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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]

Abstract: Background: Human group B rotavirus was first identified as causative agent of a large outbreak of severe gastroenteritis affecting more than 1 million people, predominantly adults in China in 1982–1983. In spite of serological evidences for the presence of group B rotavirus in many countries of the world, the virus has been detected only from China, India and Bangladesh, where most of the cases were from adults. Objectives: To ascertain the role of group B rotavirus as an aetiological agent of diarrhoea among children in Kolkata, India. Study design: An active surveillance was conducted for rotavirus infection in children in a leading referral paediatric hospital and a few samples were also collected from adults of another hospital in Kolkata, India over a period of 3 years (2002–2004). After primary screening of rotaviruses by RNA electrophoresis in polyacrylamide gel, 200 of 412 samples negative by PAGE were screened by reverse transcription polymerase chain reaction for group B rotaviruses. The group B rotavirus positives samples were also confirmed by dot-blot hybridization. Result: During the study period, we detected 37 (18.5%) sporadic cases of human group B rotavirus infection in children below 3 years of age of which 15 (7.5%) showed mixed infection with group A rotaviruses by RT-PCR. In dot-blot hybridization studies the RNA of all rotavirus positive samples hybridized with the nonisotopic psoralen-biotin labeled total RNA probe generated from a human group B rotavirus CAL-1 strain confirming the samples as group B rotaviruses. Conclusion: The shift in age preference of group B rotavirus infection from adult to children and mixed infection of group B and group A rotaviruses reveals the importance of group B rotavirus as an etiological agent of childhood diarrhoea. Therefore, future vaccination strategy should include both group A and B rotaviruses to control rotavirus diarrhoea. [Copyright &y& Elsevier]

Abstract: Background: Rotavirus genotypes, G1–G4 and G9 are associated with childhood diarrhoea throughout the world. In our previous study, we detected G1, G2, G4 and three G12 strains from Kolkata, India. Objectives: To study the prevalence of G- and P-genotypes of rotaviruses associated with dehydrating diarrhoea in children admitted to two leading hospitals in eastern India. Study design: An active surveillance was conducted for elucidation of rotavirus infection in two leading hospitals in Kolkata, West Bengal and Berhampur (GM), Orissa, India, separated by 603km from January 2003 to April 2005. The rotaviruses were detected by RNA electrophoresis in polyacrylamide gels. G- and P-typing of the positive samples were accomplished by amplifying VP7 and VP4 genes by RT-PCR and genotyped by seminested multiplex PCR methods. Sequencing, sequence analysis and phylogenetic analysis of VP7 genes of G12 strains were carried out to understand the variations between the strains isolated from different parts of the world. Results: The genotypic distribution varied remarkably from our earlier study period (1998–2001) with G1 (53.8%) being the most predominant strain followed by G2 (22.5%), G12 (17.1%), G9 (2.1%) and not a single G3 or G4 isolate was detected separately. 35.2% samples exhibited mixed P-types followed by P[4] (31.7%), P[8] (21.8%) and P[6] (9.8%). The phylogenetic analysis of G12 strains revealed that the G12 strains detected from different parts of the world clustered into three different lineages. Though VP7 sequences of G12 strains isolated from Kolkata and Berhampur are conserved, their P-types were different. Conclusion: During this study period we reported emergence of G12 strains as an important pathogen among children in eastern India, thus necessitating its inclusion in future polyvalent vaccine to control rotavirus diarrhoea. [Copyright &y& Elsevier]

Abstract: Background: : In recent years, hepatitis C virus (HCV) infection is gaining importance in Asian countries. Recent studies conducted in different parts of the world revealed that there is a genotypic correlation of disease severity and treatment outcome. Objectives: : A detailed study was carried out to delineate the genotypic distribution of HCV among acute and chronic liver disease patients in Kolkata, a city in eastern India. Study design: : Acute and chronic liver disease was diagnosed among patients attending hepatitis clinics in the city. Anti-HCV ELISA was performed on the blood samples of the cases and positive samples were tested for presence of HCV–RNA and genotyping of the samples were carried out by reverse transcription and polymerase chain reaction (RT-PCR) and sequencing. Results: : Seroprevalence of HCV infection among acute (11.0%) and chronic (25.3%) hepatitis patients were high and among them 97 (75.8%) and 323 (86.1%) were HCV–RNA positive for acute and chronic hepatitis patients, respectively. Genotyping by PCR showed that the predominant genotype was 3b (42.3%) followed by 3a (28.9%) among acute hepatitis group whereas among chronic hepatitis group, the most prevalent genotypes were 3a (34.7%) and 3b (47.7%). Sequence analysis of the untypeable isolates revealed the presence of a rare subtype 6b. Conclusions: : The study revealed very high prevalence of HCV among acute and chronic hepatitis patients with predominance of genotype 3. Subtype 6b was commonly found in Thailand but not in India. The detection of this rare strain of Thai origin reveals the spread of HCV infection from Thailand to other parts of Asia. This observation necessitates further intensive surveillance of HCV infection in India to unravel the distribution of genotypes in the country and to correlate disease severity and treatment outcome to the genotype prevalence. [Copyright &y& Elsevier]