Whole Genome Sequence Analysis Of An ALV-J Isolate And Evalution On The Effects Of A Recombinant Adeno Virus Mediated RNAi And A DNA Vaccine Against ALV-J

Avian leukosis (AL) caused by avian leukosis virus (ALV) is one of common tumor diseases that could also cause immune suppression in poultry. Subgroup J ALV (ALV-J) is the most common and most harmful subgroup prevailing now in the industry. The infection of ALV in chickens may result in the reduction of egg production, immunosuppression, bleeding, tumor occurrence and growth retardation. However there is no yet effective vaccine or drug for the prevention and treatment for the disease. Eradication has been the major measure for the control of disease. A recombinant DNA vaccine and a RNAi recombinant adenovirus had been developed in the laboratory for the purpose to provide supplemental measures to the eradication program of AL. And the efficacy of these two products on chickens was evaluated in the study here. Also an isolated ALV-J strain was studied by the complete genome sequencing and analysis.The samples of tumor tissues of diseased birds were taken and cultivated onto DF-1 cell cultures, and a strain of ALV-J was isolated by the confirmation of IFA, ELISA and PCR on the cultured cells and named as GDMM. The complete genome sequencing of GDMM with specific primers was done and compared the sequences with those of another 14 strains in the origins of Guangxi, Guangdong and other places in China. The results showed that GDMM had higher similarity with Guangxi strains GX14ZS14, GX14HG03 and Guangdong strain GDQY1201, and the similarity between GDMM and GX14ZS14 was the highest by 96.8% and they were in the same minor cluster.To evaluate the efficacy of the RNAi recombinant adenovirus pAd-gp85-shRNA2, breeding hens that were positive of p27 antigen in egg white samples and positive of viraemia condition were chosen for the experiment. The experimental group with 28 hens was inoculated with 100μL recombinant adenovirus (1.6×109PFU per bird) and the control group with another 28 hens was injected with 100μL PBS. The body-weight of the hens and positive rate of the meconium p27 antigen of their offsprings were measured pre-and post-vaccination. The p27 antigen in egg white samples and the viraemia condition of the hens were detected during the experiment. And the blood and secreta samples were taken from 5 birds in each group every week post-vaccination for the quantitative detection of ALV-J and the recombinant adenovirus respectively. The results showed that there was no significant difference in body-weight between the experimental and the control groups.25% hens with both the egg-white p27 antigen and viraemia negative were found in the experimental group post-vaccination, and 19% of their offsprings were also become the meconium p27 antigen negative. The copy-number of ALV-J in the blood of hens was decreased post-vaccination and with the lowest at 2 weeks post-vaccination, at that moment the interference efficacy of the recombinant adenovirus against ALV-J was at its highest of 68%. Recombinant adenovirus could still be detected at 4 weeks post-vaccination, which was consistent with the result that the copy-number of ALV-J at 4 weeks post-vaccination in experimental group was less than that of the control group. The results showed that recombinant adenovirus could still interfere the replication of ALV-J as long as 4 weeks post-vaccination.The efficacy of the constructed recombinant DNA vaccine pVAX1-Ub1-gp85-p27-p10 was evaluated in the study. Breeding hens that were negative of both egg-white p27 antigen and serum-antibody against ALV-J were chosen for the experiment.25 hens in the experimental group were vaccinated with the recombinant DNA vaccine and 25 hens in the control group were injected with PBS, and the birds were boosted at 2 weeks later. The body-weight and the positive rate of the serum-antibody of the hens and the maternal-antibody of their offsprings against ALV-J were detected pre-and post-vaccination. The results showed that there was no significant difference in body-weight between the experimental group and the control group, while 28% of the hens in the experimental group was become ALV-J antibody positive and 8%of hens with their offsprings also maternal-antibody positive.The results of the study demonstrated that a strain of ALV-J was successfully isolated and identified and the whole genome sequencing and analysis was completed. RNAi recombinant adenovirus could interfere the replication of ALV-J effectively and the recombinant DNA vaccine could induce the production of antibody in the hens and the maternal antibody in their offsprings.