CONSTRUCTION AND CHARACTERIZATION OF CHIMERIC TICK-BORNE ENCEPHALITIS DENGUE TYPE-4 VIRUSES
CONSTRUCTION AND CHARACTERIZATION OF CHIMERIC TICK-BORNE ENCEPHALITIS DENGUE TYPE-4 VIRUSES
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DOI：
10.1073/pnas.89.21.10532
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发表时间：
1992-11-01
影响因子：
11.1
通讯作者：
LAI, CJ
中科院分区：
文献类型：
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作者：
PLETNEV, AG;BRAY, M;LAI, CJ
以登革4型病毒（DEN 4）cDNA为载体表达远亲蜱传脑炎病毒（TBEV）的基因。全长嵌合体TBEV/DEN 4 cDNA通过将编码蛋白质如衣壳（C）、前膜（其是膜的前体）（M）、包膜（E）或非结构蛋白NS 1的TBEV基因替换为相应的DEN 4序列来构建。从cDNA制备的RNA转录物用于转染允许的猿猴细胞。回收了两种含有TBEV CME或ME基因的活嵌合病毒。与DEN 4相比，嵌合TBE（ME）/DEN 4病毒[命名为vTBE（ME）/DEN 4]在猴细胞中产生更大的噬斑，并且生长至更高的滴度。相比之下，vTBE（ME）/DEN 4在蚊子细胞上产生更小的噬斑，并且生长至比DEN 4更低的滴度。对vTBE（ME）/DEN 4和DEN 4感染的蚊子或猿猴细胞中产生的病毒RNA和蛋白质的分析表明，嵌合体进入蚊子细胞并在其中复制的能力受到限制。相比之下，vTBE（ME）/DEN 4有效地进入猿猴细胞，并且其RNA在这些细胞中比亲本DEN 4 RNA更快地复制。脑内接种后，vTBE（ME）/DEN 4在乳鼠和成年小鼠中均引起致命性脑炎，而几乎所有通过相同途径接种DEN 4的小鼠均未发生疾病。与野生型TBEV不同，当成年小鼠通过外周途径接种时，vTBE（ME）/DEN 4不会引起脑炎。先前通过外周途径接种嵌合体的成年小鼠对随后用10（3）倍半数致死剂量的TBEV的腹膜内攻击完全耐受，而先前接种DEN 4的小鼠不受保护。这些发现表明：（i）嵌合病毒的TBEV M和E基因是主要的保护性抗原，并诱导对致死性TBEV攻击的抗性，以及（ii）TBEV基因组的其他区域对于该病毒从外周部位传播到大脑的能力是必需的。成功构建保留TBEV的保护性抗原但缺乏其外周侵袭性的活的TBEV/DEN 4嵌合体为开发活的减毒TBEV疫苗提供了策略。
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 chimerk TBEV/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C); pre-membrane, which is the precursor of membrane (M); envelope (E); or nonstructural protein NS1 for the corresponding DEN4 sequences. RNA transcripts prepared from cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared with DEN4, chimeric TBE(ME)/DEN4 virus [designated vTBE(ME)/DEN4] produced larger plaques and grew to higher titer in simian cells. In contrast, vTBE(ME)/DEN4 produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in vTBE(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, vTBE(ME)/DEN4 entered simian cells efficiently and its RNA was replicated more rapidly in these cells than was parental DEN4 RNA. Following intracerebral inoculation, vTBE(ME)/DEN4 caused fatal encephalitis in both suckling and adult mice, while nearly all mice inoculated by the same route with DEN4 did not develop disease. Unlike wild-type TBEV, vTBE(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 10(3) times the median lethal dose of TBEV, whereas 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 resistance 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 viable TBEV/DEN4 chimera that retains the protective antigens of TBEV but lacks its peripheral invasiveness provides a strategy for the development of live attenuated TBEV vaccines.