Circulation of unusual and diverse enteric virus strains in adults with acute gastroenteritis: a study from Pune (Maharashtra), Western India.

In India, studies on the epidemiological and genetic characteristics of enteric viruses in adults with acute gastroenteritis (AGE) are lacking. In this study, fecal samples (n = 110) from adults with acute gastroenteritis in Pune, Western India, were tested for six enteric viruses, and the prevalence of these viruses was as follows: rotavirus A (RVA), 38.5%; enterovirus (EV), 23.1%; astrovirus (AstV), 23.1%; adenovirus (AdV), 7.7%; human bocavirus (HBoV), 7.7%; norovirus (NoV), 0%. Circulation of the RVA G1P[8], G3P[8], G9P[4], CVA-10, echovirus E13, EVC-116, AstV-5, AstV-2, HBoV-1, and AdVC-2 types was observed. When compared to the RotaTeq, Rotarix, and RotaVac vaccine strains, antigenic changes were found in the A, B, C, and F regions of the RVA strains. The circulation of genetically diverse, unusual enteric virus strains, reported here for the first time in adults with acute gastroenteritis, warrants multi-center hospital-based surveillance studies across the country.

Rotavirus G9P[4], G9P[6] and G1P[6] strains isolated from children with acute gastroenteritis in Pune, western India, 2013-2015: evidence for recombination in genes encoding VP3, VP4 and NSP1.

Species A rotaviruses (RVAs) are genetically diverse pathogens. These are the most evolutionarily adaptable organisms, with a multitude of mechanisms for evolutionary change. To date, full-genome classification has been proved to be an excellent tool for studying the evolution of unusual rotavirus strains. As limited data are available from Pune (Maharashtra), western India, the current study was undertaken with the aim of understanding the genetic diversity in three (G1P[6], G9P[4] and G9P[4]) unusual RVA strains circulating in Pune, India during 2013-2015. Full-genome analysis of these strains classified them as G1-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1, G9-P[4]-I2-R2-C2-[M1-M2_R]-[A1-A2_R]-N2-T2-E6-H2 and G9-[P4-P6_R]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequencing and phylogenetic analysis of the structural and non-structural genes of these unusual RVA strains showed nucleotide/amino acid identities of 82.3-98.5 %/77.3-99.8 % and 86.6-97.6 %/89.6-97.8 % between the strains of the study. Evidence of recombination events was found within the genes encoding VP3, VP4 and NSP1, which showed a combination of genetic information for genogroup 1 [M1/P[6]/A1] and genogroup 2 [M2/P[4]/A2] strains. This study will facilitate future investigations into the molecular pathogenesis of such RVAs as the exchange of whole or partial genetic material between rotaviruses through recombination contributes directly to their diversification, adaptation and evolution.

Detection of different enteric viruses in children with diarrheal disease: evidence of the high frequency of mixed infections.

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.

Full genome analysis of rotavirus G9P[8] strains identified in acute gastroenteritis cases reveals genetic diversity: Pune, western India.

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.

Characterisation of rotavirus strains identified in adolescents and adults with acute gastroenteritis highlights circulation of non-typeable strains: 2008-2012.

BACKGROUND: Group-A Rotavirus (RV) is the main causative agent of acute gastroenteritis in children < 5 years of age. Its role as a pathogen in adults needs to be monitored. The aim of this study was to characterise the group-A RV strains that cause infections of acute gastroenteritis in adolescents and adults and determine the temporal variations in the circulating strains during 2008-2012 in continuation of an earlier study conducted in 2004-2007, in Pune, India. METHODS: A total of 371 stool samples were tested by RV antigen capture ELISA. VP4, VP6, VP7 and NSP4 genes of all of the RV strains detected in the study were analysed using reverse transcription PCR, multiplex PCR and sequencing. RESULTS: Group-A RV was detected in 9.4% (35/371) of the stool samples examined in the study period. The frequency of detection of RV was found to decline from 18.0% (16/90) in 2008 to 3.8% (2/52) in 2012. Of the 6 strains typed for both VP7 and VP4 genes, G2P[4], G1P[8] and G9P[4] were detected in 3, 1 and 2 samples, respectively. Sequencing and phylogenetic analysis of the VP4, VP6, VP7 and NSP4 genes revealed an infrequently reported NSP4-E6 genotype and circulation of heterogenous [G2 (lineage IIC and IID), G9 (lineage 3), P[4] (lineage P[4]-5), P[8] (lineage P[8]-3), VP6 I1 / I2 and NSP4 E2] genotypes/lineages in the RV strains. Analysis of linkage within these genes showed concordance (G2-P[4]-I2-E2) and discordance (G9-P[4]-I2-E6), equally. The sequences of amplified VP6 (n = 20) and NSP4 (n = 2) genes from G and P nontypeable RV strains (80.0%, 28/35) were most homologous to human group-A RV strains. CONCLUSION: 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.

