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

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.

候选登革病毒2型（DEN-2）疫苗毒株PDK-53的基因组，与其亲本毒株DEN-2 16681存在9个核苷酸差异。本研究通过感染性cDNA克隆，构建了18株16681/PDK-53重组病毒，以分析4个由16681向PDK-53引入的突变位点，包括5′非编码区（5′ noncoding region, 5′NC）-57位C→T突变、膜前蛋白（premembrane, prM）-29位天冬氨酸→缬氨酸突变（结构蛋白区域中唯一发生的突变）、非结构蛋白1（nonstructural protein 1, NS1）-53位甘氨酸→天冬氨酸突变，以及NS3-250位谷氨酸→缬氨酸突变。

本研究对上述重组病毒的噬斑大小、在LLC-MK₂细胞中的生长速率与温度敏感性、在C6/36细胞中的生长速率，以及新生小鼠的神经毒力进行了检测。所有重组病毒在LLC-MK₂细胞中均可增殖至10^7.3 PFU/ml或更高的峰值滴度。PDK-53毒株在C6/36细胞中的复制能力受损，以及对小鼠的减毒特性，主要由5′NC-57-T与NS1-53-Asp突变决定。PDK-53毒株的温度敏感性则归因于NS1-53-Asp与NS3-250-Val突变。5′NC-57、NS1-53与NS3-250这三个位点，均与PDK-53毒株的小噬斑表型相关。若要恢复亲本16681毒株的表型特征，需在PDK-53毒株的2个或3个上述位点引入回复突变。prM-29位点对病毒表型几乎无影响。序列分析结果显示，PDK-53毒株与PDK-45毒株的遗传序列完全一致。将登革病毒减毒标记的三个主要遗传决定因子限定在非结构基因组区域，使得PDK-53毒株的基因型成为开发嵌合登革病毒疫苗候选株的理想选择。