人巨细胞病毒蚀斑实验条件的优化及在抗病毒物质活性测定中的应用

Study on the optimization of plaque assay for human cytomegalovirus and detection antiviral activity of anti-virus substance

目的探讨人巨细胞病毒(HCMV)的蚀斑形成实验最适工作条件,建立稳定易行的HCMV蚀斑技术,用于HC-MV的定量测定及体外抗病毒物质活性测定的研究。方法HCMV病毒悬液接种至人胚成纤维细胞(HF)单层,37℃吸附1h,分别用含0.463‰、0.925‰NaHCO3及0.5%、1%DMSO琼脂糖覆盖,1周后加盖第2层;2周后用0.5%结晶紫染色5min,计数、计算pfu/ml,采用PCR法对蚀斑培养物进行鉴定;分别用患者血清及HCMVAD169株兔免疫血清进行蚀斑抑制实验,计算中和抗体滴度;蚀斑减少实验测定不同浓度更昔洛韦(GCV)的抗HCMV活性。结果HCMV对照孔在5d出斑,蚀斑为圆形、实心、不透明状;两种浓度NaHCO3对蚀斑形成数量、大小有明显影响且病毒滴度差异有显著性(P<0.05)。在含0.463‰NaHCO3培养孔内,出斑时间提前至3d;但不同浓度的DMSO对蚀斑数量、大小以及病毒滴度的影响差异无显著性(P>0.05)。HCMV患者血清及HCMVAD169株兔免疫血清均可以抑制蚀斑形成。利用蚀斑减少实验测定GCV抗HCMV活性,可在7d内获得结果。结论HCMV蚀斑形成实验条件经优化后,可以方便稳定地对HCMV进行定量测定以及抗病毒活性物质的筛选和评价。

Objective To establish a optimal plaque assay for researching human cytomegalovirus （HCMV） by studying the infuence on HCMV plaque formation. Methods HCMV was inoculated into HF cells monolayer , adsorbed at 37 ℃, added two agarose overlays respectively with 0. 463%0,0. 925%0 or 0. 5% , 1% DMSO within two weeks. The cells were stained with 0.5% crystal violet in 10% formalin for 5 min. Plaques were counted accurately. PCR was used to identify the HCMV. Plaque-reduction neutralization test was carried out with the sera of patient and the sera of rabbits immuned by HCMV AD 169 strain. The optimal plaque reduction test can be used in evaluating antiviral activity of ganciclovir（GCV）. Results The time of a routine plaque formed was 5 days, and it＇ s round, solid, opaque. The numbers and sizes of plaque which overlay with different concentration of NaHCO3 were different, titers had signifcant difference （ P 〈 0. 05 ）, Plaque could be formed in 3 days by the low NaHCO3 concentration method than it which ever used as a routine assay in HCMV titration. The plaques were similar with different DMSO, titers also had no signifcant difference （ P 〉 0. 05 ）. Plaque formation could be neutralized not only by HCMV patient sera but also by rabbit sera. It was only 7 clays by the optimal plaque reduction test in evaluation antiviral activity of GCV. Conclusion The optimal plaque assay is stable and easy for the quantitative detection of HCMV and can be widely applied to the screening and evaluation of anti-virus substance.