肠道病毒71型毒株体外不同温度连续传代其生物学特性变化研究  

Dynamic changes of biological characteristics of EV-A71 strains continuously passaged in vitro at different temperatures

作　　者：朱凡丽 李华[1] 谢天宏[1] 杨婷[1] 宋霞[1] 王玺[1] 沈冬[1] 刘正玲[1] 岳磊[1] 谢忠平[1] ZHU Fan-li LI Hua XIE Tian-hong YANG Ting SONG Xia WANG Xi SHEN Dong LIU Zheng-ling YUE Lei XIE Zhong-ping(Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming , Yunnan 650118, China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗工程技术研究中心云南省重大传染病疫苗研发重点实验室,昆明650118

出　　处：《中国病毒病杂志》2016年第2期113-119,共7页Chinese Journal of Viral Diseases

基　　金：云南省科技计划(2015BC008)

摘　　要：目的探究肠道病毒71型(EV-A71)FY23-K株在不同温度下连续传代对其生物学特性的影响,旨在获得EV-A71的减毒候选株。方法在33℃条件下进行FY23-K株克隆筛选,之后分别在33℃及35℃下连续传代,并进行病毒生长增殖特性、乳鼠致病性及在小鼠体内的免疫原性检测,对比分析其各项生物学特性的变化。结果挑选出感染性滴度较高的6个克隆,在33℃条件下连续传代后发现,其感染性滴度逐渐降低,且相同接种条件下,达到75%病变程度的收获时间随代次的增加逐渐延长,至26代后,接种至第8天细胞病变程度只达到40%。而在35℃条件下连续传代的病毒能够稳定增殖,保持良好的生长特性,毒株感染性滴度>6.5lg CCID50/ml;35℃传代后毒株的免疫原性良好,6株克隆株有4株样品的灭活抗原免疫小鼠后抗体的阳转率可达100.0%,GMT可达1∶128,最高的中和抗体效价为1∶512;活病毒免疫小鼠抗体阳转率达88.9%以上,中和抗体效价均>1∶16;传代样品攻击乳鼠后,乳鼠无发病、死亡情况,其脑、脊髓也未产生明显病理性损伤,仅在肌肉组织细胞间隙发现少量的炎性细胞浸润,在脑前额叶出现数个胶质小结。结论 FY-23K克隆株在35℃低温连续传代后有稳定的生长特性和良好的免疫原性,对乳鼠未表现出明显致病性,可作为EV-A71减毒活疫苗进一步研究的候选毒株。Objective To observe the dynamic changes of biological characteristics of EV-71 strain FY23-K continuously passaged in vitro at different temperatures, in order to obtain an attenuated EV-A71 vaccine candidate. Methods FY23-K strain clones were screened at 33 ℃ and then passaged at 33 ℃and 35 ℃. The growth characteristics, the pathogenicity to suckling mice and the immunogenicity and genetic stability were tested, and the changes of biological characteristics were observed and compared. Results Six clones with higher infectious titers at 33 ℃ were selected, after continuous passage, the infectivity titers decreased gradually, while the harvest time was increased with the passages. After 26 passages, there was only 40% of cytopathic cells at day 8. However, when passaged at 35 ℃, viruses were able to maintain good growth with the infectious titers of more than 6.5 lg CCID50/ml. Four out of 6 clones showed higher immunogenicity with a 100.0% of seroconversion rate and GMT of 1 ： 128 when immunized mice with inactivated antigens. The highest neutralizing antibody titer was 1 ： 512. When mice were immunized with live virus, the seroconversion rate could be higher than 88.9 % with the neutralizing antibody titers of over 1 ： 16. Moreover, when suckling mice were attacked with passaged virus, mice were almost normal with no obvious pathological damage in the brain and spinal, only had light inflammatory cell infiltration in muscle tissue and a few glial nodules in front lobe of brain. Conclusions FY23-K stain grows stably passaged in vitro under 35 ℃and the immunogenicity is high with no pathogenicity to suckling mice, which makes it a good candidate for live attenuated vaccine of EV-A71.

关 键 词：手足口病 肠道病毒71型 减毒活疫苗 

分 类 号：R373[医药卫生—病原生物学]