水痘-带状疱疹病毒滴定方法的优化与应用评价

Optimization and application of titration method for varicella-zoster virus

目的对水痘-带状疱疹病毒(varicella-zoster virus,VZV)病毒滴定方法进行优化,并应用于中和抗体检测和抗病毒药物体外检测。方法采用VZV疫苗株Voka株在48孔板感染人视网膜上皮细胞(adult retinal pigment epithelial cell line-19,ARPE-19),连续7 d检测感染后病毒复制动力学与蚀斑特点;分别建立并优化活病毒滴定的蚀斑染色法和酶联免疫斑点法(enzyme linked immunospot assay,ELISPOT),同时对两种方法检测结果的相关性进行分析,并将改进后的方法应用于VZV药物抑制效果检测和血清中和抗体检测。结果VZV感染ARPE-19细胞(感染复数为0.05)后,第6天到达复制顶峰并形成肉眼可见蚀斑;蚀斑染色法中,添加0.2 mg/mL二乙胺乙基葡聚糖(diethylaminoethyl-dextran,DEAE-Dextran)后,较未添加组产生更大与边缘更加清晰的蚀斑。ELISPOT法可在感染后第3天进行计数,同时通过改变检测用抗体与显色液等,可产生更加清晰可计的斑点。两种方法可采用机器计数,检测病毒滴度或应用于体外检测抗VZV药物效力与血清中和抗体滴度,具有良好的相关性。结论优化后ELISPOT法快捷灵敏,可替代蚀斑染色法应用于VZV病毒滴度检测与相关应用。本研究为VZV的检测及疫苗药物研发提供了更简捷可靠的实验方法。

Objective To optimize the titration method for live varicella zoster virus(VZV),and apply it for neutralizing antibody and antiviral drug detections in vitro.Methods The VZV vaccine strain Voka strain was used to infect adult retinal pigment epithelial cell line 19(ARPE-19)in a 48-well plate.The virus replication dynamics and plaque characteristics were detected after infection for 7 consecutive days.The classical plaque staining(CPS)and enzyme-linked immunospot(ELISpot)assays for live virus titration were established and optimized.The correlation between the results of both assays was analyzed.The optimized assays were both applied to the testing of anti-VZV drug(cidofovir,used in this study)inhibition effect and serum neutralizing antibody titer.Result The replication peak of VZV was reached on the sixth day after infection of ARPE-19 cells(multiplicity of infection=0.05)and formed visible plaques.For CPS,addition of 0.2 mg/ml diethylaminoethyl-dextran(DEAE-Extran)resulted in larger and clearer plaque compared to the group without adding DEAE-Extran.The result of ELISpot can be read on the third day after infection.Modification of antibody and colorimetric solution created more clear and countable plaques.Both methods were suitable for machine counting and had good correlation in testing of virus titer or in-vitro detection of anti-VZV drug efficacy and serum neutralizing antibody titers.Conclusions The optimized ELISpot assay was rapid and sensitive and can be used in place of CPS assay in testing of VZV titer and related applications,The present study provided simple and reliable experimental method for the detection of VZV and the development of vaccine and drugs.