The infection of leafhoppers by Western X-disease virus: V. Properties of the infectious agent

Abstract

The concentration of extractable Western X-disease virus (WXV) in a vector, Colladonus montanus (the mountain leafhopper), was much higher than in any of several herbaceous plants tested. Leafhoppers were therefore used as virus source material for a survey of viral properties in crude extracts. A new method of bioassay was employed, based on the concentration dependence of the incubation period, in the insect, between injection of virus and transmission of WXV to plants. This bioassay permitted more accurate study of viral properties than has heretofore been possible with leafhopper-borne viruses. WXV activity was not recovered after saturation of saline extracts from leafhoppers with butanol. However, WXV survived brief ultrasonic treatments, brief emulsification with Genetron 113, and in limited trials, emulsification with chloroform. Freezing of whole viruliferous insects or saline extracts from such insects did not destroy the virus. Infectivity was present in centrifugal supernatants after 10 min at 8,000 g, but was almost completely sedimented after 10 min at 25,000 g. After rate zonal density gradient centrifugation for 20 or 25 min at 20,000 or 25,000 rpm, infectivity was found throughout the gradient column, but was most concentrated in the bottom one-third of the column. Viral infectivity was greatly reduced by overnight dialysis against saline, but not by dialysis against a buffer containing glycine, divalent cations, and cysteine, or by buffer transfer through Sephadex G-75 into a protective buffer containing Mg 2+ and glycine. Infectious WXV was recovered after brief exposure to various buffers at pH values from 5.1 to 9.7. Brief exposure to 0.01 m glycine or 0.01 m sodium borate buffers at pH 8.2 and 8.9, respectively, increased viral yield, but longer exposures to these buffers decreased the virus yield. Stability seemed best between pH 6.0 and 7.1. Recovery of virus from resuspended pellets after centrifugation at 80,000 g for 30 min was much better when 0.3 m glycine was used than any lower concentration of glycine whether the pH was 6.1, 7.5, or 8.5.