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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 analyze the biological characteristics of Ban18 and its attenuated strains (Ban18 HK20 and Ban18 HK30) of Yunnan dengue 4 virus strains and to compare and analyze the whole genome sequence of the original and attenuated strains Sites related to virulence. Methods After the passage of Vero cells, the morphology of plaque was observed and the pathogenicity and immunogenicity of the mice in the brain were detected. The purified strains were selected and cloned into plaques to detect the pathogenicity and immunogenicity of the mice. Genomic RNA was extracted from the attenuated strain. The whole genome sequence was amplified by RT-PCR and sequenced. DNAStar software package was used for sequence alignment analysis. Results The plaques of Ban18 HK20 and Ban18 HK30 strains were slightly larger than that of the original strain on Vero cells, but the boundaries were rather blurred. The two generations of attenuated strain did not cause disease in KM mice, The virulence in the brain of suckling mice was also significantly lower than that of the original strain. The mice immunized with Ban18 strain and Ban18 HK20 strain did not die after 3 doses of the international standard strain H241 strain, but Ban18 HK30 The strains showed no protective effect compared with the control group; of the 9 clones of Ban18 HK20 strain, 3 strains had lower intracerebral pathogenicity than the other 6 clones and 5 of Ban18 HK30 strains There was no significant difference in the virulence of the cloned strain in the brain of the neonatal rat. The two generations of the attenuated strain had three amino acids [A → V (C-111), I → T (E-155) I → L (E-369)] and one nucleotide [G → A (3’UTR-219)]. Conclusions Two generations (HK20 and HK30) of virus passaged by primary murine kidney cells significantly attenuated the pathogenicity of the brain in mice. The immunogenicity of Ban18 HK20 was good, and 3 The strains with lower virulence than Ban18 HK20 were closely related to the mutations of C-111, E-155, E-369 and 3’UTR-219.