Sequence analysis of VP4 genes of wild type and culture adapted human rotavirus G1P8 strains

OBJECTIVE@#To conduct a comparative analysis of the VP4 gene sequences of Indian wild type (06361, 0613158, 061060 and 0715880) and cell culture adapted (06361-CA, 0613158-CA, 061060-CA and 0715880-CA) G1P[8] rotavirus strains.@*METHODS@#Full-length VP4 genes of each of the four wild type G1P[8] rotavirus strains and their cell culture adapted counterparts displaying consistent cytopathic effect were subjected to RT-PCR amplification and nucleotide sequencing.@*RESULTS@#All four cell culture adapted G1P[8] rotavirus strains showed nucleotide and amino acid substitutions in the VP4 gene as compared to their wild type strains. The number of substitutions however, varied from 1-64 and 1-13 respectively. The substitutions were distributed in both VP5* and VP8* subunits of VP4 gene respectively of permeabilization and hemagglutinating activity. The presence of unique amino acid substitutions was identified in two of the four wild type (V377G, S387N in 061060 and I644L in 0715880) and all four cell culture adapted (A46V in 0613158-CA, T60R in 06361-CA, L237V, G389V and Q480H in 061060-CA and S615G and T625P in 0715880-CA) strains for the first time in the VP4 gene of P[8] specificity. Amino acid substitutions generated increase in the hydrophilicity in the cell culture adapted rotavirus strains as compared to their corresponding wild type strains.@*CONCLUSIONS@#Amino acid substitutions detected in the VP4 genes of G1P[8] rotavirus strains from this study together with those from other studies highlight occurrence of only strain and/or host specific substitutions during cell culture adaptation. Further evaluation of such substitutions for their role in attenuation, immunogenicity and conformation is needed for the development of newer rotavirus vaccines.

Molecular characterization of VP4, VP6, VP7 and NSP4 genes of group B rotavirus strains from outbreaks of gastroenteritis

OBJECTIVE@#To characterize VP4, VP6, VP7 and NSP4 genes of representative GBR strains (NIV-005625, NIV-04622 and NIV-094456) detected as the major etiologic agent in the outbreaks of gastroenteritis in western India.@*METHODS@#Fecal specimens collected during the outbreaks of gastroenteritis were processed for RNA isolation, RT-PCR using GBR VP4, VP6, VP7 and NSP4 gene specific primers, nucleotide sequencing of the amplicons and phylogenetic analysis of the sequences.@*RESULTS@#Phylogenetic analysis of all of the VP4, VP6, VP7 and NSP4 gene sequences revealed clustering of GBR strains in Indian-Bangladeshi lineage of genotype G2 with 95.8%-99.4% nucleotide and 97.3%-100.0% amino acid identities. However, all three strains showed the presence of unique amino acid substitutions in the VP4 protein suggesting alteration in the antigenicity of outbreak strains of GBR. The VP8* and VP5* regions of VP4 proteins showed respectively 0.5%-6.3% and 0.2%-1.1% amino acid divergence from human GBR strains of Indian-Bangladeshi lineage.@*CONCLUSIONS@#These data confirm the reported variability of VP8* region and suggest the possible role of this region in the perpetuation of GBR infections in the environment. This is the first study to document the phylogenetic relationship of VP4, VP6, VP7 and NSP4 genes of GBR strains detected in the outbreaks of gastroenteritis from India with the GBR strains from other parts of world.