【摘要】 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。更多还原

【Abstract】 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.更多还原