Plaque Formation of Influenza Virus at 25° C

Abstract

INFLUENZA virus was adapted to growth at 25° C by gradually lowering the temperature at which infected cells were incubated during serial passage until the optimum yield of infectious virus was obtained¹. Using this method it was possible to adapt various strains of type A and B influenza viruses to growth at 25° C in tissue culture or embryonic eggs. Adaptation of the virus to reproduce at 25° C has resulted in its attenuation when tested in a susceptible host¹. Infection of the host occurred without apparent disease but with good antibody response (my own unpublished results). During earlier work, purification of these variants was achieved using the limiting dilution technique, which is adequate but not highly reliable. The purpose of the study reported here was to develop plaque assay, and to select plaque progeny for more vigorous growth at 25° C. The development of plaques at 25° C was used also for clone purification of cold variants for genetic studies. Kidneys were excised from 1–6 day old chicks and were processed according to the method published earlier² and incubated at 36° C. Monolayers of chick kidney cells were formed between the fourth and fifth day after seeding, and the cultures were infected immediately thereafter and transferred to incubation at 25° C. Briefly, the cell monolayer was washed with prewarmed Hanks balanced salt solution (BSS) and infected with the appropriate dilutions of the virus contained in a small volume; for example, when using 3 oz. prescription bottles, 0.5 ml. of a given dilution was inoculated. The adsorption was allowed to proceed for 1 h at room temperature with gentle mechanical agitation. The infected chick cell monolayers were then overlayered with agar; the composition of the agar overlay used was exactly like the one devised by Sugiura and Kilbourne³ for the Chang conjunctival cell line clone l-5c-4 but with a final agar concentration of 1.2 per cent instead of 0.9 per cent. After incubation for 5 days at 25° C a second overlay of the agar medium with neutral red was added. The cultures were returned to 25° C for further incubation.