Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes.

Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.

Field Analysis of Biological Factors Associated With Sites at High and Low to Moderate Risk for Eastern Equine Encephalitis Virus Winter Activity in Florida.

Eastern equine encephalitis virus (EEEV) is the most pathogenic arbovirus endemic to the United States. Studies have demonstrated Florida's role as a regional reservoir for the virus and its ability to support year-round transmission. Previous research has developed risk index models for mapping locations most at risk for EEEV transmission. We compared vector abundance, vector feeding behavior, potential host species, and fauna presence at high versus low-moderate risk sites during the winter and spring. Predicted high-risk sites had a significantly greater abundance of mosquitoes overall, including Culiseta melanura (Coquillett) (Diptera: Culicidae), the primary enzootic vector of EEEV. Twenty host species were identified from Cs. melanura bloodmeals, with the majority taken from avian species. Culiseta melanura largely fed upon the Northern Cardinal (Cardinalis cardinalis (Passeriformes: Cardinalidae)), which accounted for 20-24.4% of the bloodmeals obtained from this species in years 1 and 2, respectively. One EEEV-positive mosquito pool (Cs. melanura) and nine EEEV seropositive sentinel chickens were confirmed during winter-spring collections from high-risk sites; no seropositive chickens nor mosquito pools were found at the low-moderate risk sites. These results suggest that high-risk sites for EEEV activity are characterized by habitats that support populations of Cs. melanura and which may also provide ample opportunities to feed upon Northern Cardinals. The overall low level of mosquito populations during the winter also suggests that control of Cs. melanura populations in winter at high-risk sites may prove effective in reducing EEEV transmission during the peak summer season.

Impact of the Southern Oscillation Index, Temperature, and Precipitation on Eastern Equine Encephalitis Virus Activity in Florida.

Eastern equine encephalitis virus (EEEV), an Alphavirus from family Togaviridae, is a highly pathogenic arbovirus affecting the eastern United States, especially Florida. Effects of the Southern Oscillation Index (SOI), precipitation, and cooling degree days on EEEV horse case data in Florida from 2004 to 2018 were modeled using distributed lag nonlinear models (DLNMs). The analysis was conducted at statewide and regional scales. DLNMs were used to model potential delayed effects of the covariates on monthly counts of horse cases. Both models confirmed a seasonal trend in EEEV transmission and found that precipitation, cooling degree days, and the SOI were all predictors of monthly numbers of horse cases. EEEV activity in horses was associated with higher amounts of rainfall during the month of transmission at the statewide scale, as well as the prior 3 mo at the regional scale, fewer cooling degree days during the month of transmission and the preceding 3 mo and high SOI values during the month and the previous 2 mo, and SOI values in the prior 2 to 8 mo. Horse cases were lower during El Niño winters but higher during the following summer, while La Niña winters were associated with higher numbers of cases and fewer during the following summer. At the regional scale, extremely low levels of precipitation were associated with a suppression of EEEV cases for 3 mo. Given the periodicity and potential predictability of El Niño Southern Oscillation (ENSO) cycles, precipitation, and temperature, these results may provide a method for predicting EEEV risk potential in Florida.