Detection of Encephalitis Viruses in Mosquitoes (Diptera: Culicidae) and Avian Tissues

Abstract

Diagnostic assays for the detection of St. Louis encephalitis (SLE) and western equine encephalomyelitis (WEE) viruses in mosquito pools and avian tissues were compared for sensitivity, accuracy and specificity. The in situ enzyme immunoassay (EIA), plaque assay on Vero cells, passage in Aedes albopictus Skuse C6/36 and C7/10 cells, antigen capture enzyme immunoassay (AC-EIA), and single and multiplex reverse transcription-polymerase chain reactions (RT-PCR) were evaluated using pools of 50 mosquitoes containing 1-2 experimentally infected individuals. RT-PCR was the most sensitive assay, with a detection limit of <0.1 plaque forming unit. AC-EIA was the fastest and most economical procedure, but was the least sensitive, detecting only 38% of positive pools. The in situ EIA included initial virus amplification on Vero cells, thereby improving assay sensitivity to detect 68% of positive pools. Passage in C6/36 and/or C7/10 cell culture revealed the presence of infectious virus in samples positive by RT-PCR, but initially negative by plaque assay on Vero cell culture, indicating that detection was related to assay sensitivity and not to the absence of intact infectious virus. Combining WEE and SLE RT-PCR assays into a multiplex assay reduced sensitivity, but stilldetected viral RNA at titers below plaque assay sensitivity. Plaque assay on Vero cells, mosquito cell passage, and several RT-PCR procedures were evaluated for their ability to detect WEE and SLE in white-crowned sparrow tissues during acute and chronic stages of infection. All assays detected virus during acute infection at times of high viremia; however, only RT-PCR assays were positive by day 7 when virus was not detected in sera. RT-PCR detected SLE RNA in spleen tissue from one bird 51 d after infection. Assay sensitivity also was compared using extracts of homogenized bird organs spiked with known titers of WEE and SLE. Trizol RNA extraction followed by Qiagen one-step RT-PCR was the most sensitive method, but occasionally resulted in the presence of secondary bands confounding interpretation and requiring confirmatory assays. A balanced surveillance program should combine systems that allow the detection of new agents and the sensitive monitoring of endemic agents to provide an early warning of pending health risks.

Experimental Inoculation of House Sparrows (Passer Domesticus) With Buggy Creek Virus

“…Our results for BCRV in house sparrows suggest a viremia profile largely consistent with that reported for other alphaviruses, in which virus is most easily detected in blood for typically 1-2 days after initial infection (Hardy and Reeves, 1990;Komar et al, 1999;Lindströ m and Lundströ m, 2000;Kramer et al, 2002;Reisen et al, 2003). That all but one inoculated sparrow seroconverted suggests that our experimental infections were successful, but, perhaps surprisingly, only one of six sparrows showed high levels of viremia, and four of the six inoculated sparrows showed no evidence of viremia by plaque assay.…”

“…Interestingly, we found virus RNA detectable in blood by RT-PCR for a much longer period of time, up to 15 days postinoculation, than expected based on similar studies with alpha-and flaviviruses. This may reflect in general the greater sensitivity of RT-PCR to detect the presence of viral RNA (Kramer et al, 2002). Birds were more likely to be positive for BCRV by RT-PCR on days 6-15 than on days 1-5, a perplexing result given that virus titers in most bird species are highest immediately after infection (e.g., Hardy and Reeves, 1990;Komar et al, 1999;Kramer et al, 2002;Reisen et al, 2003).…”

“…This may reflect in general the greater sensitivity of RT-PCR to detect the presence of viral RNA (Kramer et al, 2002). Birds were more likely to be positive for BCRV by RT-PCR on days 6-15 than on days 1-5, a perplexing result given that virus titers in most bird species are highest immediately after infection (e.g., Hardy and Reeves, 1990;Komar et al, 1999;Kramer et al, 2002;Reisen et al, 2003). The RT-PCR detections through day 15 may simply indicate low levels of virus (or viral RNA) in blood that are not sufficiently strong to be detectable by plaque assay or, more importantly, sufficiently strong or viable to infect invertebrate vectors.…”

“…For eastern (EEEV) and western equine encephalomyelitis (WEEV) viruses, for example, a variety of mostly passerine bird species exhibit sufficiently elevated viremia levels to infect mosquitoes for typically the first 1-2 days after inoculation, but viremias are frequently undetectable beyond 3 days postinoculation (Hardy and Reeves, 1990;Komar et al, 1999;Kramer et al, 2002;Reisen et al, 2003). Interestingly, the introduced, nonnative European starling (Sturnus vulgaris) was a more effective amplifying host (with higher levels of viremia) for the alphavirus EEEV in Massachusetts than were several native bird species tested (Komar et al, 1999).…”

Use of Scented Sugar Bait Stations to Track Mosquito-Borne Arbovirus Transmission in California

“…Mosquitoes were held for 10 d at 26°C. All mosquitoes surviving to 10 d, the WNV-infected sucrose solutions, and the sugar pads collected after 48 h of exposure to mosquitoes were tested individually for WNV RNA by qRT-PCR as described for experiment 1 and for infectious WNV by standard plaque assay on Vero cell culture (Kramer et al 2002). …”