Hepatitis B virus (HBV) and retrovirus(es) such as human immunodeficiency virus (HIV) have several properties in common including: 1) conserved nucleotide sequences in the GAG of HIV and core of HBV genes, 2) replication by a reverse transcriptase mechanism, 3) two direct repeats within their genomes, 4) lymphotrophism, 5) latent tissue infection, 6) frequent point mutations leading to or contributing to antigenic diversity, 7) integration into the cellular DNA of the host and 8) non-acute type oncogenic potential. We have recently detected by monoclonal antibodies and recombinant DNA techniques HBV and/or varients In alcoholics with and without conventional serologic markers that Includes hepatitis B surface antigen (HBsAg), antibodies to hepatitis B surface antigen (anti-HBs), hepatitis B core antigen, (anti-HBc) and hepatitis B e antigen (anti-HBe) as measured by polyclonal antibodies. These agents have been shown to be present In serum and are infectious since they will produce a long incubation hepatitis infection in chimpanzees. We wish to investigate alcoholic populations further and characterize such hepatitis viral agents In more detail at the molecular and antigenic level. We plan to study alcoholic subjects with and without serologic markers of present, recent or past HBV Infection. In this regard, we will employ a newly developed monoclonal based second generation immunoradiometric assay (M2-IRMA) that detects as low as 10-15 pg/mi of HBsAg-associated epitopes in serum. HBV-DNA sequences will be searched for In serum and liver by dot and Southern blot hybridization using a full genomic length HBV DNA probe. We will ,examine antigenic variability in the surface antigen protein by epitope mapping with 10 different monoclonal ,IRMAs. Comparisons will be made to the 9 known HBV subtypes and other varients already Identified by this !technique in other regions of the world. We will employ the polymerase chain reaction (PCR) to detect and amplify HBV and/or varients DNA sequences in serum, lymphocytes and liver of alcoholics. In this procedure we will: 1) capture on a solid phase support, HBV and/or varients with different high affinity monoclonal anti-HBs antibodies that recognize all known subtypes of HBV and thus bind to different it domain epitopes. 2) amplify defined regions of the captured HBV genome such as those conserved in all known hepadna virus(es) (pre-core and core region) as well as those sequences in the more variable pre-S and S gene domain. 3) determine nucleotide sequence variability of the surface antigen gene region which may be the molecular basis for a different antigenic composition or immunologic reactivity. Since the PCR developed in our laboratory in combination with monoclonal anti-HBs antibodies will amplify and thus detect DNA sequences between 2 and 4 viral particles/assays of serum, we will have the capability to clone amplified sequences directly through primer linkers that have a restriction enzyme site. These studies will provide new information on the presence and !characteristics of HBV and/or related agents in alcoholics using molecular and monoclonal antibody techniques. ,Such studies will assess the frequency of infection and the genomic organization of these agents.
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