Genetic variability of attachment (G) and Fusion (F) protein genes of human metapneumovirus strains circulating during 2006-2009 in Kolkata, Eastern India

<p>Abstract</p> <p>Background</p> <p>Human metapneumovirus (hMPV) is associated with the acute respiratory tract infection (ARTI) in all the age groups. However, there is limited information on prevalence and genetic diversity of human metapneumovirus (hMPV) strains circulating in India.</p> <p>Objective</p> <p>To study prevalence and genomic diversity of hMPV strains among ARTI patients reporting in outpatient departments of hospitals in Kolkata, Eastern India.</p> <p>Methods</p> <p>Nasal and/or throat swabs from 2309 patients during January 2006 to December 2009, were screened for the presence of hMPV by RT-PCR of nucleocapsid (N) gene. The G and F genes of representative hMPV positive samples were sequenced.</p> <p>Results</p> <p>118 of 2309 (5.11%) clinical samples were positive for hMPV. The majority (≈80%) of the positive cases were detected during July−November all through the study period. Genetic analysis revealed that 77% strains belong to A2 subgroup whereas rest clustered in B1 subgroup. G sequences showed higher diversity at the nucleotide and amino acid level. In contrast, less than 10% variation was observed in F gene of representative strains of all four years. Sequence analysis also revealed changes in the position of stop codon in G protein, which resulted in variable length (217-231 aa) polypeptides.</p> <p>Conclusion</p> <p>The study suggests that approximately 5% of ARTI in the region were caused by hMPV. This is the first report on the genetic variability of G and F gene of hMPV strains from India which clearly shows that the G protein of hMPV is continuously evolving. Though the study partially fulfills lacunae of information, further studies from other regions are necessary for better understanding of prevalence, epidemiology and virus evolution in Indian subcontinent.</p

Surveillance in eastern India (2007-2009) revealed reassortment event involving ns and PB1-F2 gene segments among co-circulating influenza a subtypes

<p>Abstract</p> <p>Background</p> <p>Influenza A virus encodes for eleven proteins, of which HA, NA, NS1 and PB1-F2 have been implicated in viral pathogenicity and virulence. Thus, in addition to the HA and NA gene segments, monitoring diversity of NS1 and PB1-F2 is also important.</p> <p>Methods</p> <p>55 out of 166 circulating influenza A strains (31 H1N1 and 24 H3N2) were randomly picked during 2007-2009 and NS and PB1-F2 genes were sequenced. Phylogenetic analysis was carried out with reference to the prototype strains, concurrent vaccine strains and other reference strains isolated world wide.</p> <p>Results</p> <p>Comparative analysis of both nucleotide and deduced amino acid sequences, revealed presence of NS gene with A/PR/8/34(H1N1)-like mutations (H4N, Q21R, A22V, K44R, N53D, C59R, V60A, F103S and M106I) in both RNA-binding and effector domain of NS1 protein, and G63E, the HPAI-H5N1-like mutation in NEP/NS2 of five A/H1N1 strains of 2007 and 2009. NS1 of other A/H1N1 strains clustered with concurrent A/H1N1 vaccine strains. Of 31 A/H1N1 strains, five had PB1-F2 similar to the H3N2 strains; six had non-functional PB1-F2 protein (11 amino acids) similar to the 2009 pandemic H1N1 strains and rest 20 strains had 57 amino acids PB1-F2 protein, similar to concurrent A/H1N1 vaccine strain. Interestingly, three A/H1N1 strains with H3N2-like PB1-F2 protein carried primitive PR8-like NS gene. Full gene sequencing of PB1 gene confirmed presence of H3N2-like PB1 gene in these A/H1N1 strains.</p> <p>Conclusion</p> <p>Overall the study highlights reassortment event involving gene segments other than HA and NA in the co-circulating A/H1N1 and A/H3N2 strains and their importance in complexity of influenza virus genetics. In contrast, NS and PB1-F2 genes of all A/H3N2 eastern India strains were highly conserved and homologous to the concurrent A/H3N2 vaccine strains suggesting that these gene segments of H3N2 viruses are evolutionarily more stable compared to H1N1 viruses.</p

Full genomic analysis of an influenza A (H1N2) virus identified during 2009 pandemic in Eastern India: evidence of reassortment event between co-circulating A(H1N1)pdm09 and A/Brisbane/10/2007-like H3N2 strains

Abstract Background During the pandemic [Influenza A(H1N1)pdm09] period in 2009-2010, an influenza A (Inf-A) virus with H1N2 subtype (designated as A/Eastern India/N-1289/2009) was detected from a 25 years old male from Mizoram (North-eastern India). Objective To characterize full genome of the H1N2 influenza virus. Methods For initial detection of Influenza viruses, amplification of matrix protein (M) gene of Inf-A and B viruses was carried out by real time RT-PCR. Influenza A positive viruses are then further subtyped with HA and NA gene specific primers. Sequencing and the phylogenetic analysis was performed for the H1N2 strain to understand its origin. Results The outcome of this full genome study revealed a unique reassortment event where the N-1289 virus acquired it’s HA gene from a 2009 pandemic H1N1 virus with swine origin and the other genes from H3N2-like viruses of human origin. Conclusions This study provides information on possibility of occurrence of reassortment events during influenza season when infectivity is high and two different subtypes of Inf-A viruses co-circulate in same geographical location.</p

