Inhibition Of Infectious Bursal Disease Virus Replication And Functional Research Of Viral Encoding Protein With RNA Interference Technology

Infectious bursal disease virus (IBDV), causing bursal atrophy and immunosuppression inyoung chickens, is a constant threat to the poultry industry world wide. IBD is a highly contagious,globally occurring viral disease of poultry. The economic importance of the disease is manifested intwo ways: first, some virus strains may cause upto 60ï¼…mortality in 3-week-old chickens and older,and second; the most important manifestation is severe prolonged immunosuppression in chickensinfected with IBDV at an early age and increased susceptibility to other diseases. Vaccination is theprinciple method to control IBD. Control of IBD has been complicated by the recognition of veryvirulent strains. IBDV is a member of genus Avibirnavirus of family Birnaviridae. The function ofvirus protein in the course of replication is, as yet, unknown. The research of this function isimportant significance to prevent IBD. As a new molecular biology method, RNA interference(RNAi) has already been applied in therapy and prevention of virus disease and research of genefunction. To effectively prevent IBD and research newly prevention and therapy strategy, researchesas follow were carried out.1. Inhibition of IBDV replication by plasmid-based RNA interferenceThree short interfering RNA(siRNAs) (siVP1⁶¹⁸,siVP1¹¹¹⁵ and siVP1²⁵⁷¹) directed against awell-conserved region of IBDV VP1 gene were selected. A non-specific siRNA sequence (siRNA^Con)was designed as non-specfic control. Vero cells were transfected with siRNA, prepared by in vitrotranscription, and infected with IBDV(0.01 MOI) after 6h post transfection. Viral suspension washarvested and virus titration was tested. The results of viral plaque formation assay showed that theinhibitoryratio of siVP1⁶¹⁸,siVP1¹¹¹⁵ and siVP1²⁵⁷¹ to IBDV replication were 84ï¼…,86.7ï¼…and93.3ï¼…compared with controls, respectively. The results of Real time PCR showed that viral mRNAlevels were reduced by 30.4ï¼…,94.7ï¼…and 98.2ï¼…compared with controls, respectively. The resultsof viral plaque formation assay and Real time PCR demonstrated that the best inhibitory effect to IBDV replication was siVP1²⁵⁷¹. In order to make siRNA long-lasting express and easily prepared,the primer including siVP1²⁵⁷¹ sequence was designed. The shRNA expression vector(pEC2571-shRNA) was constructed by amplifing mouse U6 promotor with the primer and cloningto pEGFP-C1 with opposite direction. Vero cells were transfected pEC2571-shRNA plasma andinfected with IBDV(0.01 MOI) after 6h post transfection. Replication effect of IBDV was testedviral plaque formation assay and Real time PCR. Inhibitory ratio of pEC2571-shRNA to IBDVreplication was 87.4ï¼…and viral mRNA was reduced by 90.7ï¼…compared with controls. In indirectimmunofluorescent assay (IFA), the cells treated with pEC2571-shRNA had obviously less VPlprotein positive cells compared with controls. It suggested that the replication of IBDV wasinhibited and the inhibitory effect ofpEC2571-shRNA was specific.2. Influcence of VP3 protein on replication of IBDVIn order to investigate the role of VP3 protein in the viral replication and the influence on viral titerand expression of VP2 protein by inhibition of VP3 expression by RNAi, three siRNAs (siVP3²²²,siVP3³²⁴ and siVP3⁴⁴⁸) directed against a well-conserved region of IBDV VP3 gene were selected.siRNA, prepared by in vitro transcription, and VP3-EGFP fusion protein expression plamidpEGFP-VP3 were co-transfected into Vero cells. Enhanced green fluorescent protein (EGFP)fluorescence intensity of transfected cells was monitored on an inverted fluorescent microscope andFlow cytometry. The results demonstrated that the best inhibitory effect to VP3 protein expressionwas siVP3³²⁴. The primer including siVP3³²⁴ sequence was designed. The shRNA expression vector(pEC324-shRNA) was constructed by amplifing mouse U6 promotor with the primer and cloning topEGFP-C1 with opposite direction. Vero cells were transfected pEC324-shRNA plasma andinfected with IBDV(0.01 MOI) after 6h post transfection. Replication effect of IBDV was testedviral plaque formation assay. VP2 and VP3 protein mRNA levels were tested by Real time PCR.Inhibitory ratio of pEC324-shRNA to IBDV replication was 86.9ï¼…. VP2 and VP3 protein mRNAlevels were reduced by 35.3ï¼…and 91.2ï¼…compared with controls. The results demonstrated that thereplication effect of IBDV reduced when expression of VP3 protein reduced. It was suggested thatVP3 proteins played an important role in the replication of IBDV.3. Influence of VP5 protein on replication of IBDVthree siRNAs (siVP5²⁵,siVP5⁴⁰ and siVP5²²⁷) directed against a well-conserved region of IBDV VP5 gene were selected. Veto cells were transfected with siRNA, prepared by in vitrotranscription, and infected with IBDV(0.01 MOI) after 6h post transfection. The results of viralplaque formation assay showed that Inhibitory ratio of siVP5²⁵, siVP5⁴⁰ and siVP5²²⁷ to IBDVreplication were 6.8%, 0% and 56.0% compared with controls, respectively, siRNA and VPS-EGFPfusion protein expression plamid pEGFP-VP5 were co-transfected into Vero cells. Enhanced greenfluorescent protein (EGFP) fluorescence intensity of transfected cells was monitored on an invertedfluorescent microscope. The effective siRNAs were selected rapidly. In addition, VP5 protein stablyexpressing Vero cell line was selected and the inhibitory effect of the selected siRNA wasdemonstrated on it. The results demonstrated that siVP5⁴⁰ can effectively inhibited expression ofVP5 protein and cannot effectively inhibit the replication of IBDV. The results suggested that VP5was not essential for viral replication.The replication of IBDV was specificly inhibited by RNA interference. This study provided anew approach to prevention and therapy of IBDV. Expression of VP3 and VP5 protein wereinhibited in the course of viral replication first time in this study. The results demonstrated that VP3proteins played an important role in the replication of IBDV and VP5 was not essential for viralreplication. These results help us to understand the replication mechanism of IBDV.