title: Rapid identification of vector-borne flaviviruses by mass spectrometry.
abstract: Flaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose. In this study, we developed and evaluated a broad-range Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was evaluated with a panel of 13 different flaviviruses. All samples were correctly identified to the species level. To determine the limit of detection for the mosquito-borne primer sets, serial dilutions of RNA from West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2 plaque-forming units/mL. Analysis of flaviviruses in their natural biological background included testing Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately identified the virus within infected mosquitoes, and we determined the average viral genome per mosquito to be 2.0 x 10(6). Using human blood, serum, and urine spiked with WNV and mouse blood and brain tissues from Karshi virus-infected mice, we showed that these clinical matrices did not inhibit the detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks collected from sites in New York and Connecticut. We found 16/322 (5% infection rate) ticks positive for deer tick virus, a subtype of Powassan virus. In summary, we developed a single high-throughput Flavivirus assay that could detect multiple tick- and mosquito-borne flaviviruses and thus provides a new analytical tool for their medical diagnosis and epidemiological surveillance.
authors.affiliation: United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Grant-Klein
authors.forename: Carson D
authors.initials: CD
authors.lastname: Baldwin
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Turell
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Rossi
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Robert
authors.initials: R
authors.lastname: Lovari
authors.forename: Chris D
authors.initials: CD
authors.lastname: Crowder
authors.forename: Heather E
authors.initials: HE
authors.lastname: Matthews
authors.forename: Megan A
authors.initials: MA
authors.lastname: Rounds
authors.forename: Mark W
authors.initials: MW
authors.lastname: Eshoo
authors.forename: Lawrence B
authors.initials: LB
authors.lastname: Blyn
authors.forename: David J
authors.initials: DJ
authors.lastname: Ecker
authors.forename: Rangarajan
authors.initials: R
authors.lastname: Sampath
authors.forename: Chris A
authors.initials: CA
authors.lastname: Whitehouse
mesh_terms: D000818:Animals; D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D009033:Culicidae / Q000821:virology; D017931:DNA Primers / Q000378:metabolism; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification; D004199:Disease Vectors; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics / Q000302:isolation & purification; D005416:Flavivirus / Q000235:genetics* / Q000302:isolation & purification*; D018177:Flavivirus Infections / Q000175:diagnosis / Q000821:virology; D051379:Mice; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013987:Ticks / Q000821:virology; D019562:Viral Load / Q000235:genetics; D014902:West Nile virus / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017931:DNA Primers