Rapid identification of vector-borne flaviviruses by mass spectrometry
Autor(es): Grant-Klein, Rebecca J.; Baldwin, Carson D.; Turell, Michael J.; Rossi, Cynthia A.; Li, Feng; Lovari, Robert; Crowder, Chris D.; Matthews, Heather E.; Rounds, Megan A.; Eshoo, Mark W.; Blyn, Lawrence B.; Ecker, David J.; Sampath, Rangarajan; Whitehouse, Chris A.
Resumo: 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 super(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.
Palavras-Chave: Electrospray ionization; Tick-borne; Mosquito-borne; Arbovirus
Imprenta: Molecular and Cellular Probes, v. 24, n. 4, p. 219-228, 2010.
Descritores: Aedes aegypti - Biochemistry ; Aedes aegypti - Cell ; Aedes aegypti - DNA ; Aedes aegypti - Flaviviridae ; Aedes aegypti - RNA ; Aedes aegypti - Infectious diseases ; Aedes aegypti - Viral infections ; Aedes aegypti - PCR detection ; Aedes aegypti - Virus ; Aedes aegypti - Transmission ; Aedes aegypti - Dengue ; Aedes aegypti - Public health ; Aedes aegypti - RT-PCR
Data de publicação: 2010