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

AbstractTNF 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

Whole-genome characterization of human group C rotaviruses: identification of two lineages in the VP3 gene

Group C rotavirus (GCRV) is distributed worldwide as an enteric pathogen in humans and animals. However, to date, whole-genome sequences are available only for a human strain (Bristol) and a porcine strain (Cowden). To investigate the genetic diversity of human GCRVs, nearly full-length sequences of all 11 RNA segments were determined for human GCRVs detected recently in India (v508), Bangladesh (BS347), China (Wu82 and YNR001) and Japan (OH567 and BK0830) and analysed phylogenetically with sequence data for GCRVs published previously. All the RNA segments of human GCRV strains except for the VP3 gene showed high levels of conservation (>93 % nucleotide sequence identity, >92 % amino acid sequence identity), belonging to a single genetic cluster distinct from those of animal GCRVs. In contrast, the VP3 genes of human GCRVs could be discriminated into two clusters, designated M2 and M3, that were distinguished phylogenetically from those of porcine and bovine GCRVs (clusters M1 and M4, respectively). Between M2 and M3, amino acid sequence identity of the VP3 gene was 84.1–84.7 %, whereas high identities were observed within each cluster (92.3–97.6 % for M2, 98.2–99.3 % for M3). Sequence divergence among the four VP3 clusters was observed throughout the amino acid sequence except for conserved motifs, including those possibly related to enzyme functions of VP3. The presence of obvious genetic diversity only in the VP3 gene among human GCRVs suggested that either the M2 or M3 VP3 gene of human GCRVs might have been derived through reassortment from an animal GCRV or from an unidentified human GCRV strain belonging to a novel genogroup

Comparative evaluation of real-time PCR and conventional RT-PCR during two year surveillance for Influenza and RSV among children with acute respiratory infections in Kolkata reveals distinct seasonality of infection

Acute respiratory tract infections (ARTI) are one of the most common cause of morbidity and mortality in young children all over the world. Influenza and Respiratory Syncytial viruses (RSV) are the predominant etiology during seasonal epidemics and thus rapid and sensitive molecular tests for screening &amp; timely identification of epidemics are required. In this study we report comparison of real time PCR (Q-PCR) with conventional RT-PCR for parallel identification of Influenza A or B (Inf-A or -B) and RSV. A total of 1091 respiratory samples were examined from children with suspected ARTI during January 2007- December 2008. Of these 1091 samples, 275 (25.21%) were positive for either Influenza or RSV by Q-PCR compared to 262 (24%) positives by RT-PCR. Overall Inf-A, -B and RSV were detected in a total of 121 (11.075%), 59 (5.38%) and 95 (8.68%) samples respectively. In spite of overlapping clinical symptoms, RSV and Influenza showed distinct seasonal peaks. Inf-A positively and RSV, negatively correlated with rainfall and temperature. No distinct seasonality was observed in Inf-B infections. This is the first report of a systemic surveillance of respiratory viruses with seasonal correlation and prevalence rate from Eastern India. The two year comparative analysis also confirmed feasibility of using Q-PCR in developing countries, which will not only improve scope for prevention of epidemics but also provide crucial epidemiological data from the tropical regions

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

AbstractRotaviruses 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 NFκB 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

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

AbstractIntroductionIndia 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.MethodsDuring 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.ResultsAmong 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.ConclusionThe 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-Mediated Suppression of miRNA-192 Family and miRNA-181a Activates Wnt/&beta;-Catenin Signaling Pathway: An In Vitro Study

The significance of the Wnt/&beta;-catenin signaling cascade in Rotavirus (RV) infection has not been elucidated. In this study, we attempt to elucidate the importance of the Wnt/&beta;-catenin pathway in the RV pathogenesis and investigate a miRNA-mediated approach to regulate the pathway to repress the RV infection in the host. The regulation of the Wnt signaling pathway in terms of &beta;-catenin accumulation and activation was analyzed by Western blotting and Confocal imaging analysis. The expression levels of miR-192 family members and miR-181a were enquired into using qPCR assays, whereas their targets in the Wnt pathway were confirmed using the Luciferase Reporter Assays. Members of the miR-192 family and miR-181a, which target the components of the pathway, were also found to be considerably decreased in expression during RV infection. Ectopic expression of these miRNAs could restrict the RV pathogenesis by targeting the intermediates of the Wnt signaling pathway. The miR-192 family and miR-181a were capable of suppressing the RV infection via targeting of the Wnt/&beta;-catenin pathway. The study not only highlights the role of the Wnt signaling cascade in RV infection but also suggests that miRNAs can synergistically decrease RV replication by a significant amount. Thus, the miR-192 family and miR-181a present themselves as prospective antivirals against RV infection

Viperin, an IFN-Stimulated Protein, Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis

Viral infections lead to expeditious activation of the host’s innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin’s antiviral action in addition to the intricacy of viperin–virus interaction