我国云南登革4型病毒减毒株生物学特性及全基因组序列分析  

Biological characteristics and complete genome sequence of attenuated strains of Dengue virus type 4 isolated in Yunnan,China

作　　者：陈柠[1] 俞永新[1] 徐宏山[1] 刘欣玉[1] 王志伟[1] 杨立宏[1] 岳广智[1] 贾丽丽[1] 董关木[1] 李玉华[1] 

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《中国生物制品学杂志》2014年第8期997-1001,1009,共6页Chinese Journal of Biologicals

摘　　要：目的 对我国云南登革4型病毒分离株（Ban18）及其减毒株（Ban18 HK20和Ban18 HK30）的生物学特性进行分析,测定原株和减毒株的全基因组序列并进行比对,分析与毒力相关的位点。方法 将各毒株在Vero细胞传代后进行空斑形态观察、小鼠脑内致病力和免疫原性检测;减毒株经空斑纯化后,挑选单克隆,检测其小鼠脑内致病力;提取减毒株基因组RNA,RT-PCR法分段扩增其全基因组序列并测序,应用DNAStar软件包进行序列比对分析。结果Ban18 HK20和Ban18 HK30毒株在Vero细胞上形成的空斑较其原株稍大,但边界较模糊;两个代次的减毒株对KM小鼠不致病,对2~4日龄乳鼠的脑内致病力也明显低于其原株;Ban18原株和Ban18 HK20毒株免疫的小鼠经3个剂量的国际标准株H241株脑内攻击后,均无死亡,但Ban18 HK30毒株与对照组相比,未显示出保护作用;Ban18 HK20毒株的9个克隆株中,3株对乳鼠的脑内致病力低于其他6株克隆株,Ban18 HK30毒株的5个克隆株对乳鼠的脑内致病力无明显差异;减毒株的两个代次病毒与其原株存在3个氨基酸[A→V（C-111）、I→T（E-155）、I→L（E-369）]和一个核苷酸[G→A（3’UTR-219）]的共同差异。结论 通过原代地鼠肾细胞传代的两个代次（HK20和HK30）病毒对小鼠的脑内致病力明显减弱,其中Ban18 HK20免疫原性较好,并从空斑克隆中筛选出3株毒力较Ban18 HK20更低的克隆株;C-111、E-155、E-369和3’UTR-219突变与毒力减弱密切相关。Objective To investigate the biological characteristics and complete genome seuqnences of Ban 18 strain of dengue type 4 isolated in Yunnan, China as well as its attenuated strains Banl8 HK20 and Banl8 HK30, and analyze the virulence-related sites. Methods The strains were subcultured in Vero cells, observed for morphology by plaque assay, and determined for intracerebral virulence in mice and immunogenicity. The attenuated strains were purified by plaque assay, and single clones were selected and determined for intracerebral virulence in mice. Genomic RNAs of attenuated strains were extracted, based on which the complete genomes were fragmentally amplified by RT-PCR and sequenced, and the sequencing results were analyzed by using DNAStar software. Results Ban18 HK20 and Banl8 HK30 strains formed large but fuzzy plaques on Veto ceils compared with their parent strain Banl8. The attenuated strains of two passages showed no virulence to KM mice, while the intracerebral virulence to the suckling mice at age of 2 - 4 d were significantly lower than those of strain Banl8. The mice vaccinated with Ban18 and Ban18 HK20 strains survived the challenge with international standard H241 strain at three dosages. However, strain Ban18 HK30 showed no protection as compared with placebo as control. The intracerebral virulence of three of nine clones of Banl8 HK20 strain to suckling mice were lower than thoseI of the other six clones, while the virulence of five clones of Banl8 HK30 I strain showed no significant difference. As compared with their parent strain Banl8, the two strains showed variations in three amino acids [A→V（C-111）、I→T（E-155）、I→L（E-369）] and one nucleotide [G→A（3＇UTR-219）]. Conclusion The virulence of Banl8 HK20 and Banl8 HK30 were significantly lower than those of their parent strain. Ban18 HK20 strain showed good immunogenicity, of which three clones with lower virulence were screened. The four common mutations at the sites of C111, E155, E369 and 3＇UTR219 were closely associa

关 键 词：登革4型病毒 Ban18株 减毒株 毒力 免疫原性 序列分析 

分 类 号：R373.33[医药卫生—病原生物学] Q789[医药卫生—基础医学]