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A study was undertaken to ascertain genotypic characterization of bovine group A rotavirus circulating in the Navsari region of Gujarat state. Total 157 diarrheic fecal samples were collected from bovine calves of organized farms and locally at village level from Navsari region. All the samples were initially screened by RNA-PAGE and RT-PCR followed by characterization by sequencing and G-P typing. The study revealed 10 (6.36%) samples positive by RNA-PAGE, while 16 (10.19%) samples were positive by VP4, VP6 and VP7 gene-based RT-PCR. From these positive samples, representative samples were sequenced for complete coverage of VP4, VP6, and VP7 genes followed by P and G typing on the basis of VP4 and VP7 sequence results, respectively. All the sequenced samples revealed G10 and P[11] types, showing a G10P[11] combination significantly prevailing in the bovine calves. Phylogenetic analysis of VP4, VP6, and VP7 genes was performed to determine the cross-species transmission dynamics and regional diversity, which showed similarity with other isolates circulating in India. Our findings suggested that a zoonotic potential genotype G10P[11] is the most circulating genotypes of Rotavirus A in bovine calves from Navsari region of Gujarat, India. [ABSTRACT FROM AUTHOR]

Malnourished children are at higher risk of mortality and morbidity following diarrheal illness and certain enteropathogens have been associated with malnutrition in children. Very few studies have comprehensively looked at the etiology of diarrhea in malnourished children and most have used conventional diagnostic methods with suboptimal sensitivity. We used a highly sensitive molecular approach against a broad range of pathogens causing diarrhea and examined their association with malnutrition. In addition, we looked at the pathogen diversity of pediatric diarrhea, three years after the nationwide rotavirus vaccine introduction to understand the evolving landscape of pathogens, which is crucial for planning strategies to further reduce the diarrhea burden. Clinical details and diarrheal stool samples were collected from hospitalized children aged < 5 years from three sentinel sites in India for a period of one year. The samples were tested by qPCR for 16 established causes of diarrhea using TaqMan Array Cards. A total of 772 children were enrolled, from whom 482 (62.4%) stool specimens were tested. No specific pathogen was associated with diarrhea among children with acute or chronic malnutrition compared to those with better nutritional status. Overall, adenovirus was the leading pathogen (attributable fraction (AF) 16.9%; 95% CI 14.1 to 19.2) followed by rotavirus (AF 12.6%; 95% CI 11.8 to 13.1) and Shigella (AF 10.9%; 95% CI 8.4 to 16.4). The majority of diarrhea requiring hospitalization in children aged < 2 years could be attributed to viruses, while Shigella was the most common pathogen among children aged > 2 years. These data on the prevalence and epidemiology of enteropathogens identified potential pathogens for public health interventions. [ABSTRACT FROM AUTHOR]

RNA viruses are a major group contributing to emerging infectious diseases and neonatal diarrhoea, causing morbidity and mortality in humans and animals. Hence, the present study investigated the metatranscriptomic-derived faecal RNA virome in rotavirus group A (RVA)-infected diarrheic piglets and calves from India. The viral genomes retrieved belonged to Astroviridae in both species, while Reoviridae and Picornaviridae were found only in piglets. The nearly complete genomes of porcine RVA (2), astrovirus (AstV) (6), enterovirus G (EVG) (2), porcine sapelovirus (PSV) (2), Aichivirus C (1), and porcine teschovirus (PTV) (1) were identified and characterised. In the piglet, AstVs of PAstV2 (MAstV-26) and PAstV4 (MAstV-31) lineages were predominant, followed by porcine RVA, EVG, PSV, Aichivirus C, teschovirus (PTV-17) in decreasing order of sequence reads. In contrast, AstV accounted for the majority of reads in bovines and belonged to MAstV-28 and a proposed MAstV-35. Both RVA G4P[6] strains exhibited prototype Gottfried strains like a genotypic constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. Ten out of eleven genes were of porcine origin, while the VP7 gene clustered with G4-lineage-1, consisting of human strains, suggesting a natural porcine-human reassortant. In the recombination analysis, multiple recombination events were detected in the PAstV4 and PAstV2 genomes, pointing out that these viruses were potential recombinants. Finally, the study finds diverse RNA virome in Indian piglets and calves for the first time, which may have contributed to diarrhoea. In the future, the investigation of RNA virome in animals will help in revealing pathogen diversity in multifactorial diseases, disease outbreaks, monitoring circulating viruses, viral discovery, and evaluation of their zoonotic potential. [ABSTRACT FROM AUTHOR]

The present study reports the detection and molecular characterisation of rotavirus C (RVC) in sloth bears (Melursus ursinus) rescued from urban areas in India. Based on an RVC VP6 gene-targeted diagnostic RT-PCR assay, 48.3% (42/87) of sloth bears tested positive for RVC infection. The VP6, VP7, and NSP4 genes of three sloth bear RVC isolates (UP-SB19, 21, and 37) were further analysed. The VP6 genes of RVC UP-SB21 and 37 isolates were only 37% identical. The sequence identity, TM-score from structure alignment, and selection pressure (dN/dS) of VP6 UP-SB37 with pig and human RVCs isolates were (99.67%, 0.97, and 1.718) and (99.01%, 0.93, and 0.0340), respectively. However, VP6 UP-SB21 has an identity, TM-score, and dN/dS of (84.38%, 1.0, and 0.0648) and (99.63%, 1.0, and 3.7696) with human and pig RVC isolates, respectively. The VP7 genes from UP-SB19 and 37 RVC isolates were 79.98% identical and shared identity, TM-score, and dN/dS of 88.4%, 0.76, and 5.3210, along with 77.98%, 0.77, and 4.7483 with pig and human RVC isolates, respectively. The NSP4 gene of UP-SB37 RVC isolates has an identity, TM-score, and dN/dS of 98.95%, 0.76, and 0.2907, along with 83.12%, 0.34, and 0.2133 with pig and human RVC isolates, respectively. Phylogenetic analysis of the nucleotide sequences of the sloth bear RVC isolates assigned the isolate UP-SB37 to genotype G12, I2 for RVC structural genes VP7 and VP6, and E1 for NSP4 genes, respectively, while isolates UP-SB19 and UP-SB21 were classified as genotype G13 and GI7 based on the structural gene VP7, respectively. The study suggests that the RVCs circulating in the Indian sloth bear population are highly divergent and might have originated from pigs or humans, and further investigation focusing on the whole genome sequencing of the sloth bear RVC isolate may shed light on the virus origin and evolution. [ABSTRACT FROM AUTHOR]

Asymptomatic infection with rotavirus C (RVC) was observed in pigs in India, with a detection rate of 20%. Sequencing of the VP6, VP7, and NSP4 genes of RVC strains identified the genotypes I7/I10, G1, and E5, respectively. Full genome sequencing of one of these strains revealed that the genotypes of the VP4, VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5 genes were P1, R1, C1, M3, A1, N5, T5, and H1, respectively. The detection of porcine RVC strains at two different locations in India at different time points strongly suggests that they are circulating continuously in the pig population through asymptomatic infections. [ABSTRACT FROM AUTHOR]

Rotaviruses A (RVA) are leading causes of diarrhea and dehydration in piglets and imply great economic loss to the pig farming community. In this study, the porcine RVA genotypes circulating in western and northern parts of India were determined by screening 214 fecal samples from diarrheic (n = 144) and non-diarrheic (n = 70) pigs. Subsequently, the structural (VP4 and VP7) and nonstructural (NSP3, and NSP4) genes were amplified, sequenced, and genetically characterized. The RVA positivity percentage was 7.94% (17/214) by RNA-PAGE and 10.28% (22/214) by RT-PCR. Higher RVA positivity was observed in samples from Uttar Pradesh (24.07%) followed by Maharashtra (6.77%) and Goa (2.38%). The sequence and automated genotyping software analysis confirmed the circulation of G4P[6] and G9P[13] RVA strains in porcine population. To note, the sequence similarity of the VP7 gene of Porcine/INDIA/RVA/PK-13 IVRI/Maharashtra/G4 and Porcine/INDIA/RVA/P-8/IVRI/U.P./G9 strain showed a relationship of 96.83 and 98.89% at the nucleotide level with human RVA strains indicating inter-species transmission. Additionally, the NSP3 (T1) and NSP4 (E1) genes (genotypes) also showed genetic relatedness with human RVA strains. Overall, the nucleotide sequences of VP7, NSP3, and NSP4 genes of porcine RVA indicate zooanthroponotic transmission. Further, we report the detection of G9P[13] RVA strain in porcine for the first time from India. RVA positivity was 7.94% (17/214) by RNA-PAGE and 10.28% (22/214) by RT-PCR The RVA strain G9P[13] reported for the first time in Indian piglets VP7, NSP3 and NSP4 genes analysis of porcine RVA showed genetic relatedness with human strains indicating evidence of zooanthroponotic transmission [ABSTRACT FROM AUTHOR]

