| BackgroundThe presence of hepatitis B virus genomes in the liver(with detectable or undetectable HBV DNA in the serum)of individuals testing nagetive for HBV surface antigen(HBsAg)is termed occult HBV infection(OBI).When detectable,the amount of HBV DNA in the serum is usually very low(<200IU/ml).Carriers of OBI is a source of hepatitis B virus infection in the case of blood transfusion,organ transplantation,dialysis.Many studies suggest that OBI may be a risk factor for hepatocirrhosis and hepatocellular carcinoma.Hepatitis B virus core protein is essential for virion formation including pgRNA and pol polymerase capsidization,assembly of icosahedral capsid,maturity of virion.In fact,a series of HBV mutants are associated with OBI,especially S and BCP mutation.However,the role of hepatitis B virus core region(C region)mutations in the occurrence of OBI still need to be elucidated.ObjectiveTo explore the role of hepatitis B virus core protein(C region)mutation in occult hepatitis B virus infection(OBI).MethodsIn this study,plasmids with C gene mutations were constructed by overlapping extension PCR and site-directed mutagenesis PCR method.The distribution of HBcAg in liver tissue were detected by immunofluorescence.The expression of HBcAg was detected by western blot.The levels of HBsAg and HBeAg were tested by immunohistochemistry.The distribution of HBcAg in liver tissue were detected by immunohistochemistry.The level of HBV RNA in liver tissue was tested by RT-PCR method.ResultsThe OBI strains and wild-type strain with single,double and triple mutations in HBV C gene were successfully constructed by overlapping PCR and site-directed mutagenesis PCR method.There were three types of HBcAg localization in HuH7 cells.Type A were found in wt,SZB,SZA-R62W,wt-P50H,wt-L55I,wt-W62R,wt-S74G,wt-S87G,wt-L95I,wt-I97L,wt-T142M,wt-T147A,SZA-H50P/R62W,SZA-P50H/R62W/G74S,SZA-R62W/G74S,wt-P50H/L95I.Type B were found in SZA,SZA-H50P,SZA-I55L,SZA-G74S,SZA-G87S,SZA-I95L,SZA-L97I,SZA-M142T,SZA-A147T.Type C were found in wt-P50H/W62R,wt-P50H/W62R/S74G,wt-P50H/W62R/S87G,wt-P50H/W62R/T147A,wt-W62R/L95I.The HBcAg level in SZA,SZA-G87S,SZA-L97I,SZA-M142T,SZA-A147T,wt-P50H/W62R/S74G were lower than that in wild-type.The HBcAg level in SZA-H50P,SZA-I55L,SZA-R62W,SZA-G74S and SZA-I95L was higher than that in SZA.Intracellular and extracellular HBeAg production of OBI strains SZA and SZB were lower than that of wild-type strain.For SZA-H50P,SZA-I55L,SZA-R62W,SZA-G74S and SZA-I95L,intracellular and extracellular HBeAg production were slightly higher than SZA.However,no significant differences were found in SZA,SZA-G87S,SZA-L97I,SZA-M142T and SZA-A147T.HBeAg level of SZA double and triple repaired substitution were higher than that of single repaired substitution.The wide-type single mutants wt-P50H and wt-W62R resulted in a decrease of HBeAg production.However,the corresponding HBeAg in the double mutants wt-P50H/W62R and wt-W62R/L95I,triple mutants wt-P50H/W62R/S87G,wt-P50H/W62R/T147A were lower than observed for single mutants.For OBI strain SZA,HBcAg were undetectable in the liver of Balb/c mice.SZA repaired substitution SZA-H50P,SZA-I55L,SZA-G74S,SZA-G87S,SZA-L97I,SZA-M142T and SZA-A147T did not express HBcAg as well.However,SZA-R62W,SZA-H50P/R62W,SZA-R62W/G74S,SZA-P50H/R62W/G74S resulted in a increase level of HBcAg.HBcAg of wide-type single,double and triple mutants were similar to that of wide-type.SZA-R62W resulted in a increase serum level of HBeAg in mice,no significant differences were found in SZA-H50P,SZA-I55L,SZA-G74S,SZA-G87S,SZA-I95L,SZA-L97I,SZA-M142T and SZA-A147T.The wild-type single mutants wt-P50H,wt-W62R,double mutants wt-P50H/W62R,wt-W62R/L95I,triplet mutants wt-P50H/W62R/S87G,wt-P50H/W62R/T147A were lower than observed for the other single mutants.For OBI strain SZA,HBcAg were undetectable in mice liver at week 1 and week 7.For wide-type strain,HBcAg was significantly increased at week 7.At week 1,the HBV total RNA and C region RNA levels in SZA were lower than that of wild-type.At week 8,however,the HBV total RNA and C region RNA of SZA were higher than that of wild strain.For SZA,HBeAg persisted in a low level(10-15 S/CO).For wild-type strain,HBeAg persisted in a high levels(500-1500 S/CO).The combination of P50H,W62R,S74G,S87G and L95I in the C region of OBI strain changed the expression level of HBcAg,HBeAg and HBV RNA,among which W62R played the most important role.The mutation of hepatitis B virus C region plays an important role in the formation of OBI.ConclusionThe combination mutations of P50H,W62R,S74G,S87G and L95I in the C region of OBI strain are related to the expression level of HBcAg,HBeAg and HBV RNA,among which W62R plays a major role.Hepatitis B virus core region mutations are associated with the occurrence of OBI. |
