Kinney R M, Butrapet S, Chang G J, Tsuchiya K R, Roehrig J T, Bhamarapravati N, Gubler D J
Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, USA.
Virology. 1997 Apr 14;230(2):300-8. doi: 10.1006/viro.1997.8500.
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 Ala. 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.
我们在登革2型（DEN-2）16681病毒及其疫苗衍生株PDK-53的基因组之间鉴定出9个核苷酸差异。这些差异包括5'-非翻译区核苷酸位置57处的C到T（16681到PDK-53）突变、三个沉默突变，以及prM-29位天冬氨酸到缬氨酸、NS1-53位甘氨酸到天冬氨酸、NS2A-181位亮氨酸到苯丙氨酸、NS3-250位谷氨酸到缬氨酸和NS4A-75位甘氨酸到丙氨酸的替换。未传代的PDK-53疫苗由于NS3-250处的部分突变而包含两种遗传变体。我们构建了16681病毒以及两种PDK-53变体各自的感染性cDNA克隆。DEN-2 16681克隆衍生病毒在蚀斑大小、在LLC-MK2细胞中的复制、在C6/36细胞中的复制、E和prM表位以及对乳鼠的神经毒力方面与16681病毒相同。PDK-53病毒以及两种克隆衍生的PDK-53变体在小鼠中均减毒。然而，含有NS3-250-谷氨酸的变体对温度的敏感性较低，并且在C6/36细胞中的复制比PDK-53病毒更好。相对于PDK-53病毒，含有NS3-250-缬氨酸的变体在LLC-MK2细胞中蚀斑更小、更弥散，复制减少，并且对温度的敏感性增加。相对于16681病毒，PDK-53病毒和NS3-250-缬氨酸变体在C6/36细胞中的复制均较差。未传代的PDK-53疫苗病毒以及在LLC-MK2细胞中传代一次的病毒具有相同序列的基因组，包括混合的NS3-250-谷氨酸/缬氨酸位点。尽管NS3-250-缬氨酸突变明显影响病毒在体外的复制，但它不是PDK-53病毒对乳鼠减毒的主要决定因素。
