Rotavirus activates MLKL-mediated host cellular necroptosis concomitantly with apoptosis to facilitate dissemination of viral progeny.

Reprogramming the host cellular environment is an obligatory facet of viral pathogens to foster their replication and perpetuation. One of such reprogramming events is the dynamic cross-talk between viruses and host cellular death signaling pathways. Rotaviruses (RVs) have been reported to develop multiple mechanisms to induce apoptotic programmed cell death for maximizing viral spread and pathogenicity. However, the importance of non-apoptotic programmed death events has remained elusive in context of RV infection. Here, we report that RV-induced apoptosis accompanies another non-apoptotic mode of programmed cell death pathway called necroptosis to promote host cellular demise at late phase of infection. Phosphorylation of mixed lineage kinase domain-like (MLKL) protein indicative of necroptosis was observed to concur with caspase-cleavage (apoptotic marker) beyond 6 hr of RV infection. Subsequent studies demonstrated phosphorylated-MLKL to oligomerize and to translocate to plasma membrane in RV infected cells, resulting in loss of plasma membrane integrity and release of alarmin molecules e.g., high mobility group box protein 1 (HMGB1) in the extracellular media. Moreover, inhibiting caspase-cleavage and apoptosis could not fully rescue virus-induced cell death but rather potentiated the necroptotic trigger. Interestingly, preventing both apoptosis and necroptosis by small molecules significantly rescued virus-induced host cytopathy by inhibiting viral dissemination.

Upsurge in hospitalization of pediatric patients with severe acute respiratory infections in Kolkata and surrounding districts caused by recombinant human respiratory adenovirus type B 7/3.

Globally, different genotypes of human adenoviruses are associated with outbreaks of acute respiratory infection (ARI) though such evidence is lacking from India. In the present study, we report a sudden increase in the positivity of respiratory adenovirus among hospitalized children with ARI from Kolkata and the surrounding districts of West Bengal, India, from December 2022 to date. A sharp rise in the positivity rate of respiratory adenovirus was found which ranged from 22.1% in early December 2022 to 52.6% in mid-March 2023. The overall positivity was 40.4% during the period and children in the 2 to <5 years (51.0%) age group were mostly affected. Single infection with adenovirus was found in 72.4% of cases while co-infection with rhinovirus was the maximum (9.4%). Around 97.5% of positive cases required hospitalization. Cough, breathlessness, and wheeze were the most common clinical features among positive patients. Phylogenetic analysis of the hexon and fiber gene of all the sequenced strains revealed HAdV-B 7/3 recombination with more than 99% homology within themselves. This report of a respiratory adenovirus outbreak in West Bengal, India causing severe illness in the pediatric population underscores the need for regular monitoring of the circulating strains.

Emergence of a unique SARS-CoV-2 Delta sub-cluster harboring a constellation of co-appearing non-Spike mutations.

Accumulation of diverse mutations across the structural and nonstructural genes is leading to rapid evolution of SARS-CoV-2, altering its pathogenicity. We performed whole genome sequencing of 239 SARS-CoV-2 RNA samples collected from both adult and pediatric patients across eastern India (West Bengal), during the second pandemic wave in India (April-May 2021). In addition to several common spike mutations within the Delta variant, a unique constellation of eight co-appearing non-Spike mutations was identified, which revealed a high degree of positive mutual correlation. Our results also demonstrated the dynamics of SARS-CoV-2 variants among unvaccinated pediatric patients. 41.4% of our studied Delta strains harbored this signature set of eight co-appearing non-Spike mutations and phylogenetically out-clustered other Delta sub-lineages like 21J, 21A, or 21I. This is the first report from eastern India that portrayed a landscape of co-appearing mutations in the non-Spike proteins, which might have led to the evolution of a distinct Delta subcluster. Accumulation of such mutations in SARS-CoV-2 may lead to the emergence of "vaccine-evading variants." Hence, monitoring of such non-Spike mutations will be significant in the formulation of any future vaccines against those SARS-CoV-2 variants that might evade the current vaccine-induced immunity, among both the pediatric and adult populations.

Suppression of classical nuclear import pathway by importazole and ivermectin inhibits rotavirus replication.

