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Background: This study was conducted to determine the etiology of diarrhoea in a hospital setting in Kolkata. Active surveillance was conducted for 2 years on two random days per week by enrolling every fifth diarrhoeal patient admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata. Results: Most of the patients (76.1%) had acute watery diarrhoea in association with vomiting (77.7%) and some dehydration (92%). Vibrio cholerae O1, Rotavirus and Giardia lamblia were the important causes of diarrhoea. Among Shigella spp, S. flexneri 2a and 3a serotypes were most predominantly isolated. Enteric viruses, EPEC and EAEC were common in children <5 year age group. Atypical EPEC was comparatively higher than the typical EPEC. Multidrug resistance was common among V. cholerae O1 and Shigella spp including tetracycline and ciprofloxacin. Polymicrobial infections were common in all age groups and 27.9% of the diarrhoea patients had no potential pathogen. Conclusions: Increase in V. cholerae O1 infection among <2 years age group, resistance of V. cholerae O1 to tetracycline, rise of untypable S. flexnerii, higher proportion of atypical EPEC and G. lamblia and polymicrobial etiology are some of the emerging trends observed in this diarrhoeal disease surveillance. [ABSTRACT FROM AUTHOR]

Deduced amino acid sequence and phylogenetic analyses of a group A rotavirus G9P[6] strain (designated as mcs/13-07), detected from a 3-year-old child in Eastern India, revealed a VP8* closely related to porcine P[6] strains (P[6] sublineage 1D), and the VP7 clustered with G9 lineage-III strains. To our knowledge, this is the first report of human P[6] strain clustering in sublineage Id. Thus, to further characterize the evolutionary diversity of strain mcs/13-07, all gene segments were analyzed. VP6 and NSP4 exhibited genetic relatedness to Wa-like human subgroup II strains, while VP1-3, NSP1-3 and NSP5 were closely related to porcine strains. Based on the new classification system of rotaviruses, mcs/13-07 revealed a G9–P[6]–I1–R1–C1–M1–A8–N1–T1–E1–H1 genotype with close similarity to human Wa-like and porcine Gottfried strains. Therefore, considering the porcine-like or porcine origin of multiple gene segments, it might be tempting to assume that strain mcs/13-07 represents a rare instance of whole-virus transmission from pig to human, after which the virus evolved with time. Alternatively, it is possible that strain mcs/13-07 resulted from multiple reassortment events involving human subgroup II and porcine P[6] strains. Nevertheless, detection of strain mcs/13-07 provides further evidence for complex interspecies transmission events, which are frequent in developing countries. [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: 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]

Background & objectives: Diarrhoeal disease is the fifth leading cause of all mortality globally. To this burden, rotavirus contributes over half a million deaths annually. This pilot study was conducted to determine the economic burden of diarrhoeal episodes on families from different geographical regions accessing medical facilities in India. Methods: Participants were enrolled from four study sites with eight reporting hospitals, categorized as non-profit and low cost, private and government facilities between November 2008 and February 2009. Questionnaires detailing healthcare utilization, medical and non-medical expenditure and lost income were completed by families of children < 5 yr of age hospitalized for gastroenteritis. All available faecal samples were tested for rotavirus. Results: A total of 211 patients were enrolled. The mean total cost of a hospitalized diarrhoeal episode was … 3633 (US$ 66.05) for all facilities, with a marked difference in direct costs between governmental and non-governmental facilities. Costs for rotavirus positive hospitalizations were slightly lower, at … 2956 (US$ 53.75). The median cost of a diarrhoeal episode based on annual household expenditure was 6.4 per cent for all-cause diarrhoea and 7.6 per cent for rotavirus diarrhoea. Of the 124 samples collected, 66 (53%) were positive for rotavirus. Interpretation & conclusions: Data on direct costs alone from multiple facilities show that diarrhoeal disease constitutes a large economic burden on Indian families. Affordable, effective vaccines would greatly reduce the economic burden of severe gastroenteritis on patients, families and the government. [ABSTRACT FROM AUTHOR]

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: Background: Noroviruses (NoVs) are one of the major causal agents of acute gastroenteritis among different age groups. Some of the recent studies reveal that NoV genome is highly prone to mutation and recombination which often leads to emergence of new strains. Objectives: To explore the genetic diversity of human Caliciviruses (HuCVs) among diarrhoeic children in Kolkata. Study design: The HuCVs were detected by reverse transcription-polymerase chain reaction (RT-PCR) of the partial RNA dependent RNA polymerase gene (RdRp) and capsid gene and confirmed by sequencing. The sequences were analyzed and the recombination point was detected. Results: Faecal specimens of children (n =111) visiting outpatient department of Dr B. C. Roy Memorial Hospital for Children with acute gastroenteritis were studied: 22 cases were HuCV positive with 21 NoVs. Of these, 12 NoV cases (54.5%) were GII.4 and six cases showed 99% identity with the new variant Japanese strain Hu/NoV/GII.4/OC07138/JP. Three novel NoV GII inter-genotype recombinant strains V1628/IND, V1656/IND and V1737/IND were also detected. The RdRp region of V1628 showed 96% identity with Pont de Roide 673/FRN whereas capsid region resembled GII.7/Osaka F140/JPN strain (98%); the strain V1656 showed 98% identity with RdRp region of GII.4/Monastir 375/TUN but capsid region resembled GII.8/Leverkusen 267/DE (91%); the strain V1737 showed 88% identity with RdRp of GII.5/Minato 6/N1/6/JPN whereas capsid region resembled the GII.12/Gifu 96/JPN (93%). During characterization of Caliciviruses two strains of NoV GII.b and one strain of each NoV GI.1/V1622/06/IND, GI.3/V1707/07/IND, GII.3/V1668/IND, GII.16/V1729/IND, Sapovirus GII.1/V1716/IND were also detected. Conclusions: The emergence of new variant of GII.4/2007, three novel NoV GII inter-genotype recombinant strains and various other NoVs, indicates the remarkable genetic diversity of the HuCVs as diarrhoeagenic viruses circulating in Kolkata, India. [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]

