流感病毒对聚合酶抑制剂类药物敏感性的检测方法研究

Study on Methods for Detecting Susceptibility of Influenza Virus to Polymerase Inhibitors

流感病毒(Influenza virus,Ⅳ)是引起季节性流感流行和流感大流行的主要病原体。抗流感药物是控制流感病毒感染的重要方法,其中,流感病毒的聚合酶抑制剂是最有前途的药物之一。为了对聚合酶抑制剂类药物抗流感病毒药物敏感性的检测方法进行优化,本研究确定不同型别/亚型的季节性流感病毒在不同培养板进行空斑实验的培养时间和药物稀释梯度等条件,并比较免疫染色和结晶紫染色2种方法,在此基础上,利用空斑减少实验检测季节性流感病毒对T-705的敏感性。结果显示:在12孔板中,甲型流感病毒A/Beijing/2/2018(pdmH1N1)和A/Switzerland/8060/2018(H3N2)培养2d为最佳时间,乙型流感病毒B/Beijing/1/2018(B Yamagata)培养2.5d为最佳时间;在96孔板中,A/Beijing/2/2018(pdmH1N1)和A/Switzerland/8060/2018(H3N2)培养20h、B/Beijing/1/2018(B Yamagata)培养24h为最佳时间;0.5 log10为合适的药物稀释度。12孔板和96孔板均可用于季节性流感病毒对T-705的敏感性试验。在12孔板中,2种染色方法无显著性差异;96孔板只能使用免疫染色法。本研究提示,在进行流感病毒对聚合酶抑制剂类药物的敏感性检测中,需严格控制病毒的培养时间和药物的稀释度,12孔板或96孔板均可定量流感病毒以及测定病毒的药物敏感性,微孔板较传统大孔板具有通量高、可自动读取等优势。本研究为其他实验室开展类似检测提供了参考。

Influenza virus(Ⅳ)is the main pathogen for seasonal flu epidemics and influenza pandemics. Antiinfluenza drugs are one of the critical pathways in control of influenza virus infection. Now,the polymerase inhibitors of influenza viruses are among the most promising category. To optimize the method for detecting the susceptibility of influenza virus against polymerase inhibitors,the incubation time,drug concentration and other test conditions were determined when different types/subtypes of seasonal influenza viruses were inoculated in different culture plates,and immuno-staining and crystal violet staining were compared. The susceptibility of seasonal influenza viruses to T-705 was evaluated by plaque reduction assay. Results showed that in 12-well tissue culture plates,the optimal incubation time was 2 days for influenza A viruses A/Beijing/2/2018(pdmH1N1)and A/Switzerland/8060/2018(H3N2),and 2.5 days was the best time for influenza B virus B/Beijing/1/2018(B Yamagata). In 96-well plates, the optimal incubation time for A/Beijing/2/2018(pdmH1N1)and A/Switzerland/8060/2018(H3N2)was 20 hrs. For B/Beijing/1/2018(B Yamagata),the optimal incubation time was 24 hrs. 0.5 log10 was appropriate dilution for T-705. Both 12-well and 96-well plates could be used to detect the susceptibility of seasonal influenza virus to T-705. There was no significant difference between the two staining methods in 12-well plates. 96-well plates could only be used for immuno-staining. The present study suggested that it is necessary to strictly control the incubation time of the virus and the concentration of the drug to detect the susceptibility of influenza viruses to polymerase inhibitors. Both 12-well and 96-well plates can be used to quantify the influenza virus and determine the susceptibility to polymerase inhibitors. Microplates have the advantages of high throughput and automatic reading compared with traditional large-well plates. This study provided a reference for other laboratories to conduct similar assays.