BACKGROUND: Rotavirus is the foremost cause of acute gastroenteritis among infants in resource-poor countries, causing severe morbidity and mortality. The currently available rotavirus vaccines are effective in reducing severity of the disease but not the infection rates, thus antivirals as an adjunct therapy are needed to reduce the morbidity in children. Viruses rely on host cellular machinery for nearly every step of the replication cycle. Therefore, targeting host factors that are indispensable for virus replication could be a promising strategy. OBJECTIVES: To assess the therapeutic potential of ivermectin and importazole against rotaviruses. METHODS: Antirotaviral activity of importazole and ivermectin was measured against various rotavirus strains (RV-SA11, RV-Wa, RV-A5-13, RV-EW) in vitro and in vivo by quantifying viral protein expression by western blot, analysing viroplasm formation by confocal microscopy, and measuring virus yield by plaque assay. RESULTS: Importin-ß1 and Ran were found to be induced during rotavirus infection. Knocking down importin-ß1 severely impaired rotavirus replication, suggesting a critical role for importin-ß1 in the rotavirus life cycle. In vitro studies revealed that treatment of ivermectin and importazole resulted in reduced synthesis of viral proteins, diminished production of infectious virus particles, and decrease in viroplasm-positive cells. Mechanistic study proved that both drugs perform antirotavirus activity by inhibiting the function of importin-ß1. In vivo investigations in mice also confirmed the antirotavirus potential of importazole and ivermectin at non-toxic doses. Treatments of rotavirus-infected mice with either drug resulted in diminished shedding of viral particles in the stool sample, reduced expression of viral protein in the small intestine and restoration of damaged intestinal villi comapared to untreated infected mice. CONCLUSIONS: The study highlights the potential of importazole and ivermectin as antirotavirus therapeutics.

Epidemiology of major entero-pathogenic viruses and genetic characterization of Group A rotaviruses among children (≤5 years) with acute gastroenteritis in eastern India, 2018-2020.

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.

Genetic characterization and phylogenetic variations of human adenovirus-F strains circulating in eastern India during 2017-2020.

Human adenovirus-F (HAdV-F) (genotype 40/41) is the second-most leading cause of pediatric gastroenteritis after rotavirus, worldwide, accounting for 2.8%-11.8% of infantile diarrheal cases. Earlier studies across eastern India revealed a shift in the predominance of genotypes from HAdV41 in 2007-09 to HAdV40 in 2013-14. Thus, the surveillance for HAdV-F genotypes in this geographical setting was undertaken over 2017-2020 to analyze the viral evolutionary dynamics. A total of 3882 stool samples collected from children (≤5 years) were screened for HAdV-F positivity by conventional PCR. The hypervariable regions of the hexon and the partial shaft region of long fiber genes were amplified, sequenced, and phylogenetically analyzed with respect to the prototype strains. A marginal decrease in enteric HAdV prevalence was observed (9.04%, n = 351/3882) compared to the previous report (11.8%) in this endemic setting. Children <2 years were found most vulnerable to enteric HAdV infection. Reduction in adenovirus-rotavirus co-infection was evident compared to the sole adenovirus infection. HAdV-F genotypes 40 and 41 were found to co-circulate, but HAdV41 was predominant. HAdV40 strains were genetically conserved, whereas HAdV41 strains accumulated new mutations. On the basis of a different set of mutations in their genome, HAdV41 strains segregated into 2 genome type clusters (GTCs). Circulating HAdV41 strains clustered with GTC1 of the fiber gene, for the first time during this study period. This study will provide much-needed baseline data on the emergence and circulation of HAdV40/41 strains for future vaccine development.

Rotavirus activates a noncanonical ATM-Chk2 branch of DNA damage response during infection to positively regulate viroplasm dynamics.

