Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA
Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA
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DOI：
10.1099/0022-1317-79-3-437
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发表时间：
1998-03-01
影响因子：
3.8
通讯作者：
Davidsen, AD
中科院分区：
文献类型：
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作者：
Gualano, RC;Pryor, MJ;Davidsen, AD
在低拷贝数载体中构建了与登革热病毒 2 型 (DEN-2) 新几内亚 C 株 (NGC) 的 RNA 相对应的基因组长度 cDNA 克隆。克隆的cDNA在大肠杆菌中稳定繁殖并命名为pDVWS501。使用 T7 RNA 聚合酶在体外从 cDNA 产生的 RNA 转录物在电穿孔到 BHK-21 细胞中后产生感染性病毒 (MON501)。与亲代 NGC 病毒相比，MON501 在白纹伊蚊 C6/36 细胞中复制到相似水平，并在乳鼠中表现出相似的神经毒力。相比之下，用作 pDVWS501 构建中间体的第二个基因组长度 cDNA 克隆 (pDVWS310) 产生的病毒 (MON310) 在 C6/36 细胞中复制良好，但在小鼠中不具有神经毒力。 MON310 含有 NGC 背景中非神经毒力 PUO-218 菌株的 prM 和 E 基因。 MON310和MON501的prM和E蛋白之间存在7个氨基酸差异，差异总体上是保守的，除了E残基126之外，在MON310中为Glu，在MON501中为Lys。为了检查该残基在小鼠神经毒力中的作用，分别在 MON310 和 MON501 中进行了 Glu --> Lys 和 Lys --> Glu 的取代。这些突变体的特性清楚地表明，E 残基 126 处的 Lys 是 DEN-2 小鼠神经毒力的主要决定因素。
A genomic-length cDNA clone corresponding to the RNA of dengue virus type 2 (DEN-2) New Guinea C strain (NGC) was constructed in a low copy number vector. The cloned cDNA was stably propagated in Escherichia coli and designated pDVWS501. RNA transcripts produced in vitro from the cDNA using T7 RNA polymerase yielded infectious virus (MON501) upon electroporation into BHK-21 cells. When compared with parental NGC virus, MON501 replicated to similar levels in Aedes albopictus C6/36 cells and showed similar neurovirulence in suckling mice, In contrast, a second genomic-length cDNA clone (pDVWS310) used as an intermediate in the construction of pDVWS501 produced virus (MON310) that replicated well in C6/36 cells but was not neurovirulent in mice. MON310 contained the prM and E genes of the non-neurovirulent PUO-218 strain in an NGC background. There were seven amino acid differences between the prM and E proteins of MON310 and MON501,The differences were generally conservative, with the exception of E residue 126, which was Glu in MON310 and Lys in MON501. To examine the role of this residue in mouse neurovirulence, substitutions of Glu --> Lys and Lys --> Glu were made in MON310 and MON501, respectively. The properties of these mutants clearly demonstrated that Lys at E residue 126 is a major determinant of DEN-2 mouse neurovirulence.