DNA sequencing of the hepatits B virus for genotyping, antiviral resistance and HBSAG mutant detection.

Hepatitis B Virus (HBV) is a small, partially double-stranded, DNA virus that is hepatotropic. In the United states, there are 1.25 million chronically infected people with 140.000 to 320, 000 new infections added each year. Diseases caused by HBV include liver cirrhosis and hepatocellular carcinoma. It is estimated that as many as 1 million deaths can be attributed to HBV around the world each year. Currently, chronic HBV infection is treated with pegylated interferon alpha or nucleoside analogues (NA), both of which have been shown to be effective at promoting seroconversion. However, both treatments can cause serious side effects and the NA drugs can select for resistant viral populations. Eight HBV geontypes (A-H) have been identified. Although type A virus accounts for most chronic infections in North America, an increasing numbers of non-A-HBV infections have been documented. Recent studies suggest that HBV genotype may influence a patient's response to both interferon and NA treatments. Current NA drugs used for HBV treatment include: lamivudine, adefovir, entecavir telbivuding and tenofovir. Mutations that arise in the reverse transcriptase domain of the HBV polymerase gene may confer resistance to these drugs and have been described. Mutations in the overlapping surface gene have been associated with vaccine-induced and Antibody therapy immune escape. Also, it has been reported that current HBsAg immunoassays (monoclonal) may be unable to detect mutant viral population (especially if the mutations occure in the "a" domain) causing false negative results. This thesis describes an assay that sequences 941 bp if the HBV polymerase gene. The assay covers the six conserved subdomains (A-F) of the RT region that contains antiviral resistance mutation and other domains that contain polymorphisms related to viral genotype. Also, surface antigen mutations for vaccine variants and diagnostic escape are assayed by changing the analysis reading frame. Using this assay, 1000 HBV DNA positive serum/plasma samples from diverse US populations have been processed. These data described the genotype distribution and frequencies of mutation associated with NA resistance and immune escape of HBV infections found in the US.