Quantification of infectious duck hepatitis B virus by radioimmunofocus assay

A simple method is described for the precise quantification of infectious duck hepatitis B virus (DHBV) in cell culture, using a radioimmunofocus assay (RIFA). Primary duck hepatocyte cell cultures were infected with serial dilutions of viral samples as for a plaque assay, but then maintained with liquid overlay medium. After incubation for up to 14 days, cell monolayers were fixed with acetone, then stained with a monoclonal antibody to DHBV L protein followed by secondary antibody labelled with 125I. Foci of infection (representing individual infectious particles in the inoculum) were detected by autoradiography. The number of foci recovered was increased by addition of dimethyl sulphoxide to culture medium, but was not appreciably altered by the use of semi-solid medium. The titre of virus suspensions determined by RIFA correlated well with titration in ducklings. The RIFA is a useful method for titration of DHBV, as it has a wide dynamic range and is well suited to parallel titration of large numbers of samples. This assay will have wide use for the analysis of DHBV growth kinetics, antiviral efficacy, and virus inactivation procedures.