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Highlights: [•] Novel recombinant human astrovirus strains in Kolkata, India. [•] ORF1b (RdRp) region analysis shows recombination break points (n =8) in IDH1300. [•] Single break point location was detected at 1205nt position. [•] ORF1b region of the seven human astrovirus strains formed close cluster. [•] Separate lineage is observed in comparison to human astrovirus strains worldwide. [ABSTRACT FROM AUTHOR]

Group A Rotavirus remains the leading cause of gastroenteritis in children and accounts for 0.2 million fatalities each year; out of which, approximately 47,100 deaths occur in India. In adults also, rotavirus is reported to be responsible for diarrhea severe enough to require hospitalizations. India has recently introduced rotavirus vaccine in the Universal Immunization Programme and Himachal Pradesh became the first Indian state to implement this project. This study is an attempt to provide the pre-vaccination data on rotavirus gastroenteritis burden and circulating genotypes in Himachal Pradesh, India. A total of 607 faecal specimens (247 children ≤ 5 years, 50 older children and 310 adults) from hospitalized diarrheal patients from Himachal Pradesh, India were screened for rotavirus using ELISA and RT-PCR. The positive samples were further G/P genotyped using semi-nested PCR. Rotavirus was detected in 25.2% and 28.3% of samples with ELISA and RT-PCR, respectively. In children, rotavirus frequency was significantly high with positivity in 49.0% cases whereas 14.0% adult samples have rotavirus in them. Genotyping of the positive samples revealed predominance of G1 (66.0%) and P[6] (66.7%) genotypes. The most common G and P combination was G1P[6] (62.8%) followed by G1P[8] (16.5%), G9P[6] (7.4%) and G12P[6] (5.0%). Molecular analysis reveals the belonging of P[6] strains in Lineage 1a. This pre-vaccination data on rotavirus prevalence and diversity would be helpful for assessing the affect of vaccination on the disease burden and its comparison with post-vaccination data of circulating genotypes would help in studying the effect on diversity of rotavirus strains possibly due to vaccine selection pressure. [ABSTRACT FROM AUTHOR]

Highlights: [•] 2899 (40%) out of 7285 stool samples were rotavirus positive from 2005 to 2009. [•] G12 infections rose to 39% in Northern India and to 24% in the Western India. [•] 8% of rotavirus detections had multiple G-types and 3% had multiple P-types. [ABSTRACT FROM AUTHOR]

Abstract: Although P[6] group A rotaviruses (RVA) cause diarrhoea in humans, they have been also associated with endemics of predominantly asymptomatic neonatal infections. Interestingly, strains representing the endemic and asymptomatic P[6] RVAs were found to possess one of the four common human VP7 serotypes (G1–G4), and exhibited little antigenic/genetic differences with the VP4 proteins/VP4 encoding genome segments of P[6] RVAs recovered from diarrhoeic children, raising interest on their complete genetic constellations. In the present study, we report the overall genetic makeup and possible origin of three such asymptomatic human P[6] RVA strains, RVA/Human-tc/VEN/M37/1982/G1P2A[6], RVA/Human-tc/SWE/1076/1983/G2P2A[6] and RVA/Human-tc/AUS/McN13/1980/G3P2A[6]. G1P[6] strain M37 exhibited an unusual genotype constellation (G1-P[6]-R1-C1-M1-A1-N1-T2-E1-H1), not reported previously, and was found to originate from possible intergenogroup reassortment events involving acquisition of a DS-1-like NSP3 encoding genome segment by a human Wa-like RVA strain. On the other hand, G2P[6] strain 1076 exhibited a DS-1-like genotype constellation, and was found to possess several genome segments (those encoding VP1, VP3, VP6 and NSP4) of possible artiodactyl (ruminants) origin on a human RVA genetic backbone. The whole genome of G3P[6] strain McN13 was closely related to that of asymptomatic human Wa-like G3P[6] strain RV3, and both strains shared unique amino acid changes, which might have contributed to their attenuation. Taken together, the present study provided insights into the origin and complex genetic diversity of P[6] RVAs possessing the common human VP7 genotypes. This is the first report on the whole genomic analysis of a G1P[6] RVA strain. [Copyright &y& Elsevier]

