Hollý Jaroslav, Fogelová Margaréta, Jakubcová Lucia, Tomčíková Karolína, Vozárová Mária, Varečková Eva, Kostolanský František
Biomedical Research Center, Institute of Virology, Department of Orthomyxoviruses, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
Biomedical Research Center, Institute of Virology, Department of Orthomyxoviruses, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05, Bratislava, Slovak Republic.
J Virol Methods. 2017 Sep;247:107-113. doi: 10.1016/j.jviromet.2017.06.004. Epub 2017 Jun 10.
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 TCID. However, both methods are time-consuming. Moreover, some IAV strains form hardly visible plaques, and the evaluation of TCID is subjective. Employment of immuno-staining into the classic protocols for plaque assay or TCID 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 TCID, considering the employed immuno-staining.
由高度可变的甲型流感病毒（IAV）引起的感染对人类和动物都构成了持续的威胁。对它们的监测需要可靠的定性和定量分析方法。最常用的定量方法是通过蚀斑测定法进行滴定或通过TCID估算50%组织培养感染剂量。然而，这两种方法都很耗时。此外，一些IAV毒株形成的蚀斑很难看见，并且TCID的评估具有主观性。将免疫染色应用于蚀斑测定或TCID测定的经典方案中，能够避免这些问题，而且还能缩短可靠的感染性病毒定量所需的时间。将这两种经典病毒滴定方法的替代方法所获得的结果与更新的快速培养测定法（RCA）进行了比较，在RCA中，基于感染细胞的免疫染色通过显微镜估算感染性病毒的滴定终点。在我们对比较方法的分析中，使用了H1、H3和H5亚型的五种不同IAV毒株，并对结果进行了统计学评估。我们得出结论，考虑到所采用的免疫染色，RCA在评估感染性病毒滴度方面至少与蚀斑测定法和TCID一样可靠。
