Genetic diversity of RNA segments 5, 7 and 9 of the Palyam serogroup orbiviruses from Japan, Australia and Zimbabwe.

Reverse transcriptase-polymerase chain reaction (RT-PCR) methods, based on the sequences of RNA segments 5, 7 and 9 of Chuzan virus, were established for specific detection and molecular characterization of the Palyam serogroup orbiviruses. Nucleotide sequences obtained from the amplified cDNA fragments of these three genes of 24 isolates were analyzed and compared individually to determine the intra-serogroup phylogenetic relationship of Japanese, Australian and Zimbabwean isolates. It seems that Chuzan virus isolates in Japan are genetically stable. Interestingly, mutations have occurred almost simultaneously on these three genes of Chuzan virus. In all cases, isolates from the same geographical area were closely related to each other at the molecular level, irrespective of serotype. The data suggested that the Palyam serogroup viruses can be differentiated into geographically distinct groups and that the viruses evolve independently in the different gene pools. A strain KY-115 was considered to be produced by reassortment of genome segments between different groups. Restriction fragment length polymorphism (RFLP) analysis of these PCR products is useful for rapid discrimination of isolates and for detection of genetic mutations.

Mapping the genetic determinants of pathogenicity and plaque phenotype in swine vesicular disease virus.

A series of recombinant viruses were constructed using infectious cDNA clones of the virulent J1'73 (large plaque phenotype) and the avirulent H/3'76 (small plaque phenotype) strains of swine vesicular disease virus to identify the genetic determinants of pathogenicity and plaque phenotype. Both traits could be mapped to the region between nucleotides (nt) 2233 and 3368 corresponding to the C terminus of VP3, the whole of VP1, and the N terminus of 2A. In this region, there are eight nucleotide differences leading to amino acid changes between the J1'73 and the H/3'76 strains. Site-directed mutagenesis of individual nucleotides from the virulent to the avirulent genotype and vice versa indicated that A at nt 2832, encoding glycine at VP1-132, and G at nt 3355, encoding arginine at 2APRO-20, correlated with a large-plaque phenotype and virulence in pigs, irrespective of the origin of the remainder of the genome. Of these two sites, 2APRO-20 appeared to be the dominant determinant for the large-plaque phenotype but further studies are required to elucidate their relative importance for virulence in pigs.