Studies on Japanese B Encephalitis Virus Vaccines from Tissue Culture

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Abstract
Summary: The Nakayama mouse adapted strain of JBE virus was carried 50 passages at 37°C in HKC culture in “rapid passage” of a large inoculum and 32 passages including several series of terminal dilution passages. No marked differences were noted in the ratio of the tissue culture titers to the mouse titer through either of these series, although some slight change probably occurred in the rapid passage lines. Some reduction in suckling mouse pathogenicity via the subcutaneous route occurred in this same rapid passage line. Several passages of each of these lines at lower temperatures failed to give any indication of any further substantial reduction in mouse virulence. A virus strain, OCT-541, isolated directly in HKC cultures from wild caught mosquitoes was carried from 15 to 60 passages in nine different HKC rapid passage lines, most of which included reduction of incubation temperature. Comparisons of mouse titers and HKC titers at 37°C and sometimes at 24°C were made at intervals of five passages. These revealed a somewhat greater ratio of titer between the mouse and tissue culture than that observed for the Nakayama strain. The line (35–24) carried five passages at each reduction step in temperature, i.e., 35°, 33°, 30°, 27° and 24°, then continued at 24°, gave greatest promise. A very significant change occurred in the average survival time of mice. This line lost its ability to infect or be transmitted by Culex tritaeniorhynchus by the 20th passage, but retained its ability to produce viremia in chicks. It failed to infect or to produce disease in suckling mice by the subcutaneous route by the 60th passage but retained its ability to grow in high titer in HKC and in chick embryo cell culture. The titer of this 35–24 line was usually 1 to 1.5 logs higher in cell culture at 24° than at 37°. This line was considered encouraging in respect to qualities essential for a live virus vaccine and manipulations to attempt further attenuation are being carried out.

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