The understanding of genotype distribution has shown that two widely used vaccines appear to protect against homologous and heterologous viruses. But the long term effects on virus circulation exerted by the immune pressure of a vaccinated population are as yet unknown and warrant

continued molecular surveillance at this time. Additionally, studies on virus diversity and evolution are important to understand the biology of transmission and circulation in the population. This knowledge propels the application of robust molecular methods to identify the prevalent genotypes and methods to track the emergence of novel viruses. A WHO manual describes the methods used to perform initial identification and further XAV-939 cost characterize group A rotavirus isolates [7]. Although the methods and primer sets described in the manual and by other networks appear

to identify the majority of strains based on updated WHO reports and network publications [6], [8] and [9], a proportion of strains remain untyped and require further testing. As the referral laboratory for the Indian National Rotavirus Surveillance Network which check details collected >4000 stool samples from 11 hospitals in 4 regional centers [8] and [11], we have developed an approach to handling samples initially untyped by standard methods and describe its application to samples collected over five years from 2007 to 2012. Stool samples were received for VP7 and VP4 molecular characterization

in the Wellcome Trust Research laboratory (WTRL) from 2007 to 2012, as part of the Indian Rotavirus PDK4 Strain Surveillance Network (IRSSN) or as referrals. All samples were screened by enzyme immunoassay (Premier Rotaclone, Meridian Diagnostics, Cincinnati, OH) and the antigen positive samples were genotyped as previously described elsewhere [8]. Complementary DNA (cDNA) was synthesized by reverse transcription (RT) as previously described using random primers (Pd(N)6 hexamers; Pharmacia Biotech) and 400 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies) [8]. Briefly, a first-round RT-PCR targeting VP7 and VP4 consensus regions using primers (VP7F/R and Con3/Con2, respectively) described in Table 1 were performed. The first-round product was used as a template to determine specific VP7 (G) types (G1, G2, G3, G4, G8, G9, G10 and G12) and VP4 (P) types (P[4], P[6], P[8], P[9], P[10], P[11]) in a semi-nested multiplex PCR format [8]. Of the 2226 rotavirus ELISA positive samples for which further molecular characterization was performed, 57 samples were partially genotyped and 308 samples were untyped for G and P types. These represent 2.