Mukti Kant Nayak

Detection of a novel intergenogroup recombinant Norovirus from Kolkata, India

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

A new variant of Norovirus GII.4/2007 and inter-genotype recombinant strains of NVGII causing acute watery diarrhoea among children in Kolkata, India.

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

The molecular chaperone heat shock protein-90 positively regulates rotavirus infection

Rotaviruses are the major cause of severe dehydrating gastroenteritis in children worldwide. In this study, we report a positive role of cellular chaperone Hsp90 during rotavirus infection. A highly specific Hsp90 inhibitor, 17-allylamono-demethoxygeldanamycin (17-AAG) was used to delineate the functional role of Hsp90. In MA104 cells treated with 17-AAG after viral adsorption, replication of simian (SA11) or human (KU) strains was attenuated as assessed by quantitating both plaque forming units and expression of viral genes. Phosphorylation of Akt and NFkappaB observed 2-4 hpi with SA11, was strongly inhibited in the presence of 17-AAG. Direct Hsp90-Akt interaction in virus infected cells was also reduced in the presence of 17-AAG. Anti-rotaviral effects of 17-AAG were due to inhibition of activation of Akt that was confirmed since, PI3K/Akt inhibitors attenuated rotavirus growth significantly. Thus, Hsp90 regulates rotavirus by modulating cellular signaling proteins. The results highlight the importance of cellular proteins during rotavirus infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies.

Rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection.

Background: Rotaviral nonstructural protein 4 (NSP4) disrupts Ca2+ ion homeostasis by translocating to the endoplasmic reticulum. Results: In this study, we show translocation of NSP4 to mitochondria, dissipation of mitochondrial potential, and initiation of apoptosis, which NSP1 counteracts during early infection. Conclusion: NSP4 and NSP1 regulate apoptosis during infection. Significance: Study signifies modulation of cellular survival and apoptotic machinery by rotavirus for their own benefit. Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca2+ homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61–83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.

Antiviral activity of baicalin against influenza virus H1N1-pdm09 is due to modulation of NS1-mediated cellular innate immune responses

OBJECTIVES Baicalin, a flavonoid, has been shown to have antiviral and anti-inflammatory activities, although the mechanism of action has been unknown. Therefore, attempts were made to analyse the mechanism behind the antiviral effects of baicalin using an influenza A virus (IAV) model in vitro and in vivo. METHODS Baicalins anti-influenza activity was elucidated (in vitro and in vivo) utilizing pandemic influenza strain A/H1N1/Eastern India/66/pdm09 (H1N1-pdm09). Anti-influenza activity was measured by plaque inhibition, fluorescent focus-forming units (ffu) and quantifying viral transcripts using quantitative real-time PCR following treatment with baicalin in a dose- and time-dependent manner. The role of the IAV non-structural protein 1 (NS1) gene in modulating host responses was measured by immunoblotting, co-immunoprecipitation and molecular docking. RESULTS Baicalin treatment following IAV infection revealed up-regulation of interferon (IFN)-induced antiviral signalling and decreased phosphoinositide 3-kinase/Akt (PI3K/Akt) activation compared with infected, untreated controls. Baicalin exerts its antiviral effects by modulating the function of the IAV-encoded NS1 protein. NS1 has been shown to counteract cellular antiviral responses by down-regulating IFN induction and up-regulating PI3K/Akt signalling. Baicalin disrupted NS1-p85β binding. Molecular docking predicted the binding site of baicalin in the RNA binding domain (RBD) of NS1. Site-directed mutagenesis within the RBD region of NS1 and the difference in the fluorescence quenching pattern of full-length NS1 and mutant NS1 proteins in the presence of baicalin confirmed the interaction of baicalin with the NS1 RBD. Amino acid residues 39-43 of the NS1 RBD were found to be crucial for the baicalin-NS1 interaction. CONCLUSIONS Overall, this study highlights that baicalin exerts its anti-influenza virus activity by modulating viral protein NS1, resulting in up-regulation of IFN-induced antiviral signalling and a decrease in PI3K/Akt signalling in cells.

Hospital based surveillance and genetic characterization of rotavirus strains in children (<5 years) with acute gastroenteritis in Kolkata, India, revealed resurgence of G9 and G2 genotypes during 2011-2013.

