Hanley Kathryn A, Manlucu Luella R, Gilmore Lara E, Blaney Joseph E, Hanson Christopher T, Murphy Brian R, Whitehead Stephen S
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Virology. 2003 Jul 20;312(1):222-32. doi: 10.1016/s0042-6822(03)00197-1.
An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Delta30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host.
一种可接受的减毒活登革病毒候选疫苗应具有较低的经蚊子传播的可能性。我们已经鉴定并表征了登革病毒4型（DEN4）中的一种突变，该突变降低了病毒感染蚊子的能力。对先前通过化学诱变产生的一组1248个诱变病毒克隆进行筛选，以寻找在蚊子C6/36细胞中复制减少但在猴Vero细胞中高效复制的克隆。一种病毒符合这些标准，并且包含一个单一的编码突变：核苷酸7129处的C到U突变，导致非结构4B基因（NS4B P101L）的第101位氨基酸由脯氨酸变为亮氨酸。相对于野生型DEN4，该突变导致在C6/36细胞中的复制减少，对蚊子的感染性降低，在Vero和人HuH-7细胞中的复制增强，以及在植入HuH-7细胞的SCID小鼠（SCID-HuH-7小鼠）中的复制增强。携带此突变的重组DEN4病毒（rDEN4）表现出相同的一组表型。将NS4B P101L突变添加到携带30个核苷酸缺失（Delta30）的rDEN4中，降低了双突变病毒感染蚊子的能力，但增加了其在SCID-HuH-7小鼠中的复制能力。尽管NS4B P101L突变降低了DEN4对蚊子的感染性，但其在SCID-HuH-7小鼠中增强复制的能力表明，在rDEN4疫苗中包含这种特定突变可能并无益处。NS4B P101L突变在蚊子和脊椎动物系统中的相反作用表明，NS4B蛋白参与维持在蚊子媒介和人类宿主中高效复制之间的平衡。
