The Holland strain of anatid herpesvirus (AHV) was 2- to 10-fold more efficiently plaqued under liquid medium or semisolid medium containing methylcellulose than in media containing agar, agarose, or Nobel agar. Virus adsorption was complete in 45 min, with the maximum virus titers obtained under liquid medium at 48 h postinfection. The AHV dose-response curve was linear. Pekin duck embryo cultures were more efficient than CCL-141 in plaque response and gave the maximum virus yield per cell. Virus titers were independent of avian cell passage number (to 6), fetal or newborn calf sera (to 10% v/v), and the presence of polyions during adsorption. The host range included cells from several species of Anseriformes and Galliformes.

Plaque reduction assays were used to evaluate the inhibitory effects of acyclovir (ACV), phosphonoacetate (PAA), and phosphonoformate (PFA) with a plaque-purified isolate of anatid herpesvirus (AHV-ppc3). A plaque assay employing a liquid overlay medium was developed to facilitate the drug inhibition studies. From dose-response curves, the ID₅₀ for PAA, PFA, and ACV were 20,12, and 0.14 µg/ml, respectively. From data obtained from combination dose-response curves, dose isobolograms were prepared and used to determine the types of interactions between drug pairs. Whereas the interaction between PFA and PAA was additive, synergism occurred with ACV and either PAA or PFA. Drug-resistant mutants of AHV-ppc3 resistant to 8.0 µg/ml ACV, 250 µg/ml PAA, 180 µg/ml PFA or 6.0 µg/ml AHV, and 220 µg/ml PAA were isolated.

Phosphonoacetate was found to be an effective inhibitor of the replication and cytopathic effects (CPE) associated with Anatid herpesvirus (AHV) infections in avian cells. In low multiplicity of infection (MOI) (10(-3) or less) infected Pekin duck and chicken fibroblast cultures, the dosage required for a 50% plaque reduction was approximately 20 microgram/ml. The amount of the inhibitor needed for complete prevention of CPE was found to be MOI dependent with up to 190 microgram/mL required at a MOI of 1.1. Delayed addition of phosphonoacetate to AHV-infected cultures resulted in differing CPE. When added before 25 h postinfection, the CPE were largely prevented. Added between 25 and 40 h postinfection, the CPE consisted of nonproductive, nonperforate focal areas containing viable and nonviable cells. The focal areas did not appreciably increase in size or number in the continued presence of phosphonoacetate, were chromophilic, and tended to be replaced by morphologically normal cells, provided the presence of phosphonoacetate was continued. Maintaining phosphonoacetate in the presence of infected cultures for periods of 7 days or longer resulted in curing and a complete loss of infections virus in the medium and in cell lysates.