Genetic Variation Analysis And Development Of Baculovirus Vector Inactiveted Vaccine Of Rabbit Hemorrhagic Disease Virus

Rabbit hemorrhagic disease virus(RHDV)belongs to the family Caliciviridae and is the etiological agent of the rabbit hemorrhagic disease(RHD).The capsid protein(VP60)is the exclusive structure protein and can make rabbits produce neutralised antybody and directly related to immune response.The cell culture in vitro method of rabbit hemorrhagic disease virus has not yet been established,so now the widely used vaccine of RHDV is a kind of inactived vaccine.Although inactived vaccine has a good immune effect,it has its own inevitable deficiencies.In the research,the genetic variation of RHDV prevalent strains in China was analyzed.Then recombinant baculovirus which express the VP60 gene of rabbit hemorrhagic disease virus was constructed and identified,and expressed in Sf9 insect cells.The immunoprotection effect of the expressed VP60 protein had been detected.This study should be useful for the research of genetic engineering subunit vaccine of RHD.The main research contents are as follows:1.Phylogenetic analysis of rabbit hemorrhagic disease virusTo investigate the genetic variability and evolution of rabbit hemorrhagic disease virus(RHDV)strains in China,190 RHDV strains including 41 Chinese isolates were analyzed.(1)The analysis of all Chinese RHDV isolates showed that WX/China/1984?HB/2014 and HB strain belong to RHDV G2 group,and the other 38 Chinese isolates were classified in genogroup G6(RHDVa).(2)VP60 gene sequences of eight new isolates collected from farms with RHD occurrences from six different provinces in China between 2009 and 2014 were analyzed,and compared with the reference sequence of the vaccine strain WF/China/2007.We conducted a comprehensive analysis of the Chinese RHDV strains,including hemagglutination tests,western blot and immunosassays of capsid proteins,and phylogenetic analysis,and identified a new distinct antigenic variant.Specifically,strain HB/2014 collected in North China was identified as a non-hemagglutinating strain,and belongs to the original RHDV(G1-G5)group.The other seven isolates were classified in genogroup G6(RHDVa),which was widely distributed across China before 2014,and was thought to replace the earlier groups.Antigenic characterization of the VP60 genes revealed a large degree of nucleotide sequence divergence between HB/2014 and the other Chinese strains.However,the current vaccine showed complete cross-protection against HB/2014 challenge in inoculated rabbits.(3)To gain a better understanding of molecular evolution of RHDV,we searched for recombination events by analyzing all full-length RHDV capsid VP60 sequences of Chinese isolates belonging to the genogroups 2 and 6.Our results revealed a recombinant origin for NanBu/China/2011 isolate.This recombination event occurred between G2 and G6 strains with two breakpoints located at nucleotide positions 393 and 1079 of the VP60 sequence.Phylogenetically the NanBu/China/2011 strain clustered with genogroup G6 in all the capsid except in the fragment between nucleotides 394 and 1078,where it clustered with genogroup G2.Collectively,these data provide new tools and insight for further understanding the molecular evolution of RHDV in China,and the circulation RHDV in the populations should be carefully monitored.2.Construction,identification and stability of recombinant baculovirus expressing the VP60 gene of rabbit hemorrhagic disease virusThe objective of this study is to develop genetic engineering vaccine to rabbit hemorrhagic disease.RT-PCR was used to amplify the capsid protein VP60 gene of rabbit hemorrhagic disease virus(RHDV).After being cloned and transformed,the recombinant shuttle vector,Bacmid-VP60,was constructed and transfected into Sf9 insect cells,then a recombinant baculovirus(BAC-VP60 strain)was obtained.The identification by IFA,SDS-PAGE,Western blotting,HA,HI and electronic microscope showed that the recombinant protein of VP60 was expressed in Sf9 insect cells and formed Virus-like particles(VLPs)with a diameter of approximately 40 nm.Then the stability of recombinant baculovirus on Sf9 cells was studied.The results showed that the culture time of each generation of Sf9 cell needs 48-54 hours in 60 generation.The PH of the Sf9 cell was 6.2,and almost no change of PH until cell culture.Sf9 cells are free of bacteria,mold,mycoplasma,exogenous virus and tumorigenicity.P8 generation of BAC-VP60 was inoculated in the 5th,10th and 60th generation of Sf9 insect cells.The TCID50 and HA were basically unchanged.3.Research on the protection of rabbit hemorrhagic disease virus baculovirus vector inactivated vaccineTo study the protective effect of rabbit hemorrhagic disease virus baculovirus vector inactivated vaccine through evaluating the humoral and cellular immunity,rabbits were injected with RHDV baculovirus vector inactivated vaccine and RHD inactivated vaccine,with PBS for control group.The result of experiment showed that the IgG antibody with a significant increase(P<0.05)after 7 days post immunization induced by RHDV baculovirus vector inactivated vaccine and inactivated vaccine.The IL-2,IL-4 and IFN-y titers were also significantly increased(P<0.05)compared with those of the control group 3 days later in RHDV baculovirus vector inactivated vaccine compared with PBS.But there was a significant difference in IL-4 and IFN-y between RHD inactivated vaccine and control,but not in IL-2.Rabbits immunized with RHDV baculovirus vector inactivated vaccine and RHD inactive vaccine were all survival after challenged with RHDV WF/China/2007 strain with no clinically symptoms and pathological changes.The minimum immune dose of RHDV baculovirus vector inactivated vaccine is 1 mL each rabbit(the HA titer before inactivation is 1:64).The immunity to RHDV kicks in 7 days after using the RHDV baculovirus vector inactivated vaccine.The immune-protection test shows that RHDV baculovirus vector inactivated vaccine can also provide full protection against prevalent strains of different genogroups collected in different provinces in China.In conclusion,rabbit hemorrhagic disease virus baculovirus vector inactivated vaccine could induce effective humoral and cellular immunity and completely protect rabbits against RHDV infection.