Evaluation of reverse transcriptase polymerase chain reaction for the detection of eastern equine encephalomyelitis virus during vector surveillance.
A reverse transcriptase polymerase chain reaction (RT-PCR) assay was evaluated for the detection of eastern equine encephalomyelitis virus (EEEV). EEEV was detected by amplification of a 416-bp PCR product from within the E2 gene. Internal restriction endonuclease digestion and hybridizations to EEEV RNA demonstrated that the PCR product was amplified from EEEV. PCR amplifications from serial dilutions of an EEEV isolate identified by a neutralization test and titered by an infectious assay in cell culture indicated that this RT-PCR assay detected viral RNA at concentrations below 1 plaque forming unit(PFU) per reaction. The performance of the PCR assay in detection of EEEV was compared with an infectious assay detection procedure (IA/IFA) as part of the New Jersey 1993 vector surveillance program. During 1993, 7,007 field-collected Culiseta melanura (Coquillett) were assayed in 522 pools by both RT-PCR and IA/IFA. EEEV was detected in 95 pools by RT-PCR and 17 pools by IA/IFA; all IA/IFA positive pools were also positive by RT-PCR. During the 1993 field season, RT-PCR consistently detected virus at enzootic foci earlier that IA/IFA and in greater numbers of mosquito pools. The data indicated that viral RNA may be present earlier and in more mosquitoes than indicated by IA/IFA.
Monroy
A M
AM
Department of Entomology, Rutgers University, New Brunswick, NJ 08903, USA.
Scott
T W
TW
Webb
B A
BA
D000330:Aedes / Q000821:virology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003465:Culex / Q000821:virology; D009033:Culicidae / Q000821:virology*; D004279:DNA, Viral; D004663:Encephalitis Virus, Eastern Equine / Q000235:genetics / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D005260:Female; D007303:Insect Vectors / Q000821:virology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012194:RNA-Directed DNA Polymerase
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase