STUDIES ON SOME CHEMICAL PROPERTIES OF ARBOR VIRUSES

III. EFFECT OF ENZYMES ON PARTIALLY PURIFIED ARBOR VIRUSES

Abstract

In the previous papers the author reported that Japanese B encephalitis and some other arbor viruses were partially purified by ECTEOLA-Cellulose column chromatography or fluorocarbon deproteinization.
The present paper describes that the partially purified viruses were treated with several enzymes and tested for sensitivity to the enzymes.
The enzymes used were trypsin, papain, bacterial proteinase, pancreatic lipase, fungal lipase, wheatgerm lipase, α-amylase, ribonuclease, and lysozyme. The enzymes, except for fungal lipase, were dissolved in 0.07M phosphate buffer solution (pH 7.2) at a final concentration of 0.125% and fungal lipase was diluted with 0.07M phosphate buffer solution (pH 6.0) to a final concentration of 125u/ml; one unit decomposes 1% of 2g of olive oil after exposure of 2.5 hours at pH 5.6 and temperature of 30°C. The partially purified viruses tested were Japanese B encephalitis, G1 strain; dengue fever type 1, Mochizuki strain; yellow fever, strain 17 D; Western equine encephalitis, Rockefeller Institute stock strain; and poliovirus, strain MEF-1. The viruses were mixed with enzymes, incubated at 37°C (except for fungal lipase which was incubated at 30°C) for 1 to 2 hours, and titrated for active virus contents by inoculation into either mice or tissue culture tubes. In controls, phosphate buffer solution was used in place of enzyme solution.
Results obtained are summarized as follows:
1) The infectivity of Japanese B encephalitis and other arbor viruses examined decreased markedly, or was lost completely, by trypsin or pancreatic lipase within 2 hours at 37°C. However, poliovirus was resistant to these enzymatic digestion.
2) Papain, bacterial proteinase, or fungal lipase also decreased (though less than trypsin or pancreatic lipase did) the infectivity of purified Japanese B encephalitis virus. On the other hand, wheat-germ lipase, lysozyme, α-amylase, and ribonuclease exerted little effect on the infectivity of the same virus.
3) When purified Japanese B encephalitis virus was treated with trypsin for 1 hour at 37°C and then exposed to ribonuclease for 2 hours, there was no reduction in viral infectivity.
It was finally concluded from the above results that trypsin and pancreatic lipase have an effect on substances controlling the infectivity of some arbor viruses. Although infective ribonucleic acid can be extracted from tissues infected with some arbor viruses, the intact virus particles are apparently resistant to ribonuclease. The various enzymesensitivities on the purified virus materials, in terms of the decrease of infectivity, are different from virus to another. Although the precise mechanism underlying the observed phenomena has remained unexplained, the enzymatic inactivation experiments may serve to analyse chemical structures of virus particles.