Background: Rotavirus is the leading cause of severe dehydrating gastroenteritis among children younger than 5 years in low-income and middle-income countries. Two vaccines-Rotavac and Rotasiil-are used in routine immunisation in India. The safety and immunogenicity of these vaccines administered in a mixed regimen is not documented. We therefore aimed to compare the safety and seroresponse of recipients of a mixed regimen versus a single regimen.Methods: We did a multicentre, open-label, randomised, controlled, phase 4, non-inferiority trial at two sites in India. We recruited healthy infants aged 6-8 weeks. Infants with systemic disorders, weight-for-height Z scores of less than minus three SDs, or a history of persistent diarrhoea were excluded. Eligible infants were randomly allocated to six groups in equal numbers to receive either the single vaccine regimen (ie, Rotavac-Rotavac-Rotavac [group 1] or Rotasiil-Rotasiil-Rotasiil [group 2]) or the mixed vaccine regimen (ie, Rotavac-Rotasiil-Rotavac [group 3], Rotasiil-Rotavac-Rotasiil [group 4], Rotavac-Rotasiil-Rotasiil [group 5], or Rotasiil-Rotavac-Rotavac [group 6]). Randomisation was done using an online software by site in blocks of at least 12. The primary outcome was seroresponse to rotavirus vaccine, measured using rotavirus-specific serum IgA antibodies 4 weeks after the third dose. The seroresponse rates were compared between recipients of the four mixed vaccine regimens (consisting of various combinations of Rotavac and Rotasiil) with recipients of the single vaccine regimens (consisting of Rotavac or Rotasiil only for all three doses). The non-inferiority margin was set at 10%. Safety follow-ups were done for the duration of study participation. This trial was registered with the Clinical Trials Registry India, number CTRI/2018/08/015317.Findings: Between March 25, 2019, and Jan 15, 2020, a total of 1979 eligible infants were randomly assigned to receive a single vaccine regimen (n=659; 329 in group 1 and 330 in group 2) or a mixed vaccine regimen (n=1320; 329 each in groups 3 and 4, and 331 each in groups 5 and 6). All eligible participants received the first dose, 1925 (97·3%) of 1979 received the second dose, and 1894 (95·7%) received all three doses of vaccine. 1852 (93·6%) of 1979 participants completed the follow-up. The immunogenicity analysis consisted of 1839 infants (1238 [67·3%] in the mixed vaccine regimen and 601 [32·7%] in the single vaccine regimen; 13 samples were insufficient in quantity) who completed vaccination and provided post-vaccination sera. The seroresponse rate in the mixed vaccine regimen group (33·5% [95% CI 30·9-36·2]) was non-inferior compared with the single vaccine regimen group (29·6% [26·1-33·4]); the seroresponse rate difference was 3·9% (95% CI -0·7 to 8·3). The proportion of participants with any type of solicited adverse events was 90·9% (95% CI 88·4-93·0) in the single vaccine regimen group and 91·1% (89·5-92·6) in the mixed vaccine regimen group. No vaccine-related serious adverse events or intussusception were reported during the study.Interpretation: Rotavac and Rotasiil can be safely used in an interchangeable manner for routine immunisation since the seroresponse was non-inferior in the mixed vaccine regimen compared with the single vaccine regimen. These results allow for flexibility in administering the vaccines, helping to overcome vaccine shortages and supply chain issues, and targeting migrant populations easily.Funding: Ministry of Health and Family Welfare, Government of India.Translation: For the Hindi translation of the abstract see Supplementary Materials section. [ABSTRACT FROM AUTHOR]

Aims: This study was carried out from January 2018 to March 2020 in Kolkata, eastern India to determine the prevalence rates and epidemiological patterns associated with the major viral agents of gastroenteritis among children ≤5 years of age. Molecular characterization of GARV, the predominant agent of viral gastroenteritis, was done to understand their genotype diversity. Methods and Results: 1284 of 3157 stool samples (~40%) from children (≤5 years) with acute gastroenteritis tested positive for one or more enteric viruses with positivity rates 25.11%, 8.74%, 6.62% and 6.11% for GARV, HAdV‐F, AstV and NoV respectively. Co‐infection was observed in 5.31% of cases. Associated clinical/meteorological variables like age, sex, symptoms, temperature and precipitation were assessed to find any correlation between these and enteric virus infection rates. >70% of viral gastroenteritis cases were observed in 6–24 months' age group. GARV and AstV infection occurred mostly during cooler months while HAdV‐F infection mostly occurred during warmer periods. No definite seasonality was observed for NoV infections. Clinical severity associated with GARV infection was higher compared to other enteric viruses. Genotyping of rotavirus positive samples revealed G3P[8] was the predominantly circulating GARV genotype throughout the study period. Conclusions: GARV remained the predominant viral agent of acute gastroenteritis among children though its prevalence rates in this region declined significantly compared to the previous years (2010–2016). The prevalence of other enteric viruses was below 10%. Significance and Impact of study: This study provides valuable insights regarding the current burden of viral gastroenteritis in Eastern India. The 2‐year study in children will provide the baseline data for future surveillance studies in evaluating the impact of the introduced GARV vaccine on the overall prevalence of viral gastroenteritis. [ABSTRACT FROM AUTHOR]

Introduction Diarrheal illness such as diarrheagenic Escherichia coli (DEC), apart from rotavirus, is a common etiological agent known to cause moderate-to-severe diarrhea in low-income countries where unregulated use of antibiotics is rampant, giving rise to multidrug resistant (MDR) strains. This study is an earnest effort in reflecting the resistance pattern in such isolates. Materials and Methods It is a hospital-based cross-sectional study conducted over a period of 1 year (January to December, 2015). Children aged less than 18 years presenting with (n = 170) and without (n = 47) diarrhea were included as cases and controls, respectively. Fresh stool sample from eligible participants was collected and inoculated on MacConkey agar. Based on the colony morphology and biochemical identification followed by polymerase chain reaction (PCR), different pathotypes of DEC were identified. All such isolates were subjected to antimicrobial susceptibility testing employing VITEK 2 identification system. The result of the tested antibiotics was evaluated as per Clinical and Laboratory Standards Institute 2015 guidelines. Results DEC with specific virulence genes were detected by multiplex real-time PCR in 39 and 3 children with or without diarrhea, respectively. Most common DEC pathotypes found were enteroaggregative E. coli (38%) followed by enteropathogenic E. coli (28.5%). MDR isolates comprised 35 of 42 DEC pathotypes (83.3%). Resistance among DEC pathotypes to ampicillin, amoxicillin–clavulanate, ciprofloxacin, cephalosporin, nalidixic acid, imipenem, and cotrimoxazole was found to be statistically significant in comparison to non-DEC isolates. Conclusion This study has highlighted the increased prevalence of MDR strains among DEC pathotypes. Looking for these isolates will help detect dreadful DEC pathotypes like enterohemorrhagic E. coli where early administration of a sensitive antibiotic will go a long way in preventing complication like hemorrhagic colitis and hemolytic uremic syndrome. [ABSTRACT FROM AUTHOR]

Rotaviruses by virtue of its segmented genome generate numerous genotypes. G1P[8] is the most common genotype reported globally. We intend to identify the evolutionary differences among G1P[8] strains from the study with vaccine strains. Stool samples collected from children <5 years were screened for rotavirus antigen by enzyme linked immunosorbent assay. The samples that tested positive for rotavirus were subjected to VP7 and VP4 semi‐nested RT‐PCR. Sanger sequencing was performed in randomly chosen VP7 and VP4 rotavirus strains. Phylogenetic analysis showed less homology between study strains and vaccine strains and they were placed in different lineages. The VP7 and VP4 proteins of rotavirus were analyzed by two different platforms to identify the amino acid substitutions in the epitope regions. Nine amino acid substitutions with respect to Rotarix®, RotaTeq® and Rotasiil®—V66A, A/T68S, Q72R, N94S, D100E, T113I, S123N, M217T, and I281T were observed in VP7. There were five amino acid substitutions—S145G, N/D195G, N113D, N/I78T, E150D in VP4 (VP8 portion) with respect to Rotarix® and RotaTeq® vaccine strains. M217T substitution in VP7 (epitope 7‐2) and N113D, D195G substitution in VP4 (epitope 8‐3, 8‐1) confer changes in polarity/electrical charge with respect to vaccine strains, thus indicating the need for continued surveillance. [ABSTRACT FROM AUTHOR]

Objective  Diarrheal diseases are one of the most common causes of hospitalization in children under five. Rotavirus is the most common cause of acute diarrhea in younger children, and the prevalence decreases rapidly with increasing age. The objective of the study was to estimate the burden of rotavirus infection in acute gastroenteritis among under-five children admitted to a tertiary care hospital in eastern Odisha, for the clinical profile and identity of the prevalent strains. Methods  This was a prospective observational study linked to the National Rotavirus Surveillance Network (NRSN), where 720 under-five children with diarrhea were enrolled. In total, 675 stool samples of eligible candidates were sent for rotavirus isolation, and identification of strains was done by identifying VP 7 (G-type) and VP 4 (P-type) genes by reverse transcription polymerase chain reaction. Results  Categorical variables were presented as frequency and percentage, and continuous variables were expressed as mean  ±  standard deviation. Rotavirus was detected in 256 (37.92%) samples. Males outnumbered females. The most common affected age group was 7 to 12 months, followed by 13 to 18 months. G3P[8] was the most prevalent strain in this study. Conclusion  Children between the age of 7 and 18 months were most vulnerable to rotavirus infection. The most prevalent strain varies from one region to another and continuous surveillance is needed. [ABSTRACT FROM AUTHOR]

