Inhibition of Infectious Bursal Disease Virus by Vector Delivered SiRNA in Cell Culture

Amol Ashok Sahare, Megha Kadam Bedekar, Sudhir Kumar Jain, Azad Singh, Sanjeev Singh, Bikas Chandra Sarkhel

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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 μg. 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.

Original languageEnglish (US)
Pages (from-to)58-64
Number of pages7
JournalAnimal Biotechnology
Volume26
Issue number1
DOIs
StatePublished - Jan 2015

Keywords

  • Cytopathic effects
  • Gene knockdown
  • Interleukin response
  • RNAi
  • VP2 gene
  • iBD virus

Fingerprint Dive into the research topics of 'Inhibition of Infectious Bursal Disease Virus by Vector Delivered SiRNA in Cell Culture'. Together they form a unique fingerprint.

Cite this