STUDIES ON THE BIOCHEMICAL, BIOPHYSICAL, AND IMMUNOGENIC PROPERTIES OF JAPANESE B TYPE ENCEPHALITIS VIRUS AND VACCINES.

Studies on the biochemical, biophysical, and immunogenic properties of Japanese B type encephalitis virus and vaccines have been made in order to determine whether a purified vaccine suitable for human use could be obtained by means of differential centrifugation of extracts of infected mouse brains. Studies were also made on extracts of normal mouse brains, it was found that extracts of normal as well as of infected mouse brains contained fairly large amounts of several components of high molecular weight. Components having sedimentation constants near 5 and 40 Svedberg units were found in extracts of infected brains. However the rates of sedimentation of the different components were so similar that it was found impossible, from a practical standpoint, to secure a vaccine consisting largely of virus by means of differential centrifugation. It was also found that a considerable portion of virus was lost or destroyed in the centrifugation process so that it was impossible to secure an effective degree of concentration of immunogenic potency. Although vaccines possessing about twice the immunogenic potency of the starting material were obtained, it was concluded that it was not practical to purify and concentrate Japanese B type encephalitis virus in infected mouse brain extracts by means of differential centrifugation for the production of a vaccine on a large scale. The optimum pH stability range of Japanese B type encephalitis virus activity was found to be near pH 8.5. The virus is inactivated fairly rapidly at pH 7 and very rapidly at more acid reactions. The virus is inactivated rapidly near pH 10. Extracts of infected mouse brains with buffers near pH 8 containing disodium phosphate were found to possess slightly higher titers than saline extracts near pH 7. However vaccines prepared from such extracts were found to possess essentially the same immunogenic potency, hence, although extraction at the more alkaline reaction may perhaps remove more active virus, there was no indication that more immunogenic material was removed at the more alkaline reaction. The use of different diluents in the titration of virus activity and the use of different agents in the preparation and storage of virus suspensions were investigated. It was found that low titers were obtained when Ringer's solution, phosphate buffer at pH 7, or saline-phosphate buffer at pH 8.2 were used as diluents but that high titers were obtained when 10 per cent rabbit serum in saline or in phosphate buffer, 10 per cent skim milk in saline or in phosphate buffer, or 1 per cent arginine at pH 8.3 were used. Undiluted skim milk adjusted to pH 8.4 was found to be as satisfactory as undiluted rabbit serum for the preparation of infected brain suspensions for storage at -70 degrees C. A satisfactory neutralization test was conducted with virus stored in undiluted skim milk at -70 degrees C. and subsequently diluted with 10 per cent skim milk in saline. The demonstration that skim milk can be substituted for rabbit serum in the storage, titration, and neutralization tests of Japanese B type encephalitis virus is of practical importance, for skim milk is more convenient to prepare and more readily available in many localities.