呼吸道合胞病毒培养及其病毒滴度检测方法的建立

Development of methods for culture and titer determination of respiratory syncytial virus

目的确定呼吸道合胞病毒（respiratory syncytial virus,RSV）培养条件,筛选病毒保护剂配方,并建立检测病毒滴度的微量细胞病变方法。方法分别将Hep2、Vero和293R细胞以0.01 MOI接种RSV,选择病毒培养细胞基质;将RSV分别以0.005和0.02 MOI接种Hep2细胞,在4～9 d收获病毒液,检测病毒滴度,确定最佳MOI及病毒收获时间;将6种配方病毒保护剂分别加至病毒液中,反复冻融7次,检测病毒滴度,筛选最佳配方;将病毒分别在33和37℃条件下滴定,第7、9天判定结果,确定微量细胞病变方法的培养温度和判定时间。结果确定病毒培养细胞基质为Hep2细胞,以0.02 MOI RSV接种后,37℃培养7～9 d收获病毒液;配方1（0.1%人血白蛋白）为最佳病毒保护剂配方;微量细胞病变法的实验条件为37℃滴定,7 d判定结果。结论建立了稳定可靠的呼吸道合胞病毒培养及病毒滴度检测方法。

Objective To determine the culture condition of respiratory syncytial virus（RSV）, screen the formula of virus stabilizer and develop a micro-cytopathic effect（CPE）method for determination of virus titer. Methods Hep2, Vero and293 R cells were inoculated with RSV at a MOI of 0. 01 to optimize the cell substrate for virus culture. Hep2 cells were inoculated with RSV at MOIs of 0. 005 and 0. 02 respectively, while the virus liquid was harvested 4 ～ 9 d later and determined for titer to optimize the MOI and time for virus harvest. Virus liquid was added with stabilizers of six formula respectively, then subjected to seven cycles of freezing and thawing and determined for titer to optimize the formula. The virus was titrated at 33 and 37 ℃ respectively, of which the results were judged on days 7 and 9 to optimize the temperature for virus culture and time for result judgment in micro-CPE method. Results The optimal substrate for virus culture was Hep2 cells. RSV was inoculated at a MOI of 0. 02 and cultured at 37 ℃ for 7 ～ 9 d before harvest. The optimal stabilizer was of formula 1（0. 1% human serum albumin）. In micro-CPE assay, RSV was titrated at 37 ℃, and the result was judged on day 7. Conclusion Stable and reliable methods for culture and titer determination of RSV were developed.