Studies on the multiplication of japanese encephalitis virus part 1 factors influencing plaque formation of japanese encephalitis virus
Journal of the Kansai Medical University 28(Suppl) PAGES=S62-S86): NO PAGE
The conditions under which Japanese encephalitis [JE] virus plaques are formed on [Rhesus monkey kidney] LLC-MK2 cell were studied in detail. The conditions concerned were: cellular sensitivity, virus strain, cell aging, washing of the cultured cells, adsorption time, diluent effect, culture temperature, concentration of fetal calf serum, methyl cellulose, NaHCO3 and trypsin. Various vertebrate cell lines were screened for plaquing efficiency. Vero [African green monkey kidney], LLC-MK2, [human amnion] FL and chick embryo cells all appeared to give comparable viral titers. The LLC-MK2 cell was sutiable for JE virus plaque assay, because of good growth and easy subcultivation. The optimal conditions for JE virus plaque formation in LLC-MK2 cells were: Incubation temperature of 37.degree. C; viral adsorption duration of 60 minutes; Hanks' balanced salt supplemented with 0.5% bovine plasma albumin at pH 7.2 as diluent of viral samples; employment of overlayer medium containing 1.5% methyl cellulose and incorporated with 3% inactivated fetal calf serum, NaHCO3 2.8 mg/ml; duration of 7 days for development of plaque; Mg2+ and Ca2+ for maximal virus adsorption but plaques produced in the absence of Ca2+ and Mg2+. Trypsin, virus strain, cell age and washing of the monolayer cells before and after exposure to virus had little effect on either plaque size or viral titer. The growth curves of LLC-MK2, Vero cell cultures and serological surveys of the virus were also described.