Powers A M, Kamrud K I, Olson K E, Higgs S, Carlson J O, Beaty B J
Department of Microbiology, Colorado State University, Ft. Collins 80523, USA.
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4187-91. doi: 10.1073/pnas.93.9.4187.
Introduction of genetic elements derived from a viral pathogen's genome may be used to reduce the vectorial capacity of mosquitoes for that virus. A double subgenomic Sindbis virus expression system was utilized to transcribe sequences of LaCrosse (LAC) virus small (S) or medium (M) segment RNA in sense or antisense orientation; wild-type Sindbis and LaCrosse viruses have single-stranded RNA genomes, the former being positive sense and the latter being negative sense. Recombinant viruses were generated and used to infect Aedes albopictus (C6/36) mosquito cells, which were challenged with wild-type LAC virus and then assayed for LAC virus replication. Several recombinant viruses containing portions of the LAC S segment were capable of inducing varying degrees of interference to the challenge virus. Cells infected with TE/3'2J/ANTI-S virus, expressing full-length negative-sense S RNA of LAC virus, yielded 3-6 log10TCID50 (tissue culture 50% infective dose) less LAC virus per ml than did cells infected with a double subgenomic sindbis virus containing no LAC insert. When C6/36 cells infected with TE/3'2J/ANTI-S were challenged with closely related heterologous bunyaviruses, a similar inhibitory effect was seen. Adult Ae. triseriatus mosquitoes infected with TE/3'2J/ANTI-S were also resistant to challenge by LAC virus. Organs that were productively infected by the double subgenomic Sindbis virus expressing the LAC anti-S sequences demonstrated little LAC virus or antigen. These studies indicate that expression of carefully selected antiviral sequences derived from the pathogen's genome may result in efficacious molecular viral interference in mosquito cells and, more importantly, in mosquitoes.
引入源自病毒病原体基因组的遗传元件可用于降低蚊子对该病毒的传播能力。利用双亚基因组辛德毕斯病毒表达系统，以正义或反义方向转录拉科罗斯（LAC）病毒小（S）或中（M）片段RNA的序列；野生型辛德毕斯病毒和拉科罗斯病毒具有单链RNA基因组，前者为正链，后者为负链。产生重组病毒并用于感染白纹伊蚊（C6/36）蚊细胞，用野生型LAC病毒攻击这些细胞，然后检测LAC病毒复制情况。几种含有LAC S片段部分的重组病毒能够对攻击病毒产生不同程度的干扰。用表达LAC病毒全长负义S RNA的TE/3'2J/ANTI-S病毒感染的细胞，每毫升产生的LAC病毒比用不含LAC插入片段的双亚基因组辛德毕斯病毒感染的细胞少3-6 log10TCID50（组织培养50%感染剂量）。当用密切相关的异源布尼亚病毒攻击感染TE/3'2J/ANTI-S的C6/36细胞时，也观察到类似的抑制作用。感染TE/3'2J/ANTI-S的成年三带喙库蚊对LAC病毒攻击也具有抗性。被表达LAC反义S序列的双亚基因组辛德毕斯病毒有效感染的器官几乎没有LAC病毒或抗原。这些研究表明，表达从病原体基因组中精心选择的抗病毒序列可能会在蚊细胞中，更重要的是在蚊子中产生有效的分子病毒干扰。
