Assignment of avian reovirus temperature-sensitive mutant recombination groups B, C, and D to genome segments

We recently generated a new set of avian orthoreovirus (ARV) temperature-sensitive (ts) mutants after chemical mutagenesis of wild-type strain ARV138 and described mutants in the A recombination group. Here, each prototype ts mutant from ARV recombination groups B, C, and D was crossed with wild-type ARV strain 176 to generate reassortant clones that were used to map the ts lesions in the respective mutants. Reassortant clones were identified by comparison of segment mobility to parental markers in polyacrylamide gels. An efficiency of plating (EOP) value, which measures the capacity of a virus clone to grow under non-permissive conditions, was used to assign reassortant clones to either a ts group or non-ts group. Analysis of EOP values and parental origin of genome segments in the reassortant clones revealed that the group B lesion in tsB31 was located on the M2 genome segment; the group C lesion in tsC37 was on the S3 genome segment; and the group D lesion in tsD46 was on the L2 genome segment. The assignments of tsB31 and tsC37 were further confirmed by sequence analysis and amino acid substitutions in the corresponding muB and sigmaB proteins localized within the recently determined homologous mammalian reovirus mu1/sigma3 heterohexameric crystal structure.