Increase of wound tumor virus in leafhoppers as assayed on vector cell monolayers

Abstract

Measurements of wound tumor virus (WTV) concentration in extracts from insect vectors ( Agallia constricta) were made by counting the foci of infection in monolayer cultures of the vector. Optimum conditions for performing such an assay procedure were determined. Brief exposure of insects to 50% ethanol and to certain antibiotics was necessary to avoid fungal and bacterial contamination in extracts prepared from the insects. The optimum pH value for the extraction of virus was 7.00 and for inoculation it was around 5.30 when a solution of 0.1 M histidine and 0.01 M MgCl 2 (His-MgCl 2) was used. When an incubation period of 40–46 hr at 30° was allowed, the optimal period of inoculation at 30° was 3 hr for insect extracts diluted to 10 −3.0 or higher. The focus counts of infected cells obtained by employing the optimal conditions for inoculation could be converted to absolute concentrations of virions by comparing the number of foci with those obtained from tumor extracts of known absolute virus concentration. After a 1-day acquisition period, virion concentrations in leafhoppers were determined for various days, starting from day 8, when the virus could be first assayed, to day 49. The virus concentration increased 50,000- to 100,000-fold between day 8 and day 25 after acquisition. Between day 23 and 28 the concentration reached a plateau level. After that the apparent concentration of virions was markedly affected by the extracting solution. The growth curve determined by focus assay on monolayers was similar to that determined previously by other methods except that the increase in virus concentration was followed over 1 to 1.5 additional log units. Extraction with His-MgCl 2 solution showed that the decline in virion concentration after the peak concentration, which was noticed in previous experiments, was almost entirely an artifact of extraction.