Development of disinfectant efficacy assay for duck hepatitis B virus using PCR and real-time quantitative PCR.

摘要

Duck hepatitis B virus (DHBV) is a partial double stranded DNA virus belonging to the Hepadnaviridae family, which includes human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHBV).; To improve the sensitivity of viral detection, a nested PCR was developed herein. Results showed nested PCR could enhance the limit of detection one thousand fold more than a single round of PCR.; In this study, it was demonstrated that duck embryonic hepatocytes supported DHBV propagation after freezing and thawing. DHBV was first reported to be cultivated in chicken embryonic hepatocytes, which are more available than duck embryo hepatocytes. When virus in serum was titrated using chicken embryonic hepatocytes, the titer was comparable to that obtained using duck embryonic hepatocytes.; The risk of nosocomial infection with hepatitis B virus arises from contamination of instruments or other fomites with blood or body fluids from infected patients. It is reduced by effective virus inactivation procedures and disinfectants. The DHBV model allows for quantification of disinfectant activity against hepadnaviruses, which has not been feasible with experimental systems involving the transmission of HBV to chimpanzees. It revealed similar inactivation The same virus titer was obtained from infected duck embryonic hepatocytes as sera from infected ducklings. Therefore, the hepatocyte culture system provides a faster and cheaper alternative for a disinfectant assay than in vivo systems.; The virucidal activities of two quaternary ammonium chloride disinfectants, n-alkyl dimethyl benzyl ammonium chloride and alkyl dimethyl benzyl ammonium chloride (10–12C), were compared using this system and the effective concentrations were 1200 ppm and 1800 ppm, respectively, with first order killing kinetics. When sepahadex LH-20 chromatography was used to remove residual disinfectants from the recovered mixtures, it decreased viral titer 1.5 log10. Efficacies of these disinfectants were further validated using real-time quantitative PCR. Results confirmed that the efficacy of n-alkyl dimethyl benzyl ammonium chloride was higher than alkyl dimethyl benzyl ammonium chloride (10C–12C).; Quantification is important for assessment of antiviral treatment and viral infectivity. Real-time quantitative PCR using SyBr green dye was developed. The detection limit of this system was 10 copies of DHBV DNA and the linear range for reliable quantification was 105 to 1010 copies of DHBV DNA. In this study, we propagated virus in duck embryos through yolk sacs. Results using real-time quantitative PCR showed the viral loads of experimentally infected ducks were higher than congenitally infected ducks. Therefore, real-time quantitative PCR successfully quantified DHBV from clinical samples. (Abstract shortened by UMI.)