Rotavirus, a primary contributor to severe cases of infantile gastroenteritis on a global scale, results in significant morbidity and mortality in the under-five population, particularly in middle to low-income countries, including India. WHO-approved live-attenuated vaccines are linked to a heightened susceptibility to intussusception and exhibit low efficacy, primarily attributed to the high genetic diversity of rotavirus, varying over time and across different geographic regions. Herein, molecular data on Indian rotavirus A (RVA) has been reviewed through phylogenetic analysis, revealing G1P[8] to be the prevalent strain of RVA in India. The conserved capsid protein sequences of VP7, VP4 and VP6 were used to examine helper T lymphocyte, cytotoxic T lymphocyte and linear B-cell epitopes. Twenty epitopes were identified after evaluation of factors such as antigenicity, non-allergenicity, non-toxicity, and stability. These epitopes were then interconnected using suitable linkers and an N-terminal beta defensin adjuvant. The in silico designed vaccine exhibited structural stability and interactions with integrins (αvβ3 and αIIbβ3) and toll-like receptors (TLR2 and TLR4) indicated by docking and normal mode analyses. The immune simulation profile of the designed RVA multiepitope vaccine exhibited its potential to trigger humoral as well as cell-mediated immunity, indicating that it is a promising immunogen. These computational findings indicate potential efficacy of the designed vaccine against rotavirus infection. [Display omitted] • Rotavirus causes severe infantile gastroenteritis, with G1P[8] identified as the prevalent rotavirus A strain in India. • 20 epitopes were identified from 3 conserved capsid protein sequences of rotavirus A. • Epitopes meet criteria for antigenicity, non-allergenicity, non-toxicity, and stability. • Multiepitope vaccine shows good binding with integrins and TLRs. • Immunity triggered by the vaccine was validated by immune simulation study. [ABSTRACT FROM AUTHOR]

Acute gastroenteritis (AGE) is a serious global health problem and has been known to cause millions of infant deaths every year. Rotavirus (RV), a member of the Reoviridae family, still majorly accounts for the AGE in children below 5 years of age in India and worldwide. The involvement of miRNAs in the pathogenesis of RV has been suggested to be of the proviral as well as the anti-viral nature. miRNAs that promote the RV pathogenesis are capable of targeting the cellular components to evade the host anti-viral strategies. On the other hand, miRNAs with anti-rotaviral properties are themselves incapacitated during the progression of the infection. The exploitation of the epithelial–mesenchymal transition (EMT) as a pro-rotaviral strategy has already been identified. Thus, miRNAs that proficiently target the intermediates of the EMT pathway may serve as anti-viral counterparts in the RV–host interactions. The role of microRNA-29b (miR-29b) in the majority of human cancers has been well demonstrated, but its significance in viral infections is yet to be elaborated. In this study, we have assessed the role of miR-29b in RV-induced EMT and RV replication. Our study on miR-29b provides evidence for the recruitment of RV non-structural protein NSP1 to control the trans-repression of miR-29b in a p53-dependent manner. The trans-repression of miR-29b modulates the EMT pathway by targeting tripartite motif-containing protein 44 (TRIM44) and cyclin E1 (CCNE1). SLUG and SNAIL transcription repressors (downstream of TRIM44 and CCNE1) regulate the expression of E-cadherin, an important marker of the EMT. Also, it is established that ectopic expression of miR-29b not only constrains the EMT pathway but also restricts RV replication. Therefore, miR-29b repression is a crucial event in the RV pathogenesis. Ectopic expression of miR-29b displays potential anti-viral properties against RV propagation. [ABSTRACT FROM AUTHOR]

• Differences between G9 RV strains from North India and vaccine strain 116E. • North Indian G9 rotavirus strains clustered in different lineage than vaccine strain. • Analysis of antigenic epitopes showed mutations between North Indian and vaccine strains. • Substitutions in antigenic epitopes of G9RV were responsible for neutralization escape. • Substitutions were found in the most exposed residues on 3D VP7 trimer. The parental rotavirus strain 116E (G9P[11]) used to generate Rotavac® vaccine was isolated in 1986 in New Delhi. Thenceforward, there is no comprehensive report on diversity of G9 rotavirus strains from 116E; therefore, the present study evaluates the VP7 gene sequence diversity of G9 strains (retrieved from GenBank) from different geographical regions (1987–2016). Additionally, 22 recently collected G9 strains from Himachal Pradesh and Delhi (2013–2016) were included in the phylogenetic analysis. Interestingly, unlike 116E which belong to lineage-II all other G9 rotavirus including these 22 samples clustered together in a separate lineage (III). Further, six amino acid substitutions including one novel, K143M (epitope 7-2) different from 116E were detected mostly in the neutralization epitopes of VP7 protein (neutralization escape mutants). Overall, the accumulation of identified substitutions in VP7 epitopes and evolution of G9 strains in India may have impact on Rotavac® efficacy. [ABSTRACT FROM AUTHOR]

An association between rotavirus vaccination and intussusception has been documented in post-licensure studies in some countries. We evaluated the risk of intussusception associated with monovalent rotavirus vaccine (Rotavac) administered at 6, 10 and 14 weeks of age in India. Active prospective surveillance for intussusception was conducted at 22 hospitals across 16 states from April 2016 through September 2017. Data on demography, clinical features and vaccination were documented. Age-adjusted relative incidence for 1–7, 8–21, and 1–21 days after rotavirus vaccination in children aged 28–364 days at intussusception onset was estimated using the self-controlled case-series (SCCS) method. Only Brighton Collaboration level 1 cases were included. Out of 670 children aged 2–23 months with intussusception, 311 (46.4%) children were aged 28–364 days with confirmed vaccination status. Out of these, 52 intussusception cases with confirmed receipt of RVV were included in the SCCS analysis. No intussusception case was observed within 21 days of dose 1. Only one case occurred during 8–21 days after the dose 2. Post-dose 3, two cases in 1–7 days and 7 cases during 8–21 days period were observed. There was no increased risk of intussusception during 1–7 days after the doses 1 and 2 (zero cases observed) or dose 3 (relative incidence [RI], 1.71 [95% confidence interval {CI} 0.0–5.11]). Similarly, no increased risk during 8–21 days after the dose 1 (zero cases observed), dose 2 (RI, 0.71 [95% CI, 0.0–3.28]) or dose 3 (RI, 2.52 [95% CI, 0.78–5.61]). The results were similar for 1–21 day periods after the doses separately or pooled. The risk of intussusception during the first 21 days after any dose of rotavirus vaccine (Rotavac) was not higher among the Indian infants than the background risk, based on limited SCCS analysis of 52 children. [ABSTRACT FROM AUTHOR]

Rotavirus A (RVA) causes viral gastroenteritis in humans and animals, including calves, piglets, and foals. The current study reports the genetic characterization of the full-length enterotoxin gene, NSP4, from caprine and ovine species. Upon characterizing eight full-length NSP4 genes by sequencing, it was found that the four caprine and three ovine RVAs NSP4 genes are of E2 genotype and the sole ovine RVA isolate was found to be of E1 genotype. In the sequence and phyloanalysis of the NSP4 gene the seven E2 genotypes clustered with bovine, human, and caprine isolates from India and Bangladesh, respectively. The E1 genotype of ovine RVA was closer to human RVA isolate from India. The nucleotide per cent identity analysis revealed that all E2 genotype strains of caprine and ovine species ranged from 88.4% to 90.4% and it was found common to both the reference human RVA isolates DS-1 and AU-1. Whereas, the E1 genotype ovine strain clustered with human RVA isolates with 93.1% nucleotide per cent identity. The RVA strains circulating in caprine and ovine populations may share a common origin which is usually found in artiodactyl species because humans share a common dwelling with animals. Future studies are needed to confirm these findings of their relationship with humans and large animals. [ABSTRACT FROM AUTHOR]

Despite the significant reduction in the global infantile death toll due to rotaviral diarrhea, India still contributes substantially to rotavirus‐related hospitalization as well as mortality rates. The rotavirus surveillance study conducted from 2008 through 2017 among children (≤5 years) with moderate to severe gastroenteritis seeking healthcare facilities at two hospitals in eastern India, revealed a change in the proportion of rotavirus positivity, seasonality, and age‐group specificity along with the cycling of different usual and unusual genotypes in this endemic setting. G1 strains predominated during 2008‐2010, while G2 and G9 genotypes eventually upsurged during 2011‐2013. G1 strains re‐established their lead during 2013‐2015, while G3 emerged for the first time in eastern India in 2015 and rooted itself as the cardinal strain 2016 onwards. Evolutionary analyses of all the predominant genotypes (G1, G2, G3, and G9) revealed that they were mostly phylogenetically distant to the rotavirus vaccine strains as depicted in the phylogenetic dendrogram. These decade‐long epidemiological studies during the pre‐vaccination period in West Bengal (eastern India) underscore the cocirculation of multiple rotavirus genotypes in addition to sporadic occurrence of zoonotic strains like G10P[6] and G11P[25]. Research Highlights: Decade long dynamics of circulating rotavirus strains in eastern India.Co‐circulation of multiple genotypes along with reassortant and zoonotic strains.Upsurge of polymorphic G3‐genotype in 2016‐17 for the first time in this setting.One‐fifth proportion of the strains was "unusual combination of usual genotypes".All the strains were phylogenetically distant to the rotavirus vaccine strains. [ABSTRACT FROM AUTHOR]

