title: Alphavirus transducing system: tools for visualizing infection in mosquito vectors.
abstract: 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.
authors.affiliation: Microbiology, Immunology, and Pathology, Colorado State University, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Phillips
authors.forename: Eric
authors.initials: E
authors.lastname: Mossel
authors.forename: Irma
authors.initials: I
authors.lastname: Sanchez-Vargas
authors.forename: Brian
authors.initials: B
authors.lastname: Foy
authors.forename: Ken
authors.initials: K
authors.lastname: Olson
mesh_terms: D000524:Alphavirus / Q000235:genetics*; D018354:Alphavirus Infections / Q000821:virology; D000818:Animals; D009033:Culicidae / Q000821:virology*; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007303:Insect Vectors / Q000821:virology*; D012313:RNA / Q000235:genetics; D012845:Sindbis Virus / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D012313:RNA