Immunofluorescence plaque assay for African swine fever virus

Suitably diluted cell culture adapted African swine fever virus preparations were inoculated on VERO cell monolayers and grown on coverslips. Gum tragacanth was used as an overlay. After three days incubation at 37 degrees C the infected cultures were fixed with acetone and stained with fluorescent antibody conjugate. Fluorescing plaques consisted of 20-30 infected cells. THREE STATISTICAL CRITERIA FOR A QUANTITATIVELY RELIABLE ASSAY WERE MET: the Poisson distribution for plaque counts, linearity of the relationship between the concentration of virus and the plaque count and reproducibility of replicate titrations. The method is suitable for counts up to at least 70 plaques per 5 cm(2) coverslip and computed titers are reproducible within 0.16 log units with a total of 300 plaques enumerated.