Abstract: Human astroviruses (HAstVs) have now emerged as another common cause of non-bacterial acute gastroenteritis (AGE) in humans worldwide. This study investigated the epidemiology and genetic diversity of human astrovirus strains circulating among infants, younger children (up to 6years), older children and adolescents (>6–17years) and adults (18years and above) hospitalized for diarrhea and their role in AGE in Kolkata, India. A total of 2535 fecal samples were screened for the presence of known enteric viral, bacterial and parasitic etiologies by conventional microbiological assays and molecular methods. The overall incidences of sole or mixed infection of HAstV with known enteric viral, bacterial and parasitic pathogens were detected in 60 cases (2.4%) among all age groups. The clinical symptoms of astrovirus-associated acute watery diarrhea cases were recorded for all sole and mixed infection cases. A high number of sole (n =13/60 [21.7%]) and mixed infection cases (n =22/60 [36.7%]) were observed in adults (18years old or more). Considering all age groups, 18 sole infection cases (n =18/60 [30%]) and 42 mixed infection cases (n =42/60 [70%]) with Rotavirus (n =11/25 [44%]), Vibrio cholerae O1 (n =6/24 [25%]) Cryptosporidium spp and Giardia lamblia (n =5/13 [38.4%]) were observed. Further, eleven HAstV samples from infants and children (up to 6years), children and adolescents (>6–17years) and adults (18years and above) were analyzed for their sequences of overlap region between ORF1b (RdRp) and ORF2 (capsid). Among these, ten strains were found to have close genetic relatedness to the Japanese strain HAstV_G1 [AB009985]. Additionally, the IDH2211 Kolkata strain showed a close genetic match with the Thai HAstV_G3 strain [EU363889]. Our study reports show that HAstVs as the sole agent and as mixed infection with other known enteric viral, bacterial, parasitic pathogens are also responsible for AGE among infants, children, adolescents and adults in Kolkata, India. [Copyright &y& Elsevier]

Abstract: The genus, Picobirnavirus (PBV), Spanish ‘pico’=‘small’, birna for ‘bipartite RNA’ genome, belongs to the family Picobirnaviridae under the proposed order Diplornavirales. PBV infections have been reported from diarrhoeic animal species and humans as well as from asymptomatic cases. The detection of Picobirnaviruses (PBVs) in diarrhoeic faecal specimens from children aged <5 years, suggestive of zoonotic transmission is being reported. 23 Picobirnavirus positive faecal specimens were detected by polyacrylamide gel electrophoresis (PAGE) and silver staining from a set of 1112 faecal specimens collected from an urban slum community in Kolkata between July and October 2007. The Picobirnaviruses showed either large profile (n =22) or small profile (n =1) for their bisegmented genomic double-stranded RNA (dsRNA). 13/23 positives were amplified by reverse transcription polymerase chain reaction (RT-PCR) as 201bp amplicon with genogroup I primers [PicoB25(+) and PicoB43(−) specific for RNA dependent RNA polymerase (RdRp) gene fragment encoded by genomic segment 2] and seven amplicons were sequenced [GPBV1–5, 7 and 8]. Sequence analyses showed that four PBV strains [GPBV1–3 and 8] resembled different clones of porcine PBV strains (D4, D6 and C10) reported in 2008 from Hungary and two PBV strains [GPBV4 and 7] resembled human PBV strains (P597, Kolkata and 2-GA-91, USA) with the maximum nucleotide (nt) identity ranging from 78% to 92%. One strain GPBV5 clustered with human PBVs and porcine PBVs that were reported from Hungary, Venezuela and Argentina showing close homology to human-like PBVs. Therefore, the close monitoring of their global spread as well as in-depth molecular characterization is essential for better understanding of emerging PBV strains. [Copyright &y& Elsevier]

Abstract: Picobirnaviruses (PBVs) with bisegmented small RNA genome profile (1.75 and 1.55kbp for segment 1 and 2, respectively) were detected from 1999 to 2003 in faecal specimens of acute watery diarrhoea cases, largely children (n =20) and an adult in Kolkata, India. Varying degrees of dehydration necessitated their visit to hospital for further treatment and management of acute watery diarrhoea. PBV was associated with rotavirus (n =3) or astrovirus (n =3) and with both in one case. No co-infection with norovirus, sapovirus or adenovirus was detected in the picobirnavirus positive cases. No co-infection with parasites (Cryptosporidium spp., Giardia spp., Entamoeba spp., helminths) or bacteria (Vibrio spp., Shigella spp., Escherichia coli) was detected among the picobirnavirus positive cases. There was a single instance of co-infection with Salmonella spp. (n =1). PBVs not associated with serious diarrhoea illness and showing large genome profile (2.3–2.6 and 1.5–1.9kbp for segment 1 and 2, respectively) have earlier been reported in adult individuals and recently among children from a slum community in Kolkata, India. The short genome profile PBVs associated with acute watery diarrhoea may be another emerging diarrhoeagenic virus in Kolkata, India. Molecular characterization using reported primers PicoB25–PicoB43 for Genogroup I and PicoB23–PicoB24 for Genogroup II in RT-PCR showed the presence of Genogroup I PBVs (n =6) and Genogroup II PBVs (n =5), while some could not be amplified (n =3) with these primers. Sequence analysis of Genogroup I amplicons indicated remarkable sequence heterogeneity. After more than a decade, four PBV positives of Genogroup II were detected during this study. Phylogenetic analysis showed varying degree of genetic diversity amongst PBV strains from Kolkata and other countries. [Copyright &y& Elsevier]

