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

项目摘要 登革热病毒(DENV)、寨卡病毒(ZIKV)、基孔肯雅病毒(CHIKV)和马亚罗病毒(MAYV)都是 蚊媒核糖核酸病毒。它们是公共卫生问题，因为(1)DENV和CHIKV导致数百人 每年有数百万人感染，在受影响地区造成重大负担；(2)巴西寨卡病毒的爆发 2015/2016年，由于与新生儿小头畸形症有关，引起了普通民众的焦虑，以及 (3)MAYV最近在中南美洲出现，具有流行传播的潜力。 因为这些病毒感染具有几乎相同的临床表现，而且它们经常在 同时，重要的是要有一个护理点(POC)测试平台来准确识别病毒感染 用于患者的临床管理，包括这些病毒的不同并发症。 根据疾病控制和预防中心(CDC)的说法，目前授权用于 评估DENV、ZIKV和CHIKV感染的方法包括逆转录聚合酶链式反应(RT- 聚合酶链式反应(PCR)和酶联免疫吸附试验(EL ISA)。然而，这些化验是在 实验室，而不是在POC、诊所或受感染的田野。同样重要的是要注意到，病毒感染可以 导致无症状感染，高达80%的ZIKV患者、50%的DENV患者和28%的CHIKV患者 分别为患者。因此，现场的POC检测对于筛查无症状患者将更有价值 患者和监测可能的病毒传播比实验室检测要好，因为只有有症状的患者 去医院或诊所寻求医疗帮助或接受筛查。 为了满足这一需求，我们建议开发一种名为Valve-Enabled Lysis的PoC诊断平台。 基于RNA浓缩和RNA扩增设备(VLEAD)。VLEAD将整合样品准备- 包括病毒裂解和RNA浓缩-通过核酸扩增同时检测这些 病毒。为了实现这一目标，我们的目标是(1)开发用于同时检测ZIKV的多路VLEAD， DENV、CHIKV和MAYV；(2)使用不同的样本对VLEAD进行优化，并比较设备的适用性 用于尿液、唾液和血液样本；以及(3)使用临床样本验证VLEAD，并与 基准方法包括常规RT-PCR。 本研究的意义在于以下几个方面。首先，这些由蚊子传播的RNA病毒是 这是一个公共卫生问题。POC的准确和样本到答案的病毒检测平台将有助于 临床护理和病人管理。其次，这些病毒感染中有很大一部分是没有症状的， 因此，一个非侵入性的POC平台将非常有利于对受感染的普通人群进行筛查 区域和监测病毒传播。第三，VLEAD平台可以被适配为检测其他 感兴趣的病原体，有可能产生更多社会影响。

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 preparation— including virus lysis and RNA enrichment—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.