An analysis of the plaque assay of herpes simplex virus in chick embryo monolayers

Summary

Various factors were examined for their influences upon the plaque assay of herpes simplex virus in chick embryo monolayers. The factors analysed included the process of monolayer preparation, constituents of growth medium, conditions of virus adsorption and conditions of maintenance of cells after infection, and eventually a standard procedure was established. An important difference from the conventional method was the use of a small inoculum size, based on the fact that when the inoculum size was 0.05 ml. per 60-mm dish a higher titer was obtained than when larger inocula were used. Virus adsorption was practically over in one hour at 37° C leaving approximately 30% of the inoculated virus unadsorbed. This adsorption rate could not be improved even when adsorption was allowed to proceed in a heavy cell suspension. The titration efficiency as well as the plaque size was greatly influenced by the passage history of the virus. An HF strain which had gone through numerous egg passages showed equal plaque and pock titers but serial passage in chick embryo fibroblasts resulted in a ten times higher plaque titer than the pock titer. Similar trends appeared also with other less egg-adapted strains. The present chick embryo plaque assay was much more sensitive thanRussel'a plaque assay in HeLa cells even for a highly HeLa-adapted subline of the HF strain. The possibility that one chick embryo plaque-forming unit of the HF strain represents close to one infectious virus particle is discussed.