痘苗病毒天坛广-9分离株的生物学特性初步研究  

Preliminary study on the biological characteristics of the isolate of vaccinia virus Tiantan strain VG9

作　　者：王云鹏[1] 曹守春[1] 吴小红[1] 李加[1] 石磊泰[1] 赵丹华 李长贵[1] 叶强[1] Wang Yunpeng;Cao Shouchun;Wu Xiaohong;Li Jia;Shi Leitai;Zhao Danhua;Li Changgui;Ye Qiang(National Institutes for Food and Drug Control,Research Units of Innovative Vaccine Quality Evaluation and Standardization,Chinese Academy of Medical Sciences,Beijing 102629,China)

机构地区：[1]中国食品药品检定研究院、中国医学科学院创新疫苗质量评价与标准化研究创新单元,北京102629

出　　处：《中华微生物学和免疫学杂志》2024年第12期1059-1066,共8页Chinese Journal of Microbiology and Immunology

基　　金：中国医学科学院医学与健康科技创新工程项目资助(2021-I2M-5-005)。

摘　　要：目的进一步研究痘苗病毒天坛株(vaccinia virus Tiantan strain,VTT)中传代获得的广-9株(VG9)的生物学特性。方法取冻干VG9株复溶后接种Vero细胞进行复苏并连续传代,通过间接免疫荧光法和PCR法对该毒株进行鉴定。使用二代测序技术对该毒株进行全基因组测序,将测序结果与天坛株参考基因组序列及其他33株正痘病毒基因组序列进行比对,并绘制系统发育树。通过终末稀释法获得1株VG9克隆纯化株(VG9-V3-3),对该克隆株的病毒滴度、兔皮内毒力等生物学特性进行初步研究。结果经间接免疫荧光鉴定,VG9株可以与VTT兔免疫血清特异性结合,PCR结果证明该病毒株含有痘苗病毒特征性TK基因片段。全基因组测序结果显示该VG9株基因组全长183596 bp,其中编码区165150 bp,将测序结果在NCBI核心核苷酸数据库内进行比对,结果显示VG9株与现有多个VTT株的序列高度同源(一致性>99.5%)。经序列比对发现VG9株与VTT TP5分离株的基因序列共存在749个核苷酸位点差异。VG9-V3-3分离株的病毒滴度为6.3 lg(PFU/ml),兔皮内毒力试验显示其毒力较VTT株明显减弱。结论对VG9株生物学特性进行了初步研究并获得1株纯化克隆株VG9-V3-3,该分离株病毒滴度基本稳定,且毒力较VTT株明显减弱,后续还将对该毒株的免疫原性进行深入研究,以探索其作为疫苗生产毒株的可行性。ObjectiveTo further study on the biological characteristics of vaccinia virus VG9 strain obtained by passaging from vaccinia virus Tiantan strain(VTT).MethodsThe freeze-dried vaccinia virus VG9 strain was reconstituted and inoculated into Vero cells for recovery and sequential passaging.The strain was identified by indirect immunofluorescence and PCR amplification.The whole genome of VG9 was sequenced using next-generation sequencing technology.The sequencing results were compared with the reference sequence of VTT and the genome sequences of other 33 orthopoxviral strains,and a phylogenetic tree was drawn.A purified clone of VG9,namely VG9-V3-3,was obtained by terminal dilution method.Preliminary studies were conducted to characterize the biological properties of this clone,such as virus titer and intradermal virulence in rabbit.ResultsVG9 could specifically bind to the rabbit antibody of VTT by indirect immunofluorescence identification,and the PCR amplification results proved that this strain contained the characteristic TK gene fragment of vaccinia virus.Whole-genome sequencing showed that the genome length of VG9 was 183596 bp,including 165150 bp in coding region.The sequencing results were compared with the NCBI core nucleotide database,and showed that the sequences of VG9 and multiple existing Tiantan isolates were highly homologous(consistency>99.5%).Sequence comparison revealed a total of 749 nucleotide site differences between the gene sequences of VG9 and TP5 isolate of VTT.The viral titer of the VG9-V3-3 isolate was 6.3 lg(PFU/ml),and the rabbit intradermal virulence assay showed that its virulence was significantly weakened compared with that of VTT.ConclusionsThe biological characteristics of vaccinia virus VG9 strain are preliminarily studied and a purified clone strain named VG9-V3-3 is obtained.The viral titer of the isolate is basically stable,and the virulence is significantly weakened compared with that of VTT.In-depth study on the immunogenicity of VG9-V3-3 will be carried out in the future t

关 键 词：痘苗病毒 生物学特性 免疫荧光 二代测序 生物信息学分析 

分 类 号：R392-33[医药卫生—免疫学]