The RGD integrin-binding motif within VP1 is highly conserved among FMDV field isolates, and is generally considered essential for virus viability via its interaction with cellular integrin heterodimers [24–26]. Biochemical evidence that small peptides containing the RGD sequence inhibited the adsorption of the virus to tissue culture cells , and genetically engineered virions containing either mutations or deletions of the RGD sequence were unable to bind to cells or cause disease in susceptible animals [12, 25, 27]. However, the RGD triplet may be dispensable upon short-term evolution of the virus harboring it in a constant environment [21, 28, 29]. In the present report, we have documented that non-RGD derivatives arose serendipitously during short-term passage of a FMDV field isolate in vitro and in vivo. One derivative containing an RDD triplet in the receptor-binding site was obtained from the serotype Asia 1 field isolate after a single cattle-to-pig transmission and subsequent BHK-21 in vitro passage. Sequence analysis of 10 biological clones of the VP1 encoding region of this population demonstrated that RDD viruses instead of the original RGD virus became predominant at an early phase of Asia1/JS/CHA/05 quasispecies evolution. Unexpectedly, however, both RGD and RSD viruses were obtained from the Asia1/JSM4 population that were generated after four serial passages of the Asia1/JS/CHA/05 field isolate in suckling mice, via intraperitoneal inoculation. The population equilibrium of RSD mutant and ancestor viruses was maintained after 20 passages of the Asia1/JSM6 population in BHK-21 cells. Although RDD- or RSD-containing FMDV are unusual, they were genetically stable upon extended replication in cell culture. Our results suggest that, in the context of the capsid proteins of Asia1/JS/CHA/05, a highly conserved RGD motif is not essential for replication in vitro and in vivo, suggesting functional flexibility of FMDV to enter cells in response to environmental modifications.
Like other RNA viruses, FMDV exists as closely related but non-identical genomes, termed viral quasispecies [30, 31]. Genetic diversity is an intrinsic property of the quasispecies, which arise due to the lack of proofreading activity during viral genome replication, a short replication cycle, and other environmental selective pressures [32, 33]. Our observations showed that evolution of FMDV population exhibited receptor binding motif diversity (genetic diversity) subjected to short-term passage of field isolate in different environments. From the standpoint of RNA virus population evolution, one possible scenario could explain this observation. The early interactions between viruses and host cells exert major selective force on virus populations, thus, the variants (RSD- and RDD-containing viruses) may already be present at low frequency in the natural population that are possibly more fit in new environments and become dominant strains. While this presumption is contrary to the view that the RGD triplet is highly conserved among natural isolates of FMDV, there is direct evidence that an RDD containing field virus was isolated from pigs during a type Asia 1 FMD outbreak in China. RDD-containing FMDV VP1 genes were amplified from sheep oesophageal-pharyngeal fluids (OP-fluids) collected during 2006 from a sheep herd in the region of China that had endemic Asia 1 serotype FMDV [34, 35]. The emergence of these non-RGD mutants in nature is likely to be influenced by specific epidemiological and immunological aspects of host-virus interaction as well as the quasispecies composition of the viral population [36–39].
The molecular basis for this apparent relaxation of the requirement for the precise RGD sequence is not well understood, but functional replacements within the RGD triplet allow the use of an alternative receptor for entry of FMDV in BHK-21 cells. Since other FMDV lack the RGD motif, host cell recognition may be mediated through another integrin receptor or a non-integrin pathway, or use a third receptor (neither integrin-based nor HS) for entry into the host cell [18, 21, 40]. Further studies are required to analyze the interaction of these mutants with the major FMDV integrin receptors αvβ3, αvβ6, αvβ1 and αvβ8 identified to date, and to understand whether these viruses obtain alteration of cell tropism, antigenicity, and virulence.
To examine the influence of single amino acid substitutions in the receptor binding site of RDD-containing FMD viral genome on virus viability and the ability of non-RGD viruses to cause disease in susceptible animals, we constructed an FMDV Asia1/JS/p1c8 full-length clone and derived mutant molecules with RGD or RSD receptor recognition motifs. Following transfection of BSR cells with these clones, three recombinant viruses were rescued, in particular, six other amino acid differences in the P1 capsid region of Asia1/JS/CHA/05 and Asia1/JSM4 (compared with Asia1/JS/p1c8) did not affect rescue of viable RGD- and RSD-harboring viruses. Furthermore, in vitro growth properties of these viruses did not differ significantly. Our results showed that Asia1/JS/p1c8 viral genome can tolerate substitutions in the receptor binding site with no other changes in the capsid. The ability of the Asia1/JS/p1c8 viral genome to tolerate substitution of receptor binding sites may depend on the capsid sequence, because the Asp-143 Gly change of receptor recognition site was lethal in the context of the capsid proteins of FMDV C-S8c1. However, the same replacement yielded viable viruses in the context of the capsid protein of FMDV C-S8c1p100 and C-S8c1p213 [21, 41].
To assess the ability of non-RGD FMD viruses to cause disease in naturally susceptible animals, we performed experiment infections of cattle and pigs using the Asia1/JS/p1c8 and two non-RGD recombinant viruses. Subsequent experiments showed that all viruses were able to cause disease in cattle and pigs and produce rapid onset of clinical signs, characteristic of infection with RGD field strains. The disease was characterized by viremia in all inoculated animals, including the individuals that did not generate vesicular lesions. Amongst these viruses, the RSD virus produced less tissue damage at the inoculation sites and induced fever and vesicles a day later than in the animals inoculated with RDD-containing viruses, which indicated a different degree of disease severity. The different virulence of these viruses was also supported by the maintenance of original receptor recognition sequence in vesicle samples obtained from infected animals. The reason for low virulence of RSD virus, which may bear on receptor usage, needs to be further explored. These observations are particularly interesting, since the presence of an RGD motif is believed to be the main determinant to direct FMDV to integrin-containing target tissues during infection in the natural host . In addition, information currently available indicates that FMDV utilizes integrins for entry in the natural host, and there is no evidence of the use of alternative receptors in vivo [5, 14, 28]. Therefore, our results further support the possibility that a non-RGD-integrin interaction could be responsible for the generation of FMD in the natural host. Our study was the first to demonstrate the ability of an RDD containing natural isolate to cause disease in naturally susceptible animals, and will provide knowledge about the in vivo pathogenesis of non-RGD viruses.