Cloning And Sequence Analysis Of The S1 Gene Of Avian Viral Arthritis Virus Isolates And Detection Of AVAV By RT-PCR

The Avian Viral Arthritis Virus (AVAV) isolated in Inner Mongolia and Tianjin(AVAVB-98,C-98,G-98,T-98) was propagated,and the total RNA of virus was extracted.According to the published S1 gene sequence of ARV-S1133 strain in GeneBank,two pairs of primer were designed,one is sequencing primer,the other is diagnostic primer.The S1 gene of virus was amplificated with sequencing primer by RT-PCR and then cloned into pGEM-TEasy plasmids and then sequenced.Each of the acquired sequences contains 1643bp,and all congtain three ORFs(ORF1,ORF2 and ORF3) and the nontranslated region.P10,P17andσ3 protein was encoded by ORF1,ORF2 and ORF3 respectively.The result of sequence comparison indicated:there aren't differences in P10 protein and P17 protein among the four isolates of AVAV,and only three nucleotides difference inσ3 gene;their homology is above 95%,compared with the four isolated AVAV strains with other reference strains except ARV-138 and Nelson Bay Virus(NBV).This indicated that the S1 gene's dependablity is high between the AVAV isolated from China and reference strains.The virus RNA was amplificated with diagnostic primer by one step RT-PCR,then established RT-PCR diagnostic method,and detected this method's specificity and sensitivity.The results indicated that the one step RT-PCR method can detect the nucleic acid of standard strain virus (ARV–S1133) and locality isolated strains(B-98,C-98,G-98,T-98),which were propagated in chick embryo.We get a 435bp DNA fragment,which is the same with we expected,while the detection results of other reference etiological agents(NDV,IBDV,IBV,ILTV,EDS76V) were negative;the minimum ARV RNA's detection level of this RT-PCR method is 0.5ng, this illustrated that this method is specific and sensitive for the detection of Avian reovirus.