Evidence of discordant genetic linkage in the VP4, VP6, VP7 and NSP4 encoding genes of rotavirus strains from adolescent and adult patients with acute gastroenteritis.

The vast diversity within rotavirus strains circulating in the developing countries continues to be a major challenge for the efficacy of currently used preset rotavirus vaccines. The sequence analysis and phylogeny of multiple genes of rotavirus strains enable identification of reassortant strains and their human or animal origin. The objective of this study was to monitor the genetic linkage between the rotavirus VP4(P), VP6(I), VP7(G) and NSP4(E) encoding genes. The G, P, I and E genotypes of a total of 80 rotavirus strains isolated from adolescent and adult cases of acute gastroenteritis at the two time points [1993-1996 (n=67) and 2004-2007 (n=13)] were determined by nucleotide sequencing and phylogenetic analysis. The rotavirus strains from the 1990s and 2000s revealed common combinations of genotypes (G1-P[8]-I1-E1, G2-P[4]-I2-E2, G3-P[8]-I1-E1 and G4-P[8]-I1-E1) in 47.8% and 30.8%, unusual combinations of the same genotypes (G2-P[8]-I2-E2, G9-P[6]-I1-E1, G9-P[6]-I1-E2, G9-P[6]-I2-E1 and G4-P[4]-I1-E2, G1-P[4]-I2-E1, G9-P[4]-I1-E1) in 7.5% and 23% and mixed infections of different G and P genotypes in 31.3% and 46.2%, respectively. Discordance in the association of I with E, G with I and E and P with I and E genotypes was found to be contributed respectively by 23.8-38.5%, 40.3-69.8% and 49.3-61.5% of the rotavirus strains at the two time points. The data suggest relatively high occurrence of intergenogroup reassortment in circulating rotavirus strains emphasizing the need for continuous surveillance and whole genome sequence based characterization of rotavirus strains for better understanding of their evolution and ecology.

Diversity in the VP7 encoding genes of rotavirus strains isolated from adolescent and adult cases of acute gastroenteritis.

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.

Sequence and phylogenetic analysis of the VP6 and NSP4 genes of human rotavirus strains: evidence of discordance in their genetic linkage.

NSP4 and VP6 genes of a total of 118 rotavirus strains detected in adolescent and adult cases of acute gastroenteritis (AGE) in 1993-1996 and 2004-2007 were characterized to determine their diversity and genetic linkage. Eighty-two percent and 89% of the strains showed amplification of NSP4 and VP6 genes respectively in RT-PCR. Sequencing and phylogenetic analysis of the VP6 genes showed distribution of genogroups in the lineages I-1 (1.4%), I-2 (50.7%) and II-4 (47.9%) in the 1990s and I-2 (73.5%) and II-4 (26.5%) in 2000s, indicating diversity in genogroups at both time points. Amino acid divergence within the genogroup II strains from 1990s and genogroup I strains from the 2000s was noteworthy (4.7-6.7%). Sequencing and phylogenetic analysis of the NSP4 genes showed almost equal distribution (45.0-55.0%) of genotypes A and B however, higher amino acid divergence within the genotype B strains (up to 9.3%) than in genotype A strains (up to 2.9%) at the two-time points. Nearly 70% of the strains showed NSP4-A-VP6-I or NSP4-B-VP6-II genetic linkage. The discordance in the linkage noted in 29.7% of the strains was predominated by NSP4-B and VP6-I combination and appeared strikingly high in the infections caused by unusual and mixed rotavirus strains. This is the first report to describe the phylogenetic analysis of rotavirus NSP4 and VP6 genes and their discordance in adolescent and adult cases with AGE from India. The extensive diversity within the rotavirus genes and their relationship revealed by this study emphasizes the need for evaluation of the rotavirus vaccines being used currently.

Characterization of group A rotavirus infections in adolescents and adults from Pune, India: 1993-1996 and 2004-2007.

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.

High frequency of rotavirus viremia in children with acute gastroenteritis: discordance of strains detected in stool and sera.

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.