Analysis of Reverse Transcriptase Gene Mutations in the Hepatitis B Virus at a University Hospital in Korea

BACKGROUND: Most mutations in the reverse transcriptase (RT) gene of the hepatitis B virus (HBV) are related to resistance to antiviral agents. Cross-sectional studies on the mutations of this gene are rare. Thus, we analyzed the mutation patterns of RT genes and their biochemical parameters. METHODS: From 2009 to 2012, 301 blood specimens from patients with chronic hepatitis B at Daegu Catholic University Medical Center were retrospectively analyzed for the RT gene sequence of HBV, ALT, hepatitis B e antigen (HBeAg), and HBV DNA. The mutation patterns of the RT gene were compared with the biochemical parameters. RESULTS: Of the 301 patients, 100 (33.2%) had no RT gene mutations. The remaining showed the following mutation patterns: rtM204I/V (50.2%), rtL180M (39.2%), and rtA181T/V (19.6%). Combined mutations were found in 146 cases (48.5%). Of these, the combination of amino acid changes at rt180+rt204 (49.3%) was most frequently detected, followed by rt181+rt236 (11.0%) and rt173+rt180+rt204 (9.6%). In the mutated group, HBV DNA and HBeAg positive rates were significantly higher (P<0.05 for both). Phenotypic analysis showed that lamivudine resistance was most frequently detected (34.6%), followed by adefovir resistance (15.6%). Multidrug resistance was detected in 48 cases (15.9%). The adefovir-resistant group had a higher proportion of cases with HBV loads greater than 2,000 IU/mL. CONCLUSIONS: We found correlations between the mutation status of the RT domain and biochemical parameters such as HBV DNA and HBeAg positive rate. The presence of RT gene mutations could therefore be utilized to predict clinical status.

Human Monoclonal Antibody Inhibiting Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Protein / 대한소화기학회지

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.

Exposición ocupacional al virus de la hepatitis B del personal hospitalario del centro médico naval " Cirujano mayor Santiago Távara " / Ocupational exposition to hepatitis B virus in Hospital Personnel at Naval Medical Center \"Cirujano Mayor Santiago Tavara\"

En el presente estudio se realizó una encuesta seroepidemiológica para hepatitis B en 400 trabajadores de salud aparentemente sanos de todas las áreas del Centro Médico Naval " Cirujano Mayor Santiago Távara ". Se determinaron mediante la técnica de enzimo-inmunoanálisis ( ELISA ), el antígeno de superficie ( AgHBs ), el anticuerpo al antígeno core tipo IgG ( anti-HBc-IgG ) y el anticuerpo al antígeno core tipo IgM ( anti-HBc-IgM ). Los resultados demuestran que el 11.75 por ciento tienen anticuerpos contra el antígeno core-IgG; en éstos el anti HBc IgM y el AgHBs fueron negativos. Se encontró asociación entre la exposición a sangre y la prevalencia del anti-HBc-IgG en la categoría icupacional de médicos. El anti-HBc-IgG fue encontrado positivo con mayor frecuencia entre los que tuvieron historia previa de hepatitis, mayor tiempo en la ocupación y mayor edad. Los resultados obtenidos confirman el mayor riesgo de infección por el virus de la hepatitis B ( VHB ) entre el personal hospitalario que en la población general. El estudio demuestra que el personal hospitalario el Centro Médico Naval tiene una prevalencia menor de infección por el VHB que la hallada por otros autores en el personal de otros centros hospitalarios en el Perú

Properties of Hepatitis B Virus Associated DNA Polymerase

The nature of hepatitis B virus (HBV) particle associated DNA polymerase was studied in relation to various enzyme inhibitors including antiviral agents. HBV DNA polymerase required high concentration of MgCl2(> 30 mM) and neutral pH for its full activity. p-chloromercuribenzoate was a strong inhibitor (85% inhibition at 1 mM) but N-ethylmaleimide had much less inhibitory effect (20% inhibition at 10 mM). Phosphonoformic acid showed the greatest inhibitory effect on HBV-DNA polymerase (almost complete inhibition at 100 microM) among phosphocompounds tested. Adenine arabinoside triphosphate (ara-ATP) and cytosine arabinoside triphosphate (ara-CTP) were competitive inhibitors with respect to their respective deoxyribonucleoside triphosphate (dATP and dCTP). Ara-CPT was a stronger inhibitor of HBV-DNA polymerase compared to ara-ATP. Ki values for ara-ATP and ara-CTP were 15.0 microM and 11.7 microM , respectively. HBV-DNA polymerase is characteristic in its ionic requirements and susceptibilities to certain inhibitors.