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Eighteen temperature-sensitive (ts) mutants of rabbitpox virus were examined for defects in synthesis of DNA and protein. Two mutants (ts-3 and ts-16) were defective in DNA synthesis (DNA-), since both incorporated significantly less than wild-type amounts of labelled thymidine into acid-precipitable material when infected cells were incubated at the restrictive temperature. Both these mutants gave only the 'early' class of virus polypeptides when infected cell extracts were examined by SDS--polyacrylamide slab gel electrophoresis following incubation at 40 degrees C. Nine of the remaining sixteen DNA+ ts mutants (ts-1, ts-2, ts-6, ts-12, ts-15, ts-17, ts-31, ts-32 and ts-33) synthesized wild-type levels of most virus polypeptides at 40 degrees C; six DNA+ ts mutants (ts-7, ts-8, ts-9, ts-11, ts-23 and ts-24) were defective in the post-translational cleavage of the polypeptides involved in membrane stabilization and particle assembly; one DNA+ ts mutant (ts-14) synthesized only the 'early' class of virus polypeptides, implying that either replicated DNA was not fully functional or that a specific early function was required for late transcription.

The original foot-and-mouth diseases virus recombination map (Lake, Priston & Slade, 1975), which include 35 mutagen-induced ts mutants, has been extended both in detail and size by the mapping of a further 33 ts mutants (9 mutagen-induced and 24 spontaneous). The size increase from 0.57% to 3.27% maximum recombination fequency was principally due to the use of a new standardization technique for recombination fequencies but, in addition, the original map distance was increased by approx. 30% due to the mapping of new mutations. As in the original map, there was a marked concentration of mutations near the guanidine (gs) locus, i.e. 83% of the mutants had mutations in the third of the map adjacent to the gs locus.

Sixty ts mutants were isolated from the Pacheco strain of type O foot-and-mouth disease virus after treatment with either 5-fluorouracil or hydroxylamine. The conditions affecting recombination and assay of the ts+ recombinants were standardized. Using two ts mutants resistant to guanidine, three-factor crosses, supported by two-factor crosses, located 34 of the mutations in a linear arrangement. The recombination frequencies between certain pairs of mutations were additive. The guanidine character of the two resistant mutants mapped as a single site mutation and was located near the middle of the map.

The DNAs of wild-type rabbitpox virus (u+.p+), and selected u (white pock, u.p+) and p (white pock, PK-15 cell non-permissive, u.p) mutants were compared by restriction enzyme analysis. The cleavage fragments produced by digestion with HindIII or EcoRI endonucleases were separated by agarose gel electrophoresis. Deletions of approx. 10 x 10(6) were recognized at the right-hand terminus of each u mutant (three isolates), whereas deletions of 5 x 10(6) to 20 x 10(6) were detected at the left-hand terminus of each p mutant (nine isolates), Rapidly renaturing terminal restriction fragments, indicative of the covalent cross-links, were only found at the terminus unaffected by deleted sequences. Recombinant viruses that were wild-type in both genetic characters and in restriction pattern were recovered from mixed infections involving u and p mutants, but no genetic interaction was detected between crosses involving only u or p mutants. The p mutants were divided into two non-permissive classes dependent on their biochemical expression in PK-15 cells: the 'early' class which failed to replicate virus DNA and synthesized only the 'early' polypeptides and the 'late' class which appeared to be normal both in virus DNA and polypeptide synthesis. There was no correlation between the extent of the left-hand terminal deletion and the extent of permissiveness of the p mutants in PK-15 cells.

Twenty-eight mutations, representing mutation in five different polypeptide-coding regions of the foot-and-mouth disease genome, were examined for their effect on the virulence of the virus for suckling mice. Five types of mutation were examined: temperature-sensitive (ts), electrophoretic (e), co-variant temperature-sensitive and electrophoretic (ts/e), guanidine-resistant (gs+) and putative co-variant guanidine-resistant and electrophoretic (gs+/e). All the ts mutations and three out of the 11 non-ts mutations produced some reductions in virulence. In the majority of cases this reduction in virulence was shown to co-vary with the mutation. No correlation was observed between the site of a mutation or its 'cut-off' temperature and the extent of the reduction in virulence. Studies of the growth in vivo of a small selection of ts mutants suggested that for most mutants their reduced virulence was a trivial effect of their slow growth rate. With one exception they all eventually grew to parental virus levels, the resulting virus being temperature-sensitive and the disease indistinguishable from that caused by the parental virus. The one exception was an avirulent ts mutant which only grew to one-thousandth the titre of the parent virus. This mutant did not cause disease and was therefore considered to be the only avirulent mutant. Its mutation was in the coat protein-coding region of the genome, probably the region coding for VP3.