Surveillance for maintaining genomic pristineness, a protective safeguard of great onco-preventive significance, has been dedicated in eukaryotic cells to a highly conserved and synchronised signalling cascade called DNA damage response (DDR). Not surprisingly, foreign genetic elements like those of viruses are often potential targets of DDR. Viruses have evolved novel ways to subvert this genome vigilance by twisting canonical DDR to a skewed, noncanonical response through selective hijacking of some DDR components while antagonising the others. Though reported for many DNA and a few RNA viruses, potential implications of DDR have not been addressed yet in case of infection with rotavirus (RV), a double-stranded RNA virus. In the present study, we aimed at the modulation of ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2) branch of DDR in response to RV infection in vitro. We found activation of the transducer kinase ATM and its downstream effector Chk2 in RV-SA11-infected cells, the activation response being maximal at 6-hr post infection. Moreover, ATM activation was found to be dependent on induction of the upstream sensor Mre11-Rad50-Nbs1 (MRN) complex. Interestingly, RV-SA11-mediated maximal induction of ATM-Chk2 pathway was revealed to be neither preceded by occurrence of nuclear DNA damage nor transduced to formation of damage-induced canonical nuclear foci. Subsequent investigations affirmed sequestration of MRN components as well as ATM-Chk2 proteins away from nucleus into cytosolic RV replication factories (viroplasms). Chemical intervention targeting ATM and Chk2 significantly inhibited fusion and maturation of viroplasms leading to attenuated viral propagation. Cumulatively, the current study describes RV-mediated activation of a noncanonical ATM-Chk2 branch of DDR skewed in favour of facilitated viroplasm fusion and productive viral perpetuation.

Comprehensive analysis of genomic diversity of SARS-CoV-2 in different geographic regions of India: an endeavour to classify Indian SARS-CoV-2 strains on the basis of co-existing mutations.

Accumulation of mutations within the genome is the primary driving force in viral evolution within an endemic setting. This inherent feature often leads to altered virulence, infectivity and transmissibility, and antigenic shifts to escape host immunity, which might compromise the efficacy of vaccines and antiviral drugs. Therefore, we carried out a genome-wide analysis of circulating SARS-CoV-2 strains to detect the emergence of novel co-existing mutations and trace their geographical distribution within India. Comprehensive analysis of whole genome sequences of 837 Indian SARS-CoV-2 strains revealed the occurrence of 33 different mutations, 18 of which were unique to India. Novel mutations were observed in the S glycoprotein (6/33), NSP3 (5/33), RdRp/NSP12 (4/33), NSP2 (2/33), and N (1/33). Non-synonymous mutations were found to be 3.07 times more prevalent than synonymous mutations. We classified the Indian isolates into 22 groups based on their co-existing mutations. Phylogenetic analysis revealed that the representative strains of each group were divided into various sub-clades within their respective clades, based on the presence of unique co-existing mutations. The A2a clade was found to be dominant in India (71.34%), followed by A3 (23.29%) and B (5.36%), but a heterogeneous distribution was observed among various geographical regions. The A2a clade was highly predominant in East India, Western India, and Central India, whereas the A2a and A3 clades were nearly equal in prevalence in South and North India. This study highlights the divergent evolution of SARS-CoV-2 strains and co-circulation of multiple clades in India. Monitoring of the emerging mutations will pave the way for vaccine formulation and the design of antiviral drugs.

Genetic characterization and evolutionary analysis of norovirus genotypes circulating among children in eastern India during 2018-2019.

Noroviruses are significant etiological agents of acute gastroenteritis (AGE) across all age groups, especially in children under 5 years of age. Although the prevalence of norovirus infection is known to have increased in various countries, in India there are few reports pertaining to the norovirus disease burden. We investigated the epidemiology and molecular characteristics of noroviruses in children seeking health care at two hospitals in Kolkata, Eastern India. Faecal specimens were collected between January 2018 and December 2019 from 2812 children under 5 years of age with acute gastroenteritis. Noroviruses were detected in 6.04% (170/2812) of the samples, and 12.9% (22/170) of these were cases of coinfection with rotavirus. Among children (≤5 years), a higher infection rate (8.2%, n = 94/1152) was observed in the 6 to 12 month age group. GII.4 Sydney 2012 was the dominant norovirus capsid genotype (n = 75/90, 83.3%), followed by GII.3 (n = 10/90, 11.1%). Other capsid types GII.13 (n = 4/90, 4.4%) and GII.17 (n = 1/90; 1.1%) were also detected at low frequency. Phylogenetic analysis showed that the GII.P16 polymerase of strains in this region clustered with those of the phylogenetically distinct monophyletic clade of GII.P16 strains, whose members have been circulating worldwide since 2014. Inter-genotypic norovirus recombinants such as GII.P16-GII.3 (n = 10) and GII.P16-GII.13 (n = 4) were also observed among the circulating strains. In comparison to previous studies from eastern India, the present study shows a higher detection rate of norovirus infection in the paediatric population suffering from acute gastroenteritis. Continuous surveillance is required for predicting the emergence of novel genotypes and recombinant strains and for future vaccine development.

