Implications of a quasispecies genome structure: effect of frequent, naturally occurring amino acid substitutions on the antigenicity of foot-and-mouth disease virus.

Abstract

We provide evidence that the quasispecies nature (extreme genetic heterogeneity) of foot-and-mouth disease virus is relevant to the virus evading an immune response.A monoclonal antibody neutralizing the viral infectivity ( Foot-and-mouth disease virus (FMDV) is a picornavirus that causes the economically most important viral disease of cattle and other cloven-hooved animals (1, 2). As for most other RNA genomes, FMDV populations are genetically heterogeneous (3-5) and show the potential for very rapid evolution (6, 7). A direct consequence of the genetic variability of FMDV is its remarkable antigenic diversity (1, 2). Attempts to quantify such diversity and classify FMDV isolates have included the early serological subtyping (8) and the more recent analyses of reactivity with monoclonal antibodies (mAbs) (9)(10)(11). Knowledge of the types and frequency of occurrence of amino acid substitutions that lead to variations in viral epitopes is relevant to the design of new synthetic vaccines, whose efficacy may be hampered by the rapid antigenic variation of RNA viruses in nature (7,(10)(11)(12) In recent years, we have emphasized that FMDV shows a quasispecies structure (3-5), probably shared by most other RNA genetic elements (6, 7, 12, 24). This concept, which originated in theoretical work by Eigen and his colleagues (25)(26)(27), was first shown experimentally to adequately describe populations of phage QP3 (28) and later other RNA genomes as well (reviewed in refs. 6, 7, 12, and 24). According to the quasispecies structure, each FMDV genome population includes one or several "master" sequence(s)-which may nevertheless represent a small proportion of molecules (4, 24, 28)-and a "mutant spectrum" consisting of a distribution of single and multiple residue mutants (4,12,24,27,28). The proportion of each mutant depends on the frequency with which it arises by mutation and on its competitive growth with all other variants (present and arising) in the replicating population (26-28). Mutant viruses with chemically conservative amino acid substitutions, prone to behave in a quasineutral fashion, are more likely to be represented in a viral quasispecies than variants with mutations that adversely affect fitness even if viable, as documented with phage QB (28). In the present study we show that chemically conservative amino acid substitutions fixed at the main antigenic determinant of FMDV C during one epizootic-and that are also found frequently among other FMDV isolatesgreatly affected the interaction with one neutralizing mAb (n-mAb), SD6. This antibody recognizes an epitope surrounding a conserved amino acid sequence of VP1, proposed to be a receptor binding site (17, 18, 44). The effect of these naturally occurring amino acid changes has been mimicked with substituted synthetic peptides, which bind n-mAb SD6 to the same extent as complete variant viruses, thus providing an in vitro assay for epitopic variation of FMDV. MATERIALS AND METHODSViruses. The origin of the FMDV field isolates (C-S isolates) has b...

Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus

“…1), previously proposed to be essential for the viability of FMDV. The 86 mutants isolated from FMDV C-S8c1 (Table 1), together with many field viruses and laboratory variants sequenced (29,36,(38)(39)(40)(41)(42) should be sufficient to consider FMDV of serotype C highly intolerant to amino acid substitutions at the RGD motif and several contiguous residues. Work with peptides indicates that, in addition to the RGD triplet, neighboring residues 144, 145, and 147 are also involved in receptor recognition of C-S8c1 (13), thus explaining their high degree of conservation.…”

“…A 1:1 dilution of the supernatant of an SD6 hybridoma culture suppressed plaque formation of 100 pfu of FMDV C-S8c1 without any significant decrease of plaque numbers of resistant mutants previously selected with SD6 (29).…”

“…It must be emphasized that contrary to persistent infections in which the BHK-21 cells coevolved with FMDV (28), in the present experiments BHK-21 cells did not evolve, since a fresh monolayer from the same clonal stock of cells was used for each viral passage. mAb SD6 recognizes an extensively characterized epitope within the G-H loop of VP1 (29).…”

“…Selection of plaque-purified viruses ensured that each MARM analyzed originated from an independent mutational event. About 10 5 -10 6 pfu of virus were incubated for 2 hr at 4°C with a 1:1 dilution of the mAb (supernatant of the SD6 hybridoma culture), and plated with a 1:50 dilution of the same mAb in the agar overlay, as previously described (29). Virus from a single plaque (about 10 5 pfu) was isolated and its resistance to the antibody (no detectable reduction in the number of plaques) tested in the neutralization assay (29).…”

“…mAb SD6, which recognizes a continuous epitope located within amino acids 136-147 of VP1 of FMDV C-S8c1 (23,29,32,33), was used to isolate resistant mutants. Viruses C-S8c1 and its derivative C-S8c1p100, obtained by propagating C-S8c1 in cell culture (see Materials and Methods) were incubated with mAb SD6 and resistant mutants were selected.…”