Faecal specimens collected from two outbreaks of acute gastroenteritis that occurred in southern Mumbai, India in March and October, 2006 were tested for seven different enteric viruses. Among the 218 specimens tested, 95 (43.6%) were positive, 73 (76.8%) for a single virus and 22 (23.2%) for multiple viruses. Single viral infections in both, March and October showed predominance of enterovirus (EV, 33.3% and 40%) and rotavirus A (RVA, 33.3% and 25%). The other viruses detected in these months were norovirus (NoV, 12.1% and 10%), rotavirus B (RVB, 12.1% and 10%), enteric adenovirus (AdV, 6.1% and 7.5%), Aichivirus (AiV, 3% and 7.5%) and human astrovirus (HAstV, 3% and 0%). Mixed viral infections were largely represented by two viruses (84.6% and 88.9%), a small proportion showed presence of three (7.7% and 11%) and four (7.7% and 0%) viruses in the two outbreaks. Genotyping of the viruses revealed predominance of RVA G2P[4], RVB G2 (Indian Bangladeshi lineage), NoV GII.4, AdV-40, HAstV-8 and AiV B types. VP1/2A junction region based genotyping showed presence of 11 different serotypes of EVs. Although no virus was detected in the tested water samples, examination of both water and sewage pipelines in gastroenteritis affected localities indicated leakages and possibility of contamination of drinking water with sewage water. Coexistence of multiple enteric viruses during the two outbreaks of gastroenteritis emphasizes the need to expand such investigations to other parts of India.

Recently, rotavirus antigenemia and viremia have been identified in patients with acute gastroenteritis. This study examined rotavirus viremia in children hospitalized for acute gastroenteritis in order to establish its association with fecal shedding of rotavirus, infecting genotypes and antibody marker of acute infection. Thirty-one pairs of stool-serum specimens were collected from November 2004 to February 2005 together with clinical information. All paired specimens were screened for rotavirus RNA by RT-PCR using the VP6 gene primers. All stool and serum specimens were tested for rotavirus antigen and anti-rotavirus IgM respectively by ELISA. Sixteen of 31 stool-serum pairs showed the presence of rotavirus RNA. Nine stool and two serum specimens were positive only by RT-PCR. The total positivity in rotavirus RNA was significantly higher in both stools (80.6%) and sera (58.1%) than that of stool antigen (38.7%) and anti-rotavirus IgM (25.8%) (P < 0.01). All PCR positive paired specimens were typed for the VP7 (G) and VP4 (P) genes. Five of sixteen pairs could be typed for both genes. Three of the five pairs showed concordance (G2P[4]/G2P[4]) while two showed discordance (G12P[8]/G2P[4], G8P[4]/G2P[4]) in the genotypes detected in stool and serum specimens respectively. The study documents a high frequency of rotavirus viremia in patients with acute diarrhea. The discordance of rotavirus strains at the genotypic level in the serum and stool of individual patients with diarrhea suggests the susceptibility of extra-intestinal sites for rotavirus infection and the possibility of differential dissemination of rotavirus strains from the intestine.

A total of 1,591 fecal specimens were collected in 1993-1996 and 2004-2007 from adolescents and adults with acute gastroenteritis in Pune, India for detection and characterization of rotavirus. At the two time points, group A rotavirus was detected in 8.6% and 16.2% of the adolescents and 5.2% and 17.2% of the adults, respectively. Reverse transcription-PCR with consensus primers followed by multiplex genotyping PCR detected common strains G1P[8], G2P[4], G3P[8], and G4P[8] in a total of 53.1% of the samples from 1993 to 1996, while the only prevalent strain identified in 2004-2007 was G2P[4] (23.5% of total). Uncommon rotavirus strains (G1P[4], G2P[8] G9P[6]/P[4]) increased from 7.8% (1993-1996) to 41.2% (2004-2007), while the prevalence of mixed rotavirus infections was high (39%/35%) at both time points. Mixed infections detected by multiplex PCR were confirmed by sequencing two or more individual genotype-specific PCR products of the VP7 and VP4 genes from the same sample. Phylogenetic analysis of the sequences showed circulation of a heterogeneous rotavirus strain population comprising genotypes G1 (lineages I and IIb), G2 (lineages I and IIb), G4 (lineage Ia), P[4] (lineages P[4]-5 and P[4]-1), P[8] (lineages P[8]-II and P[8]-III), and P[6] (M37-like lineage). The VP6 gene sequences of the nontypeable strains were most homologous to animal strains. This study documents the molecular epidemiology of rotavirus strains in adolescents and adults in India, and suggests that it may be important to monitor these strains over time for the potential impact on rotavirus vaccines under development for use in the Indian population. J. Med. Virol. 82:519-527, 2010. (c) 2010 Wiley-Liss, Inc.

