ASSAY OF WOUND TUMOR VIRUS BY FLUORESCENT CELL COUNTING TECHNIQUE

The fluorescent cell counting technique was used for assaying the infectivity of wound tumor virus (WTV) in cell cultures of the vector leafhopper, Agallia constricta. Monolayers of the leafhopper cells were prepared on 15-mm coverslips and were inoculated the next day when confluent. Nearly maximal virus adsorption was obtained in about 2 hours at 30° or 3 hours at 24°. Although specific fluorescence was first seen in some of the infected cells after subsequent incubation for 12 hours at 30°, the number of fluorescent cells increased with incubation time. There occurred a 3-6-hour period in which cells infected from the inoculum were most suitably stained. This period began at about 27 hours after inoculation, if adsorption was allowed for 2 hours at 30°; or at about 30 hours, if adsorption was allowed for 3 hours at room temperature (24°). Under such conditions, a linear relationship was obtained between the number of fluorescent cells and the relative virus concentration for a dilution range of 1.8 log units. The distribution of infected cells in a monolayer culture covering 0.075 mm2 was found to follow the Poisson distribution pattern. In attempts to correlate the actual number of virus particles to cell infecting units (CIU), a ratio of 405 to 1 was obtained after a factor of 1.27 was applied to correct for multiplication of infected cells during incubation. The number of CIU corresponded satisfactorily to the 50% dilution end-point determination. Cell cultures of the nonvector leafhopper Aceratagallia sanguinolenta were also susceptible to WTV, though at a lower susceptibility level. The use of the A. sanguinolenta cells as host system gave a titer of the virus 1 or 2 log units lower than when assay was made on cells of A. constricta. © 1969.