Such

studies are also essential in higher age groups for better understanding of RV spread in the community. In our earlier study carried out to characterise RV infections in adolescents and adults, a rise in RVA infections in BMS-354825 2004–2007 as compared to 1993–1996 was reported [15]. Infections with uncommon G-P and mixed infections were higher in these age groups when compared to those in children. In the present study, the surveillance of RV infections was continued in the same age groups of patients with acute gastroenteritis to understand the temporal variations in the rate of RV infections and the strains during the 5 year period, 2008–2012. A total of 371 stool specimens were collected buy GW3965 from adolescent (10–18 years) and adult (>18 years) cases of acute gastroenteritis, admitted to or visiting out-patient departments

of local hospitals from Pune city during 2008–2012. The study was approved by the ethical committee of the National Institute of Virology. Epidemiologic data including age, gender, dates of diarrhoea onset and specimen collection were available from all patients. Ten percent (w/v) stool suspension of each of the specimens was prepared in 0.01 M phosphate buffered saline (PBS), pH 7.2 containing 0.01 M CaCl2. The suspensions were centrifuged at 805 g for 15 min to remove debris. The supernatants were stored in aliquots these at -70 ̊C until tested for RVA antigen and genotypes. All specimens were tested for the presence of RV by using Generic Assay ELISA kit for rotavirus (Cat. No. 6001, Germany) as per manufacturer’s instructions. Specimens with optical density (OD) values above the cut-off value (0.2 + mean value of OD of negative control wells) were considered positive for rotavirus antigen. RVA dsRNA was extracted from stool specimens by using TRIZOL®LS reagent (Invitrogen, Carlsbad, CA) as per the manufacturer’s protocol. The VP7 and VP4 genes were genotyped by multiplex reverse transcription

(RT)-PCR using the methods described earlier [16] and [17] and modified thermal cycling programme [18]. The full-length NSP4 genes (751 bp) and VP6 gene subgrouping region (379 bp) were amplified using the NSP4-F and NSP4-R primers [19] and forward (F) VP6 and reverse (R) VP6 primers [20], respectively, with the one step RT-PCR kit (Qiagen, Hilden, Germany). The PCR conditions involved initial reverse transcription step of 30 min at 45̊C and 95̊C for 15 min followed by 40 cycles of 94̊C for 1 min, 50̊C for 1 min, 70̊C for 2.5 min with a final extension at 70̊C for 7 min. All PCR products, including those from the first-round and multiplex PCRs, were analysed by electrophoresis using Tris acetate EDTA (TAE) buffer, pH 8.3 on 2% agarose gels, containing ethidium bromide (0.5 ug/ml) and visualised under UV illumination.