Development of viral disinfectant assays for duck hepatitis B virus using cell culture/PCR

Abstract

Human hepatitis B virus (HBV) is a worldwide public health problem with chronic carriers at risk for developing cirrhosis and hepatocellular carcinoma. Accidental nosocomial infections from inadequately disinfected equipment or exposure to blood and body fluids from patients are major routes. To solve such problems, disinfectants to inactivate HBV must be validated. Duck hepatitis B virus (DHBV) is accepted as a surrogate for HBV, due to their similar sensitivities to disinfectants and its safety. Ducklings are used for disinfectant efficacy assays; however, the same virus titer is obtained using duck embryonic hepatocytes. Viral titration in disinfectant efficacy assay is conducted using Southern hybridization of infected duck serum. However, this test requires radioisotopes. Therefore, disinfectant assessment protocols were developed using duck embryonic hepatocytes with polymerase chain reaction (PCR) or nested PCR. The ease of handling, lowered cost and enhanced sensitivity make PCR desirable. Chicken embryonic hepatocytes were applied to DHBV disinfectant efficacy assay. Results were consistent and could be used under certain conditions. The virucidal activities of two quaternary ammonium chloride disinfectants, n-alkyl dimethyl benzyl ammonium chloride and alkyl dimethyl benzyl ammonium chloride (10C–12C) were compared and effective concentrations were 1200 and 1800 ppm, respectively. Efficacies of these disinfectants were 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). This assay was useful for rapid discrimination of killing potentials of disinfectants. In conclusion, these assays can be applied to other viruses that are unable to cause CPE in cell cultures and broadened the utility of DHBV as animal model for HBV.