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SUMMARYSeven neutralizing monoclonal antibodies were used to characterize 30 escape mutants of a type O foot-and-mouth disease (FMD) virus (O1 Kaufbeuren) selected with the five most active antibodies. Three non-overlapping antigenic sites were found by ELISA and cross-neutralization studies. Within two of the sites the epitopes of two or more monoclonal antibodies overlapped. Two of the sites were conformationdependent and could not be detected on virus subunits or isolated denatured polypeptides. The third site was less conformation-dependent since the appropriate monoclonal antibodies were able to bind to 12S subunits, isolated VP1 protein and a synthetic peptide containing residues 141 to 160 of VP1 in ELISA. Electrofocusing of mutants of that site showed a high frequency of electrophoretic alterations in VP 1.

The aphthovirus genome consists of a single molecule of single-stranded RNA that encodes all the virus-induced proteins. We isolated recombinant aphthoviruses from cells simultaneously infected with temperature-sensitive mutants of two different subtype strains. Analysis of the proteins induced by 16 independently generated recombinants revealed two types of protein pattern, which were consistent with single genetic crossovers on the 5' side and 3' side, respectively, of the central P34-coding region. Recombinants invariably inherited all four coat proteins from the same parent, and novel recombinant proteins were not observed. RNAase T1 fingerprints of virus RNA, prepared from representatives of each recombinant type, confirmed the approximate crossover sites that had been deduced from the inheritance of proteins. These fingerprints provide molecular evidence of recombination at the level of RNA and demonstrate the potential of RNA recombination for producing genetic diversity among picornaviruses.

SUMMARYA single radial haemolysis in gel technique has been developed for the detection and measurement of antibody to influenza haemagglutinin. The method combines the sensitivity of haemagg]utination-inhibition with the accuracy of single radial diffusion. It is simple, quick, reproducible, does not require purified or concentrated virus, and is unaffected by non-specific inhibitors. The method is particularly suitable for the routine screening of large numbers of serum samples, and may have application also to viruses other than the influenza group.

SUMMARYNeutralizing monoclonal antibodies raised against type 0 foot-and-mouth disease virus have been characterized on the basis of their reactivity with a panel of single site monoclonal antibody-resistant mutants which had defined three antigenic sites. Five antibodies neutralized all these mutants, but by selecting further single site mutants with one of these antibodies it was possible to define a fourth site involved in virus neutralization. Two monoclonal antibodies still neutralized these mutants and all multiple site resistant mutants. One multiple site resistant mutant was resistant to neutralization at each of four antigenic sites but was still efficiently neutralized by type 0 convalescent cattle sera. The relationship between sites recognized by different monoclonal antibodies generated in different laboratories is discussed. INTRODUCTIONStudies on the antigenic structure of two picornaviruses, poliovirus and rhinovirus, have been considerably enhanced by the solving of their three-dimensional crystal structure (Hogle et al., 1985;Rossmann et al., 1985) and the isolation and sequence analysis of monoclonal antibody (MAb) escape mutants (Minor et al., 1986;Sherry et al., 1986). Crystallographic studies on footand-mouth disease virus (FMDV), another picornavirus, are also in progress (Fox et al., 1987) and a number of laboratories have generated MAbs capable of neutralizing type O virus. characterized some 30 MAb-resistant mutants of the O1 Kaufbeuren strain of FMDV isolated using five different neutralizing MAbs. Three distinct antigenic sites were involved in virus neutralization. In this context the term antigenic site is used to describe an area of the virus surface which may contain several MAb epitopes; if one epitope is changed and this affects the ability of a second MAb to neutralize the virus then it is considered that the second MAb recognizes an epitope in the same antigenic site as that changed.Recently Stave et al. (1988) have used different MAbs to define three sites of neutralization. In this communication we now use the panel of mutants generated by Xie et al. (1987) to map the binding sites of MAbs generated in different laboratories and then use these antibodies to show the existence of further sites involved in the neutralization of type O FMDV. We have also generated multiple site mutants which are resistant to neutralization at each of two, three or four sites.