Development and application of an optimized focus-forming assay for quantitation of influenza A virus titer
10.3760/cma.j.cn112309-20240626-00226
- VernacularTitle:甲型流感病毒病灶形成滴定方法的建立与应用
- Author:
Jia LI
1
;
Qiaohong CHU
;
Lingfang ZHANG
;
Xuchang SHAN
;
Tangqi WANG
;
Ruiwen HAN
;
Yujie JIANG
;
Donghong WANG
;
Baoying HUANG
;
Yao DENG
;
Wenjie TAN
Author Information
1. 温州医科大学检验医学院 生命科学学院，浙江省医学遗传学重点实验室，温州 325035
- Publication Type:Journal Article
- Keywords:
Influenza A virus;
Plaque forming assay;
Focus forming assay;
Hemagglutination inhibition test;
Antiviral drugs;
Neutralizing antibody
- From:
Chinese Journal of Microbiology and Immunology
2025;45(1):45-52
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish and optimize a novel method, focus forming assay (FFA), for quantitation of influenza A virus (FluA) and compare its application performance with traditional plague forming assay (PFA).Methods:The foci chromogenic effects of three peroxidase substrates in immunostaining were compared. The PFA and FFA methods were used to explore FluA incubation times and plaque morphology on 12-well plates, and to determine optimal incubation times and virus adsorption volumes for different FluA subtypes on 96-well plates. The correlation between FFA and PFA was evaluated, and the optimized FFA was applied to the in vitro antiviral efficacy analysis of Favipiravir and neutralization test against different subtypes of FluA. Results:TRUEBLUE substrate was identified as the optimal substrate for foci visualization. Compared with the PFA, the FFA showed improved sensitivity and reduced detection time in FluA titration, and good correlation was shown between the two methods′ results. By replacing the 96-well plate with the 12-well plate for FFA titration of different subtypes of FluA, the detection time was shortened, and the amount of serum samples used could be further reduced by optimizing the virus adsorption volume. The half-maximal effective concentration of favipiravir against influenza viruses assessed by the FFA and PFA methods showed no significant difference, and was consistent with the results obtained from quantitative PCR. Additionally, the focus reduction neutralization test and hemagglutination inhibition assays demonstrated strong correlation in determining antibody titers against FluA in serum neutralization assays.Conclusions:The improved FFA method developed here provides a more efficient experimental tool for FluA titration, antiviral drug screening and broad-spectrum vaccine evaluation.
