DEVELOPMENT OF NEW APPROACHES TO VACCINES AGAINST TICK-BORNE ENCEPHALITIS VIRUS

登革4型病毒（DEN 4）cDNA被用作表达载体， 蜱传脑炎病毒（TBEV）。 全长 TBE/DEN 4嵌合cDNA通过替换TBEV基因构建， 对于蛋白质如衣壳（C）、前膜（M）、包膜（E）或 非结构蛋白NS 1的DEN 4的相应序列。 充分 从这些cDNA制备的长度的RNA转录物被用来进行PCR扩增。 允许的猿类细胞 两种含有TBEV的活嵌合病毒 CME或ME基因被回收。 与DEN 4相比，嵌合TBE（ME）/DEN 4 病毒在猿猴细胞中生长到更高的滴度并产生更大的噬斑。 在 相反，嵌合TBE（ME）/DEN 4病毒在蚊子上产生较小的噬斑 细胞并生长至比DEN 4更低的滴度。 病毒RNA分析和 TBE（ME）/DEN 4和DEN 4感染的蚊子或猿猴中产生的蛋白质 细胞显示，嵌合体的进入能力受到限制， 在蚊子细胞中复制。 与此相反，TBE（ME）/DEN 4进入猿类细胞， 细胞有效，其病毒RNA在这些细胞中复制得更快， 与亲本DEN 4病毒RNA相比， 脑内注射后 接种TBE（ME）/DEN 4病毒可导致两种哺乳动物的致死性脑炎 而通过相同途径接种DEN 4的小鼠则没有 发展疾病。 然而，与野生型TBEV不同，嵌合TBE（ME）/DEN 4不 当成年小鼠接种外周血淋巴细胞时， 路线 成年小鼠先前通过外周免疫接种嵌合体， 途径完全抵抗随后的腹腔内攻击 TBEV的LD 50为103，而之前接种DEN 4的小鼠则没有 保护. 这些发现表明：（i）TBEV的M和E基因， 嵌合病毒是主要保护性抗原， 致死性TBEV攻击，和（ii）TBEV基因组的其他区域， 对于这种病毒从外周部位传播到 大脑 成功构建保留TBE/DEN 4嵌合体的TBE/DEN 4嵌合体 TBEV的保护性抗原，但缺乏其外周侵袭性， TBEV减毒活疫苗开发的新策略。

Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full length chimeric TBE/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C), premembrane (M), envelope (E), or nonstructural protein NS1 for the corresponding sequences of DEN4. Full length RNA transcripts prepared from these cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared to DEN4, chimeric TBE(ME)/DEN4 virus grew to higher titer and produced larger plaques in simian cells. In contrast, chimeric TBE(ME)/DEN4 virus produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in TBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, TBE(ME)/DEN4 entered simian cells efficiently and its viral RNA was replicated more rapidly in these cells than was parental DEN4 viral RNA. Following intracerebral inoculation TBE(ME)/DEN4 virus caused fatal encephalitis in both suckling and adult mice, while mice inoculated by the same route with DEN4 did not develop disease. However, unlike wild-type TBEV, chimeric TBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 103 LD50 of TBEV, while mice previously inoculated with DEN4 were not protected. These findings indicate that: (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistant to lethal TBEV challenge, and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain. Success in constructing a TBE/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a new strategy for the development of live attenuated TBEV vaccines.