Inhibition Of Infectious Bursal Disease Virus Replication By RNA Interference Technology

Infectious Bursal Disease (IBD) is an acute, high contact, virulent infectious disease. It is oneof the most important causes of economic loss to poultry industry worldwide. The etiological agentis Infectious Bursal Disease Virus (IBDV). IBDV has so strictly tropism to the host lymphocyte andhas so variable genome that it is hard for the traditional vaccine design strategies to have the idealimmunity effect. Therefore, exploring the new preventing and controlling method has the vitalsignificance.As a new effective antiviral tool, RNA interference (RNAi) has a different mechanism fromclassic vaccine which is based on the immunogenicity of proteins. It is a post-transcriptionalmechanism of sequence-specific gene silencing that initiated by double-stranded RNA (dsRNA). Inmammalian cells, short interfering RNA (siRNA) (21-25nt) can specifically silence the expressionof the corresponding gene. It provides a new method in anti-virus research fields.This research in view of IBDV in RNA polymerase (VP1) the coding gene, main structureprotein VP2, the VP3 coding gene, used http://www.ambion.com the target site screening and thedesign tool, separately selected 3 specific siRNA sequence (VP1:siRNA618, siRNA1115 andsiRNA2571, VP2: siRNA989, siRNA1143 and siRNA1311, VP3: siRNA222, siRNA324 andsiRNA448). Simultaneously a non-specific siRNA sequence (siRNA-CON) was designed asnon-specfic control. These siRNAs were synthesized by transcription in vitro.In this study, Vero cells were infected by IBDV after being transfected with these siRNA byliposomes. To research the inhibition effect of VP1-siRNA, VP2-siRNA, VP3-siRNA, real-timePCR was used to detect the synthesis of IBDV RNA and virus titration was measured by PFU(Plague-forming unit). VP2-siRNA, VP3-siRNA were transfected with recombinating plasmidpEGFP-VP2, pEGFP- VP3 that expressing Green fluorescent protein (GFP) into Vero cells. Theratio of expressing Green fluorescent protein cells were detected by fluorescent microscope. Thismethod was used to screen rapidly the effective VP2-siRNA, VP3-siRNA. Eventually, the real-timePCR, PFU and flow cytometry were used to detect the inhibiting result confirmed gene silencingresult.This study provided a primary material for anti-IBDV infection by RNAi and provided a newapproach to study of the function of viral proteins in IBDV infection.