In quest of small-molecules as potent non-competitive inhibitors against influenza.

A series of scaffolds namely aurones, 3-indolinones, 4-quinolones and cinnamic acid-piperazine hybrids, was designed, synthesized and investigated in vitro against influenza A/H1N1pdm09 virus. Designed molecules adopted different binding mode i.e., in 430-cavity of neuraminidase, unlike sialic acid and oseltamivir in molecular docking studies. All molecules reduced the viral titer and exhibited non-cytotoxicity along with cryo-protective property towards MDCK cells. Molecules (Z)-2-(3'-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2f), (Z)-2-(4'-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2g) and 2-(2'-Methoxy-phenyl)-1H-quinolin-4-one (3a) were the most interesting molecules identified in this research, endowed with robust potencies showing low-nanomolar EC50 values of 4.0 nM, 6.7 nM and 4.9 nM, respectively, compared to reference competitive and non-competitive inhibitors: oseltamivir (EC50 = 12.7 nM) and quercetin (EC50 = 0.56 µM), respectively. Besides, 2f, 2g and 3a exhibited good neuraminidase inhibitory activity in sub-micromolar range (IC50 = 0.52 µM, 3.5 µM, 1.3 µM respectively). Moreover, these molecules were determined as non-competitive inhibitors similar to reference non-competitive inhibitor quercetin unlike reference competitive inhibitor oseltamivir in kinetics studies.

A decade-long temporal analyses of human group-A rotavirus among children with gastroenteritis: Prevaccination scenario in West Bengal, eastern India.

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].

Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2.

RNA interference (RNAi) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. Viruses have also gained counter-defensive measures by producing virulence determinants called viral-suppressors-of-RNAi (VSRs). Interestingly, in spite of dominance of interferon-based immunity over RNAi in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of RNAi in mammalian cells. The present study focuses on the modulation of intracellular RNAi during infection with rotavirus (RV), an enteric virus with double-stranded RNA genome. Intriguingly, a time point-dependent bimodal regulation of RNAi was observed in RV-infected cells, where short interfering RNA (siRNA)-based RNAi was rendered non-functional during early hours of infection only to be reinstated fully beyond that early infection stage. Subsequent investigations revealed RV nonstructural protein 1 to serve as a putative VSR by associating with and triggering degradation of Argonaute2 (AGO2), the prime effector of siRNA-mediated RNAi, via ubiquitin-proteasome pathway. The proviral significance of AGO2 degradation was further confirmed when ectopic overexpression of AGO2 significantly reduced RV infection. Cumulatively, the current study presents a unique modulation of host RNAi during RV infection, highlighting the importance of antiviral RNAi in mammalian cells.

Diversity of rotavirus genotypes circulating in children < 5 years of age hospitalized for acute gastroenteritis in India from 2005 to 2016: analysis of temporal and regional genotype variation.

BACKGROUND: From 2016, the Government of India introduced the oral rotavirus vaccine into the national immunization schedule. Currently, two indigenously developed vaccines (ROTAVAC, Bharat Biotech; ROTASIIL, Serum Institute of India) are included in the Indian immunization program. We report the rotavirus disease burden and the diversity of rotavirus genotypes from 2005 to 2016 in a multi-centric surveillance study before the introduction of vaccines. METHODS: A total of 29,561 stool samples collected from 2005 to 2016 (7 sites during 2005-2009, 3 sites from 2009 to 2012, and 28 sites during 2012-2016) were included in the analysis. Stools were tested for rotavirus antigen using enzyme immunoassay (EIA). Genotyping was performed on 65.8% of the EIA positive samples using reverse transcription- polymerase chain reaction (RT-PCR) to identify the G (VP7) and P (VP4) types. Multinomial logistic regression was used to quantify the odds of detecting genotypes across the surveillance period and in particular age groups. RESULTS: Of the 29,561 samples tested, 10,959 (37.1%) were positive for rotavirus. There was a peak in rotavirus positivity during December to February across all sites. Of the 7215 genotyped samples, G1P[8] (38.7%) was the most common, followed by G2P[4] (12.3%), G9P[4] (5.8%), G12P[6] (4.2%), G9P[8] (4%), and G12P[8] (2.4%). Globally, G9P[4] and G12P[6] are less common genotypes, although these genotypes have been reported from India and few other countries. There was a variation in the geographic and temporal distribution of genotypes, and the emergence or re-emergence of new genotypes such as G3P[8] was seen. Over the surveillance period, there was a decline in the proportion of G2P[4], and an increase in the proportion of G9P[4]. A higher proportion of mixed and partially typed/untyped samples was also seen more in the age group 0-11 months. CONCLUSIONS: This 11 years surveillance highlights the high burden of severe rotavirus gastroenteritis in Indian children < 5 years of age before inclusion of rotavirus vaccines in the national programme. Regional variations in rotavirus epidemiology were seen, including the emergence of G3P[8] in the latter part of the surveillance. Having pre-introduction data is important to track changing epidemiology of rotaviruses, particularly following vaccine introduction.

