Nested PCR for Detecting Duck Hepatitis B Virus
Chi-Yong J. Wang,
Joseph J. Giambrone,
Teresa V. Dormitorio
Lois B. Allen
Hepadnaviridae is family of DNA-containing viruses that include human hepatitis B virus (HBV), woodchuck hepatitis virus (WhbV) and duck hepatitis B virus (DHBV). DHBV infection occurs in domestic and migratory wild ducks. HBV is an important health problem with chronic carriers at risk for developing cirrhosis and liver cancer. Medical staff adn scientistis working with HBV must be vaccinated, because of its contagious nature. DHBV is a safe surrogate for HBV, because of their similar sensitivity to drug and siinfectants. Due to the inability of DHBV to case cytopathic effect, immunoflorescent staining and Southern blot hybridization are used to detect DHBV. In this study, a sensitive nested pCR was developed to detect DHBV DNA in cell cultures including duck hepatocytes, duck embryonic hepatocytes, freeze-thawed duck embryonic hepatocytes, and chicken embryonic hepatocytes. A quick serum preparation for detection of DHBV was also demonstrated. We detected DHBV DNA in all cell tyhpes and duck serum using PCR and nested PCR. This was the first report using chicken embryonic hepatocytes to cultivate DHBV. Congenital infection was mimicked in ducks by inoculation of DHBV containing serum into duck embryos by yolk sac. Detection limits for Southern blot, PCR and nested PCR were 10mg, 100pg and 100fp of DHBV DNA, respectively. Nested PCR incrased sensitivity of detection a thousand fold more than PCR and was useful for anit-viral treatment and disinfectants efficacy tests. It broadened the utility of DHBV as model for HBV.