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Publications (3)2.94 Total impact

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    ABSTRACT: Infectious Bursal Disease (IBD) is major threat to poultry industry. It causes severe immunosuppression and mortality in chicken generally at 3 to 6 weeks of age. RNA intereference (RNAi) emerges as a potent gene regulatory tool in last few years. The present study was conducted to evaluate the efficiency of RNAi to inhibit the IBD virus (IDBV) replication in-vitro. VP2 gene of virus encodes protein involved in capsid formation, cell entry and induction of protective immune responses against it. Thus, VP2 gene of IBDV is the candidate target for the molecular techniques applied for IBDV detection and inhibition assay. In this study, IBDV was isolated from field cases and confirmed by RT-PCR. The virus was then adapted on chicken embryo fibroblast cells (CEF) in which it showed severe cytopathic effects (CPE). The short hairpin RNA (shRNAs) constructs homologous to the VP2 gene were designed and one, having maximum score and fulfilling maximum Reynolds criteria, was selected for evaluation of effective inhibition. Selected shRNA construct (i.e., VP2-shRNA) was observed to be the most effective for inhibiting VP2 gene expression. Real time PCR analysis was performed to measure the relative expression of VP2 gene in different experimental groups. The VP2 gene was less expressed in virus infected cells co-transfected with VP2-shRNA as compared to mock transfected cells and IBDV+ cells (control) at dose 1.6 µ g. The result showed ∼95% efficient down regulation of VP2 gene mRNA in VP2-shRNA treated cells. These findings suggested that designed shRNA construct achieved high level of inhibition of VP2 gene expression in-vitro.
    Animal Biotechnology 01/2015; 26(1):58-64. · 0.88 Impact Factor
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    ABSTRACT: RNAi is an evolutionary conserved, highly efficient, and cost effective technique of gene silencing. It holds considerable promise and success has been achieved both in vitro and in vivo experiments. However, it is not devoid of undesirable side effects as dsRNA can trigger the immune response and can also cause non-specific off-target gene silencing. In the present study, silencing of myostatin gene, a negative regulator of myogenesis, was evaluated in caprine fetal fibroblasts using three different shRNA constructs. Out of these three constructs, two constructs sh1 and sh2 showed, 72% and 50% reduction (p<0.05) of myostatin mRNA, respectively. Efficient suppression (42-86%) of MSTN gene (p<0.05) was achieved even by reducing the concentration of shRNA constructs. The induction of classical interferon stimulated gene (Oligoandenylate Synthetase-1, OAS-1) was studied to analyze the immune response against shRNAs. Notably, a reduction in the potency of shRNAs to induce interferon response was observed at lower concentration for OAS1 gene. The results obtained in the study would be helpful in the abrogation of the bystander effects of RNAi for long term stable expression of anti-MSTN expression constructs in the muscle.
    Animal Biotechnology 07/2012; 23(3):174-83. · 0.88 Impact Factor
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    ABSTRACT: In order to detect infectious bursal disease virus (IBDV), bursal tissue was collected from 10 IBD-suspected birds from a 30-day-old, IBDV-vaccinated commercial broiler chicken flock of 2000 birds exhibiting clinical signs suggestive of infectious bursal disease (IBD). The presence of IBDV was confirmed by partial amplification of the VP2 gene by reverse transcription and polymerase chain reaction. Isolates were identified as very virulent strains of IBDV (vvIBDV) by nucleotide sequence analysis. The comparison of the VP2 nucleotide sequences among the isolates revealed the presence of single-nucleotide polymorphisms in the VP2 gene of IBDV in the same flock. The comparative analysis indicated that these viruses were genetically close to the vvIBDVs previously detected in India. Our analysis provided information about the existence of vvIBDV in Central India.
    Acta Veterinaria Hungarica 03/2012; 60(1):165-74. · 1.17 Impact Factor