Detection Of Avian Leukosis Virus By Real-time PCR And Study On Inhibition ALV-J Replication By RNA Interference

Avain leukosis(AL) is a neoplastic avian disease infecting most species of birds caused by Avain leukosis virus(ALV),which is one member of the family retrovirus, belongs to C type retrovirus group. Vertical transmission is main form of the virus. It is immunotolerance for chicken and without immune response to vaccines,and the antibody can't provide cross protection between subtypes of ALV, so vaccines and antiviral drugs are inefficient, development of a feasible method is needed. RNA interference(RNAi) reside in living nature,which is viewed as a conserved ancient mechanism protecting genomes from nucleic acid invaders. RNAi has a different mechanism from classic vaccine which is based on the immunogenicity of proteins.It is a post-translational mechanism of sequence-sepecific gene silencing that initiated by double-stranded RNA(dsRNA).From procaryote to eumycete, plants and invertebrate .In current years, this technique has been used to suppress viral infection in cell culture or animal experiment, which including human immunodeficiency virus, influenza virus, hepatitis B virus,foot-and-mouth disease and so on,which has been raised expectations about the use of RNAi as a new antiviral strategy.In this study,we have established method on detection ALV and ALV-J by real-time PCR.Sensitivity of this method is 100 fold common PCR.This establishes foundation for early diagnosis of AL pathogeny. Meanwhile the method is utilized to evaluate effect of inhibiting virus replication in cellular level by miRNAs bargeting to gag,pol,env gene of ALV-J.In the study, 13 miRNAs targeting to gag,pol,env gene of ALV-J were designed and synthesized, which based on the principle of miRNA designing .They were cloned to eukaryotic expression vector pcDNA6.2.Those coloned plasmids were transfected into DF-1 cells, ALV-J(Shang dong strain,SD strain) inoculation was conducted the coloned plasmids transfection in 24 hours. Indirect Immunofluorescence (IFA) was conducted ,which ALV-J inoculated DF-1 cell at 120h. The cells culture and supernatant of containing the ALV-J were collected at 72 hours after infection. Inhibition effect of virus is detected by Western-blot and Real-time PCR in cellular level. However, Most virus can escape from RNAi mediated antiviral therapy by selection of mutations in the targeted sequence. To prevent viral escape, several RNAi sequences may be combined. So we screened three cloned plasmids which have been verificated efficient to viral replication. we made the multitarget series connection miRNAs expressing vector, which could express two or three miRNA sequences of gag, pol and env at the same time once the vector were transcribed. The recombinated miRNAs plasmids were tested to detect their co-inhibition effect by compared with single miRNA plasmid.The result of the experiment illustrated we screeded seven miRNA expression plasmids targeting to gag,pol,env gene of ALV-J,which had apparente inhibition action on viral,replication.Inhibition efficiency of virus replication is 17.5%~86.0%.We successfully constructed the multitarget series connection miRNAs expressing vector,Inhibition efficiency of virus replication is 85.0%~91.2%which have the stronger inhibition effect than the single miRNA expressing vector, so multitarget miRNA strategy may be a hoped therapeutic approach to attack escape prone viral pathogens.Thereby this study settled foundation for screening of resistance gene and breeding for disease resistance.