SOUTHERN-BLOT ANALYSIS AND SIMULTANEOUS IN-SITU DETECTION OF HEPATITIS-B VIRUS-ASSOCIATED DNA AND ANTIGENS IN PATIENTS WITH END-STAGE LIVER-DISEASE

To gain new insights into the pathogenesis of hepatitis B virus-induce d chronic liver disease, we have used nonisotopic in situ detection me thods for the simultaneous analysis of hepatitis B virus DNA and antig ens at the single-cell level. Paraffin-embedded liver specimens from 2 3 cirrhotic patients (12 HBsAg positive and 11 HBsAg negative) who und erwent liver transplantation were evaluated by in situ hybridization w ith a digoxigenin-labeled DNA probe and digoxigenin detection system a nd by immunohistochemistry with an enhanced biotin-streptavidin techni que. DNAs extracted from liver and serum specimens were analyzed by So uthern- and slot-blot hybridization, respectively. Using the in situ t echniques, we detected hepatitis B virus-specific DNA and antigens in 11 of 12 HBsAg-positive patients and in none of the 11 HBsAg-negative individuals. Replicative intermediates of hepatitis B virus DNA were d etected by Southern-blot analysis in the same 11 HBsAg-positive patien ts, 6 of whom had no serological markers of hepatitis B virus replicat ion. Therefore a good correlation was found between the results obtain ed by the in situ and Southern-blot hybridization analyses of tissue s pecimens. However, a lack of correlation was found between serum- and tissue-associated markers of viral replication. In addition, the simul taneous in situ detection analyses revealed that some hepatocytes cont aining high levels of viral DNA were devoid of detectable HBcAg, sugge sting a mechanism by which the virus may escape immunological surveill ance. These data provide evidence that liver-associated HBV replicatio n may persist in the absence of serological markers of active hepatiti s B virus replication in cirrhotic patients with advanced liver diseas e and demonstrate that the evaluation of liver- rather than serum-asso ciated markers of viral replication provides a more accurate assessmen t of the virological events occurring in HBsAg-positive individuals.