Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5′ noncoding region and nonstructural proteins 1 and 3
Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5′ noncoding region and nonstructural proteins 1 and 3
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DOI：
10.1128/jvi.74.7.3011-3019.2000
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发表时间：
2000-04-01
影响因子：
5.4
通讯作者：
Kinney, RM
中科院分区：
文献类型：
--
作者：
Butrapet, S;Huang, CYH;Kinney, RM
一株登革2型（DEN - 2）候选疫苗病毒株PDK - 53的基因组与其亲本DEN - 2 16681相比有9个核苷酸差异。我们利用感染性cDNA克隆构建了18株重组16681/PDK - 53病毒，以分析4个从16681到PDK - 53的突变，包括5'非编码区（5'NC）第57位的C→T、前膜蛋白（prM）第29位的天冬氨酸→缬氨酸（结构蛋白中唯一的突变）、非结构蛋白1（NS1）第53位的甘氨酸→天冬氨酸以及NS3第250位的谷氨酸→缬氨酸。我们研究了这些病毒在LLC - MK₂细胞中的蚀斑大小、生长速率和温度敏感性，在C6/36细胞中的生长速率以及对新生小鼠的神经毒力。所有病毒在LLC - MK₂细胞中复制达到的峰值滴度为10⁷.³PFU/ml或更高。PDK - 53病毒在C6/36细胞中复制受阻以及对小鼠的减毒主要由5'NC - 57 - T和NS1 - 53 - 天冬氨酸突变决定。PDK - 53病毒的温度敏感性归因于NS1 - 53 - 天冬氨酸和NS3 - 250 - 缬氨酸突变。5'NC - 57、NS1 - 53和NS3 - 250位点都对PDK - 53病毒的小蚀斑表型有贡献。PDK - 53病毒在这三个位点中的两个或三个位点发生回复突变才能重建亲本16681病毒的表型特征。prM - 29位点对病毒表型几乎没有影响。序列分析表明PDK - 53病毒在基因上与PDK - 45病毒相同。将减毒标记的三个主要遗传决定因素限制在非结构基因组区域，使得PDK - 53病毒基因型对开发嵌合登革病毒候选疫苗具有吸引力。
The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly to-Asp, and NS3-250 Glu-to-Val, The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10(7.3) PFU/ml or greater in LLC-MK(2) cells, The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the small-plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristics of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype, Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.