Direct broad-range detection of alphaviruses in mosquito extracts.
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.
Eshoo
Mark W
MW
Ibis Biosciences, 1891 Rutherford Rd., Carlsbad, CA 92008, USA. meshoo@isisph.com
Whitehouse
Chris A
CA
Zoll
Scott T
ST
Massire
Christian
C
Pennella
Thuy-Trang D
TT
Blyn
Lawrence B
LB
Sampath
Rangarajan
R
Hall
Thomas A
TA
Ecker
Joseph A
JA
Desai
Anjali
A
Wasieloski
Leonard P
LP
Li
Feng
F
Turell
Michael J
MJ
Schink
Amy
A
Rudnick
Karl
K
Otero
Glen
G
Weaver
Scott C
SC
Ludwig
George V
GV
Hofstadler
Steven A
SA
Ecker
David J
DJ
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
D017931:DNA Primers; D012367:RNA, Viral