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Muhasin Asaf V.N.,Indian Veterinary Research Institute | Kumar A.,Indian Veterinary Research Institute | Raut A.A.,ICAR National Institute of High Security Animal Diseases | Bhatia S.,ICAR National Institute of High Security Animal Diseases | Mishra A.,ICAR National Institute of High Security Animal Diseases
Theory in Biosciences | Year: 2015

Avian influenza is a highly contagious viral infection caused by avian influenza virus type A of the family Orthomyxoviridae primarily affecting the avian species. The non-structural protein 1 (NS1) encoded by the NS1 gene of the virus is critical in establishing the infection. NS1 protein acts to suppress the virus-induced host interferon response and also inhibit Protein kinase R activation thereby helping the virus to establish the infection. MicroRNAs (miRNA) are small regulatory endogenous non-coding RNAs of ~22 nucleotides in length located within introns of coding and non-coding genes, exons of non-coding genes or inter-genic regions. miRNAs can target the gene at various sites and effectively reduce or shut down its expression. In this study, set of differentially expressed chicken miRNA identified by deep sequencing H5N1 infected and SPF chicken lung were computationally analyzed, to identify targets in the NS1 gene. 300 differentially expressed miRNAs were then analyzed individually for target sites in gi|147667147|gb|EF362422.1| influenza A virus (A/chicken/India/NIV33487/06(H5N1)) segment 8, complete sequence using RNAhybrid 2.2. The analysis yielded gga-miR-1658* as the potential miRNA which is targeting the NS1 gene of H5N1 genome. © 2015, Springer-Verlag Berlin Heidelberg. Source

Dash S.K.,ICAR National Institute of High Security Animal Diseases | Kumar M.,ICAR National Institute of High Security Animal Diseases | Kataria J.M.,ICAR Central Avian Research Institute | Nagarajan S.,ICAR National Institute of High Security Animal Diseases | And 3 more authors.
Microbial Pathogenesis | Year: 2016

Low pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 106 EID50 of H9N2 virus were challenged with 106 EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co-circulating. © 2016 Elsevier Ltd. Source

Venkatesh G.,ICAR National Institute of High Security Animal Diseases | Vanamayya P.R.,IVRI Campus | Sharma N.,ICAR National Institute of High Security Animal Diseases | Kulkarni D.D.,ICAR National Institute of High Security Animal Diseases | Dubey S.C.,ICAR National Institute of High Security Animal Diseases
Journal of Pure and Applied Microbiology | Year: 2015

A gene fragment (1008bp) of Porcine Parvovirus (PPV) non-structural protein NS1 was amplified by PCR from tonsillar tissue sample. The amplicon was cloned and sequenced. The deduced amino acid sequences of the gene fragment showed more than 98% homology with published sequences. The NS1 gene fragment was subcloned into prokaryotic expression vector pET28a (+) and designated as pET-NS1. The recombinant plasmid was transformed into E.coli BL21(DE3) pLysS cells and the expression of the truncated recombinant NS1 (rNS1) protein with a size of 43kDa was induced with ImM IPTG for four hours. The rNS1 protein was purified by affinity column chromatography under denaturing conditions and characterised by SDS-PAGE and Western blot. It reacted with PPV positive serum but not with PPV negative serum or Porcine Circovirus serum. The rNS1 protein can be used to develop tests to detect PPV antibodies in infected animals and also to differentiate it from animals vaccinated with inactivated vaccine as it is found only in virus-infected cells but not in mature virion. Source

Kumar S.,Indian Institute of Science | Ingle H.,Indian Institute of Science | Mishra S.,Indian Institute of Science | Mahla R.S.,Indian Institute of Science | And 7 more authors.
Cell Death and Disease | Year: 2015

RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and proinflammatory cytokines through a sole adaptor IFN-β promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners. © 2015 Macmillan Publishers Limited All rights reserved. Source

Dubey P.,ICAR National Institute of High Security Animal Diseases | Mishra N.,ICAR National Institute of High Security Animal Diseases | Behera S.P.,ICAR National Institute of High Security Animal Diseases | Prakash A.,ICAR National Institute of High Security Animal Diseases | Prakash A.,Barkatullah University
Indian Journal of Animal Sciences | Year: 2015

Present knowledge on bovine viral diarrhoea virus (BVDV) entry, morphogenesis and release in host cells is incomplete. This study reports the results of effect of drug cytochalasin D and nystatin on BVDV entry in bovine and ovine cells and effect of Brefeldin-A (BFA), on BVDV release in ovine cells. The bovine (MDBK) and ovine (SFT-R) cells were treated with various concentrations of cytochalasin D or nystatin before infection with BVDV and at 16 h post infection, the number of infected cells was determined by immunochemistry. The ovine cells were infected with BVDV before addition of different concentration of BFA at 8 h post infection and the supernatants at 24 hpi were subjected to RT-PCR or immunochemistry. The results showed that only a minor inhibitory effect was observed on the entry and infectivity of BVDV in both ovine and bovine cells even at highest concentration of cytochalasin D or nystatin. As revealed by RT-PCR and virus titration, BFA treatment inhibited the BVDV secretion in a dose-dependent manner with complete inhibition obtained by 2μg/ml of BFA. However, the intracellular virus particles in BFA treated cell pellet were infectious. Taken together, our results showed that entry of BVDV into bovine or ovine cells is not dependent on macropinocytosis or claveolae-mediated endocytosis and provided evidence of existence of a common mechanism of BVDV release in bovine and ovine cells. © 2015, Indian Council of Agricultural Research. All rights reserved. Source

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