title: Direct broad-range detection of alphaviruses in mosquito extracts.
abstract: Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 36 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype IIID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses.
authors.affiliation: Ibis Biosciences, 1891 Rutherford Rd., Carlsbad, CA 92008, USA. meshoo@isisph.com
authors.forename: Mark W
authors.initials: MW
authors.lastname: Eshoo
authors.forename: Chris A
authors.initials: CA
authors.lastname: Whitehouse
authors.forename: Scott T
authors.initials: ST
authors.lastname: Zoll
authors.forename: Christian
authors.initials: C
authors.lastname: Massire
authors.forename: Thuy-Trang D
authors.initials: TT
authors.lastname: Pennella
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Blyn
authors.forename: Rangarajan
authors.initials: R
authors.lastname: Sampath
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Hall
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Ecker
authors.forename: Anjali
authors.initials: A
authors.lastname: Desai
authors.forename: Leonard P
authors.initials: LP
authors.lastname: Wasieloski
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Turell
authors.forename: Amy
authors.initials: A
authors.lastname: Schink
authors.forename: Karl
authors.initials: K
authors.lastname: Rudnick
authors.forename: Glen
authors.initials: G
authors.lastname: Otero
authors.forename: Scott C
authors.initials: SC
authors.lastname: Weaver
authors.forename: George V
authors.initials: GV
authors.lastname: Ludwig
authors.forename: Steven A
authors.initials: SA
authors.lastname: Hofstadler
authors.forename: David J
authors.initials: DJ
authors.lastname: Ecker
mesh_terms: D000330:Aedes / Q000821:virology*; D000524:Alphavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003465:Culex / Q000821:virology*; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
chemical_list: D017931:DNA Primers; D012367:RNA, Viral