Reduction of infectious bursal disease virus replication by shRNAs targeting the VP1 and VP2 genes driven by chicken U6 promoter |
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| Authors: | Wei Ouyang Jin-rong Ma Yong-qiang Wang Li-ting Qin Jie-yuan Jiang Hong-jie Fan Xiao-mei Wang Yong-shan Wang |
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| Institution: | 1. Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;2. State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China;3. Department of Microbiology and Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China |
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| 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 24 h before infection with IBDV B87 reduced 50% tissue culture infectious doses (TCID50) from 108.75 TCID50/0.1 mL to 103.75–101.0 TCID50/0.1 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. |
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| Keywords: | IBDV infectious bursal disease virus VP1-shRNA2550 shRNA targeted to the position 2550 of VP1 gene of IBDV VP2-shRNA392 shRNA targeted to the position 392 of VP2 gene of IBDV |
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