两种方法制备的水痘-带状疱疹病毒(Oka株)毒种感染2BS细胞的敏感性

Sensitivity of varicella-zoster virus Oka strain prepared by two methods to 2BS cells

目的用现有方法及新方法分别制备水痘-带状疱疹病毒（varicella-zoster virus,VZV）（Oka株）毒种,比较两种方法制备的毒种对2BS细胞的感染复数及増殖动态差异。方法用现有方法（用含2%小牛血清的病毒维持液于-70℃条件下冻存Oka株毒种）和新方法（用含90%小牛血清的病毒冻存液于-196℃条件下冻存Oka株毒种）制备第47代Oka株VZV毒种,采用不同的MOI（0.000 5、0.001、0.005、0.01、0.05、0.1）感染第37代2BS细胞,观察细胞病变（CPE）情况,收获不同MOI接种和培养不同时间点病毒制备疫苗,采用噬斑法检测疫苗原液、成品滴度以及37℃稳定性,确定两种方法制备的毒种对2BS细胞感染的最适MOI。结果现有方法制备毒种以0.01~0.1 MOI感染2BS细胞72 h后,病变达到高峰期;新方法制备毒种以0.001~0.01 MOI感染2BS细胞72 h后,病变达到高峰期。两种方法收获的病毒液制备的成品疫苗的病毒滴度为4.4~4.6 lg PFU/ml,37℃放置1周,病毒滴度3.8~4.0 lg PFU/ml;疫苗原液、成品滴度以及37℃稳定性无差异,均达到本公司水痘减毒活疫苗制造与检定规程制定的相关标准。结论现有方法和新方法制备的毒种最适MOI分别为0.01~0.1和0.001~0.01,新方法制备的毒种更适合水痘疫苗的生产。

Objective To prepare varicella-zoster virus（VZV） Oka strain by the current and novel methods respectively and compare their multiplicities of infection（MOIs） and proliferation dynamic difference in 2BS cells. Methods VZV Oka strain of passage 47 prepared by the current（storage in frozen at-70 ℃ in maintain medium containing 2% calf serum）and novel（storage in frozen at-196 ℃ in virus freezing medium containing 90% calf serum） methods respectively,with which the 2BS cells of passage 37 were infected at various MOIs（0. 000 5,0. 001,0. 005,0. 01,0. 05 and 0. 1）and observed for CPE. Viruses inoculated at various MOIs and cultured for various hours were harvested and prepared into vaccines,of which the titers in bulk and final product as well as stability at 37 ℃ were determined by plaque assay to optimize the MOIs of virus seeds prepared by two methods to 2BS cells. Results The CPE of 2BS cells reached a peak value 72 h after infection with virus seed prepared by the current method at MOIs of 0. 01 ~ 0. 1 and with that prepared by the novel method at MOIs of 0. 001 ~ 0. 01. The titers of final products of vaccines prepared by the virus harvested by two methods were 4. 4 ~ 4. 6 lg PFU / ml,which decreased to 3. 8 ~ 4. 0 lg PFU / ml after storage at 37 ℃ for one week.However,the titers in bulk,final product and after storage at 37 ℃ showed no significant difference,which met the requirements for live attenuated varicella vaccine manufactured by Changchun Keygen Biological Products Co.,Ltd.Conclusion The optimal MOIs of VZV Oka strain prepared by the current and novel methods were 0. 01 ~ 0. 1 and0. 001 ~ 0. 01 respectively. However,the virus seed prepared by the novel method was more suitable for the production of varicella vaccine.