The Establishment Of A Woodchuck Model Infected By The Chimeric WHV-HBV Virus Expressing HBsAg

Objective: 1.To construct WHV-HBV chimeric viruses by taking place of the homologous fragment in the woodchuck virus genome with the genomic fragment encoding hepatitis B virus surface antigen.2.To establish a woodchuck model infected by the chimeric WHV-HBV virus which contains the ‘a’ determinant of HBV S gene and expresses HBs Ag.Methods: 1.Construct the chimeric plasmids WHV-HBV-Sa(Sa-M1,Sa-M2)based on the 1.3 fold WHV genome with the ‘a’ determinant of HBV S gene replacing the corresponding fragments.2.Transfect WHV-HBV chimeric plasmids into Huh7 cells,detect the replication ability of the chimeric viruses in vitro and the HBs Ag expression level in the supernatant of the transfected cel s.3.Hydrodynamically inject the WHV-HBV chimeric viruses into BALB/c mice.Detect the virus DNA in serum by real-time PCR,detect the HBs Ag expression in serum by ELISA,detect viral replication intermediates in liver tissues by Southern blot,and detect the WHc Ag expression in liver by IHC.4.Inject the mouse sera containing chimeric viruses into woodchucks.Detect the viral DNA in serum by PCR,detect the WHc Ab and HBs Ag expression in serum by ELISA.Results: 1.The sequencing results indicated that the chimeric plasmids WHV-HBV-Sa: Sa-M1,Sa-M2 were successfully constructed.2.Transfect plasmids p BS-WHV1.3,M1,M2,Sa,Sa-M1,Sa-M2 into Huh7 cells,respectively.The results showed that the productions of encapsidated viral DNA in the supernatant and in the transfected cells by M1,M2,Sa-M1,Sa-M2 were fewer than p BS-WHV1.3.And the encapsidated DNA levels in the Sa-M1 and Sa-M2 transfected cells were lower than M1 and M2 transfected cells,respectively.HBs Ag expression in the Sa,Sa-M1,Sa-M2 transfected cel s could not be detected by ELISA.3.The chimeric plasmids p BS-WHV1.3,M1,M2,Sa-M1,Sa-M2 were hydrodynamically injected into BALB/c mice,respectively.The serum viral DNA in M1 and M2 challenged BALB/c mice were obviously higher than in p BS-WHV1.3 challenged mice,and M1 and M2 could persistently replicate for day 63 in mice.The serum viral DNA in Sa-M1 and Sa-M2 injected mice were slightly fewer than in p BS-WHV1.3 injected mice.HBs Ag expression was detectable in Sa-M1 and Sa-M2 injected mice.4.Two woodchucks were intravenously injected by the mouse sera containing chimeric WHV-HBV viruses: M2 and Sa-M1,respectively.The viral DNA of M2 and Sa-M1 could be detected in the serum of the challenged woodchuck.WHc Ab and HBs Ag was detectable in the serum of the M2 and Sa-M1 challenged woodchuck,respectively.Conclusion: 1.WHV-HBV chimeric plasmids Sa-M1,Sa-M2 were successfully constructed.2.In the hydrodynamically injected mice with Sa-M1 and Sa-M2,HBs Ag was persistently expressed in the serum.However,the replication ability of the chimeric viruses was lower than p BS-WHV1.3.3.The woodchuck could be infected by the chimeric viruses of M2 and Sa-M1 through the intravenous injection of the mouse sera containing high levels of viral DNA.In the serum of the infected woodchuck,the viral DNA of chimeric viruses was detectable.And WHc Ab and HBs Ag was detectable in the serum of the M2 and Sa-M1 infected woodchuck,respectively.