Context: Rotavirus is the most common cause of severe diarrhea requiring hospitalization among infants and young children worldwide. The prevalence of Rotavirus diarrhea in India has been found to vary from 5% to 71% in hospitalized children less than 5 years of age with acute gastroenteritis. The seasonal variation of Rotavirus diarrhea in India varies in different geographical regions with high incidence in winter months, at low relative humidity in north India. Aim: To estimate the prevalence of Rotavirus diarrhea among the hospitalized children of age less than 5 years. To know the genotypic distribution of Rotavirus causing diarrhea. Settings and Design: Study design: Hospital-based cross-sectional study. Study setting: Pediatrics Department of Sri Venkateswara Ram Naraian Ruya Government General Hospital, Tirupati. Study period: 20th September 2012 to 19th September 2013. Methods and Materials: Study units: Children less than 5 years of age presenting with diarrhea. Stool specimens from all hospitalized children of age less than 5 years who had presented with acute watery diarrhea were collected and tested for Rotavirus by enzyme-linked immunosorbent assay (ELISA). Positive samples were tested for G and P typing by reverse transcription polymerase chain reaction technique. Statistical Analysis Used: Percentage and Chi-square analysis. Results: Among the study sample, 68.7% of children were in the age group between 1 and 12 months and 25.6% of children showed positive result for Rotavirus by ELISA. Of the Rotavirus positives, 50% were G1P8 viruses. Conclusion: Rotavirus is an important cause of diarrhea in hospitalized children. [ABSTRACT FROM AUTHOR]

• Vaccination against diarrhea, pneumonia and meningitis results in immediate savings. • 88% of savings are attributable to productivity loss due to premature death. • Scaling vaccines against Hib, PCV and RV save US$1 billion and 90,000 deaths/year. Background: To support vaccine decision-making we estimated from the societal perspective the potential health impact and costs averted through immunization with three vaccines – Haemophilus influenzae type b (Hib), pneumococcal conjugate vaccine (PCV) and rotavirus vaccine (RVV). Methods: Based on variability in disease burden, strength of health system and economic status, we selected four states in India: Bihar, New Delhi, Maharashtra and Tamil Nadu. We used secondary data sources to estimate the number of under-5 deaths averted from Hib, pneumococcus and rotavirus in each state and back-calculated the total cases averted. We synthesized available data to estimate the disease burden, treatment cost, caretaker productivity loss and vaccine coverage in each state. A Delphi Survey and roundtable among Indian experts was conducted to reach consensus on model inputs. Results: By scaling up coverage of Hib, PCV and RVV, India could save over US$1 billion (uncertainty range: US$0.9–US$2.4 billion) in economic benefits and avert more than 90,000 needless child deaths each year. An estimated US$1 billion (US$0.9–US$2 billion) or 88% of the total amount of cost savings would be attributable to lost productivity due to premature pneumococcal death. Another US$112.8 million (US$105–297 million), or 10% of the total cost would be accounted by costs related to loss of productivity due to disability as a result of these diseases. Treatment costs of Hib, pneumococcal disease and rotavirus gastroenteritis, would account for US$8.4 million (US$4–12 million) or <1% of the total costs of these diseases. Finally, caretaker productivity loss from seeking care would represent US$1.5 million (US$ 1–4.9 million). Cost savings varied by vaccine, coverage scenarios and states. Conclusions: Hib, PCV and RVV vaccine introduction in India can result in immediate benefits to the government and households in terms of savings. [ABSTRACT FROM AUTHOR]

• Systemic surveillance of rotavirus in children (≤5 years) hospitalized with diarrhea. • Rotavirus positively correlates with dehydration, mean hospital stay and vomiting. • Dynamicity of rotavirus genotypes among rural and urban settings during 2014–16. • Phylogenetic analyses and variation in antigenic epitopes of prevailing strains. Group-A human rotaviruses (GARV) are among the major cause of childhood diarrhea worldwide. In lieu of monitoring the circulatory GARV strains and underscoring the burden of GARV related hospitalization, a systematic surveillance was conducted in three hospitals of eastern India. In this hospital-based diarrheal disease surveillance (2014–2016), GARV was the most common cause of acute infantile gastroenteritis. The strains were genotyped and characterized to understand their prevalence and phylodynamics prior to the introduction of vaccine in eastern India. A total of 3652 stool samples were screened from children (≤5 years) hospitalized with acute diarrhea during 2014–2016. Initial screening for VP6 antigen was done by ELISA. GARV positive samples were genotyped by multiplex semi-nested PCR and DNA sequencing and phylogenetic analyses were based on the capsid proteins VP4 and VP7. Of 3652 samples, 1817 (49.8%) were GARV positive. G1, G2, G3 and G9 in conjunction with P[4], P[6]and P[8]genotypes were seen to co-circulate in the population. A sharp deflection from G1 to G3 occurred since 2016; upsurge of G9 strains was seen in alternate years, whereas G2 strains had a low frequency. All the circulating genotypes depicted a low phylogenetic relatedness to the vaccine strains. Differences in antigenic epitopes of VP4 and VP7 proteins in local strains were seen when compared to the vaccine strains. A significant difference in the degree of dehydration, duration of mean hospital stay and frequency of vomiting/24 h between GARV positive and negative children was evident. The study provides a relevant set of base-line data on high burden of rotaviral gastroenteritis and the varied genotypic diversity among children prior to the introduction of GARV vaccine in this endemic region. Continuous monitoring during post-vaccination era will be required to assess the impact of vaccination in this region. [ABSTRACT FROM AUTHOR]

Background: Rotavirus (RVA) causes severe gastroenteritis in under-five children, and there are many diverse strains of the virus that are localized to different parts of the world. Objectives: To study the burden and molecular epidemiology of RVA causing gastroenteritis among children from Eastern India. Materials and Methods: This hospital-based cross-sectional study included children under-five with gastroenteritis. Demographic and clinical parameters were recorded in a predesigned pro forma. Stool samples collected from these children were initially screened for RVA VP6 antigen by enzyme immunoassay (EIA). Each EIA-positive sample was then subjected to RNA extraction, followed by reverse transcription, and heminested multiplex polymerase chain reaction for genotyping of RVA strains. Results: Of 320 included children, RVA was detected in 30.62% (98/320) cases by EIA. The highest incidence for RVA-positive cases (34.61%) was observed among children in the age group of 24–36 months, followed by 0–12 months (33.04%). Of the 97 completely typed samples, single genotype was detected in 85 (87.62%) samples with either G (VP7) or P (VP4) types. However, mixed genotypes were detected in 12 (11.21%) samples. G3P[8] (44.09%) was the most common genotype, followed by G1P[8] (32.65%), G2[P4] (5.10%), G1[P6] (3.06%), and G9[P4] (1.02%). Conclusions: The present study found RVA positivity in 30.62% of children with gastroenteritis, with the highest burden among 24–36 months old. The predominant genotypes were G1, G3, and P[8]. Further large-scale/multicentric studies should be conducted to document the diversity of circulating RVA genotypes in this region for giving inputs for vaccination strategy. [ABSTRACT FROM AUTHOR]

• India became one of the first countries in Asia to introduce rotavirus vaccine. • Rotavirus vaccine is being expanded to the entire country in a phase wised manner. • The new vaccine introduction strengthened the programme rather than burdening it. In 2016, India became one of the first countries in Asia to introduce an indigenously manufactured rotavirus vaccine. However, any new vaccine introduction needs to be meticulously planned to allow for strengthening of the existing immunization systems instead of burdening them. The process of rotavirus vaccine introduction in India started with the establishment of National Rotavirus Surveillance Network in 2005 which generated relevant evidence to inform policy level decisions to introduce the vaccine. The preparatory activities started with assessment of health systems and closing any gaps. This was followed by development of vaccine specific training packages and cascade training for programme managers and health workers. The introduction was complemented with strong communications systems and media involvement to allow for good acceptability of the vaccine on the ground. Each step of introduction was led by the government and technically supported by development partners. India introduced rotavirus vaccine in a phased wise manner. In the first two phases the vaccine has been introduced in nine states of the country accounting for nearly 35% of the annual birth cohort of the country. From March 2016 to November 2017, approximately 13,260,000 rotavirus vaccine doses were administered in the country. The vaccine was well accepted by both the health workers and parents/caregivers. Rotavirus vaccine introduction in India is an excellent example of how government stewardship with well-defined roles for development partners can allow a new vaccine introduction to be used as a system strengthening activity. [ABSTRACT FROM AUTHOR]

