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To investigate the HBV infection and its replication in Chinese patients with chronic liver disease, polymerase chain reaction (PCR) was employed to detect hepatitis B virus DNA in sera of 410 patients with chronic liver disease and liver samples from 188 patients. The HBV DNA detectability in all serum samples was 58%. Among them 100% HBeAg-positive and 58% anti-HBe cases were HBV DNA detectable respectively. However, HBV DNA was also found in 23% HBsAg-negative/anti-HBc positive chronic cases. Furthermore, 30% anti-HBs positive chronic cases who had neutralizing antibody against HBV infection, continued to contain HBV DNA. Our findings indicate that HBV infection and its replication are dominant cause of chronic liver disease and some HBV variants may escape from the protective antibody to induce chronic liver disease, even anti-HBs antibody circulated.

We demonstrated the constitutive polypeptides (PP) of Dane particles employing Western Blot and investigated the antibody response ability of HBV infected subjects to PreS1 PP in comparison with other serum markers from HBV infected individuals. The results indicated that 1) the major reason for discrepant results may be related to the detergents used in the sample solutions and the degree of denaturation the samples had undergone; 2) there are 12 bands in the PAGE-graph of Dane particles. By Western Blot it was confirmed that 5 PP (P24, P27, P36, P39, P42) are derived from S-open reading frame (S-ORF), P21 is associated with C-ORF, P24-25 possesses some epitopes of Pol protein, and P45 and P76 express similar epitopes to human IgG and IgM; and 3) the prevalence of anti-PreS1 PP was 17.24% in the group of healthy persons following latent HBV infection, much higher than that of HBV infected patients (1.21%). The above findings imply that antibody response ability of the host to PreS1 PP is attributing to the outcome of HBV infection. It may play an important role in the elimination of the virus.

To assess the relationship between pre-S proteins of HBV and polymerized human serum albumin (PHSA), a labeled avidin biotin ELISA was used to detect pre-S1, S2 and PHSA receptor (PHSAR) activity. PHSAR activity was only present in the samples positive for pre-S1 and/or S2, but not in the samples positive only for HBsAg. Pre-S1, S2 and PHSAR could in some degree be blocked by preincubating serum with PHSA, and the blocking efficiency of PHSA with respect to pre-S2 and PHSAR was similar, suggesting that pre-S2 is the dominant site for binding PHSA in vitro. We also found that PHSAR activity was detectable in 2 cases positive only for pre-S1, but not pre-S2. Furthermore, PHSA could selectively block pre-S1 and PHSAR activity in 2 cases negative for pre-S2, revealing that pre-S1 also possesses binding ability to PHSA, at least in a small number of cases. Using sandwich ELISA, we demonstrated the existence of complexes of HBV envelope proteins and human serum albumin (HSA) in some HBV infected serum samples. The possible significance of these complexes is discussed.

Intrahepatic HBVDNA of patients with chronic hepatitis B was detected by in situ cytohybridization assay in combination with demonstration of HBsAg and HBcAg expression, and correlated with the foci of hepatic necrosis. It was found that HBsAg and HBcAg expressing sites appeared in the same areas where HBVDNA replicated; when HBsAg and HBcAg simultaneously expressed in the liver, one antigen usually dominated, the other declined. This unbalanced expression may be caused by the gene or transcripts variance which encodes the surface or core antigen, or by the activity of each replication. The other observation is that HBVDNA-positive hepatocytes outnumbered liver cells containing HBsAg and HBcAg in a few cases. It is supposed that a part of cytoplasmic HBVDNA was on replicative phase with gene production expression, on the contrary, another part of cytoplasmic HBVDNA may be out of replication with no gene product expression during chronic hepatitis B. Finally HBsAg and HBcAg expressing sites corresponding to HBVDNA-positive hepatocytes were closely attached to the foci of hepatic necrosis. There was no significant difference in the frequency of HBsAg and HBcAg expression correlating to hepatic necrosis.

To assess the significance of intrahepatic expression of pre-S1 and S2 proteins of hepatitis B virus (HBV) in patients with HBV infection, an indirect immunoperoxidase technique employed monoclonal antibodies to pre-S proteins was used to detect pre-S1 and -S2 proteins in 80 liver specimens. The frequency of pre-S1 and -S2 proteins was 61.3% and 51.3%, respectively, and the co-expression of pre-S and HBsAg occurred in most specimens. The preferential expression of pre-S1 and -S2 in HBcAg-positive specimens suggests that pre-S proteins are associated with HBV replication. Membranous expression of both pre-S1 and -S2 is associated with inflammatory activity and liver cell necrosis. Furthermore, our results show that T cells, not NK or B cells, were the predominantly infiltrating cells in necrotic foci with pre-S expression. Almost all of these T cells may express HLA-DR antigen simultaneously; therefore, they are activated. In conjunction with these data, we conclude that, as the essential components of HBV envelope proteins, pre-S proteins may play an important role in resulting in liver cell necrosis.

To assess the significance of pre-S proteins expression during chronic hepatitis B virus (HBV) infection, we developed a sensitive labeled avidin biotin ELISA to detect pre-S1 and pre-S2 proteins. In serum specimens from 80 patients with chronic liver disease, the frequency of pre-S1 and S2 proteins was 53.7% and 47.5%, respectively. Furthermore, it reached 87.8% and 77.6%, respectively, in cases with chronic HBV infection, indicating that pre-S proteins are usually expressed in sera with chronic HBV infection. We found that the expression of pre-S proteins is closely associated with HBV replicating markers, such as HBV DNA, HBcAg and HBeAg, in sera of patients with chronic HBV infection. Both pre-S1 and S2 proteins were often concurrently expressed in liver and serum with chronic HBV infection. However, the frequency was slightly higher in liver than in serum, suggesting that it may be clinically valuable to detect pre-S proteins in serum and liver simultaneously to determine the status of HBV infection. Our results also indicated that pre-S proteins expression in serum can serve as markers of HBV infection, but cannot be used to estimate the severity and activity of hepatic pathology.