Tissue Culture Studies on Japanese B Encephalitis Virus

II. Viral propagation in plasma-embedded roller-tube cultures of human and animal tissues

Abstract

Tissues employed were: Human adult lung, kidney, spleen, thyroid, adrenal, lymph node, embryonic brain (cerebrum or cerebellum); puppy lung, kidney, spleen, liver, heart, lymph node; kitten kidney, testicle; rabbit lung, testicle, etc. The plasma-embedded roller-tube method was applied. Coverslip cultures were additionally prepared for purpose of morphological examinations. Constituents of culture medium were 0.5% lactalbumin hydrolysate. 10% bovine serum in Hanks' balanced salt solution, supplemented with 100 u/ml penicillin, 100 γ/m1 streptomycin, as well as phenol red as a pH indicator. For nervous tissues, a medium, consisting of 50% human cancerous ascitic fluid, 5% chick embryo extract, 45% Gey's salt solution, 300 mg% glucose and 1, 000 u/ml penicillin, was also used. After being incubated for 4 to 7 days at 37°C, tubes in which good cellular growth or survival was observed were selected for virus inoculation. Virus used was Gl strain JBE virus which had been passed through white mice intracerebrally for about 240 generations. At intervals following inoculation of the virus of a given concentration, portions of the culture fluid were taken out and measured for active virus contents. Temperature for incubation after virus inoculation was 34-35°C.
The viral growth patterns revealed could be divided into three categories:(1) “Convex” curves, indicating good multiplication of virus;(2) “Flattened” curves with few peaks, suggesting a low grade multiplication or a significantly prolonged persistence of virus; and (3) “Continuously declining” curves, indicating no growth of virus. The majority of the tissues thus far investigated was shown to belong to the category of either (1) or (2). It was noted that human adult spleen and adrenal tissues did not support the growth of JBE virvs, at least under the conditious adapted, although further experiments are required. No CP was found in any of the tissues tested. Virological and biological sigdificance of the results obtained is discussed and extended studies along the same line are being undertaken.