ROTAVAC® (nHRV), derived naturally from the human 116E rotavirus (RV) neonatal strain, was licensed in India in 2015 based on promising results of a phase 3, safety and efficacy vaccine trial. As a pre-requisite for WHO prequalification, we compared the immunogenicity and safety of ROTAVAC® to those of a WHO-prequalified, Rotarix®. We conducted a multicentre, open-labeled, randomized phase 4 clinical trial where 464 infants, 6–8 weeks of age were equally randomized to receive as licensed, the complete regimen of ROTAVAC® (3 doses; Group I) or Rotarix® (2 doses; Group II). Antibody responses (serum anti-RV Immunoglobulin A [IgA]) were measured by enzyme-linked immunosorbent assay (ELISA). The primary analysis was an assessment of non-inferiority of ROTAVAC® to Rotarix® for geometric mean concentration (GMC) for infants who received the complete regimen of either vaccine. The GMC for Group I was 20.4 (95%CI: 17.6, 23.6) and that for Group II was 24.8 (95%CI: 20.3, 30.3), the GMC ratio was 0.82 (95% CI: 0.64, 1.05), thus meeting the non-inferiority criterion. Site-wise analysis of GMC titres revealed that one site had a peculiar pre-vaccination titre affecting only ROTAVAC® post-vaccination GMCs. Seroconversion rates were 35.3% (95%CI: 29.0, 41.9) and 31.0% (95%CI: 25.1, 37.4) for Groups I and Group II, respectively. There was no substantive difference in safety profiles between both vaccines. The complete regimen of ROTAVAC® demonstrated immunological non-inferiority to the complete regimen of Rotarix® with a clinically acceptable safety profile. Because the demand for RV vaccines is increasing as more countries are expanding their immunization schedules, the lack of need of a buffering agent, low dose volume (0.5 mL), non-interference with other concomitantly administered vaccines, and conformance with WHO-prequalification requirements provide ROTAVAC® the potential for widespread global usage. Post completion of this study, ROTAVAC® is now a WHO-prequalified vaccine. Clinical Trials Registration: (CTRI Number: CTRI/2015/12/006428). [ABSTRACT FROM AUTHOR]

Abstract Rotavirus infections in neonates are generally nosocomial, and differ from pediatric infections both clinically and epidemiologically. These infections are predominantly asymptomatic and often associated with unusual strains. Globally, so far limited data is available on rotavirus infections in neonates admitted at Neonatal Intensive Care Unit. The aim of the present study is to determine the prevalence of rotavirus among neonates and to study their genetic characteristics. Stool specimens (n = 701) collected from neonates (n = 621) admitted during April 2016 to March 2018 mainly for prematurity, low birth weight and associated respiratory distress syndrome from two hospitals from Pune were tested for rotavirus, genotyped and representative strains were sequenced for the genes encoding outer capsid proteins, VP7 and VP4. Rotavirus was detected in 24.31% neonates. Majority of rotavirus infected neonates (98.68%) were asymptomatic. Peak rotavirus antigen detection (91.38%) occurred during the first 2 weeks of admission. Low, very low and normal birth weight neonates with gestational age ≥28 weeks had significantly higher rotavirus infection than those with extreme low birth weight with gestational age <28 weeks. Rotaviral infections occurred almost evenly throughout the year without an apparent peak in colder months. Predominance of unusual G12P[11] strains (97.1%) was observed. Phylogenetic analysis of the partial VP7 coding gene revealed all G12 strains clustered in lineage III and shared 96.94%–100% (nucleotide) and 96.26%–100% (amino acid) identities among themselves, and 95.69%–98.98% (nucleotide) and 94.77%–98.98% (amino acid) with other lineage III G12 strains respectively. Similarly VP4 partial gene sequences of P[11] study strains shared 97.5%–100% (nucleotide and amino acid) identities among themselves and highest 93.34%–94.53% (nucleotide) and 93.57%–94.64% (amino acid) identity with vaccine strain 116E, G9P[11]. The study highlights high frequency of unusual G12P[11] strains among neonates for the first time in western India and reaffirms limited strain diversity in this population. The knowledge of neonatal strains is important for estimating the efficacies of rotavirus vaccines. Highlights • Rotavirus G12P [11], a human-bovine-like reassortant strain is prevalent in neonates. • Rotavirus infections in neonates are predominantly asymptomatic. • A limited strain diversity is observed in neonates. • First report of G12P [11] in neonates in western India • Monitoring early natural infections is important for estimation of vaccine efficacy. [ABSTRACT FROM AUTHOR]

Abstract Background A lyophilized bovine-human rotavirus reassortant pentavalent vaccine (BRV-PV, Rotasiil®) was licensed in 2016. A liquid formulation of this vaccine (LBRV-PV, Rotasiil - Liquid) was subsequently developed and was tested for non-inferiority to Rotasiil® and for lot-to-lot consistency. Methods This Phase II/III, open label, randomized study was conducted at seven sites across India from November 2017 to June 2018. Participants were randomized into four arms; Lots A, B, and C of LBRV-PV and Rotasiil® in 1:1:1:1 ratio. Three doses of study vaccines were given at 6, 10, and 14 weeks of age. Blood samples were collected four weeks after the third dose to assess rotavirus IgA antibody levels. Non-inferiority of LBRV-PV to Rotasiil was proven if the lower limit two-sided 95% confidence interval (CI) of geometric mean concentration (GMC) ratio was at least 0.5. Lot-to-lot consistency was proven if 95% CI of the GMC ratios of three lots were between 0.5 and 2. Solicited reactions were collected by using diary cards. Results Of the 1500 randomized infants, 1436 infants completed the study. The IgA GMC ratio of LBRV-PV to Rotasiil® was 1.19 (95% CI 0.96, 1.48). The corresponding IgA seropositivity rates were 60.41% (57.41, 63.35) and 52.75% (47.48, 57.97). The IgA GMC ratios among the three LBRV-PV lots were: Lot A versus Lot B: 1.34 (1.03, 1.75); Lot A versus Lot C: 1.22 (0.93, 1.60); and Lot B versus Lot C: 0.91 (0.69, 1.19). The 95% CIs for the GMC ratios were between 0.69 and 1.75. The incidence of solicited reactions was comparable across the four arms. Only one serious adverse event of gastroenteritis event in the Rotasiil® group was causally related. Conclusion The immunological non-inferiority of LBRV-PV against Rotasiil® as well as lot-to-lot consistency of LBRV-PV was demonstrated. LBRV-PV had safety profile similar to Rotasiil®. Trial registration number : Clinical Trials.Gov [NCT03474055] and Clinical Trial Registry of India [CTRI/2017/10/010104]. [ABSTRACT FROM AUTHOR]

The current global coronavirus disease 2019 pandemic has shown us once again how important vaccination is in controlling and preventing the spread of deadly diseases. Vaccinations are one of the most tried and tested public health measures aimed at the prevention and eventual eradication of various diseases. Many debilitating diseases like polio have been eradicated in countries like India due to effective vaccination strategies. Just like with any other public health initiative, there do exist various challenges for vaccination. Efficacy and correlate of protection studies are crucial in determining which vaccine works best. The rotavirus vaccine (ROTAVAC; Bharat Biotech International Ltd., Hyderabad, India) is one such example where efficacy seen in one geographical and ethnic population is not replicated elsewhere. This has prompted various researchers and pharmaceutical companies to think about customizing vaccines to the individual needs of a particular geographic and ethnic group. In this brief communication, we look at the rotavirus vaccination story and see how it laid down the idea for customized vaccination development and what the future of vaccine development looks like. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Infectious diarrhea is leading infectious cause of childhood morbidity, hospitalizations, and mortality particularly in children living in developing countries like India. The etiological agents differ depending on geographical area, and recent data suggest increase in drug resistance to various enteropathogens. AIMS AND OBJECTIVES: The aim of the study was to investigate emerging diarrheal agents and antimicrobial resistance profile of bacterial pathogens from children (<12 years of age) hospitalized with acute diarrhea. MATERIALS AND METHODS: A cross-sectional, hospital-based observational study was conducted over 1 year in which 100 children <12 years who were hospitalized due to diarrhea were recruited. Diarrhea was defined as the passage of three or more liquid stools in a 24-h period using the World Health Organization guidelines. Samples were processed for detection of various bacterial, viral, and parasitic agents by standard microbiological, serological, and molecular tests. Antimicrobial resistance testing was performed with the Kirby-Bauer disk diffusion method. ELISA was performed for Rotavirus and Escherichia coli O157. Multiplex polymerase chain reaction test was performed to detect diarrheagenic E. coli (DEC). RESULTS: Pathogenic diarrheal agents were found in 63% patients. Rotavirus was identified in 52.5%, DEC in 29%, Vibrio cholerae in 4%, Shigella flexneri in 3%, Aeromonas sp. in 1%, Giardia lamblia in 4%, and Entamoeba histolytica in 1% cases. Enteropathogenic E. coli (EPEC) in 19 (65.5%) cases was the most common agent followed by Enteroaggregative E. coli (EAEC) in 5 (17.2%), Enterotoxigenic E. coli (ETEC) in 2 (6%), and Enteroinvasive E. coli (EIEC) in 3 (10.3%) cases. Resistance rates of DEC to first-line therapeutic drugs were high, 97.3% to ampicillin and 95.95% to co-trimoxazole. DEC was susceptible to chloramphenicol in 58.11%, gentamicin in 48.19%, and amikacin in 58.11% cases. Shigella sp. and V. cholerae isolates were 100% sensitive to gentamicin and ofloxacin. CONCLUSION: EPEC is the most common DEC pathotype and EAEC, ETEC, and EIEC are also emerging as dominant diarrheal agents. Rotavirus was the most common causative agents of diarrhea especially in children <5 years. Most of the bacterial isolates showed high level of drug resistance to first-line empirical drugs and were multidrug resistant making them unsuitable for empiric treatment. Laboratory monitoring of drug susceptibility of stool isolates appears necessary to formulate antibiotic policy for treating diarrheal illness at the local level. There is an urgent need to strengthen diarrheal surveillance to monitor susceptibility to commonly prescribed antibiotics. [ABSTRACT FROM AUTHOR]