Rotaviral nonstructural protein 4 triggers dynamin-related protein 1-dependent mitochondrial fragmentation during infection.

Dynamic equilibrium between mitochondrial fission and mitochondrial fusion serves as an important quality control system within cells ensuring cellular vitality and homeostasis. Viruses often target mitochondrial dynamics as a part of their obligatory cellular reprogramming. The present study was undertaken to assess the status and regulation of mitochondrial dynamics during rotavirus infection. Distinct fragmentation of mitochondrial syncytia was observed during late hours of RV (SA11, Wa, A5-13) infection. RV nonstructural protein 4 (NSP4) was identified as the viral trigger for disrupted mitochondrial morphology. Severance of mitochondrial interconnections was found to be a dynamin-related protein 1 (Drp1)-dependent process resulting synergistically from augmented mitochondrial fission and attenuated mitochondrial fusion. Cyclin-dependent kinase 1 was subsequently identified as the cellular kinase responsible for fission-active Ser616 phosphorylation of Drp1. In addition to its positive role in mitochondrial fission, Drp1 also resulted in mitochondrial translocation of E3-ubiquitin ligase Parkin leading to degradation of mitochondrial fusion protein Mitofusin 1. Interestingly, RV-NSP4 was found to interact with and be involved in recruiting fission-active pool of Serine 616 phosphoDrp1 (Ser616 pDrp1) to mitochondria independent of accessory adaptors Mitochondrial fission factor and Fission protein 1 (Fis1). Inhibition of either Drp1 or Ser616 pDrp1 resulted in significant decrease in RV-NSP4-induced intrinsic apoptotic pathway. Overall, this study underscores an efficient strategy utilised by RV to couple apoptosis to mitochondrial fission facilitating dissemination of viral progeny.

Tyrosine phosphorylation modulates mitochondrial chaperonin Hsp60 and delays rotavirus NSP4-mediated apoptotic signaling in host cells.

Phosphoproteomics-based platforms have been widely used to identify post translational dynamics of cellular proteins in response to viral infection. The present study was undertaken to assess differential tyrosine phosphorylation during early hours of rotavirus (RV) SA11 infection. Heat shock proteins (Hsp60) were found to be enriched in the data set of RV-SA11 induced differentially tyrosine-phosphorylated proteins at 2 hr post infection (hpi). Hsp60 was further found to be phosphorylated by an activated form of Src kinase on 227th tyrosine residue, and tyrosine phosphorylation of mitochondrial chaperonin Hsp60 correlated with its proteasomal degradation at 2-2.5hpi. Interestingly, mitochondrial Hsp60 positively influenced translocation of the rotaviral nonstructural protein 4 to mitochondria during RV infections. Phosphorylation and subsequent transient degradation of mitochondrial Hsp60 during early hours of RV-SA11 infection resulted in inhibition of premature import of nonstructural protein 4 into mitochondria, thereby delaying early apoptosis. Overall, the study highlighted one of the many strategies rotavirus undertakes to prevent early apoptosis and subsequent reduced viral progeny yield.

Species A rotaviruses isolated from hospitalized patients over 5 years of age in Kolkata, India, in 2012/13.

