David Rowlands collection

Chymotrypsin cleaves only one of the four major polypeptides of foot-and-mouth disease virus (FMDV serotype O) in situ. This polypeptide (VP1, mol. wt. 29 × 103) was first cleaved into fragments of mol. wt. 20 and 9 × 103 and further cleavage could be prevented by the addition of a large excess of bovine serum albumin. The infectivity of the virus particles at this stage was the same as that of the intact virus although the rate of attachment to BHK 21 cells was slower and the immunogenic activity was reduced. If hydrolysis was allowed to continue, VP1 was cleaved into fragments with mol. wt. r8 and < 9 × 103, similar to those obtained with trypsin, and the virus particles then had a greatly reduced infectivity and a lower immunogenicity. Treatment of strains from five other serotypes of the virus with the two enzymes cleaved only VP1 in each instance and there was a corresponding loss of infectivity. The results are discussed in relation to the location and biological activity of the virus polypeptides.

In addition to the major infective component, which bands at a density of 1.34 g/ml in caesium chloride (‘light component’), a component with a density of 1.44 g/ml (‘heavy component’) has been found in harvests of poliovirus (type 1), Coxsackie B5 virus, a bovine enterovirus (VG-5-27) and swine vesicular disease virus (SVDV). With SVDV about 98% of the infectivity equilibrated at 1.34 g/ml but approx. 2% was present as a peak at 1.44 g/ml. The morphology of the two forms was similar but the heavy component had a smaller diameter (28 nm) than the light component (30 nm). No inter-conversion of the two forms was observed on re-cycling in fresh caesium chloride gradients and the two components had the same proportions of RNA and protein and the same polypeptide composition. Each component gave a similar proportion of the light and heavy forms on replication, but the light component had a specific infectivity about fourfold higher than that of the heavy component and was also much more efficient in eliciting the formation of neutralizing antibodies in guinea pigs. Although these results suggest that the two particles are alternative stable configurations of the virus, iodination failed to reveal any differences in the extent or pattern of labelling of the polypeptides in the two forms.

Treatment of foot-and-mouth disease virus with 4% glutaraldehyde increases the diam. of the particles by 25% and makes them permeable to phosphotungstic acid so that they appear empty. The treated particles also resemble naturally-occurring empty particles in their low sedimentation coefficient (about 75S) but, in contrast to empty particles, they have a normal content of RNA and a higher than normal buoyant density in caesium chloride. The RNA can be removed from fixed particles by ribonuclease. Two models are suggested which account for these alterations in the structure of the virus particles. These results show that fixation with glutaraldehyde, far from maintaining the structural integrity of the virus particles, leads to considerable alterations in the arrangement of the RNA and protein subunits.

Several of the physico-chemical properties of representative members of the Picornaviridae family have been examined. On the basis of their buoyant density in caesium chloride, stability at pH 3 to 7 and the base composition of the virus RNA, a division of this family of viruses into six subgroups is suggested.

Further evidence has been obtained which confirms that foot-and-mouth disease virus contains several structural proteins. By electrophoresis in urea-polyacrylamide gels, virus of type O gave six distinct bands. In sodium dodecyl sulphate-polyacrylamide gels four proteins with molecular weights of 34, 30, 26 and 13.5 × 103 were clearIy demonstrated. When virus preparations were labelled with a single amino acid, in both sodium dodecyl sulphate-polyacrylamide and urea-polyacrylamide gel electrophoresis, the fastest migrating protein contained no arginine and only traces of cysteine. This protein also stained differently from the other bands with Coomassie Blue and was absent from the 12s protein subunit prepared by mild acid (pH 6.5) disruption of the virus. This protein was separated from the 12s subunit by sucrose gradient centrifugation and by ion exchange chromatography on AmberIite IRC-50.

Unfractionated harvests of foot-and-mouth disease virus grown in baby hamster kidney cells fixed complement with both heterotypic and homotypic antisera but the freshly prepared intact virus (25 nm. component) from these harvests fixed complement only with the homotypic antiserum. Storage at 4° or heating at 37° released an antigen from the 25 nm. component which fixed complement with heterotypic serum. This antigen could also be prepared by mixing the 25 nm. component with baby hamster kidney cells but it was obtained in greatest yield by disrupting with guanidine. It had a sedimentation coefficient of 14S in sucrose gradients. Serum from hyperimmunized infected guinea pigs which had been absorbed with excess homotypic 25 nm. component fixed complement with the disrupted virus but not with intact virus. The disrupted virus also reacted with heterotypic antiserum produced by inoculation of guinea pigs with inactivated 25 nm. component, providing further evidence that the antigen is a structural component of the virus particle.