Abstract Acute gastroenteritis due to Rotavirus (RV) infection is a major cause of morbidity and mortality in infants and young children worldwide. In India, around 0.1 million death reported annually due to RV illness. So, to assess the disease burden continuous surveillance of the circulating genotypes is needed. This study aimed to ascertain the genetic variance of 429 rotavirus positive specimens observed during the period October 2013–September 2014 at four study centers from North India. Out of 1057 patients enrolled, 1018 stool samples were collected at four centers in four different states of North India. Children aged <5 years who showed the symptoms of severe diarrhea and needed hospitalization were enrolled. The stool samples collected were screened by Enzyme Immuno Assay (EIA), and the RV positive samples were characterized by semi-nested PCR. During the study period October 2013 through September 2014, ~42% patients were found to be rotavirus positive of 1018 collected specimen. In Delhi, Rohtak and Meerut, we observed that Rotavirus is seasonal compared to Tanda (HP). The rate of rotavirus detection was significantly higher among children aged below 2 years, and a total of 21.5% of rotavirus infections comprised children aged <6 months. Genotype G1(48.0%) was predominant and frequently circulating strain whereas G12 (16.8%) and G9 (10.0%) was second and third prevalent strain in the four states of North India. High frequency of G1 genotypes was detected under the age group of 6–11 months which is followed by G12, similarly high rate severe disease was observed due to G1 genotypes followed by P[8], P[6] and G12. The most common types of strains were G1P[8] (27.73% of strains), G12P[6] (13.28%), G9P[4] (7.23%) and G1P[6] (6.75%). The rare strain reported were G1P[9]; P[11] strain was detected in combination with G1, G2, and G12. These data emphasized G12 is the second most predominant strain circulating among Northern Indian children highlights the needs for inclusion in the future polyvalent vaccine to break the burden of rotavirus infection. Highlights • Rotavirus was detected 42.1% (n = 429) in diarrheal children aged ≤ 5 years during the study period in North India. • The incidence of rotavirus infection in children was peaked at the age of ≤12 months (44.98%). • We have detected rare rotavirus strains G1P[9] (4/429, 0.9%), this is the first report in North India. • In this study we observed that G12 was the second most prevalent strain circulating in North Indian children. [ABSTRACT FROM AUTHOR]

All over the world, children and adults are severely affected by acute gastroenteritis, caused by one of the emerging enteric pathogens, rotavirus C (RVC). At present, no extensive surveillance program is running for RVC in India, and its prevalence is largely unknown except cases of local outbreaks. Here, we intended to detect the presence of RVC in diarrheic children visiting or admitted to hospitals in Haldwani (state of Uttarakhand, India), a city located in the foothills of the Himalayas. During 2010-2013, we screened 119 samples for RVC by an RVC VP6 gene-specific RT-PCR. Of these, 38 (31.93%) were found positive, which is higher than the incidence rates reported so far from India. The phylogenetic analysis of the derived nucleotide sequences from one of the human RVC (HuRVC) isolates, designated as HuRVC/H28/2013/India, showed that the study isolate belongs to genotype I2, P2 and E2 for RVC structural genes 6 and 4 (VP6, and VP4) and non-structural gene 4 (NSP4), respectively. Furthermore, the VP6 gene of HuRVC/H28/2013/India shows the highest similarity to a recently-reported human-like porcine RVC (PoRVC/ASM140/2013/India, KT932963) from India suggesting zoonotic transmission. We also report a full-length NSP4 gene sequence of human RVC from India. Under the One-health platforms there is a need to launch combined human and animal RVC surveillance programs for a better understanding of the epidemiology of RVC infections and for implementing control strategies. [ABSTRACT FROM AUTHOR]

Background: Observational studies in socioeconomically distinct populations have yielded conflicting conclusions about the strength of naturally acquired immunity against rotavirus gastroenteritis (RVGE), mirroring vaccine underperformance in low-income countries. We revisited birth cohort studies to understand naturally acquired protection against rotavirus infection and RVGE.Methods: We reanalyzed data from 200 Mexican and 373 Indian children followed from birth to 2 and 3 years of age, respectively. We reassessed protection against RVGE, decomposing the incidence rate into the rate of rotavirus infection and the risk of RVGE given infection, and tested for serum antibody correlates of protection using regression models.Results: Risk for primary, secondary, and subsequent infections to cause RVGE decreased per log-month of age by 28% (95% confidence interval [CI], 12%-41%), 69% (95% CI, 30%-86%), and 64% (95% CI, -186% to 95%), respectively, in Mexico City, and by 10% (95% CI, -1% to 19%), 51% (95% CI, 41%-59%) and 67% (95% CI, 57%-75%), respectively, in Vellore. Elevated serum immunoglobulin A and immunoglobulin G titers were associated with partial protection against rotavirus infection. Associations between older age and reduced risk for RVGE or moderate-to-severe RVGE given infection persisted after controlling for antibody levels.Conclusions: Dissimilar estimates of protection against RVGE may be due in part to age-related, antibody-independent risk for rotavirus infections to cause RVGE. [ABSTRACT FROM AUTHOR]

Background: Group C Rotavirus (RVC) is an enteric pathogen responsible for acute gastroenteritis in children and adults globally. At present there are no surveillance studies on group C Rotaviruses in India and therefore their prevalence in India remains unknown. The present study aimed to evaluate group C rotavirus infection among <5 years old children hospitalized with acute gastroenteritis in New Delhi. Methods: A total of 350 fecal specimens were collected during September 2013 to November 2014 from <5 years old diarrheal patients admitted at KSCH hospital, Delhi. The samples found negative for group A rotavirus (N = 180) by Enzyme immunoassay were screened for group C rotavirus by RT-PCR with VP6, VP7 and VP4 gene specific primers. The PCR products were further sequenced (VP6, VP7, VP4) and analyzed to ascertain their origin and G and P genotypes. Results: Six out of 180 (group A rotavirus negative) samples were found positive for group C rotavirus by VP6 gene specific RT-PCR, of which 3 were also found positive for VP7 and VP4 genes. Phylogenetic analysis of VP7 and VP4 genes of these showed them to be G4 and P[2] genotypes. Overall, the nucleotide sequence data (VP6, VP7 and VP4) revealed a close relationship with the human group C rotavirus with no evidence of animal ancestry. Interestingly, the nucleotide sequence analysis of various genes also indicated differences in their origin. While the identity matrix of VP4 gene (n = 3) showed high amino acid sequence identity (97.60 to 98.20%) with Korean strain, the VP6 gene (n = 6) showed maximum identity with Nigerian strain (96.40 to 97.60%) and VP7 gene (n = 3) with Bangladeshi and USA strains. This is true for all analyzed samples. Conclusion: Our study demonstrated the group C rotavirus as the cause of severe diarrhea in young children in Delhi and provides insights on the origin of group C rotavirus genes among the local strains indicating their source of transmission. Our study also highlights the need for a simple and reliable diagnostic test that can be utilized to determine the disease burden due to group C rotavirus in India. [ABSTRACT FROM AUTHOR]

Human adenoviruses (HAdVs) are the viral agents responsible for a wide spectrum of acute and chronic diseases. HAdVs are the most important etiological agents of acute gastroenteritis (AGE) and are identified as the major contributor to the deaths of diarrheal children globally. The significant rise in HAdV infections in rotavirus-vaccinated children documented in multiple studies demands continuous monitoring of HAdV strains. After the inclusion of rotavirus vaccines in the immunization schedule of India, public health research regarding prevalence, etiology, and risk factors is highly necessary for evidence-based policies and their implementation to sustain diarrhea prevention programs. In the present study, children admitted for AGE between 2013 and 2016 in seven different hospitals in Maharashtra and Gujrat states of Western India were subjected for investigation. HAdVs were found in 5.2% of the fecal specimens with the dominance of species-F (52.4%) strains, followed by the occurrence of non-enteric adenoviruses of species A (17.4%), C (11.4%), B (8.2%), and D (3.2%). The species-F strains were predominant in Ahmadabad (78.5%), Mumbai (61.5%), and Surat (57.1%) cities, followed by species-A strains. In Pune city, species B strains were detected in all HAdV patients, with none of the species A strains. Clinically, patients infected with enteric and non-enteric HAdV strains were indistinguishable. However, a high viral load was observed in species-F specimens as compared to non-species-F. The present study on fecal specimens collected in the pre-rotavirus vaccination era from hospitalized AGE patients will be important for future comparative analysis to know the exact impact of vaccination in children of Western India. • In AGE patients. • Dominance of adenovirus Species F followed by A, C, B and D. • Adenovirus Species F and non-species F strains were clinically indistinguishable. • High viral load in Species F specimens as compared to non- species-F. [ABSTRACT FROM AUTHOR]

