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Studies on Japanese B Encephalitis Virus Vaccines from Tissue Culture¹

III. Further Selection and Testing of Attenuated Virus Lines from OCT-541

From the Department of Epidemiology and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania

Abstract

Methods were developed by which plaques and supposedly the progeny of single virus particles could be selected from the partially attenuated OCT-541 strain. At first these were by relatively "blind" picking methods from HKC at 24°C, subsequently visually from CEC at 28°.

Beginning with the 30th rapid passage of line 35–24 (24°C) of OCT-541 a series of four plaque pickings was undertaken. Each plaque was prepared as a pool and titrated i.c. in weanling mice and in HKC at 37° and at 24°C. A virulence ratio was calculated when possible (log TCID₅₀ minus log LD₅₀).

First passage plaques of which there were seven selected had relatively high virulence with a range of virulence ratios of 1.0 to 2.7 logs. The second passage plaques, 13 in number, from a low virulence plaque 4 possessed a virulence range of from <1.5 to 5.3. Progeny of one of these, plaque 4–5, were tested extensively in a series of three pools and found to have the following properties:

1. I.c. weanling mouse log LD₅₀ of 3.0 to 4.2/ml.

2. HKC log TCID₅₀ at 37°C of 7.8 to 9.1 and at 24°C of 8.5 to 9.4.

3. Virulence ratio of 5.2 to 6.2 logs.

4. Identified as JBE virus.

5. Greatly prolonged AST in mice, and mice which became paralyzed frequently lived and occasionally recovered.

6. Suckling mouse s.c. log LD₅₀ about 1.0 but survivors were present in the undiluted inoculum.

7. No detectable pathogenicity for adult hamsters by i.c. or s.c. routes.

8. One of two monkeys inoculated intrathalamically with a 10^-1 dilution had a mild encephalitis with long incubation period. Monkeys receiving higher dilutions and the other at 10^-1 were without illness.

9. Ten monkeys given undiluted or 10^-1 dilution s.c. were without signs of illness and had no viremia.

10. Only the monkeys receiving 10^-1 virus inoculum i.c. developed significant antibody responses, except for the controls receiving original OCT-541 s.c.

11. Mexican burros given virus s.c. were without viremia, fever or illness and they failed to develop antibody.

12. Guinea pigs inoculated s.c. were without illness or viremia.

13. Proved to be stable in passage at 24° either by the rapid or the terminal dilution method but reversion to virulence was observed to be occurring at 37°C.

Third and fourth plaque passages, of which 46 were tested, were of more uniform character than those selected earlier. All had so little pathogenicity for mice that the LD₅₀ was considered <10⁰. The virulence ratio was estimated at >8.5 logs. Surviving mice of low dilution inocula frequently survived a subsequent challenge of 100 LD₅₀ of the Nakayama strain, but others failed to develop such immunity.

For comparison with the above four serial series of plaques were single series made from other lines. Plaques of passage 60 of line 35–24 gave a wide range of virulence ratios but in general they were less virulent than the first plaques of passage 30, many with a virulence ratio of >7.0 logs. Passage 50 of line 37 (37°C) gave plaques with virulence ratios less desirable than those of the lower temperature line. Plaques from the original pool of OCT-541 previous to serial HKC passage gave plaques of high virulence ratios and the best plaque had a ratio of only 2.7 logs.

It was concluded that plaque 4-5 progeny of the 24° line have most of the properties which are theoretically considered as essential for an attenuated vaccine strain. Inability of this virus to produce infection and immunity in many laboratory animals with normally high temperatures and a tendency to regain partial virulence during serial passages at 37°C in vitro lead to certain reservations regarding its usefulness in man which will require further study.

Footnotes

¹ This work was carried out under the sponsorship of the Commission on Viral Infections, Armed Forces Epidemiological Board, and was supported by the U. S. Army Medical Research and Development Command, Department of the Army, under Contract No. DA-49-193-MD-2042.

² Dr. Skon Rohitayodhin was a postdoctoral fellow of this department on leave of absence from the Pasteur Institute, Bangkok, Thailand, supported for 2 years by a fellowship from the National Academy of Sciences and 1 year under a training grant in Microbiology, National Institutes of Health.

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