Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing 英文参考文献.docVIP
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Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing 英文参考文献
ArbovirusDetectioninInsectVectorsbyRapid,High-
ThroughputPyrosequencing
KimberlyA.Bishop-Lilly1*,MichaelJ.Turell2,KristinM.Willner1,AmyButani1,NicholeM.E.Nolan1,
ShannonM.Lentz1,AryaAkmal1,AlMateczun1,TruptiN.Brahmbhatt1,ShanmugaSozhamannan1,
ChrisA.Whitehouse2,TimothyD.Read1¤
1Biological Defense Research Directorate, Naval Medical Research Center, Silver Spring, Maryland, United States of America, 2United States Army Medical Research
InstituteforInfectiousDiseases,FortDetrick,Maryland,UnitedStatesofAmerica
Abstract
Background:Despitetheglobalthreatcausedbyarthropod-borneviruses,thereisnotanefficientmethodforscreening
vectorpopulationstodetectnovelviralsequences.Currentviraldetectionandsurveillancemethodsbasedonculturecan
be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific
oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative
agentofanoutbreakisunknown.
Methodology/PrincipalFindings:Inthisstudyweexploredtheuseofhigh-throughputpyrosequencingforsurveillanceof
arthropod-borneRNAviruses.Denguevirus,amemberofthepositivestrandRNAFlavivirusfamilythatistransmittedby
severalmembersoftheAedesgenusofmosquitoes,wasusedasamodel.Aedesaegyptimosquitoesexperimentallyinfected
withdenguevirustype1(DENV-1)werepooledwithnoninfectedmosquitoestosimulatesamplesderivedfromongoing
arbovirus surveillance programs. Using random-primed methods, total RNA was reverse-transcribed and resulting cDNA
subjectedto454pyrosequencing.
Conclusions/Significance:Intwotypesofsamples,onewith5adultmosquitoesinfectedwithDENV-1-andtheotherwith1
DENV-1infectedmosquitoand4noninfectedmosquitoes,weidentifiedDENV-1DNAsequences.DENV-1sequenceswere
not detected in an uninfected control pool of 5 adult mosquitoes. We calculated the proportion of the Ae. aegypti
metagenome contributed by each infecting Dengue virus genome (pIP), which ranged from 2.7561028 to 1.0861027.
DENV-1RNAwassufficie
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