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Hepadnaviridae is a family of DNA-containing viruses that include human hepatitis B virus (HBV), woodchuck hepatitis virus (WHBV), and duck hepatitis B virus (DHBV). DHBV causes a nonpathogenic infection in domestic ducks and several species of migratory wild ducks. HBV is a worldwide public human health problem because chronic carriers are at high risk of developing cirrhosis and liver cancer. It is dangerous to work with HBV in laboratories, because of its highly contagious nature. All lab workers, who work with this virus, must receive a vaccination against HBV. DHBV has been accepted as the best surrogate for HBV
for use in laboratory experiments because these two viruses share similar
sensitivity to drugs and disinfectants. Because of the inability of DHBV
to cause cytopathic effect in cell culture, immunoflorescent analysis
and southern blot hybridization are used to detect DHBV. Immunoflorescent
analysis uses a florescent dye attached to an antibody against DHBV. When
the antibody attaches to the virus, the virus-antibody complex can be
viewed under a microscope containing an ultraviolet light. In southern
blotting, the DNA from the virus genome is extracted and electrophoretically
separated in an agarose gel. The DNA is then transferred to a nitrocellulose
strip by diffusion. A piece of DNA that is homologous (similar structure)
to the viral DNA is tagged with a reporter compound. This tagged DNA is
called a probe and will attached to the target DHBV DNA. This probe-viral
DNA complex then can be detected using various chemical reactants. An AAES study developed a sensitive polymerase chain reaction (PCR) and nested PCR to detect DHBV in several culture systems including duck hepatocytes, duck embryonic hepatocytes, and freeze-thawed duck hepatocytes. Hepatocyctes are the functioning cells that make up the liver. PCR is an enzymatic in vitro method for rapid amplification of DNA. “Nested” PCR is a second round of PCR, which further amplifies the template DNA. It uses a second internal “nested” set of primers. PCR could detect DHBV from serum of congenitally infected ducks with a quick DNA preparation. Researchers successfully detected DHBV in all cell types and duck serum using PCR. Congenital infection was successfully mimicked in ducks by inoculation of a DHBV-containing serum into duck embryos via the yolk sac. Nested PCR increased the limit of detection to 100 more than PCR (Figure 1). This nested PCR was used to test the efficacy of a common commercial disinfectant (n-alkyl dimethyl benzyl ammonium chloride) against DHBV. Figure 2 shows effective concentrations of a disinfectant against DHBV in various cell culture systems. All preparations showed some inactivation of DHBV. When the concentration of disinfectant was below 200 parts per million (ppm), it only caused one log10 reduction (viral titer = 5 ´´ 103.5 TCID50/ml). If the concentration of disinfectant was increased above 1,200 ppm, it caused a reduction in titer _ 4 log10. The U.S. government requires that the chemical show at least a three-fold decrease in viral amount for it to be efficacious. The AAES nested PCR system can be applied to drug and disinfectant assays for both DHBV and HBV and is an improvement over previously developed assays.
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