CAJ | 학술논문

目的应用构建的水痘-带状疱疹病毒(VZV)报告细胞系MV9G建立一种筛选抗VZV药物的新方法。方法将VZV疫苗株(vOka)的无细胞病毒液(CFV)直接感染MV9G细胞2h后移除（CFV直接感染法），或将感染vOka株的MeWo细胞（含带细胞病毒，CAV）与MV9G细胞共培养48 h（CAV共培养法），以激发MV9G细胞表达报告基因萤火虫荧光素酶。在培养基中加入肝素、磷酸甘露糖(M-6-P)、阿昔洛韦(ACV)、白藜芦醇、roscovitine等抗病毒药物，通过比较加药前后MV9G细胞荧光素酶活性变化来分析药物对VZV的作用。结果抗病毒药物各浓度组不同程度地抑制CFV直接感染和/或CAV共培养激发的MV9G细胞荧光素酶活性，荧光素酶活性的降低与平行对照组中病毒蚀斑数的降低一致。抗病毒药物中肝素、M-6-P和roscovitine 2.5 μmol/L组抑制CFV激发荧光素酶的作用强于抑制CAV激发荧光素酶的作用，而ACV和白藜芦醇抑制CAV激发荧光素酶的作用强于抑制CFV激发荧光素酶的作用。ACV抗药株Kanno和rOka YSR的CAV与MV9G细胞共培养均激发其强表达荧光素酶，但ACV抑制抗药株激发荧光素酶50％时浓度(IC50)显著高于抑制敏感株pOka和CaGu的IC50。结论 CFV直接感染法和CAV共培养法分别有助于筛选针对VZV感染早期和后期的药物，利用MV9G细胞建立的报告细胞法可为抗VZV药物筛选和作用机制研究提供一种简便、快速、敏感和高通量的新方法。
목적 응용구건적수두-대상포진병독(VZV)보고세포계MV9G건립일충사선항VZV약물적신방법。방법 장VZV역묘주(vOka)적무세포병독액(CFV)직접감염MV9G세포2h후이제（CFV직접감염법），혹장감염vOka주적MeWo세포（함대세포병독，CAV）여MV9G세포공배양48 h（CAV공배양법），이격발MV9G세포표체보고기인형화충형광소매。재배양기중가입간소、린산감로당(M-6-P)、아석락위(ACV)、백려호순、roscovitine등항병독약물，통과비교가약전후MV9G세포형광소매활성변화래분석약물대VZV적작용。결과 항병독약물각농도조불동정도지억제CFV직접감염화/혹CAV공배양격발적MV9G세포형광소매활성，형광소매활성적강저여평행대조조중병독식반수적강저일치。항병독약물중간소、M-6-P화roscovitine 2.5 μmol/L조억제CFV격발형광소매적작용강우억제CAV격발형광소매적작용，이ACV화백려호순억제CAV격발형광소매적작용강우억제CFV격발형광소매적작용。ACV항약주Kanno화rOka YSR적CAV여MV9G세포공배양균격발기강표체형광소매，단ACV억제항약주격발형광소매50％시농도(IC50)현저고우억제민감주pOka화CaGu적IC50。결론 CFV직접감염법화CAV공배양법분별유조우사선침대VZV감염조기화후기적약물，이용MV9G세포건립적보고세포법가위항VZV약물사선화작용궤제연구제공일충간편、쾌속、민감화고통량적신방법。
Objective To establish a novel method to screen for anti-varicella-zoster virus (VZV) compounds with our previously generated reporter cell line for VZV, MV9G. Methods MV9G cells were directly infected with cell-free virus of Oka vaccine strain (vOka) for 2 hours( CFV direct-infection) or cocultured with vOka-infected MeWo cells containing cell-associated virus for 48 hours (CAV co-culture) to promote expression of the reporter gene firefly luciferase. Antiviral compounds including heparin, mannose-6-phosphate( M-6-P), acyclovir( ACV ), resveratrol and roscovitine were added in the medium before or after the virus infection. Inhibitory effects( IC50 ) of the antiviral compounds were analyzed by comparing firefly luciferase activities of MV9G cells in the presence of antiviral compounds with those in the absence. Results Antiviral compounds inhibited luciferase activities of MV9G cells activated by CFV direct-infection and/or CAV co-culture in different levels. The reductions of luciferase activities statistically correlated with those of viral foci shown by immunostaining with a monoclonal antibody against VZV immediate early 62 antigens (IE62) in controls. Among these compounds, heparin, M-6-P, and 2.5 μmol/L of roscovitine inhibited CFV-activated more strongly than CAV-activated luciferase activities, whereas ACV and resveratrol inhibited CAV-activated more strongly than CFV-activated luciferase activities. Cell-associated ACV-resistant strains,Kanno and rOka YSR, activated luciferase activities of MV9G cells, too. However, the inhibitory concentrations (IC50) of ACV to the ACV-resistant strains were much higher than those to the ACV-sensitive strains,pOka and CaGu. Conclusion The CFV direct-infection and CAV co-culture assays were useful to screen for antiviral compounds targeting the early and late phases of VZV infection, respectively. The VZV reporter cell-based assays may provide a simple, rapid, sensitive, and high-throughput method to screen for anti-VZV compounds.