Powers A M, Olson K E, Higgs S, Carlson J O, Beaty B J
Department of Microbiology, Colorado State University, Fort Collins 80523.
Virus Res. 1994 Apr;32(1):57-67. doi: 10.1016/0168-1702(94)90061-2.
A cDNA of the small RNA genome segment of La Crosse (LAC) virus was inserted, in an antisense orientation, into a double subgenomic Sindbis (dsSIN) virus expression vector generating pTE/3'2J/ANTI-S (15,000bp). In vitro transcription of the pTE/3'2J/ANTI-S template generated genomic RNA that was electrotransfected into BHK-21 cells to produce virus. Northern blot analysis of RNA isolated from infected Aedes albopictus (C6/36) cells showed that the TE/3'2J/ANTI-S virus produced a subgenomic mRNA of the appropriate size, indicating transcription of the LAC cDNA segment. C6/36 cells were infected with either TE/3'2J/ANTI-S, TE/3'2J (a dsSIN virus with no LAC insert), or wild type Sindbis (SIN, strain AR339) viruses and subsequently challenged with LAC virus. LAC virus titers were determined using a capture antibody ELISA. Mosquito cells infected with TE/3'2J/ANTI-S virus yielded at least 4 log10 TCID50/ml less LAC virus than cells infected with either TE/3'2J or AR339 SIN viruses. The use of the infectious SIN virus expression vectors provides a novel approach for high level cytoplasmic expression of genes or sequences of interest in arthropod cells, and for evaluating strategies for intracellular immunization against arboviruses.
将拉克罗斯（LAC）病毒小RNA基因组片段的cDNA以反义方向插入到双亚基因组辛德毕斯（dsSIN）病毒表达载体中，构建出pTE/3'2J/ANTI-S（15,000碱基对）。对pTE/3'2J/ANTI-S模板进行体外转录，生成基因组RNA，将其电转染到BHK - 21细胞中以产生病毒。对从感染的白纹伊蚊（C6/36）细胞中分离的RNA进行Northern印迹分析表明，TE/3'2J/ANTI-S病毒产生了大小合适的亚基因组mRNA，表明LAC cDNA片段发生了转录。用TE/3'2J/ANTI-S、TE/3'2J（一种无LAC插入的dsSIN病毒）或野生型辛德毕斯（SIN，AR339株）病毒感染C6/36细胞，随后用LAC病毒进行攻击。使用捕获抗体ELISA测定LAC病毒滴度。感染TE/3'2J/ANTI-S病毒的蚊子细胞产生的LAC病毒比感染TE/3'2J或AR339 SIN病毒的细胞至少少4个对数10 TCID50/毫升。感染性SIN病毒表达载体的使用为在节肢动物细胞中高水平细胞质表达感兴趣的基因或序列，以及评估针对虫媒病毒的细胞内免疫策略提供了一种新方法。
