Molecular characterization of VP6 genes of human rotavirus isolates: correlation of genogroups with subgroups and evidence of independent segregation\rAmino acid substitution within the VP7 protein of G2 rotavirus strains associated with failure to seroty

Abstract

A reverse transcription-PCR (RT-PCR) was established to amplify a 379-bp cDNA fragment (nucleotides 747 to 1126, coding for amino acids 241 to 367) of the VP6 gene of group A rotaviruses associated with subgroup (SG) specificity. Thirty-eight human rotavirus strains characterized with SG-specific monoclonal antibodies were subjected to VP6-specific RT-PCR, and PCR amplicons were used for sequencing. Nucleic acid sequencing and phylogenetic analysis of the VP6 amplicons revealed two clusters, or genogroups. Two genetic lineages were distinguished within genogroup I, consisting of strains serologically characterized as SG I, and three genetic lineages were distinguished within genogroup II, composed of strains serologically characterized as SG II, SG I + II, and SG non-I, non-II. Subgrouping of rotaviruses by means of serological methods may result in strains not being assigned the correct SG or in a failure of strains to subgroup. Molecular characterization of the SG-defining region of VP6 provided evidence for independent segregation of the rotavirus genes encoding VP4, VP6, and VP7. Rotavirus strains collected in the United Kingdom during the 1995-1996 season and genotyped as G2 by reverse transcription-PCR failed to serotype in enzyme-linked immunosorbent assays using three different G2-specific monoclonal antibodies. The deduced amino acid sequences of the antigenic regions A (amino acids 87 to 101), B (amino acids 142 to 152), and C (amino acids 208 to 221) of VP7 revealed that a substitution at position 96 (Asp-->Asn) correlated with the change in ability to serotype these G2 strains. The G and P genotypes of 3,601 rotavirus strains collected in the United Kingdom between 1995 and 1999 were determined (M. Iturriza-Gomara et al., J. Clin. Microbiol. 38:4394-4401, 2000). In 95.4% of the strains the most common G and P combinations, G1P[8], G2P[4], G3P[8], and G4P[8], were found. A small but significant number (2%) of isolates from the remaining strains were reassortants of the most common cocirculating strains, e.g., G1P[4] and G2P[8]. Rotavirus G9P[6] and G9P[8] strains, which constituted 2.7% of all viruses, were genetically closely related in their G components, but the P components of the G9P[8] strains were very closely related to those of cocirculating strains of the more common G types (G1, G3, and G4). In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.