Construction of infectious cDNA clones for dengue 2 virus: Strain 16681 and its attenuated vaccine derivative, strain PDK-53
Construction of infectious cDNA clones for dengue 2 virus: Strain 16681 and its attenuated vaccine derivative, strain PDK-53
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DOI：
10.1006/viro.1997.8500
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发表时间：
1997-04-14
期刊：
影响因子：
3.7
通讯作者：
Gubler, DJ
中科院分区：
文献类型：
--
作者：
Kinney, RM;Butrapet, S;Gubler, DJ
我们鉴定了登革热-2 (DEN-2) 16681病毒及其衍生疫苗菌株PDK-53的基因组之间的9个核苷酸差异。这些突变包括5'-非翻译区57核苷酸位置的C-to-T （16681-to-PDK-53）突变，三个沉默突变，以及prM-29 Asp到Val， NS1-53 Gly到Asp， NS2A-181 Leu到Phe， NS3-250 Glu到Val和NS4A-75 Gly到Afa的替换。非传代PDK-53疫苗由于NS3-250的部分突变而含有两种遗传变异。我们构建了16681病毒和PDK-53两种变体的感染性cDNA克隆。den2 - 16681克隆衍生病毒在斑块大小和在lc - mk2细胞中的复制、在C6/36细胞中的复制、E和prM表位以及对哺乳小鼠的神经毒性方面与16681病毒相同。PDK-53病毒和两种克隆衍生的PDK-53变体在小鼠中被减毒。然而，与PDK-53病毒相比，含有NS3-250-Glu的变异对C6/36细胞的温度敏感性较低，复制效果更好。与PDK-53病毒相比，含有NS3-250-Val的变异在LLC-MK2细胞中具有更小、更弥漫性斑块、复制减少和温度敏感性增加。PDK-53病毒和NS3-250-Val变体在C6/36细胞中的复制都比16681病毒差。未传代的PDK-53疫苗病毒与在LLC-MK2细胞中传代一次的病毒具有相同的基因组序列，包括混合的NS3-250-Glu/Val位点。尽管NS3-250-Val突变明显影响病毒在体外的复制，但它并不是PDK-53病毒在哺乳小鼠体内衰减的主要决定因素。
We identified nine nucleotide differences between the genomes of dengue-2 (DEN-2) 16681 virus and its vaccine derivative, strain PDK-53. These included a C-to-T (16681-to-PDK-53) mutation at nucleotide position 57 of the 5'-untranslated region, three silent mutations, and substitutions prM-29 Asp to Val, NS1-53 Gly to Asp, NS2A-181 Leu to Phe, NS3-250 Glu to Val, and NS4A-75 Gly to Afa. Unpassaged PDK-53 vaccine contained two genetic Variants as a result of partial mutation at NS3-250. We constructed infectious cDNA clones for 16681 virus and each of the two PDK-53 variants. DEN-2 16681 clone-derived viruses were identical to the 16681 virus in plaque size and replication in LLC-MK2 cells, replication in C6/36 cells, E and prM epitopes, and neurovirulence for suckling mice. PDK-53 virus and both clone-derived PDK-53 variants were attenuated in mice. However, the Variant containing NS3-250-Glu was less temperature sensitive and replicated better in C6/36 cells than did PDK-53 virus. The variant containing NS3-250-Val had smaller, more diffuse plaques, decreased replication, and increased temperature sensitivity in LLC-MK2 cells relative to PDK-53 virus. Both PDK-53 virus and the NS3-250-Val variant replicated poorly in C6/36 cells relative to 16681 virus. Unpassaged PDK-53 Vaccine virus and the Virus passaged once in LLC-MK2 cells had genomes of identical sequence, including the mixed NS3-250-Glu/Val locus. Although the NS3-250-Val mutation clearly affected virus replication in vitro, it was not a major determinant of attenuation for PDK-53 virus in suckling mice.