title: Reduction of infectious bursal disease virus replication by shRNAs targeting the VP1 and VP2 genes driven by chicken U6 promoter.
abstract: Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease in young chickens and results in considerable economic losses for the poultry industry. To suppress the replication of IBDV, two short hairpin RNAs (shRNAs) were designed for targeting the VP1 and VP2 genes of IBDV. Recombinant plasmids carrying each shRNA or two shRNAs were constructed based on vector pSilencer2.1-U6 in which the human U6 promoter was replaced with chicken U6 promoter. In chicken embryo fibroblasts, transfection with these shRNA plasmids 24h before infection with IBDV B87 reduced 50% tissue culture infectious doses (TCID(50)) from 10(8.75) TCID(50)/0.1 mL to 10(3.75)-10(1.0) TCID(50)/0.1 mL. In 10-day old specific pathogen-free (SPF) chicken embryos, incubation with a mixture of IBDV B87 and a shRNA plasmid via the allantoic cavity resulted in 100% mortality and high IBDV virus titer in the control group but 25-0% mortality and near normal embryo development in the specific shRNA groups; additionally, IBDV VP1 and VP2 mRNA levels were reduced by 72-95% in the shRNA groups as compared with the control groups. When challenged with a virulent strain IBDV GX8/99, 14-day-old chickens pre-treated with the single shRNA plasmids or the dual shRNA plasmid showed approximately 70% or 90% survival at 5 days post-challenge while those pre-treated with control plasmid or saline had less than 5% survival. The current study suggests that two IBDV shRNAs expressed by a plasmid under chicken U6 promoter could effectively and synergistically reduce IBDV replication in vitro and in vivo.
authors.affiliation: Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ouyang
authors.forename: Jin-rong
authors.initials: JR
authors.lastname: Ma
authors.forename: Yong-qiang
authors.initials: YQ
authors.lastname: Wang
authors.forename: Li-ting
authors.initials: LT
authors.lastname: Qin
authors.forename: Jie-yuan
authors.initials: JY
authors.lastname: Jiang
authors.forename: Hong-jie
authors.initials: HJ
authors.lastname: Fan
authors.forename: Xiao-mei
authors.initials: XM
authors.lastname: Wang
authors.forename: Yong-shan
authors.initials: YS
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D002645:Chickens; D015316:Genetic Therapy / Q000662:veterinary*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007243:Infectious bursal disease virus / Q000235:genetics / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D011201:Poultry Diseases / Q000628:therapy / Q000821:virology*; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics*; D013047:Specific Pathogen-Free Organisms; D014162:Transfection / Q000662:veterinary; D015678:Viral Structural Proteins / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; C107669:VP1 protein, infectious bursal disease virus; C082513:VP2 protein, infectious bursal disease virus; D015678:Viral Structural Proteins