David Rowlands collection

Equine rhinitis A virus (ERAV) shares many features with foot-and-mouth disease virus (FMDV) and both are classified within the genus Aphthovirus of the family Picornaviridae. ERAV is used as a surrogate for FMDV research as it does not require high-level biosecurity. In contrast to FMDV, which uses integrins as cellular receptors, the receptor for ERAV has been reported to involve the sugar moiety sialic acid. This study confirmed the importance of sialic acid for cell entry by ERAV and reports the crystal structure of ERAV particles complexed with the receptor analogue 3′-sialyllactose. The receptor is attached to the rim of a capsid pit adjacent to the major immunogenic site and distinct from the sialic acid binding site used by a related picornavirus, the cardiovirus Theiler's murine encephalitis virus. The structure of the major antigenic determinant of the virus, previously identified from antibody escape mutations, is also described as the EF loop of VP1, which forms a hairpin stretching across the capsid surface close to the icosahedral fivefold axis, neighbouring the receptor-binding site, and spanning two protomeric units.

Synthetic peptides incorporating the derived amino acid sequence of VP2 residues 156 to 170 of human rhinovirus type 2 (HRV2) have previously been shown to elicit antibodies that neutralize virus infectivity. The proportion of virus-reactive antibodies present in polyclonal antisera to these peptides is, however, very low. Moreover, neutralization titrations of such antisera correlate poorly with other assays of either anti-virus or anti-peptide activity, suggesting the presence of antibodies with different specificities. To investigate these findings further, we produced a panel of monoclonal antibodies (MAbs) to VP2 peptides of residues 156 to 170 and characterized their reactions with a range of antigens in ELISA, precipitation and neutralization titrations. All the MAbs obtained recognized the homologous peptide, but could be divided into four main reaction groups according to their specificity for viral antigens. Antibodies in the first group recognized and neutralized native virus, apparently by preventing attachment to cells. A second group of MAbs bound to intact particles with similar affinities to the first group, but failed to neutralize infectivity. Antibodies in the third group recognized virus only after capsid distortions incurred by heating or by previous reaction with polyclonal antibodies. The fourth group comprised MAbs that were mainly peptide-specific. Some possible applications of anti-peptide MAbs to improving the design of peptide immunogens are considered.

The amino acid sequence RGD (arginine-glycine-aspartic acid) is highly conserved in the VP1 protein of foot-and-mouth disease virus (FMDV), despite being situated in the immunodominant hypervariable region between amino acids 135 and 160. RGD-containing proteins are known to be important in promoting cell attachment in several different systems, and we report here that synthetic peptides containing this sequence are able to inhibit attachment of the virus to baby hamster kidney (BHK) cells. Inhibition was dose-dependent and could be reversed on removal of the peptide. A synthetic peptide corresponding to a portion of the same hypervariable region but not containing the RGD sequence did not inhibit virus attachment under the same conditions. Antibody against the RGD region of VP1 blocked attachment of the virus to BHK cells, and neutralizing monoclonal antibodies, which neutralize virus by preventing cell attachment, were blocked by RGD-containing peptides from binding virus in an ELISA test. Cleavage of the C-terminal region of virus VP1 in situ with proteolytic enzymes reduced cell attachment, and antiserum against a peptide corresponding to this region was also able to inhibit attachment of virus to BHK cells. These results indicate that the amino acid sequence RGD at positions 145 to 147 and amino acids from the C-terminal region of VP1 (positions 203 to 213) contribute to the cell attachment site on FMDV for BHK cells.

In addition to the four major polypeptides VP1 to VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 × 103 (P40) and 52 × 103 (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.