Rotaviruses C (RVCs) circulate worldwide as an enteric pathogen in both humans and animals. Most studies of their genetic diversity focus on the VP7 and VP4 genes, but the complete genomes of 18 human RVCs have been described in independent studies. The genetic background of the Far East Asian RVCs is different than other human RVCs that were found in India and Bangladesh. Recently, a RVC detected in 2010 in South Korea had genetic background similar to the Indian–Bangladeshi RVCs. This study was undertaken to determine the whole genome of eight Japanese RVCs detected in 2005–2012, and to compare them with other human and animal global RVCs to better understand the genetic background of contemporary Far East Asian RVC. By phylogenetic analysis, the human RVCs appeared to be distinct from animal RVCs. Among human RVCs, three lineage constellations had prolonged circulation. The genetic background of the Far East Asian RVC was distinguished from Indian–Bangladeshi RVC as reported earlier. However, we found one Japanese RVC in 2012 that carried the genetic background of Indian–Bangladeshi RVC, whereas the remaining seven Japanese RVCs carried the typical genetic background of Far East Asian RVC. This is the first report of the Indian–Bangladeshi RVC in Japan. With that observation and the reassortment event of human RVCs in Hungary, our study indicates that the RVCs are spreading from one region to another. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Shilajit, a herbomineral substance exuded from rocks in steep mountainous regions, has been used for thousands of years by the Indian Ayurvedic and Siddha systems of traditional medicine to relieve ailments and enhance quality of life. Although a large number of therapeutic properties have been ascribed to Shilajit, its therapeutic potential is still largely unexplored by modern research and many of its claimed bioactivities lack scientific validation. The present study was undertaken to investigate the antiviral activity of Shilajit against a panel of viruses including herpes simplex type 1 and 2 (HSV-1, HSV-2), human cytomegalovirus (HCMV), human respiratory syncytial virus (RSV), human rotavirus (HRV), and vesicular stomatitis virus (VSV). Materials and methods The antiviral activity of Shilajit was assayed in vitro by plaque reduction and virus yield assays and the major mechanism of action was investigated by virucidal and time-of-addition assays. Results Shilajit exhibited a dose-dependent inhibitory activity against HSV1, HSV2, HCMV, and RSV infectivity in vitro (EC 50 values: 31.08 μg/ml, 12.85 μg/ml, 34.54 μg/ml, and 30.35 μg/ml, respectively), but was inactive against HRV and VSV. Humic acid, a constituent of Shilajit, displayed the same spectrum of activity. Partial virus inactivation and interference with virus attachment were both found to contribute to the antiviral activity of Shilajit. Conclusions The results of the present study demonstrate that Shilajit is endowed with broad, yet specific, antiviral activity in vitro and constitutes a natural source of antiviral substances. Further work remains to be done to assess its efficacy in vivo . [ABSTRACT FROM AUTHOR]

Burden of rotavirus gastroenteritis (RVGE) in outpatient setting in India is not fully understood. A prospective study was undertaken to describe RVGE among Indian children less than 5 years of age presenting in outpatient departments with acute gastroenteritis (AGE). This study was conducted at 11 outpatient departments (OPDs) of private pediatric clinics in urban areas of India. A total of 605 eligible children were enrolled at OPDs. Stool samples of the subjects were collected and tested for presence of rotavirus antigen by enzyme immune assay (EIA) and were typed by reverse-transcriptase polymerase chain reaction (RT-PCR). Physician examined the children and documented the disease particulars. In addition, parents/guardians were interviewed for AGE related symptoms, health care utilization and cost incurred due to AGE, and parental stress associated with AGE. After OPD, parents/guardians completed diary cards and questionnaires to capture the information for 14 days following the enrollment. Complete data for analysis including stool sample results was available from 552 subjects. 23% (127/552; [CI 19.5, 26.5]) of stool samples were rotavirus (RV) positive. RT-PCR was done for 85.8% (109/127) of RV positive samples. G1, G2, G9, and G12 types were identified in 34.9% (38/109), 37.6% (41/109), 8.3% (9/109), and 6.4% (7/109) stool samples, respectively. P[4] and P[8] were identified in 36.7% (40/109) stool samples each, followed by P[6] identified in 15.6% (17/109) stool samples. At the time of enrollment, all three symptoms (vomiting, diarrhea, and fever) were observed concurrently in higher proportion of RV positive subjects compared to RV negative subjects (60.6% [77/127] vs. 42.8% [182/425], p = 0.0004). Healthcare resource utilization, costs incurred due to disease, and parental stress were higher for RV positive subjects compared to RV negative subjects. In conclusion, RVGE was found to be a definite burden in AGE cases attending pediatric outpatient clinics in urban areas and it was associated with substantial economic and psychological burden. Introduction of rotavirus vaccine in India may help in reducing this disease burden. [ABSTRACT FROM AUTHOR]

Background A vast diversity in rotaviruses at inter- and intra-genotypic level underscores the need for monitoring of circulating rotavirus strains. The aim of this study was to update the data on rotavirus disease and strains for the period from January 2009 to December 2012 in Pune, western India which has been one of the sites of the Indian Rotavirus Strain Surveillance Network since November 2005. Methods Children aged <5 years admitted for acute gastroenteritis in three different hospitals from Pune city were included in the study. The stool specimens were collected and tested for rotavirus antigen by a commercial enzyme immunoassay. The rotavirus strains were genotyped by multiplex reverse transcription polymerase chain reaction. Results During the study period, we found 35.1% of 685 stool specimens contained rotavirus antigen. Frequency of rotavirus detection was greatest (58.5%) among children aged 7-12 months. The G1P[8] (31.4%), G2P[4] (20.2%) and G9P[8] (11.8%) strains were the most common types. We noted predominance of G1P[8] strains (39.6%-46.1%) in all the years of study except 2009 wherein G9P[8] strains scored highest level (15.3%). Subsequent to this, we identified G9P[8] strains at the second highest position in 2010, their sudden decline and rise in G9P[4] strains in 2011-2012. We detected G12 strains in combination with P[6] and P[8] at variable rates (0-10.2%) and highest level (27.1%) of mixed rotavirus infections in 2009 as compared to 2010-2012 (0-3.8%). Conclusion The study highlights the huge burden of rotavirus disease and changing profile of circulating rotavirus strains displaying emergence of G9P[4] reassortant strains in Pune, western India and emphasizes the need to analyze the entire genomic constellation of rotavirus strains for better evaluation of the impact of rotavirus. [ABSTRACT FROM AUTHOR]

Highlights: [•] BRV-TV manufactured in India, was non-reactogenic, safe and immunogenic. [•] Choice of antigen is important for immunogenicity assays. [•] Human and bovine rotavirus antigens resulted in different IgA estimates in the same samples. [Copyright &y& Elsevier]

Highlights: [•] A 5-year comparative study on acute diarrhea due to NPEV and rotavirus was conducted. [•] NPEVs and rotavirus were detected in 17.2% and 13.8% diarrheal stools, respectively. [•] During the study, NPEVs were detected in 17% diarrheic and 6.16% healthy children. [•] 37 NPEV serotypes with E11 being more prevalent were detected in diarrheal children. [•] NPEV-associated diarrhea is as significant as that of rotavirus but with contrasting seasonality. [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: In a community based case–control study in Kolkata, India, in 2009, two human rotaviruses with uncommon genotypes G6P[14] and G11P[25] were identified, having bovine and porcine characteristics respectively. Strain N-1/2009 with G6P[14] and strain N-38/2009 with G11P[25] genotypes, were isolated from a 13months aged boy who was asymptomatic and a 10months old girl with severe diarrhea respectively. The remaining 9 gene segments of these two strains were analyzed to find the exact origin of these unusual rotaviruses, and the origin of these two strains from bovine/porcine rotaviruses was apparent. This study identifies zoonotic transmission and single and multiple reassortment events as mechanisms driving the diversity of human rotaviruses. This study indicates interspecies transmission between human and animal rotaviruses causes single or multiple reassortment and thus contribute to the genetic diversity of rotavirus. [Copyright &y& Elsevier]

Rotavirus is the most common cause of severe diarrhoea worldwide, affecting over 125 million young children every year in developing countries. The present study is a part of ongoing childhood diarrhoeal surveillance to determine the strain diversity of rotaviruses prevalent in Andaman and Nicobar Islands. From October 2010 to February 2012, 296 stool samples from children (age group 6-60 months) with gastroenteritis were obtained from different referral hospi-tals/primary health centres and community health centres in Andaman and Nicobar Islands. A total of 47 samples were found positive for GARV. Among these, 21 (44.7%) samples belong to G2P[4], 12 (25.5%) samples were G1P[8], 10 (21.2%) samples belong to G9P[4], 2 (4.3%) samples belong to Gl P[4] and 2 (4.3%) samples had a mixed genotype. Rotavirus G2 genotype remains the most common genotype in these is-lands. The prevalence of G9 rotavirus reported in the present study is higher than that reported from mainland India. The results emphasize the role of the unusual serotype G9 as an epidemiological important genotype and the need to in-clude G9 specificity in a rotavirus vaccine. [ABSTRACT FROM AUTHOR]