In hospital-based diarrhoeal disease surveillance at Infectious Diseases & Beliaghata-General Hospital (May-2012 to April-2013), Kolkata, India, stool samples were collected from patients < 5 years (n = 830) and > 5 years of age (n = 728) hospitalized with diarrhea. Group-A rotavirus (GARV) was identified by ELISA followed by multiplex RT-PCR. In children < 5 years of age, 53.4% of the samples were positive for GARV. In patients > 5 years to 90 years old, only 6.04% (n = 44) tested positive for GARV. G2P[4] strains (n = 16 [36.36%]) were the most prevalent, followed by G9P[4] strains (n = 13 [29.54%]), while P[4]-(n = 30 [68.18%]) was most prevalent among the P genotypes. The GARV strains G2, G9 and P[4] detected in adults clustered together in the phylogenetic tree with the GARV strains identified in children (< 5 years) during the same period. Rotavirus positivity was high among female patients (75%), suggesting that caregivers (mother/grandmother/older-siblings) may get infected through young children or may act as carriers for transmission.

Molecular characterization of enteric adenovirus genotypes 40 and 41 identified in children with acute gastroenteritis in Kolkata, India during 2013-2014.

Human enteric adenovirus (HAdV) belonging to species F is one of the most common pathogens responsible for infantile gastroenteritis worldwide. This study was initiated to estimate prevalence and types of HAdV among children below 5 years of age seeking health care facility for treatment of diarrhea in Kolkata, Eastern India. A total of 1,562 stool specimens were tested during 2013-2014 and among them, 185 (11.8%) were positive for enteric HAdV. Maximum number of positive cases were observed among children between 6 and 12 months of age (13.9%). HAdV infection occurred at a low frequency throughout the year, with an increased incidence in the month of March-July in both the years. Among HAdV positive samples (n = 185), 44.8% showed coinfection with rotavirus. Genotyping based on hypervariable region of hexon and partial shaft region of fiber genes, revealed prevalence of HAdV-40 over HAdV-41 genotype during this study period. Nucleotide sequence analysis of HAdV-40 strains exhibited more than 99% similarity among themselves and 98.5% with the prototype strain Dugan. Sequence analysis of six hypervariable regions (HVRs) of hexon genes from all the HAdV-41 strains revealed co-circulation of both genome type cluster 1(GTC1) and GTC2. The presence of both types of GTCs reflects accumulation of amino acid (aa) mutations in HVR of hexon gene. A recombination event was evident in a subset of HAdV-41 strains where hexon gene belonged to GTC1 whereas, fiber gene clustered with GTC2. Sequence analysis of fiber gene shaft region of HAdV-41 strains revealed 15 aa deletion from the 15th repeat motif. J. Med. Virol. 89:606-614, 2017. © 2016 Wiley Periodicals, Inc.

Rotavirus-induced miR-142-5p elicits proviral milieu by targeting non-canonical transforming growth factor beta signalling and apoptosis in cells.

MicroRNA (miRNA) expression is significantly influenced by viral infection, because of either host antiviral defences or proviral factors resulting in the modulation of viral propagation. This study was undertaken to identify and analyse the significance of cellular miRNAs during rotavirus (SA11 or KU) infection. Sixteen differentially regulated miRNAs were identified during rotavirus infection of which hsa-miR-142-5p was up-regulated and validated by quantitative polymerase chain reaction. Exogenous expression of miR-142-5p inhibitor resulted in a significant reduction of viral titer indicating proviral role of miR-142-5p. Functional studies of hsa-miR-142-5p identified its role in transforming growth factor beta (TGFß) signalling as TGFß receptor 2 and SMAD3 were degraded during both hsa-miR-142-5p overexpression and rotavirus infection. TGFß is induced during rotavirus infection, which may promote apoptosis by activation of non-canonical pathways in HT29 cells. However, up-regulated miR-142-5p resulted in the inhibition of TGFß-induced apoptosis suggesting its anti-apoptotic function. Rotavirus NSP5 was identified as a regulator of miR-142-5p expression. Concurrently, NSP5-HT29 cells showed inhibition of TGFß-induced apoptosis and epithelial to mesenchymal transition by blocking non-canonical pathways. Overall, the study identified proviral function of hsa-miR-142-5p during rotavirus infection. In addition, modulation of TGFß-induced non-canonical signalling in microsatellite stable colon cancer cells can be exploited for cancer therapeutics.