SV40感染滴度测定方法的建立及高滴度病毒的制备

Development of Determination Method for Infectious Titer of SV40 and Preparation of High Titer Virus

目的建立稳定的SV40感染滴度测定方法,制备高滴度SV40,用于生物制品病毒清除/灭活工艺的验证。方法通过分析不同细胞感染SV40后出现病变的时间、病变程度及产毒量,确定SV40敏感细胞株。分析维持液、细胞培养时间、病毒吸附时间等对病毒滴定测定的影响,建立SV40滴度测定的方法。并分析病毒感染后不同时间及细胞不同部位SV40滴度的差异,制备大量的高滴度SV40。结果与Vero、Vero76及VeroE6细胞相比,CV-1细胞对SV40高度敏感,细胞病变出现时间最早,病变最明显,产毒量最高。SV40滴度与病毒接种前细胞的培养时间、细胞接种量和维持液无明显相关,但吸附时间对病毒滴度有一定的影响。病毒的最佳吸附时间为120 min。接种病毒后48 h收集细胞沉淀,所获得的SV40滴度最高,平均为8.81 lg CCID50/ml。结论已建立了稳定的SV40滴度测定方法,并制备了高滴度SV40,为病毒清除/灭活工艺验证研究奠定了基础。

Objective To develop a stable determination method for infectious titer of SV40 and prepare high titer SV40 used for the validation of virus removal/inactivation procedure of biologics. Methods Analyze the time when CPE appears, the degree of CPE and virus yield of various cells after infection with SV40 to screen SV40-sensitive cell strain. Analyze the effects of maintain medium, time for culture and time for virus adsorption on the virus titration to develop a method for determination of SV40 titer. Analyze the variation of SV40 titers at various hours after infection and at various sites of cells to prepare high titer SV40 in a large quantity. Results Compared with Vero, Vero 76 and VeroE6 cells, CV-I cells were more sensitive to SV40,in which the CPE appeared earlier and was more significant,and the virus yield was higher. SV40 virus titer showed no significant relationship to the time for culture of cells before inoculation, concentration of cells inoculated and cell maintain medium. However, the time for virus adsorption showed a certain influence on virus titer. The optimal time for virus adsorption was 120 min. The mean titer of SV40 harvested 48 h after inoculation was 8.81 CCID50/ml. Conclusion A stable method for determination of SV40 titer was developed,and high titer SV40 was prepared, h laid a foundation of validation of virus removaL/inaetivation procedure of biologies.