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A Study of Rice Dwarf Virus in Vector Cell Monolayers by Fluorescent Antibody Focus Counting

Department of Cell Biology, National Institute of Agrobiological Resources, Yatabe, Tsukuba, Ibaraki 305, Japan

Infectivity assays of rice dwarf virus (RDV) were done by the fluorescent antibody focus counting technique on vector cell monolayers of the green rice leafhopper, Nephotettix cincticeps. The focus count method was shown to be an accurate and quantitative method for determining RDV infectivities. The optimal pH value for inoculation was about 6.0 in a solution containing 0.1 M-histidine HCl and 0.01 M-MgCl₂. Below pH 5.5 and above 6.5, the infectivity of RDV dropped rapidly. The optimal adsorption period at 28 °C was dependent upon the RDV concentration. Optimal periods for adsorption with relative RDV concentrations of 10^-3, 10^-4 and 10^-4.5 were about 60, 90 and 120 min, respectively. The period from virion adsorption to penetration into the cell was about 90 to 120 min. Infective progeny virions were first detected 12 h after the initial inoculation. From 12 to 20 h, the growth rate of the virus in the cells was exponential with a doubling time of about 96 min, and then from 20 to 28 h there was little or no further increase in infective virus. When the infectivities of the same inocula were compared by using the focus count and vector insect injection methods, the dilution endpoints were approx. 10^-6 and 10^-4, respectively. The focus count assay method was thus about 100 times more sensitive than vector injection.

Keywords: RDV, vector cell monolayer, fluorescent antibody focus counting

Received 20 March 1986; accepted 23 June 1986.

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