Human astroviruses (HAstVs) associated with acute watery diarrhea among hospitalized infants, children and adults as sole or mixed infection, were earlier reported from Kolkata, India. Further, novel recombinations have been detected through sequencing of the highly conserved ORF1b (RdRp) region of seven human astrovirus strains in Kolkata, India. Primers were designed and the ORF1b region was amplified by RT-PCR and sequenced. To examine the evolutionary pressures influencing the evolution of human astroviruses we implemented evolutionary genetics analysis. Maximum recombination break points detected in Kolkata strain IDH1300 were 8 and a single break point location was detected at 1205nt position. Partition-wise phylogenetic analyses of the IDH1300 Kolkata strain did not show close homology to the reference strains. Further phylogenetic analyses of full length ORF1b region of the seven human astrovirus strains showed that they formed a close cluster with each other and displayed a separate lineage in comparison to reference human astrovirus strains worldwide. This study shows the emergence of novel recombinant human astrovirus strains in Kolkata, India, warranting stringent surveillance to monitor the genetic diversity of human astrovirus strains infecting different age groups., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Group B rotavirus (GBR) is a rare enteric pathogen that causes severe diarrhoea, primarily in adults. Nearly full-length sequences of all 11 RNA segments were determined for human GBRs detected recently in India (IDH-084 in 2007, IC-008 in 2008), Bangladesh (Bang117 in 2003) and Myanmar (MMR-B1 in 2007), and analysed phylogenetically with the sequence data of GBRs reported previously. All RNA segments of GBR strains from India, Bangladesh and Myanmar showed >95 % nucleotide sequence identities. Among the 11 RNA segments, the VP6 and NSP2 genes showed the highest identities (>98 %), whilst the lowest identities were observed in the NSP4 gene (96.1 %), NSP5 gene (95.6 %) and VP8*-encoding region of the VP4 gene (95.9 %). Divergent or conserved regions in the deduced amino acid sequences of GBR VP1-VP4 and NSP1-NSP5 were similar to those in group A rotaviruses (GARs), and the functionally important motifs and structural characteristics in viral proteins known for GAR were conserved in all of the human GBRs. These findings suggest that, whilst the degree of genetic evolution may be dependent on each RNA segment, human GBR may have been evolving in a similar manner to GAR, associated with the similar functional roles of individual viral proteins.

Mutation and recombination are recognized as important driving forces of evolution among RNA viruses. An intergenogroup recombinant norovirus strain [Hu/Kol/NLV/L8775/AB290150/2006/India] was detected in the faecal specimen of a 17 year old male, who had suffered from acute watery diarrhea and severe dehydration. Sequence analysis confirmed that this novel recombinant strain had a polymerase gene fragment that closely resembled a Norovirus (NoV) genogroup-I genotype-3 virus (HuCV/NLV/GI.3/VA98115/AY038598/1998/USA) and a capsid gene resembling NoV genogroup-II genotype-4 virus (NoV/Hu/GII.4/Terneuzen70/EF126964/2006/NL). The crossing over and recombination was observed at nucleotide (nt) 790 of NoV GI VA98115 strain and nt808 of NoV GII Terneuzen70 strain. In both parent strains conserved nucleotide sequence and hairpin structure (DNA secondary structure) were reported at the junction point of ORF1 and ORF2, exhibiting the mechanism of recombination in these viruses. Thus this novel recombinant NoV is another step in evolution among NoVs, indicating that constant surveillance is important to successfully monitor emergence of these strains.

Long RNA electropherotype rotavirus strains with subgroup I specificity predominated the infantile gastroenteritis outbreak in Manipur, India, in 1987-88. One such strain (RMC321) was found to possess porcine characteristics in 7 out of 8 genes sequenced. Partial characterization of its remaining VP1, VP2 and VP3 genes along with a porcine rotavirus strain (HP140) uncovered their close genetic relation to porcine strains. VP7 was the only gene segment of this strain with significant genetic identity to human strains. This indicates that a rotavirus reassortant strain with most of its genetic material derived from a porcine strain may cause symptomatic infection in a human host.

An epidemiological study was conducted in Eastern and Northern India to determine the genomic diversity of rotaviruses in these parts of the country. In 2001, a total of 126 Group A rotavirus positive samples were detected from children below 4 years of age with diarrhoea from Kolkata, Dibrugarh and Bhubaneswar in Eastern India, and Chandigarh, a city in Northern India. All the samples were genotyped for VP7 (G-type) and VP4 (P-type) gene by reverse transcription (RT) and multiplex PCR using different type specific primers. The strains with G1P[8] (32.5%) was predominant as reported earlier [Das et al. (2002) J Clin Microbiol 40:146-149] followed by G2P[4](4.7%) and only one sample was of G4P[8] specificity. Along with these common types some rare strains like G1P[6], G2P[8], G2P[6], G4P[4], and G4P[6] were also detected in 14.3% of cases. Thirty percent of samples in this study were mixed infections and 21 (16.7%) specimens remained untypeable either for the VP7 or for the VP4 gene. After sequencing of the VP7 gene, two G9 strains (RMC321 and ISO-3) were identified with P[8] and P[19] specificities. Sequence analysis revealed that they have much lower homology to the G9 strains (116E, INL1, and G16) isolated earlier from Indian subcontinent, but have much higher homology to isolates from Argentina, Brazil, Malawi, Taiwan, and USA suggesting a separate progenitor for these strains., (Copyright 2004 Wiley-Liss, Inc.)