Abstract: During an ongoing surveillance for diarrheal pathogens, an unusual human group A rotavirus strain G3P[10] (RVA/Human-wt/IND/mcs60/2011/G3P[10]) was detected in a stool sample of a 14months old girl child with acute diarrhea in Kolkata, eastern India. The VP7 nucleotide sequence of this strain revealed a close phylogenetic relationship to the prototype G3 strain AU-1 and Australian feline strain Cat2, whereas, the VP4 gene segment was closely related to the G8P[10] rotavirus 69M from Indonesia. Analysis of 11 gene segments of this unusual G3P[10] strain demonstrates a complex evolutionary pattern, with genes possibly derived from the group A rotaviruses of human DS-1-like and AU-1-like strains of simian and caprine host species. To our knowledge, this is the first complete genotyping report of any G3P[10] rotavirus, worldwide. [Copyright &y& Elsevier]

The present study describes the genotypic distribution of rotaviruses (RVs) in an Indian bovine population with unexpectedly higher proportions of G3 alone or in combination of G8[G10. PCR-genotyping confirmed that 39.4 % (13/33) of the prevalent RVs were the G3 type while 60.6% (20/33) were dual G3G10 or G3G8 types. P typing revealed that 93.9 % (31/33) of the samples were P[11] while 6.1% (2/33) possessed a dual P[1]P[11] type. Sequence analysis of the VP7 gene from G3 strains viz. B-46, 0970, and BR-133 showed that these strains had sequence identities of 90.5% to 100% with other bovine G3 strains. The highest identity (98.9% to 100%) was observed with RUBV3 bovine G3 strains from eastern India. The G3 strains (B-46, 0970, and BR-133) showed 97.5% to 98.8% sequence homologies with the Indian equine RV strain Erv-80. Phylogenetic analysis demonstrated that G3 strains clustered with bovine RUBV3 and J-63, and equine Erv-80 G3. Overall, these results confirmed that the incidence of infection by RVs with the G3 genotype and mixed genotypes in the bovine population was higher than previously predicted. This finding reinforces the importance of constantly monitoring circulating viral strains with the G3 genotype in future surveillance studies. [ABSTRACT FROM AUTHOR]

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: Introduction: According to the World Health Organization in 2008, pneumonia accounted for 20% of deaths and diarrheal diseases accounted for 13% of deaths among children under 5 in India. Vaccines are available for Streptococcus pneumoniae (pneumococcal conjugate vaccine (PCV)), Haemophilus influenzae type b (Hib vaccine), and rotavirus. Barriers to including these vaccines in routine immunization schedule in India include potential negative impacts on fragile existing immunization programs and cost. Pediatricians who are members of the Indian Academy of Pediatrics (IAP) are important stakeholders for vaccine delivery and maintaining public confidence in vaccines. Methods: A random sample of 785 pediatricians belonging to IAP was selected for the survey conducted from June 2009 to June 2010. Descriptive analyses using sampling weights were performed to evaluate the distributions of variables assessing vaccine-related attitudes and behaviors among pediatricians. Logistic regression was used to assess factors associated with routine vaccine use. Results: The majority of pediatricians reported administering PCV (85.6%), Hib (95.9%), and rotavirus (80.2%) vaccine selectively or routinely. Pediatricians who had high perceived disease susceptibility were 2.42 times more likely to report routine administration of Hib vaccine (OR 2.42, 95% CI 1.24, 4.74). Pediatricians who had high perceived Hib vaccine efficacy were 4.74 times more likely to administer Hib vaccine routinely (OR 4.74, 95% CI 2.09, 10.74). Perceptions of disease susceptibility and severity or of vaccine safety and efficacy were not associated with routine administration of PCV or rotavirus vaccine. Conclusions: Understanding predictors of routine use of a new vaccine could help focus interventions to improve the routine use of other vaccines. The importance of perceived susceptibility to and severity of diseases caused by S. pneumoniae, Hib, and rotavirus and perceived efficacy and safety of the vaccines by pediatricians presents an opportunity to design strategies to build support for new vaccine introduction and may have important implications for national immunization policy in India. [Copyright &y& Elsevier]

Abstract: G10 rotaviruses, which are usually found in cattle, have also been reported in neonatal infections in recent years. During the rotavirus surveillances of children less than 4years of age between 2003 and 2006 in Kolkata, eastern India, 60 out of 1153 samples could not be typed. All 60 samples gave usual electropherotype pattern in polyacrylamide gel. Thirty-one out of these 60 G and P untypable rotavirus strains were successfully characterized during the study. Among 31 samples, G9P[4] (n =8), G12P[8] (n =8), G1P[8] (n =6), G10P[4] (n =6), and G2P[4] (n =3) genotypes were identified. In this study we report genetic analysis of the six G10 strains, which revealed close relations with Turkish (E29TR) bovine strains, as well as with bovine-like-equine strain (Erv2) from India. SimPlot of the VP7 gene segment suggested possible recombination event between the bovine and the bovine-like-equine rotaviruses in these human rotavirus infections. [Copyright &y& Elsevier]

Abstract: A total of 78 fecal specimens were collected from both apparently healthy (n =71) and diarrheic (n =7) cattle from an organized farm in Pune, western India in December 2007–January 2008. Three specimens tested positive for group A rotavirus (RV) by antigen capture ELISA were subjected to RT-PCR for amplification of entire coding regions of three structural (VP4, VP6 and VP7) and one nonstructural (NSP4) genes. All three strains were genotyped as G8P[14]. Phylogenetic analysis of the VP7 and VP4 genes showed clustering of the VP7 gene with G8 strains of bovine origin and VP4 gene with P[14] strains of human origin. The identification of VP6 and NSP4 genes to have I2 (subgroup I) and E2 (genotype A) specificity, respectively of bovine and human origin indicated independent segregation of genes in bovine RV strains. This study indicates circulation of a rare RV genotype, G8P[14] in western India. To our knowledge, this is the second report on RV G8[14] isolated from bovine species after bovine group A RV strain, SUN9 from Japan. [Copyright &y& Elsevier]

Abstract: Rotavirus G1P[8] strains are the most predominant cause of rotavirus diarrhea, worldwide and are an important component of currently licensed RotaTeq and Rotarix vaccines. Despite a significant contribution of these strains in causing diarrhea in Indian children, none of them has been characterized completely, to date. This issue was addressed in the present study by sequencing and phylogenetic analysis of complete genomes of 3 Indian rotavirus strains (06361, 0613158 and 061060) of G1P[8] specificity. Genotype of G1P[8] I1R1C1M1A1N1T1E1H1 respectively, for the VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4 and NSP5 genes was assigned to all of the three strains. The sequence analysis of structural and nonstructural genes indicated genetic relatedness (94–99.5%) with recently circulating strains and divergence (2.4–15.6%) with old prototype strains. Phylogenetic analysis revealed that new strains (Western Indian rotavirus strains and recently isolated strains – Dhaka16-03 (G1P[8]), Dhaka25-02 (G12P[8]), Matlab13-03 (G12P[6]), B3458 (G9P[8]), Matlab36-03 (G11P[8]), and B4633-03 (G12P[8]) and old prototype strains (KU and Wa) clustered in the same lineages of VP1, VP2, VP3, NSP2 and NSP4 genes however, grouped separately in VP6, NSP1 and NSP5 genes with 10–11%, 15.6–16.7% and 6.3–7.5% nucleotide sequence divergence, respectively. These results suggest that the rotavirus VP6, NSP1 and NSP5 genes of Wa-like rotaviruses are more prone to temporal mutations. Both structural and nonstructural genes of the Western Indian rotavirus strains shared nucleotide and amino acid substitutions with the Bangladeshi strain, Dhaka16-03 (G1P[8]) in the year 2003. This study documents for the first time the phylogenetic and evolutionary relationships of Indian G1P[8] rotavirus strains with the rotavirus strains from other parts of world and provides data useful for the evaluation of rotavirus vaccine programs. [Copyright &y& Elsevier]

Aims: To determine the genetic diversity of group A rotaviruses in bovine calves in Kashmir, India. Methods and Results: Of 200 diarrhoeic faecal samples collected from calves, aged between 0 and 6 months and screened by polyacrylamide gel electrophoresis (PAGE), 31 were detected positive for group A rotaviruses. On G and P genotyping by reverse transcriptase-polymerase chain reaction (RT-PCR), G10P[11] turned out to be predominant (80·64%) combination followed by G8P[11] (7·7%). One (3·84%) sample carried mixed infection of G8 + G10P[11]. Two (7·7%) samples belonged to P[11] genotype, but their G genotype specificity could not be established. This study revealed the ambiguity in RT-PCR typing method. All the samples that turned out to be G10 by Isegawa et al. (1993; Mol Cell Probes 7, 277) primers could be amplified by G3 specific primers of Gouvea et al. (1990; J Clin Microbiol 32, 1338). However, on homology study of their VP7 gene sequence, the strains turned out to be G10. Conclusions: Rotavirus is prevalent in diarrhoeic calves in Kashmir, India, and G10P[11] is the predominant genotype in circulation. There is evidence of mixed infection. Even though RT-PCR method is the quick way to type the strains, there is need to generate more sequence data to improve the specificity of typing primers. Significance and Impact of the Study: Rotavirus is a significant cause of diarrhoea in calves. RT-PCR typing method needs to be supported by the sequence data, and there is need to re-evaluate the primers used for typing. [ABSTRACT FROM AUTHOR]