Comparison of cell culture systems for duck hepatitis B virus using SyBr green quantitative PCR.
The Hepadnaviridae family contains DNA viruses such as human hepatitis B virus (HBV), woodchuck hepatitis B virus (WHV), and duck hepatitis B virus (DHBV). DHBV is distributed in both wild and domestic ducks. HBV is a worldwide health problem with carriers at risk of developing cirrhosis and liver cancer. All medical staff and scientists working with HBV must be vaccinated, because of its highly contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Several cell culture systems have been developed to study anti-DHBV drugs and disinfectants. However, differences in their capabilities to support DHBV propagation have not been reported. Therefore, a sensitive and reproducible quantitative PCR based on SyBr green dye was developed. This system does not need electrophoresis for analysis of PCR products, thus reducing processing time and potential for cross-contamination. It allowed precise quantification of DHBV over 8-logarithm dynamic range with a good correlation (R(2) = 0.9689) and showed minimal run-to-run deviation. Sensitivity was 820 copies of DHBV genome and specificity was confirmed by melting curve analysis. It demonstrated good repeatability in quantification of DHBV loads from serum of infected ducks. This assay compared DHBV yields from different cultured cells. All cells had similar kinetic curves for DHBV replication and replication peaks appeared 4 days post-infection. Duck embryonic hepatocytes showed the highest (P > 0.05) replication peak for DHBV. Therefore, duck embryonic hepatocytes and quantitative PCR based on SyBr green dye are a good choice for anti-DHBV drug and disinfectant testing.
Wang
Chi-Young J
CY
Department of Poultry Science, Poultry Annex Building, Auburn University, Auburn, AL 36849-5416, USA.
Giambrone
Joseph J
JJ
Smith
Bruce F
BF
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004372:Ducks; D005456:Fluorescent Dyes; D015611:Hepatitis B Virus, Duck / Q000254:growth & development*; D022781:Hepatocytes / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014776:Virus Cultivation
D005456:Fluorescent Dyes