Due to the error-prone nature of FMDV′s RNA-dependent RNA polymerase and high replication rate in vivo, FMDV exhibits a high degree of antigenic variability [19, 20]. Studies of the antigenic diversity and determination of amino acid changes involved in this diversity are important to the design of broadly protective new vaccines . Although extensive studies have been carried out to examine the epitopes in the viral capsids of serotype O [8, 10, 21] and serotype A FMDV [11, 22, 23], there are few reports on Asia1 serotype FMDV . In this study, we explored the molecular basis of the antigenic difference between two viruses, Asia1/YS/CHA/05 from cows and Asia1/1/YZ/CHA/06 from pigs, which react differently against MAb 1B4. We detected three amino acid substitutions in the P1 coding region of Asia1/YS/CHA/05 by sequencing and alignment of these two viruses, including a Ser-to-Asn substitution at position 154 (S154N) in VP1, an Asp-to-Gly substitution at position 72 (D72G) in VP2 and a Val-to-Ile substitution at position 107 (V107I) in VP2 (Table 1). We expected that the S154N substitution in the VP1 G-H loop would be related to the variation in 1B4 recognition; however, single amino-acid substitutions based on an FMDV reverse genetics system revealed that the D72G substitution in VP2 contributed to this antigenic variation. Moreover, we found that the virus containing an Asp-to-Asn substitution (D72N) had a reduced ability to replicate in BHK-21 cells and was 10-fold less virulent in suckling mice. These results provide evidence that amino acid substitutions in the VP2 B-C loop of serotype Asia1 FMDV not only mediate significant antigenic diversity in the field but also alter the replicative ability and pathogenicity of the virus.
X-ray crystallographic data have shown that the B-C loop of VP2 lies in the vicinity of the G-H loop of VP1 . In addition, amino acid changes in the B-C loop of VP2 in type O  and type A [11, 23] FMDV have been shown to contribute to an immunodominant site in VP1. In this study, an Asp-to-Gly substitution or an Asp-to-Asn substitution at position 72, which comprises part of the B-C loop of VP2, inhibited the ability of the Asia1 FMDV strain to react with MAb 1B4. Thus, the Asp-to-Gly or Asp-to-Asn substitutions at position 72 of VP2 may affect the conformation and/or orientation of the VP1 G-H loop, which could impact the ability of MAb 1B4 to recognize its epitope.
Interestingly, two closely related viruses from type A22, which showed different behavior in cell attachment assays and had different host range properties, did not have any differences in the VP1 coding region; however, they did have three amino acid substitutions in VP2. One of these substitutions (Glu82 to Gly) on the surface of VP2 perturbed the structure of the VP1 G-H loop, which determined the cell-binding properties of the variants . This report adds support to the idea that VP2 is important for the cell tropism of the virus. The natural hosts of most serotype Asia1 FMDV strains are cattle, and there is only one report of pigs infected with Asia1 serotype FMDV (i.e., IND17/91 [GenBank: AF390682]). Here, the Asia1/1/YZ/CHA/06 virus from pigs and ano-ther porcine isolate, Asia1/WHN/CHA/06 [GenBank: FJ906802], shared the same Asp-to-Gly substitution at position 72 of VP2. The loss of the aspartic acid side chain on the surface of VP2 in the porcine variants of Asia1 FMDV may perturb the G-H loop of VP1. Because a key determinant of the cellular tropism of FMDV is also located in the G-H loop of VP1 [27, 28], we cannot exclude that the Asp-to-Gly substitution changed the host tropism of viral isolates from a similar lineage, which may explain the difference in host tropism of Asia1/YS/CHA/05 and Asia1/1/YZ/CHA/06. Further investigations are needed to elucidate the effects of these amino acid changes on FMDV infection of various species.
The D72N mutation decreased the growth rate of Asia1/YS/CHA/05 in BHK-21 cells. The results of in vitro growth analysis were consistent with those of in vivo virulence experiments, which suggest that the decreased growth activity of the virus caused by the D72N mutation may contribute to the decreased virulence of the rD72N mutant. A single Asp-to-Asn substitution at position 72 of VP2 may influence the structure of the virus capsid, which would account for the low replication ability and reduced virulence of the virus. Further studies are needed to clarify the mechanisms of these changes.