Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide.

Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulfide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194) (VP=viral protein). Guinea pig antisera to the peptides recognized the viral particle and competed with site D-specific monoclonal antibodies, while inoculation with a simple (not covalently joined to one another) admixture of the three VP1-VP3 sequences yielded no detectable virus-specific serum conversion. Similar results have been reproduced in two bovines. Antisera to the peptides also moderately neutralize FMDV in cell cultures and partially protect guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent, peptide-based vaccine against FMDV.

Resistance to extinction of low fitness virus subjected to plaque-to-plaque transfers: diversification by mutation clustering.

Plaque-to-plaque transfers of RNA viruses lead to accumulation of mutations and fitness decrease. To test whether continuing plaque-to-plaque transfers would lead to viral extinction, we have subjected several low fitness foot-and-mouth disease virus (FMDV) clones to up to 130 successive plaque transfers, and have analyzed the evolution of plaque titers and genomic nucleotide sequences. No case of viral extinction could be documented. Some low fitness clones that posses an internal poly(A) tract evaded extinction by modifying the length or base composition of the poly(A) tract. The comparison of entire genomic sequences of FMDV clones at increasing plaque transfer number revealed that mutations accumulated at a uniform rate, and that they were distributed unevenly along the genome. Clusters of mutations were identified at different genomic sites in two plaque transfer lineages. Mutation clustering appears to occur stochastically and could not be related to fixation of compensatory mutations. The results document resistance of viral clones to extinction, and suggest that mutation clustering may be a mechanism of genetic diversification of low fitness virus.

Antigenic properties and population stability of a foot-and-mouth disease virus with an altered Arg-Gly-Asp receptor-recognition motif.

The antigenic properties and genetic stability of a multiply passaged foot-and-mouth disease virus (FMDV) clone C-S8c1 with an Arg-Gly-Gly triplet (RGG) instead of the Arg-Gly-Asp (RGD) integrin-recognition motif at positions 141 to 143 of capsid protein VP1 are described. Clear antigenic differences between FMDV RGG and clone C-S8c1 have been documented in ELISA, enzyme-linked immunoelectrotransfer (Western) blot and neutralization assays using site A-specific monoclonal antibodies and anti-FMDV polyclonal antibodies from swine and guinea pigs. The results validate with a live virus the role of the RGD (in particular Asp-143) in recognition of (and neutralization by) antibodies, a role previously suggested by immunochemical and structural studies with synthetic peptides. The FMDV RGG was genetically stable in a large proportion of serial infections of BHK-21 cells. However, a revertant virus with RGD was generated in one out of six passage series. Interestingly, this revertant FMDV did not reach dominance but established an equilibrium with its parental FMDV RGG, accompanied by an increase of quasispecies complexity at the sequences around the RGG triplet. FMDV RGG exhibited a selective disadvantage relative to other RGD-containing clones isolated from the same parental FMDV population. The results suggest that large antigenic variations can be prompted by replacements at critical capsid sites, including those involved in receptor recognition. These critical replacements may yield viruses whose stability allows them to replicate efficiently and to expand the sequence repertoire of an antigenic site.

EVOLUTIONARY DYNAMICS OF FITNESS RECOVERY FROM THE DEBILITATING EFFECTS OF MULLER'S RATCHET.

The great adaptability shown by RNA viruses is a consequence of their high mutation rates. The evolution of fitness in a severely debilitated, clonal population of the nonsegmented ribovirus vesicular stomatitis virus (VSV) has been compared under five different demographic regimes, ranging from severe serial bottleneck passages (one virion) to large population passages (105 virions or more) under similar environmental conditions (cell culture type and temperature). No matter how small the bottleneck, the fitness of the evolved populations was always higher than the fitness of the starting population; this result is clearly different from that previously reported for viruses with higher fitness. The reattainment of fitness under a regime of serial population passages showed two main characteristics: (1) the rate of adaptation was higher during early passages; and (2) a maximum fitness value was reached after a large number of passages. The maximum fitness reached by this initially debilitated clone was similar to the fitness of wild-type virus. The practical implications of these findings in the design of vaccines using attenuated viruses are also discussed.