严重发热伴血小板减少综合征病毒培养以及病毒滴度检测方法的建立

Development of Methods for Culture and Titer Determination of Severe Fever with Thrombocytopenia Syndrome Virus

目的:建立严重发热伴血小板综合征病毒(SFTSV)培养和病毒滴度检测的方法,为其致病机制研究奠定基础。方法:选取生长良好的Vero细胞铺六孔板接种SFTSV,每隔24 h收集培养病毒上清检测SFTSV病毒复制拷贝数,从而选取最佳时间点收获病毒液并用浓缩离心管浓缩病毒液,保存备用。将SFTSV病毒液进行10倍比稀释,每梯度200μL接种生长良好的单层Vero细胞,用3 m L高压灭菌的甲基纤维素-DMEM覆盖物覆盖每孔Vero细胞,约9 d后温和去除甲基纤维素-DMEM覆盖物,经预冷的4%甲醛固定细胞后,用结晶紫染色,PBS洗脱3次,计算空斑数。结果:根据病毒繁殖生长情况,病毒在第4 d后达复制高峰并随后进入平台期,第8天病毒复制开始下降。参照甲基纤维素-结晶紫空斑法实验结果,病毒滴度为6×106PFU/m L。结论:SFTSV病毒培养以及滴度检测方法成功建立,选取4天后收获病毒上清最佳,甲基纤维素-结晶紫空斑法形成的空斑清晰可数。

Objective： To develop methods for culture and titer determination of severe fever with thrombocytopenia syndrome virus（SFTSV） and provide basis for the pathogenesis research. Methods： SFTSV was cultured in well-grown Vero cells plated in six well plate. Culture supernatant was collected to test copies of viral genomes every 24 h and the optimal harvest time was selected. Virus was condensed with Millipore centrifuge tube and conserved for next use. A series of 10-fold dilutions of virus were added into well-grown Vero cells with 200 μL every well. Vero cells were overlaid with 3 m L autoclaved methylcellulose-DMEM covering every well, the covering was removed gently by PBS after 9 days. Then, the Vero cells were fixed with 4 % pre-cooling formaldehyde and stained with crystal violet. After washing three times by PBS, the plaques were counted. Results： During the process of severe fever with thrombocytopenia syndrome virus culture, virus reached the peak of replication at 4 th day and then enter the plateau. Finally, replication of virus started to declined from 8 thday. According to plaque assay, the viral titer was 6×106 PFU/m L. Conclusion： A successive method to culture SFTSV and detect virus titer was developed. Culture supernatant was harvested at 4 th days post-inoculated. Plaques were clearly visible by methylcellulose plaque assay.