Abstract: The study is aimed to determine the seasonal distribution and clinical characteristics of astroviruses associated with acute watery diarrhoea among children in Kolkata and characterize them at the molecular level. Method of study: Faecal specimens of acute watery diarrhoea cases (n =857) and non-diarrhoeic samples (n =211) from the hospitals and a nearby field community were screened with IDEIA Astrovirus detection kit; astrovirus co-infections with rotavirus and/or picobirnavirus were detected by RNA-PAGE and silver staining. Further RT-PCR was carried out using specific primers, viz. Mon340 (+) and Mon348 (−) targeting a highly conserved domain of ORF1a (289bp) of human astroviruses. Results: Astrovirus infection was detected in 50 cases (50/857); astroviruses were detected mostly in children aged 6–12 months (50%); all non-diarrhoeic samples (n =211) were negative for astrovirus. In 52% of astrovirus positive cases, the virus was detected as the sole agent; mixed infections were also detected with other diarrhoeic pathogens such as rotavirus (32%), picobirnavirus (2%), rotavirus and picobirnavirus (2%), picobirnavirus and Enterotoxigenic E. coli (ETEC) (2%), rotavirus and ETEC (2%), rotavirus and Enteroaggregative E. coli (EAEC) (2%), Enteropathogenic E. coli (EPEC) (2%), Shigella flexneri type 3a (2%) and Ascaris (2%). RT-PCR and sequencing of amplicons of astroviruses from Kolkata, with specific primers targeted to the conserved domain of ORF1a (289bp) of the astrovirus genome, showed maximum homology to the astrovirus strain (“5–158”) from Seoul (98%). Results and Conclusions: Clinical characteristics of the diarrhoeic children in Kolkata indicated that astrovirus infections were detected throughout the year and were associated with varying degree of dehydration and acute watery diarrhoea. In-depth molecular epidemiological surveillance of astroviruses in Kolkata is essential for better understanding of their overall genetic nature. [Copyright &y& Elsevier]

Abstract: Picobirnaviruses are a group of unclassified, non-enveloped, small spherical viruses, 35–41nm in diameter without any apparent surface morphology. They have characteristic bisegmented double stranded RNA genome of two types namely large profile (2.3–2.6kbp for the larger and 1.5–1.9kbp for the smaller segment, respectively) or small profile (1.75 and 1.55kbp for segments 1 and 2, respectively). Human picobirnaviruses (n =12 positives; 2/56 diarrhoeic children and 10/607 non-diarrhoeic children) with large (n =11) or small (n =1) genome pattern were observed in faecal specimens of children from a slum community by silver stained PAGE gels. Faecal specimen from four asymptomatic cases (P597_02_IND, K135_02_IND, A373_03_IND, A356_03_IND) and one diarrhoeic case (K135_03_IND) had genogroup I picobirnaviruses (1-CHN-97 like) showing amplicons within the 201bp region, with primers PicoB25–PicoB43, targeting the conserved domain of RNA-dependent RNA polymerase (RdRp) gene. It was interesting to note that only the PBV strain P597_02_IND from Kolkata with large genome was closely related to a reported strain (similarity with 2-GA-91 from USA was 87% at the nucleotide level and 90% at the amino acid level). Sequence analysis showed three conserved amino acid domains as well as a highly conserved D-S—D motif, characteristic of RNA-dependent RNA polymerase gene of bisegmented, double stranded RNA viruses. Sequence data of the picobirnavirus A356_03_IND indicated strong heterogeneity with all other picobirnavirus strains sequenced till date. After nearly a decade a genogroup II picobirnavirus strain (R227_03_IND) was isolated from a diarrhoea case in the community, with small genome profile and amplified with specific primers PicoB23–PicoB24; but the sequence data showed that it was divergent from the hitherto reported prototype strain 4-GA-91 of genogroup II human picobirnaviruses. [Copyright &y& Elsevier]