Development Of The Real-Time PCR Methods For The Detection Of ALV And The Study On The Inhibition Of ALV-J With The Recombinant Adenovirus Mediated RNAi

Subgroup J avian leukosis virus (ALV-J) is one of the common exogenous avian leukosis virus (ALV). Since the 1999's first isolation in the commercial broilers in China, ALV-J infections have been reported all over the country. ALV infection could cause big economic losses in poultry industry by the growth retardation, the reduction of performance such as the decreases of egg production and their fertility, tumors and immunosuppression in birds. Since the lack of effective medicines to treat and control ALV infection in the supplemental to the eradication program at present, there is in badly need of an effective method for the antiviral infection.Five specific pairs of primers were designed for the Real-time PCR detection for the transcriptions of genes protease PR, reverse transcriptase RT, integrase IN, envelop protein gp85 and gp37, respectively based on the sequences of gag, pol and env genes of ALVs in GenBank. The specific PCR productions were recovered and cloned, and prepared for the standard plasmid samples. Ten-fold series dilutions of the plasmids were prepared for establishing standard curves and melting curve by SYBR Green I Real-time PCR. The results showed that the amplification efficiency of the standard curves were 92.6% to 100.5% with the correlation coefficient greater than 0.994. The analysis of melting curve and specificity assay demonstrated that a good specificity was shown as only ALV could be detected by the designed primers and the results of the sensibility assay showed that at least one order of magnitude could be detected. Therefore, the developed Real-time PCR methods could be utilized to detect the mRNA transcription level of ALV genes.In the study, RNAi recombinant adenoviruses were constructed to inhibit the replication of ALV-J HG03. The relative expression of mRNA of PR, RT, IN, gp85 and gp37 were measured and corrected by the detection of internal control of GAPDH gene. And the inhibition effect of the RNAi could be evaluated by comparing to the control group. Three pairs of siRNAs were designed based on the sequences of gp85 gene of HPRS103 (Z46390.1) and an ALV-J isolate HG03, with the reference to the design rule of siRNA and the demand of the shuttle vector pHBAd-U6-RFP. Restriction sites of EcoR I and BamH I were inserted into the siRNAs to help to the ligation with the shuttle vectors. Then, RNAi vectors and backbone plasmids were co-transfected into HEK 293 cells, and the recombinant vectors named pAd-gp85-shRNA1,pAd-gp85-shRNA2, pAd-gp85-shRNA3 and the none-inserted blank vector pAd-N were obtained and confirmed by the sequencing of the vectors. The vector seeds were then max-prepared and tittered, and the final concentrations of 1.26×1010PFU/mL, 1.58×1010PFU/mL,2.6×1010PFU/mL and 2.0×1010PFU/mL were harvested respectively for pAd-gp85-shRNA1, pAd-gp85-shRNA2, pAd-gp85-shRNA3 and pAd-N.The inhibition effects of RNAi of the recombinant adenovirus were evaluated on DF-1 cells by both the mRNA levels of the target genes and the expression of envelope protein gp85 of ALV. Firstly, the multiplicity of infection (MOI) of the recombinant adenovirus on was tested, and the value of 500 was got. Secondly,500 MOI pAd was infected onto the monolayer DF-1 cells,8 hours later, the cells were infected with 1 X 103 TCIDso of HG03. The mRNAs of HG03 genes were detected by the developed Real-time PCR methods. The results showed that the Ads could inhibit the gene transcriptions of HG03 by the inhibition efficiency on gp85 transcription were 51.6% to 82.0%, and with the highest inhibition efficiency of 82.0% by ppAd-gp85-shRNA2. The different inhibition efficiencies on the other four genes were also detected. The result of IFA against the envelope protein gp85 demonstrated that the fluorescence of RNAi group was weaker than that of the control group, and no significant difference was found between the positive group and the non-inserted blank vector group, and consistent with the results of Real-time PCR detections.The study successfully constructed the RNAi recombinant adenoviruses that targeted on the gp85 of ALV-J strain HPRS103, and a good inhibition effect on the replication of ALV-J isolate HG03 was observed while it was evaluated on the DF-1 cells.