Approach to molecular characterization of partially and completely untyped samples in an Indian rotavirus surveillance program

AbstractSurveillance networks for rotavirus document the burden of the disease using the proportion of children hospitalized with gastroenteritis positive for rotavirus by enzyme immunoassay. They also describe genotypes of circulating viruses by polymerase chain reaction for the VP7 and VP4 genes, which determine G and P types, respectively. A proportion of samples cannot be genotyped based on initial testing and laboratories need to assess further testing strategies based on resources and feasibility. To 365 samples obtained from an Indian rotavirus strain surveillance program, we applied an approach to determine the G and P types in antigen positive samples that failed to type initially with the standard laboratory protocol. Fifty-eight samples (19%) were negative for the VP6 gene, indicating that the antigen test was likely to have been false positive. Alternative extraction and priming approaches resulted in the identification of G and P types for 264 strains. The identity of one strain was determined by sequencing the first-round amplicons. Thirty-five strains were partially typed and seven strains could not be typed at all. The distribution of G and P types among strains that had initially failed to type, except one strain, did not differ from that in strains that were typed using the standard laboratory protocol