Construction And Immunogenicity Study On Recombinant Canine Adenovirus Type 2 Expressing The Vp60 Gene Of Rabbit Hemorrhagic Disease Virus

Rabbit hemorrhagic disease(RHD), caused by rabbit hemorrhagic disease virus(RHDV), is an acute, lethal and highly contagious disease in adult rabbits. The disease is characterized by acute necrotising hepatitis and respiratory system hemorrhage and results in significant losses in the rabbit meat and fur industry in many countries. First reported in China in 1984, the disease rapidly spread worldwide. Currently, RHD prevention and control in rabbits relies mainly on vaccination. As this virus does not replicate in cell culture systems, until now, tissue-source vaccine is still common used to control the disease. Although the vaccine is safe and effective, its application has been restricted because of high cost, biosecurity and animal welfare. Therefore, safe, effective, and affordable RHDV vaccines are still being sought. The capsid protein VP60 is the major structure protein and protective antigen of RHDV, which plays an important role in the induction of immune response against viral infection. Therefore, the VP60 is the target protein for developing novel vaccines against RHDV.Canine adenovirus type 2(CAV2) is a good vector and has most of the attractive features including easy culture, high virus and protein production. CAV2 is not pathogenic in rabbits and is safe for rabbits. Therefore, CAV2 can be used as a vector for developing vaccine against RHDV. This research was aimed at constructing a recombinant CAV2 expressing RHDV vp60 gene as a candidate vaccine strain to develop a novel vaccine against RHDV.In order to construct recombinant canine adenoviruses using ligation in vitro method, a backbone vector containing the full-length genome of CAV2 and a shuttle vector with a deletion of 1478 bp in the nonessential E3 region were constructed firstly. The backbone plasmid p CAV2 was constructed by cloning the complete genome of CAV2 into a modified p Shuttle-CMV using homologous recombination in E.coli BJ5183. The shuttle plasmid p UC-ΔE3-CMV was generated by subcloning a fusion fragment containing the flanking sequences of the CAV2 E3 region and expression cassette sequences from pc DNA3.1(+) into modified p UC18. The backbone vector and the shuttle vector had unique Nru I and Sal I sites that were used to insert exogenous genes into the CAV2 E3 region. Using this system, an egfp gene was inserted into the shuttle plasmid and cloned into the backbone plasmid using two unique Nru I and Sal I sites. Transfection of MDCK cells with the recombinant adenovirus genome containing the egfp expression cassette resulted in recombinant virus r CAV2-EGFP. Green fluorescence was observed in the cytoplasm of MDCK cells by fluorescence microscope. Growth curves of r CAV2-EGFP and CAV2 were similar. This result indicated that the strategy of constructing recombinant adenovirus type 2 using p CAV2 and p UC-ΔE3-CMV by ligation in vitro is feasible. It provided a technique platform for construction of r CAV2-VP60.The RHDV vp60 gene was amplified by PCR from the plasmid pc DNA-VP60 and cloned into the shuttle vector p UC-ΔE3-CMV with the right orientation. Then the expression cassette containing vp60 gene was cloned into the backbone vector p CAV2 using two unique Nru I and Sal I sites. The recombinant plasmid p CAV2-VP60 was transfected into MDCK cells after linearized by Asc I. Then a recombinant virus r CAV2-VP60 was obtained. PCR result showed that the vp60 gene was successfully inserted into r CAV2-VP60. After 20 times of passages, the vp60 gene can be still detected, which showed that r CAV2-VP60 had stable genetic character. The result of Western blot and IFA assay indicated that r CAV2-VP60 can express VP60 protein in MDCK cells. The growth curve showed that r CAV2-VP60 had similar growth characteristic compared with CAV2 and reached peak titers of 107.5 TCID50/0.1m L.RHDV-free rabbits were immunized with r CAV2-VP60、CAV2 and RHDV inactive vaccine at a titer of 107 TCID50/1m L and boost after 2 weeks. The result showed that RHDV specific antibody can be detected at 1 week after prime-boost and reached highest level at 3 weeks after prime-boost in r CAV2-VP60 and RHDV inactive vaccine group. r CAV2-VP60 induced significantly T Lymphocyte proliferation and cytokines(IFN-γ and IL-4) secretion. Rabbits immunized with r CAV2-VP60 and RHDV inactive vaccine were all survival after a lethal challenge with the RHDV HYD strain and no clinically symptoms and pathological changes appeared.In sum, we successfully constructed a recombinant virus r CAV2-VP60 expressing RHDV vp60 gene. r CAV2-VP60 had similar growth characteristic compared with CAV2 and had stable genetic character. The result of rabbits experiment showed that r CAV2-VP60 was able to induce a significant humoral and cellular responses against RHDV. The rabbits immunized with r CAV2-VP60 were completely protected against RHDV HYD strain challenge. This research provided a foundation for development a new recombinant vaccine against RHDV.