Purification of arboviruses grown in tissue culture.

Abstract

Arboviruses (Chikungunya, dengue type 1, and Japanese encephalitis) cultivated in monolayer cultures of BHK-21 cells grown with Eagle's MEM supplemented with bovine serum albumin were purified by the following procedures: The viruses were precipitated from infectious tissue culture fluids by zinc acetate (0.05 M) and were resuspended in saturated ethylenediaminetetraacetate. The suspension was filtered through a Sephadex G200 [(lower) 10 cm]-Sepharose 6B [(upper) 40 cm] column (2.5 cm in diam) by upward elution. The filtrate was concentrated in a collodion bag in vacuo. The final step was sucrose density gradient centrifugation. The procedures were comparatively short and straightforward, and about 100 to 500 times purification factors were obtained consistently. The CHIK virus preparation after the sucrose density gradient centrifugation was demonstrated as a single peak, the infectivity and HA activity coinciding with each other; whereas the DEN-1 and JE viruses were shown to be heterogeneous. The problems of whether such a difference reflects some basic characteristics of groups A and B viruses need further investigation. Electron microscopic pictures of the preparations revealed spherical particles of (mμ): 50 to 60 (CHIK), 50 to 55 (DEN-1), and 45 to 50 (JE). These images are compatible, in general, with those previously reported by Igarashi et al. (CHIK virus) (13, 14); Matsumura and Hotta (DEN-1 virus) (15); Smith et al. (DEN-2 virus) (16); Takaku et al. (JE virus) (17); and Nozima et al. (JE virus) (18); although the origin of the viruses and the purification methods are not necessarily the same. The present purification procedures can probably be applied with success to other arboviruses in general. Further studies on the biochemical and biophysical nature of the purified virions are underway and will be reported later.