Comparison of infectious influenza A virus quantification methods employing immuno-staining

Infections caused by highly variable influenza A viruses (IAVs) pose perpetual threat to humans as well as to animals. Their surveillance requires reliable methods for their qualitative and quantitative analysis. The most frequently utilized quantification method is the titration by plaque assay or 50% tissue culture infectious dose estimation by TCID₅₀. However, both methods are time-consuming. Moreover, some IAV strains form hardly visible plaques, and the evaluation of TCID₅₀ is subjective. Employment of immuno-staining into the classic protocols for plaque assay or TCID₅₀ assay enables to avoid these problems and moreover, shorten the time needed for reliable infectious virus quantification. Results obtained by these two alternatives of classic virus titration methods were compared to the newer rapid culture assay (RCA), where titration endpoint of infectious virus was estimated microscopically based on the immuno-staining of infected cells. In our analysis of compared methods, five different IAV strains of H1, H3 and H5 subtypes were used and results were statistically evaluated. We conclude that the RCA proved to be at least as reliable in assessment of infectious viral titer as plaque assay and TCID₅₀, considering the employed immuno-staining.