Phillips Aaron, Mossel Eric, Sanchez-Vargas Irma, Foy Brian, Olson Ken
Microbiology, Immunology, and Pathology, Colorado State University, USA.
J Vis Exp. 2010 Nov 24(45):2363. doi: 10.3791/2363.
Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect. ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP(2,9). Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.
甲病毒转导系统（ATSs）是在感染过程中表达目的基因（GOI）的重要工具。ATSs源自蚊媒RNA病毒（甲病毒属；披膜病毒科）的cDNA克隆。甲病毒属包含约30种不同的蚊媒病毒。甲病毒是包膜病毒，含有单链RNA基因组（约11.7 Kb）。甲病毒转录一种亚基因组mRNA，该mRNA编码病毒基因组包装和病毒成熟所需的病毒结构蛋白。甲病毒通常在脊椎动物细胞中具有高度溶解性，但能以最小的细胞病变持续感染易感蚊细胞。这些特性使其成为蚊媒中基因表达的优秀工具。目前使用最广泛的ATSs源自辛德毕斯病毒（SINV）。SINV广泛的宿主嗜性使其能够感染昆虫、鸟类和哺乳动物细胞8。然而，ATSs也源自其他甲病毒。通过插入额外的病毒亚基因组RNA起始位点或启动子，可实现外源基因的表达。外源基因序列位于病毒结构基因5'端的ATSs用于昆虫中的稳定蛋白表达。基因序列位于结构基因3'端的ATSs用于触发RNAi并沉默昆虫中该基因的表达。ATSs已被证明是理解媒介-病原体相互作用、病毒复制分子细节和维持感染周期的有价值工具。特别是，荧光和生物发光报告基因的表达有助于追踪病毒在媒介中的感染和病毒传播。此外，使用两种经过基因工程改造以表达绿色荧光蛋白（GFP）的SINV菌株描述了媒介免疫反应（2,9）。在此，我们展示了一种从cDNA感染性克隆生产含荧光报告基因（GFP）的SINV的方法。从线性化的cDNA克隆中转录出感染性全长RNA。通过电穿孔将感染性RNA导入允许的靶细胞。转染的细胞产生表达GOI的感染性病毒颗粒。收获的病毒用于感染蚊子，如下所述，或其他宿主物种（本文未展示）。通过检测中肠外的荧光或监测病毒传播来评估媒介能力。使用荧光报告基因作为GOI可方便地估计病毒在整个细胞培养物中的传播情况，确定感染率、在暴露蚊子中的传播情况、蚊子的病毒传播情况，并提供媒介能力的快速评估指标。
