Infectivity assays for the Autographa californica nuclear polyhedrosis virus using Spodoptera littoralis cells

Tissue culture ID50 and plaque assays for the Autographa californica nuclear polyhedrosis virus, using the Spodoptera littoralis cell line HPB-SL, were developed. Direct comparison using these assay methods showed that these cells were as susceptible to infection as the more commonly used Spodoptera frugiperda cell line IPLB-SF-21. Both infectious tissue culture supernatants or virus isolated directly from polyhedra could be titrated. It was important to use low cell seeding densities in the assays so that clear centres of infection formed. Dose-response curves indicated that one infectious particle was capable of initiating an infection. Virus could be cloned using either method even though, for the plaque assay, plates had been stained. The tissue culture ID50 assay was performed using 96-well plates and required an incubation period of about 10 days. The plaque assay used a simple nutrient agarose overlay and an incubation period of 5-6 days. Easily countable plaques of 0.3-1.2 mm diameter were detected after staining with iodonitrote-trazolium chloride. The plaques comprised areas of inhibited cell division and round or dead cells. Most plaques contained only some cells with polyhedra and yields averaged about 1/cell. Occasionally plaques or infected wells were found in which no polyhedra could be seen. These infectivity assays are therefore not dependent on polyhedra formation.