INTRODUCTION India accounts for an estimated 457,000-884,000 hospitalizations and 2 million outpatient visits for diarrhea. In spite of the huge burden of rotavirus (RV) disease, RV vaccines have not been introduced in national immunization programme of India. Therefore, continuous surveillance for prevalence and monitoring of the circulating genotypes is needed to assess the disease burden prior to introduction of vaccines in this region. METHODS During January 2011 through December 2013, 830 and 1000 stool samples were collected from hospitalized and out-patient department (OPD) patients, respectively, in two hospitals in Kolkata, Eastern India. After primary screening, the G-P typing was done by multiplex semi-nested PCR using type specific primers followed by sequencing. Phylogenetic analysis for the VP7 gene of 25 representative strains was done. RESULTS Among hospitalized and OPD patients, 53.4% and 47.5% cases were positive for rotaviruses, respectively. Unlike previous studies where G1 was predominant, in hospitalized cases G9 rotavirus strains were most prevalent (40%), followed by G2 (39.6%) whereas G1 and G12 occurred at 16.4% and 5.6% frequency. In OPD cases, the most prevalent strain was G2 (40.3%), followed by G1, G9 and G12 at 25.5%, 22.8%, 9.3%, respectively. Phylogenetically the G1, G2 and G9 strains from Kolkata did not cluster with corresponding genotypes of Rotarix, RotaTeq and Rotavac (116E) vaccine strains. CONCLUSION The study highlights the high prevalence of RV in children with gastroenteritis in Kolkata. The circulating genotypes have changed over the time with predominance of G9 and G2 strains during 2011-2013. The current G2, G9 and G1 Kolkata strains shared low amino acid homologies with current vaccine strains. Although there is substantial evidence for cross protection of vaccines against a variety of strains, still the strain variation should be monitored post vaccine introduction to determine if it has any impact on vaccine effectiveness.

Rotavirus-encoded nonstructural protein 1 modulates cellular apoptotic machinery by targeting tumor suppressor protein p53.

ABSTRACT p53, a member of the innate immune system, is triggered under stress to induce cell growth arrest and apoptosis. Thus, p53 is an important target for viruses, as efficient infection depends on modulation of the host apoptotic machinery. This study focuses on how rotaviruses manipulate intricate p53 signaling for their advantage. Analysis of p53 expression revealed degradation of p53 during initial stages of rotavirus infection. However, in nonstructural protein-1 (NSP1) mutant strain A5-16, p53 degradation was not observed, suggesting a role of NSP1 in this process. This function of NSP1 was independent of its interferon or phosphatidylinositol 3-kinase (PI3K)/AKT modulation activity since p53 degradation was observed in Vero cells as well as in the presence of PI3K inhibitor. p53 transcript levels remained the same in SA11-infected cells (at 2 to 14 h postinfection), but p53 protein was stabilized only in the presence of MG132, suggesting a posttranslational process. NSP1 interacted with the DNA binding domain of p53, resulting in ubiquitination and proteasomal degradation of p53. Degradation of p53 during initial stages of infection inhibited apoptosis, as the proapoptotic genes PUMA and Bax were downregulated. During late viral infection, when progeny dissemination is the main objective, the NSP1-p53 interaction was diminished, resulting in restoration of the p53 level, with initiation of proapoptotic signaling ensuing. Overall results highlight the multiple strategies evolved by NSP1 to combat the host immune response.

Rotavirus NSP1 inhibits interferon induced non-canonical NFκB activation by interacting with TNF receptor associated factor 2.

TNF receptor associated factor 2 (TRAF2) plays a very important role in cellular innate immune as well as inflammatory responses. Previous studies have reported TRAF2 mediated regulation of TNF and Interferon (IFN) induced canonical and non-canonical activation of NFκB. In this study, we show that rotavirus NSP1 targets TRAF2 to regulate IFN induced non-canonical NFκB activation. Here we found that rotavirus Non-Structural Protein-1 (NSP1) interacts with TRAF2 and degrades it in a proteasome dependent manner. C-terminal part of NSP1 was sufficient for interacting with TRAF2 but it alone could not degrade TRAF2. This inhibition of interferon mediated non-canonical NFκB activation by NSP1 may modulate inflammatory cytokine production after rotavirus infection to help the virus propagation.

Detection of human G10 rotavirus strains with similarity to bovine and bovine-like equine strains from untypable samples

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.

Identification of Cellular Calcium Binding Protein Calmodulin as a Regulator of Rotavirus A Infection during Comparative Proteomic Study

Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5years age) worldwide. In cellular environment, viruses constantly adapt and modulate to survive and replicate while the host cell also responds to combat the situation and this results in the differential regulation of cellular proteins. To identify the virus induced differential expression of proteins, 2D-DIGE (Two-dimensional Difference Gel Electrophoresis) based proteomics was used. For this, HT-29 cells were infected with RV strain SA11 for 0 hours, 3 hours and 9 hours post infection (hpi), differentially expressed spots were excised from the gel and identified using MALDI-TOF/TOF mass spectrometry. 2D-DIGE based proteomics study identified 32 differentially modulated proteins, of which 22 were unique. Some of these were validated in HT-29 cell line and in BALB/c mice model. One of the modulated cellular proteins, calmodulin (CaM) was found to directly interact with RV protein VP6 in the presence of Ca2+. Ca2+-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca2+ chelator (BAPTA-AM) resulted in decreased viral titers. This study not only identifies differentially modulated cellular proteins upon infection with rotavirus in 2D-DIGE but also confirmed positive engagement of cellular Ca2+/CaM during viral pathogenesis.