Enteric viruses play a major role in causing diarrhea in children. Early identification of the causative pathogen is still a challenge in the clinical laboratory. A multiplex PCR assay is a useful tool to screen a large number of clinical samples especially in an outbreak situation. In this study, a multiplex reverse transcription (RT)-PCR assay was developed to detect nine enteric viruses such as group A rotavirus, norovirus GGII, sapovirus, adenovirus, astrovirus, aichivirus, parechovirus, bocavirus and enterovirus in clinical samples of diarrheal cases. Stool samples ( n =185) collected from infants and children with acute gastroenteritis cases in Pune, western India were analysed for nine different enteric viruses by currently developed multiplex RT- PCR. Predominance of group A rotavirus (76%) followed by enterovirus (11.5%), astrovirus (4.5%), adenovirus (2.7%) and norovirus GII (1.6%) was observed. A total of 44.8 % (82/185) samples analysed by this method showed high frequency of mixed infections. These results highlighted high prevalence and diversity of different enteric viruses in children. The multiplex PCR showed good concordance with monoplex RT-PCR for detection of these enteric viruses in clinical samples. This is the first report on the development of a multiplex RT-PCR assay for detection of multiple enteric viruses in diarrheal diseases from India.

The study underscores the significant temporal variations in RV strains, identifies circulation of intergenogroup reassortants among adolescent and adult patients with acute gastroenteritis and emphasizes the need for continued surveillance and whole genome analysis of emerging rotavirus strains.

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Group A rotaviruses (RVA) are the major enteric etiological agents of severe acute gastroenteritis among children globally. As G9 RVA now represents as one of the major human RVA genotypes, studies on full genome of this particular genotype are being carried out worldwide. So far, no such studies on G9P[8] RVAs have been reported from Pune, western part of India. Keeping in view of this, the study was undertaken to understand the degree of genetic diversity of the commonly circulating G9P[8] RVA strains. Rotavirus surveillance studies carried out earlier during the years 2009-2011 showed increase in the prevalence of G9P[8] RVAs. Representative G9P[8] RVA strains from the years 2009, 2010, and 2011 were selected for the study. In general, all the G9 RVA strains showed clustering in the globally circulating sublineage of the VP7 gene and showed nucleotide/amino acid identities of 96.8-99.7%/96.9-99.8% with global G9 RV strains. Full genome analysis, of all three RVAs in this study indicated Wa-like genotype constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Within the strains nucleotide/amino acid divergence of 0.1-3.4%/0.0-4.1% was noted in all the RVA structural and non-structural genes. In conclusion, the present study highlights intra-genotypic variations throughout the RVA genome. The study further emphasizes the need for surveillance and analysis of the whole genomic constellation of the commonly circulating RVA strains of other regions in the country for understanding to a greater degree of the impact of rotavirus vaccination recently introduced in India.

A study was conducted to examine the diversity in the VP7 genes of rotavirus strains circulating in adolescent and adult cases of acute gastroenteritis during two different time periods, 1993–1996 and 2004–2007. The multiplex RT‐PCR carried out on 131 rotavirus positive fecal specimens detected 65 (49.6%) single and 48 (36.6%) mixed infections of VP7 genotypes that included 43G1 (38.1%), 37G2 (32.7%), 8G3 (7.1%), 15G4 (13.3%), and 10G9 (8.8%) specificities. Sequencing and phylogenetic analysis of the VP7 gene amplicons revealed the presence of G1‐IA (4.7%), G1‐IB (69.8%), and G1‐IC (25.5%) lineages within the G1 strains, G2‐IIb1 (70.3%) and G2‐IIb2 (29.7%) lineages within G2 strains, G3‐3S1 (12.5%) and G3‐3S4 (87.5%) lineages within G3 strains, G4‐Ia (6.7%) and G4‐Ib (93.3%) lineages within G4 strains, and G9‐III lineage within G9 strains. The variability within VP7 genotypes was evident by 1.4–8.0% and 1.3–3.9% amino acid divergence respectively from the prototype strains and between the groups of strains at the two time points. This is the first report describing the phylogenetic analysis of VP7 genes of rotaviruses from adolescent and adult cases of acute gastroenteritis in India. Since adults infected with rotavirus could act as a source of infection and affect the epidemiology of rotaviruses in children, genetic analysis of the rotavirus strains circulating in adults is required. The intragenotypic diversity within VP7 genes demonstrated by the present study highlights the need for constant surveillance of rotavirus infections to understand better the evolution and transmission of group A rotaviruses in the community. J. Med. Virol. 84:1481–1488, 2012. © 2012 Wiley Periodicals, Inc.