Development of a novel molecular method for detection of La Crosse virus from vectors and reservoirs

La Crosse virus (LACv) is transmitted via the bite of primary vector, Aedes triseriatus, and secondary vectors Ae. albopictus and Ae. japonicus. The virus is maintained in the environment by reservoir hosts, squirrels (Sciurus carolinensis) and chipmunks (Tamias striatus) and is the leading cause of arboviral encephalitis in children. Most severe cases occur in individuals under 15-years-old. The standard molecular method for LACv screening is reverse transcriptase polymerase chain reaction (RT-PCR), an effective yet somewhat time consuming and expensive procedure. No vaccines or antivirals exist, making accurate surveillance and prevention methods crucial for protecting human health. Additionally, mosquito abatement districts and county health departments often lack resources for performing these molecular tests. We hypothesize that an economical, isothermal (eliminating the need for PCR machines), sensitive, specific, and faster molecular method for the detection of LACv from vectors and reservoirs can be developed. Consequently, positive controls obtained from the CDC&P were tested using the known/standard RT-PCR protocol and compared with a newly designed procedure. Varying concentrations of reagents, including primers, buffer, and enzymes, in the novel reaction mix were tested against RT-PCR for efficacy and sensitivity. Once virus was routinely detected from positive controls the novel method was compared to RT-PCR for detection of LACv from vectors and reservoirs collected from sites around Knox County, Tennessee. The development of a new method for LACv detection provides a rapid and cost-effective monitoring tool for mosquito abatement districts and county health departments, which may not have the resources for expensive RT-PCR equipment.