Bioinformatics Analysis Of Capsid Protein And Construction Of Bivalent Virus-like Particles Of Rabbit Hemorrhagic Disease Virus

Rabbit hemorrhagic disease virus(RHDV)can lead to a fatal infectious disease,called Rabbit hemorrhagic disease,which only infects Oryctolagus cuniculus.All RHDV strains are of the same single serotype,but different genetic subtypes of RHDV have been reported around the world,which exhibit different antigenicity and pathogenicity..At present,not only is the antigenic variation mechanism of RHDV between different subtypes unclear,but there is also a lack of a safe and effective vaccine against both classic RHDV and RHDV2.In view of these reasons,we firstly analyzed the differences between the sequences of classic RHDV and RHDV2,and the amino acid sequences of VP60 with the help of variety of bioinformatics soft wares.Then we constructed a recombinant baculoviruses which carries capsid proteins of both classic RHDV VP60 and RHDV2.Finally,we evaluated the immunogenicity of recombinant baculovirus in animal experiments,which established a foundation for the development of a new bivalent vaccine against both classic RHDV and RHDV2.The research content of this thesis mainly includes the following three aspects:(1)Bioinformatics Analysis of RHDV Genomes: We selected 184 partial and/or complete genome sequences of RHDV and 46 genome sequences of RCV and analyzed those sequences by several bioinformatics soft wares.The mutations of amino acid sites of VP60 lead to the classification of different subtypes of RHDV.And those potential functional divergence-related sites in VP60 consisted of seven variable regions.The potential N-linked glycosylation and phosphorylation sites of VP60 were also predicted using online websites,which may result in biological differences between classic RHDV and RHDV2.(2)Construction of Virus-Like Particles of RHDV: Classic RHDV VP60 and RHDV2 VP60 genes were amplified and inserted into the p Fast Bac vector,and the recombinant bacmid was generated by transforming the recombinant plasmid into DH10 Bac.Subsequently,the recombinant bacmid DNA was used to transfect SF9 insect cells to generate the recombinant baculoviruses.SF9 insect cells infected with the recombinant baculoviruses could both express classic RHDV VP60 and RHDV2 VP60 by western blot and IFA analyses.And the expression reached the highest level at 72 h post-infection.The recombinant VP60 proteins could assemble into Virus-Like Particles by the TEM analysis.(3)Immunogenicity and immunization efficiency test of Bivalent Virus-Like Particles of RHDV: In order to evaluate the immunogenicity and protection against RHDV challenge of the recombinant baculoviruses.Firstly,the recombinant baculoviruses were inactivated and emulsified with an adjuvant to make an inactivated vaccine.SPF rabbits of experimental groups were immunized with the bivalent vaccine and those of control group were immunized with SFM900 II.Serum samples of rabbits were collected at 0,7,14,21 days post-immunization and used to detect the level of specific antibodies,IFN-? and IL-4 by direct ELISA.The outcomes suggested that the humoral and cellular immune responses of the rabbits immunized with the bivalent vaccine were efficiently induced and differed significantly from those of rabbits immunized with the control group.The results of HI assay showed that specific antibodies induced by the bivalent vaccine had the excellent abilities to neutralize viruses.Challenge protection test demonstrated that the bivalent VLPs vaccine provides rabbits with full protection against classic RHDV challenge.The results of IHC and RT-PCR analysis showed that the bivalent vaccine could inhibit the spread of classic RHDV in rabbits.Compared with the control group,rabbits of immunized group didn't show any typical clinical symptoms and pathological changes of RHD,and classic RHDV couldn't also be detected in all organs.By detection the duration of specific antibodies in immunized rabbits,results presented that specific antibodies induced by the bivalent vaccine in rabbits could last at a high level for more than 5 months,which demonstrated the bivalent vaccine provided rabbits with a long-term protection against RHDV infection for long time.In conclusion,according to the results of bioinformatics analysis between a variety of sequences of classic RHDV and RHDV2,we found that the mutations of amino acid sites of VP60 lead to the biological differences of different subtypes of RHDV.We also constructed the recombinant baculoviruses which could express both classic RHDV VP60 and RHDV2 VP60.The results of animal experiment showed that the bivalent vaccine conferred full protection against classic RHDV challenge in rabbits.Besides,it could induce high level of humoral and cellular immune responses and and has effective ability to neutralize both classic RHDV and RHDV2.From these,we speculated that the bivalent VLPs vaccine would also protect rabbits against RHDV2 as well as classic RHDV.In a word,this study laid a foundation for understanding the mechanism of differences between classic RHDV and RHDV2 and the development of novel bivalent vaccines.