Objective: To observe the effects of HBV genotypes on the level of HBsAg in serum and hepatocytes in chronic hepatitis B patients without antiviral therapy.
Methods: Seventy-six chronic hepatitis B inpatients were enrolled into this study, and liver biopsies and histologic diagnosis were performed, and serum samples were collected at the time point of liver biopsy. PCR-RFLP method was adopted to determine the genotype of hepatitis B virus and Abbott Architect HBsAg assay was used to quantify the serum HBsAg. Immunostaining for antigens in liver tissues with monoclonal antibody (for HBsAg) or polyclonal antibodies (for HBcAg) was carried out in consecutive slides. The percentages of hepatocytes for HBsAg stain, hepatocytes for HBcAg nuclear stain and hepatocytes for HBcAg cytoplasm stain were estimated in the ranges of 0 (negative), < or = 1%, 1+; 2% - 5%, 2+; 6% - 25%, 3+; 26% - 50%, 4+; and > 50%, 5+. The distributions of positive cells in slides are described as single or isolated, cluster or widespread. Surface gene was directly sequenced with the serum HBV DNA from 6 patients with genotype B and 8 with genotype C HBV infection, respectively.
Results: Four HBV genotypes were detected in 76 patients: 47 patients with B, 21 with C, 3 with D and 5 were infected by genotype B mixed with C HBV infection. Age, gender, serum HBV DNA level, ALT, AST or histological evaluation (grades and stages scores) were not different between the patients infected with genotype B or C HBV. The level of serum HBsAg was not significantly different between the patients infected with genotype B or C HBV, but the proportions of hepatocytes stained with HBsAg was greater in patients with C type HBV infection than B (P < 0.01). In the liver slides from the patients infected HBV genotype B, HBsAg was stained frequently in single or isolated hepatocytes (22/47), and widespread HBsAg-positive hepatocytes were often seen in the patients with C type HBV (8/21), P < 0.01. In the patients with B type HBV, serum HBsAg was positively correlated with serum HBV DNA (r = 0.674, P = 0.000), proportion of hepatocytes with HBcAg in nucleus (r = 0.534, P = 0.000) and in cytoplasm (r = 0.405, P = 0.004). In the patients with C type HBV infection, serum HBsAg had positive correlation only with serum HBV DNA (r = 0.503, P = 0.017). Proportion of HBsAg positive hepatocytes was positively correlated only with the proportion of HBcAg cytoplasm positive hepatocytes in the patients with B type HBV (r = 0.318, P = 0.029) and no correlation with serum HBsAg, HBV DNA, or proportions of hepatocytes with HBcAg in nucleus. Analysis of the first 40 amino acid sequences of surface antigen showed that variations most existed at amino acid 3, 4, 5 and 8.
Conclusion: Proportion of HBsAg in hepatocytes is significantly greater in the patients with C type HBV than those with B type HBV. Positive correlation between serum HBsAg and viral replication was seems to be more significant in the patients with HBV genotype B infection.