Graduation Year

2021

Document Type

Thesis

Degree

M.S.P.H.

Degree Name

MS in Public Health (M.S.P.H.)

Degree Granting Department

Public Health

Major Professor

Thomas Unnasch, Ph.D.

Committee Member

Lynn Martin, Ph.D.

Committee Member

Michael Teng, Ph.D.

Keywords

arbovirus, colorimetric, LAMP, mosquito control districts

Abstract

Eastern Equine Encephalitis virus (EEEV) is a neurotrophic alphavirus for which there is no effective treatment or vaccine for humans. Periodic outbreaks in the Eastern United States represent an ongoing public health problem; Florida serves as the reservoir for EEEV for the rest of the country. Reverse transcription-polymerase chain reaction (RT-PCR) is the current gold standard for molecular diagnostic testing of the presence of EEEV in vectors. However, RT-PCR is technically complex and can be difficult for mosquito control districts to utilize. In order to provide a simple, cost-effective alternative for mosquito surveillance and control, a novel one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, designed by New England BioLabs® for the detection of EEEV, was evaluated. The assay utilizes synthetic positive controls and three primer sets targeting distinct portions of the EEEV genome. This LAMP assay was tested initially using diluted EEEV from viral culture, followed by experimentally spiked mosquito pools, known positive mosquito pools, and known negative mosquito pools. The LAMP assay was found to be capable of detecting virus titers equivalent to those present in 10% of an infectious mosquito in a mosquito homogenate, based on the dilution process that occurs during homogenization following RNA extraction. No false positive results were obtained from RNA isolated from mosquito homogenates prepared from 23 different EEEV vector mosquito species previously shown to be EEEV negative by RT-PCR. The LAMP assay is technically simple to perform, provides a visual readout and requires no sophisticated equipment. These results suggest that the EEEV LAMP represents an attractive alternative to RT-PCR for vector surveillance by mosquito control districts.

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