title: Inhibition of infectious bursal disease virus by vector delivered SiRNA in cell culture.
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.
authors.affiliation: a Animal Biotechnology Center, JNKVV Campus, Adhartal , Jabalpur , Madhya Pradesh , India.
authors.forename: Amol Ashok
authors.initials: AA
authors.lastname: Sahare
authors.forename: Megha Kadam
authors.initials: MK
authors.lastname: Bedekar
authors.forename: Sudhir Kumar
authors.initials: SK
authors.lastname: Jain
authors.forename: Azad
authors.initials: A
authors.lastname: Singh
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Singh
authors.forename: Bikas Chandra
authors.initials: BC
authors.lastname: Sarkhel
mesh_terms: D000818:Animals; D018175:Birnaviridae Infections / Q000662:veterinary* / Q000821:virology; D002478:Cells, Cultured; D002642:Chick Embryo; D005347:Fibroblasts / Q000473:pathology / Q000821:virology; D055785:Gene Knockdown Techniques; D007243:Infectious bursal disease virus / Q000187:drug effects*; D011201:Poultry Diseases / Q000235:genetics / Q000628:therapy* / Q000821:virology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D015678:Viral Structural Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; C082513:VP2 protein, infectious bursal disease virus; D015678:Viral Structural Proteins
keywords: Cytopathic effects; Gene knockdown; Interleukin response; RNAi; VP2 gene; iBD virus