Laboratory Testing of Vectors

Mosquitoes should be identified to species or lowest taxonomic unit. Specimens are placed into pools of 50 specimens or less based on species, sex, location, trap-type, and date of collection. Larger pool sizes can be used in some assays with loss of sensitivity (Sutherland and Nasci 2007). If resources are limited, testing of mosquitoes for surveillance purposes can be limited to the primary vector species.

After adding an appropriate homogenization buffer (cell culture medium, viral transport medium, PBS), mosquito pools can be macerated or ground by a variety of techniques including mortar and pestle, vortexing sealed tubes containing one or more copper clad BBs, or by use of tissue homogenizing apparatuses that are commercially available. After grinding, samples are centrifuged, and an aliquot is removed for testing. Because mosquito pools may contain arboviruses and other pathogenic viruses which may be aerosolized during processing, laboratory staff should take appropriate safety precautions including use of a Class II Type A biological safety cabinet, appropriate personal protective equipment (PPE) and biosafety practices.

EEE virus is an HHS Select Agent, and therefore, subject to strict regulations regarding its possession and use. Those intending to conduct EEE virus testing must be familiar with the complete information and specific guidance found at the Federal Select Agent Program website before conducting EEE virus testing.

Briefly, samples determined to be positive for EEE virus must be documented and reported to the Federal Select Agent Program via Form 4 (https://www.selectagents.gov/form4.html) within 7 calendar days of identification, and, if not diagnosed at a registered entity, they much then be transferred to a registered Select Agent facility or destroyed.

Multiple diagnostic methods can be used to identify EEE virus in mosquitoes including virus isolation, molecular assays, or immunoassays. Virus isolation in Vero cell culture (Armstrong et al. 2011) remains the standard for confirmation of positive pools but is time consuming and requires specialized laboratory facilities. For virus isolation, mosquito pool homogenates are added to Vero cell cultures, monitored for cytopathic effect, and identified using appropriate diagnostic assays. Aliquots are stored at -70°C to retain virus viability for future testing. Vero cell culture has an additional benefit of detecting other viruses in the mosquitoes, a feature lost using test procedures that target virus-specific nucleotide sequence or proteins (Andreadis et al. 1998; Andreadis et al. 2004). Molecular assays detect viral RNA or nucleic acids in mosquito pools quickly, with high sensitivity and specificity, and do not require cold chain or high levels of biocontainment (Lanciotti et al. 2000; Callahan et al. 2001; Lambert et al. 2003; Armstrong et al. 2012). Methods include rapid molecular assays, RT-PCR, and other nucleic acid amplification tests (e.g., multi-target RT-PCR). Assays may use different primer sets for detection and confirmation of virus in mosquito pools (Lambert et al. 2003). Antigen detection assays are less sensitive than molecular assays, but for some arboviruses (e.g., WNV) can provide valuable infection rate data when employed consistently in a mosquito surveillance program. For additional details see: Human Diagnostic Detection and Annex 1 Real Time RT-PCR Protocol for Mosquito Pools.

References

Andreadis TG, et al. Multiple isolations of eastern equine encephalitis and highlands J viruses from mosquitoes (Diptera: Culicidae) during a 1996 epizootic in southeastern Connecticut. J Med Entomol 1998;35:296–302.

Andreadis TG, et al. Epidemiology of West Nile virus in Connecticut, USA: A five-year analysis of mosquito data, 1999-2003. Vector Borne Zoonotic Dis 2004;4:360–378.

Armstrong PM, et al. Detection of infectious virus from field-collected mosquitoes by Vero cell culture assay. J Vis Exp 2011;pii2889.

Armstrong PM, et al. Development of a multi-target TaqMan assay to detect eastern equine encephalitis virus variants in mosquitoes. Vector Borne Zoonotic Dis 2012;12(10):872–876.

Callahan JD, et al. Development and evaluation of serotype- and group-specific fluorogenic reverse transcriptase PCR (TaqMan) assays for dengue virus. J Clin Microbiol 2001;39:4119–4124.

Lambert AJ, et al. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays J Clin Microbiol 2003 41:379–385.

Lanciotti RS, et al. Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay. J Clin Microbiol 2000;38:4066–4071.

Sutherland GL, Nasci RS. Detection of West Nile virus in large pools of mosquitoes. J Am Mosq Control Assoc 2007;23:389–395.

The choice of laboratory diagnostic tests depends on the needs, approach, and surveillance capability of a given health agency. Tests include antibody-capture ELISA, complement fixation (CF), hemagglutination inhibition (HI), and plaque reduction neutralization test (PRNT). However, few reagents are commercially available for domestic or wildlife IgM antibodies for antibody capture ELISAs. Many public health laboratories will therefore use PRNT because these are not dependent on species specific antibodies.

The same serologic techniques applied to clinically ill animals may also be used for healthy subjects for vertebrate serosurveys or for healthy sentinel animals serially-sampled as sentinels. As with human diagnostic samples, serologic results from non-human vertebrates must be interpreted with caution due to potential cross-reactivity. Cross reactions may occur between EEE and WEE antibodies in the CF and HI tests. Vaccination for EEE can also cause positive PRNT, HI, CF, and possibly IgM test results.

Specimens from horses and other domestic animals can be tested through diagnostic laboratories including U.S. Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service’s (APHIS) National Veterinary Services Laboratory (NVSL) in Ames, Iowa. Testing can take up to several weeks to complete depending upon the type of sample submitted and the testing protocol required to obtain a definitive result. Details on the diagnostic criteria for EEE for can be found at: https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/equine/eee-wee-vee/equine-encephalitis

For additional details see: Human Laboratory Diagnosis and Testing.

Methods for virus detection, isolation, and identification are the same as described for human and mosquito diagnostics. The most commonly used methods to detect EEE virus or viral RNA in animal populations are immunoassays, virus isolation, and molecular tests. Specimens typically are tissues or fluids from acutely ill or dead animals. Virus detection in apparently healthy animals is very low-yield and inefficient, and therefore not cost-effective, and should not be considered for routine surveillance programs. Some animals have few tissues with detectable virus particles or viral RNA at necropsy, such as horses. Others, such as certain bird species, may have fulminant infections with high viral loads in almost every tissue.