Optimization and Performance Evaluation of A Novel Focus-Forming Assay for Titration of the Vaccinia Virus
We aimed to establish a rapid,universal,high-throughput focus-forming assay(FFA)for vaccinia virus(VACV)research.Initially,the plaque characteristics of representative VACV strains(Western Reserve strain,WR;Tiantan strain,VTT;Modified Ankara strain,MVA)in BHK-21 cells and Vero cells were evaluated using classical plaque-staining and immunostaining methods.Experimental conditions(choice of substrate,non-specific staining,cell culture plates,and virus incubation time)were optimized to enhance the visibility and accuracy of focal detection.An improved FFA method,based on these optimizations,was used to study the growth curves of VACV in BHK-21 cells and Vero cells.The repeatability of the FFA method was assessed.BHK-21 cells were sensitive to VACV infection and suitable for the titration of various VACV strains.Optimization of experimental conditions improved the quality of focal visualization and accuracy of measurement significantly.The FFA method demonstrated effective replication of WR,VTT,and MVA strains in BHK-21 cells,whereas replication of MVA in Vero cells was limited.The FFA method showed good repeatability within the same experiment,with a coefficient of variation ranging from 0.17％to 3.50％.The improved FFA method was a rapid,versatile,and high-throughput approach suitable for VACV titration,and provided reliable technical support for the research and application of VACV.
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