title: Subgenomic reporter RNA system for detection of alphavirus infection in mosquitoes.
abstract: Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: J Jordan
authors.initials: JJ
authors.lastname: Steel
authors.affiliation: Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Alexander W E
authors.initials: AW
authors.lastname: Franz
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Irma
authors.initials: I
authors.lastname: Sanchez-Vargas
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Ken E
authors.initials: KE
authors.lastname: Olson
authors.affiliation: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America ; Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Geiss
mesh_terms: D000330:Aedes / Q000821:virology*; D000524:Alphavirus / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007303:Insect Vectors / Q000821:virology*; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins