Research On Mutual Interaction Between Rabbit Ferritin Heavy Chain And Rabbit Haemorrhagic Disease Virus

Rabbit haemorrhagic disease virus (RHDV) is a calicivirus of the genus Lagovirus that causes an acute, potent, highly contagious disease in adult rabbits, namely rabbit haemorrhagic disease (RHD). First described in China in1984, the virus rapidly spread worldwide and is nowadays considered as endemic in several countries. The lack of a cell culture system has been hampering the study of RHDV pathogenesis. The indirect strategies have been employed for the study of the pathogenesis of RHDV. HBGA H type2, for example, identified as an attachment factor for RHDV. But, hepatocytes, the main cellular target for viral replication, have been shown not to express HBGA. This indicates the existence of at least additional hepatic cellular receptors go beyond the the attachment of the virus to host cells through histo-blood group antigens. Research on the hepatic cellular attachment factors is of significance for the pathogenesis of RHDV. Previously, the eukaryotic expression library of rabbit hepatocyte was constructed by our laboratory and screened by expression cloning to search for the protein to interact with VP60, which is the capsid of RHDV. Rabbit ferritin heavy chain is one of candidate proteins VP60.To verify the interaction between VP60and rabbit ferritin heavy chain, the FTH gene were amplified by RT-PCR from total RNA of rabbit hepatic cells and cloned into pET32a(+) vector to express in E.coli. The recombinant protein (rFTH) was expressed with IPTG induction and purified by Ni-NTA agarose. The interaction between FTH and VP60was identified by GST pull down and detected by ELISA coating with VP60as the capture antigen; the interaction between FTH and RHDV was identified by co-immunoprecipitation and detected by ELISA coating with RHDV. The influence of FTH to the hemagglutination activity of RHDV was studied by hemagglutination inhibition. The mixtures of4096HAU RHDV and358.4μg,716.8μg,1075.2μg,1443.6μg FTH, respectively, named A, B, C, D group, were used to challenge the experimental animals. The viral load of the dead of experimental animals’liver was detected by fluorescence quantitative method and Hemagglutination. The titer of antibody to RHDV in the survival of experimental animals’serum was detected by Hemagglutination inhibition.The results of GST pull down and ELISA showed the interaction between FTH and VP60. The results of Co-IP and ELISA proved that FTH interacted with RHDV. As a result of Hemagglutination inhibition,1.4ug rFTH can neutralize the hemagglutination activity of8HAU RHDV. In animal experiment, there were3and4survivals in C group and D group respectively. The survival time of the dead of experimental animals was less than4days. The virus load of the dead of experimental animals’ liver was close. The virus load in the survival of experimental animals in D group was lower than in C group. And the HI titer of the experimental animals’ serum in D group was lower than in C group.In this study, the interaction between FTH and VP60was identified by GST pull down and detected by ELISA coating; the interaction between FTH and RHDV was identified by co-immunoprecipitation and detected by ELISA. FTH can neutralize the hemagglutination activity of RHDV, which was demonstrated by HI experiment. The animal experiment show that FTH can neutralize RHDV so as to inhibit the infection ability of RHDV.