Microarray hybridization for assessment of the genetic stability of chimeric West Nile/dengue 4 virus.-猫眼课题宝

DOI：

10.1002/jmv.22033

发表时间：

2011-05

期刊：

JOURNAL OF MEDICAL VIROLOGY

影响因子：

12.7

通讯作者：

中科院分区：

医学3区

文献类型：

作者：

Laassri, Majid;Bidzhieva, Bella;Speicher, James;Pletnev, Alexander G.;Chumakov, Konstantin

关键词：

flavivirus vaccine mutants screening mutants quantitation molecular consistency quality control

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410669/?report=classic

遗传稳定性是活病毒疫苗的一个重要特征，因为突变的积累可导致回复到毒力表型以及免疫原性的丧失。本研究旨在评价用西尼罗河病毒(West Nile，WN)减毒活疫苗基因替换登革4型病毒前膜蛋白基因和包膜蛋白基因的遗传稳定性。嵌合病毒在Vero、SH-SY5Y人神经母细胞瘤和HeLa细胞中连续繁殖，并使用覆盖整个基因组的重叠寡核苷酸探针微阵列杂交来筛选点突变。分析发现了几个导致氨基酸变化的自发突变，其中大部分位于包膜(E)和非结构蛋白NS4A、NS4B和NS5中。在Vero和SH-SY5Y细胞中传代的病毒有两个共同的突变：E基因的G2337C(Met457Ile)和NS4A基因的A6751G(Lys125Arg)。对这些突变体在病毒库中含量的定量评估表明，它们在细胞培养中的繁殖过程中以不同的动力学独立积累。与亲本病毒相比，突变病毒在Vero细胞中生长得更好，这表明它们具有更高的适应性。当在新生小鼠身上进行测试时，细胞培养传代的病毒没有表现出更强的神经毒力。该方法可用于疫苗毒株的分子一致性监测和质量控制。

Genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. This study was aimed at evaluating the genetic stability of a live attenuated West Nile (WN) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from WN. Chimeric virus was serially propagated in Vero, SH-SY5Y human neuroblastoma and HeLa cells and screened for point mutations using hybridization with microarrays of overlapping oligonucleotide probes covering the entire genome. The analysis revealed several spontaneous mutations that led to amino acid changes, most of which were located in the envelope (E) and non-structural NS4A, NS4B, and NS5 proteins. Viruses passaged in Vero and SH-SY5Y cells shared two common mutations: G2337C (Met457Ile) in the E gene and A6751G (Lys125Arg) in the NS4A gene. Quantitative assessment of the contents of these mutants in viral stocks indicated that they accumulated independently with different kinetics during propagation in cell cultures. Mutant viruses grew better in Vero cells compared to the parental virus, suggesting that they have a higher fitness. When tested in newborn mice, the cell culture-passaged viruses did not exhibit increased neurovirulence. The approach described in this paper could be useful for monitoring the molecular consistency and quality control of vaccine strains.