Multiplexed Detection of Mosquito-Borne Viruses at the Point-of-Care

Project Summary Dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus (CHIKV), and Mayaro virus (MAYV) are all mosquito-borne RNA viruses. They are public health concerns because (1) DENV and CHIKV cause hundreds of millions of infections each year, with significant burdens in affected areas, (2) the outbreak of ZIKV in Brazil in 2015/2016 caused anxieties to general population due to its association with microcephaly of newborns, and (3) MAYV emerged in Central and Southern America recently and has the potential for epidemic spread. Because these virus infections have virtually identical clinical presentation and they often circulate concurrently, it is important to have a point-of-care (POC) testing platform to accurately identify virus infection for clinical management of patients, including different complications from these viruses. According to the Centers for Disease Control and Prevention (CDC), the current methods authorized for assessing DENV, ZIKV, and CHIKV infections include reverse transcription polymerase chain reaction (RT- PCR) assay and enzyme-linked immunosorbent assay (ELISA). However, these assays are carried out in laboratories, not at POC in a clinic or in an infected field. It is also important to note that virus infection can cause asymptomatic infections, up to 80% of ZIKV patients, 50% of DENV patients, and 28% of CHIKV patients, respectively. As a result, POC testing in the field will be more valuable for screening asymptomatic patients and monitoring possible virus transmission than a laboratory test because only symptomatic patients go to hospitals or clinics for seeking medical help or to be screened. To address the need, we propose to develop a POC diagnostic platform called Valve-enabled Lysis, paper- based RNA Enrichment, and RNA Amplification Devices (VLEAD). VLEAD will integrate sample preparationâ€Â" including virus lysis and RNA enrichmentâ€Â"with nucleic acid amplification for simultaneous detection of these viruses. To achieve the goal, we aim to (1) develop multiplexed VLEAD for simultaneous detection of ZIKV, DENV, CHIKV and MAYV; (2) optimize VLEAD using various samples and compare the suitability of the device for urine, saliva and blood samples; and (3) validate VLEAD using clinical samples and compare VLEAD with the benchmark methods including conventional RT-PCR. The significance of the research lies in the following aspects. First, these mosquito-borne RNA viruses are a public health concern. An accurate and sample-to-answer virus detection platform at POC will be useful for clinical care and patient management. Second, a large percentage of these virus infections are asymptomatic, thus a non-invasive POC platform would be very beneficial for screening the general population in the infected area and monitoring virus transmission. Third, the VLEAD platform can be adapted for detecting other pathogens of interest, with a potential to have more societal impacts.