Autor(es): Steel J Jordan, Franz Alexander W E, Sanchez-Vargas Irma, Olson Ken E, Geiss Brian J

Resumo: 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 - 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 - 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 - 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 - in live transgenic mosquitoes.

Imprenta: PloS One, v. 8, n. 12, p. e84930, 2013

Identificador do Objeto Digital: 10.1371/journal.pone.0084930

Descritores: Chikungunya virus - Biochemistry ; Chikungunya virus - Biosynthesis ; Chikungunya virus - Cell ; Chikungunya virus - Cytopathology ; Chikungunya virus - DNA ; Chikungunya virus - Genome ; Chikungunya virus - Molecular structure ; Chikungunya virus - Pathogenesis ; Chikungunya virus - Proteins ; Chikungunya virus - RNA ; Chikungunya virus - Molecular methods ; Chikungunya Virus - Virus ; Chikungunya virus - Immunology ; Chikungunya virus - Public health

Data de Publicação: 2013