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RNA病毒它们是公共卫生问题，因为（1）DENV和CHIKV引起数百人死亡， 每年有数百万人感染，受影响地区负担沉重，（2）巴西ZIKV的爆发 在2015/2016年，由于其与新生儿小头畸形的相关性，引起了一般人群的焦虑， (3)MAYV最近在中美洲和南美洲出现，并具有流行蔓延的潜力。 因为这些病毒感染具有几乎相同的临床表现， 同时，重要的是要有一个即时（POC）检测平台，以准确识别病毒感染 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感染包括逆转录聚合酶链反应（RT-PCR）。 PCR）测定和酶联免疫吸附测定（ELISA）。然而，这些测定是在 实验室，而不是在诊所或感染现场的POC。同样重要的是要注意，病毒感染可以 引起无症状感染，高达80%的ZIKV患者，50%的DENV患者和28%的CHIKV患者 患者分别。因此，现场POC检测对于筛查无症状患者将更有价值 病人和监测可能的病毒传播比实验室测试，因为只有症状的病人 前往医院或诊所寻求医疗帮助或接受检查。 为了满足这一需求，我们建议开发一个名为Valve-enabled Lysis的POC诊断平台，论文- 基于RNA富集和RNA扩增设备（VLEAD）。VLEAD将整合样品制备- 包括病毒裂解和RNA富集-用核酸扩增同时检测这些 病毒。为了实现该目标，我们的目标是（1）开发用于同时检测ZIKV的多路复用VLEAD， DENV、CHIKV和MAYV;（2）使用各种样本优化VLEAD并比较器械的适用性 用于尿液、唾液和血液样本;和（3）使用临床样本验证VLEAD，并将VLEAD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，这些蚊子传播的RNA病毒是 一个公共健康问题在POC建立一个准确和样本到答案的病毒检测平台将有助于 临床护理和患者管理。其次，这些病毒感染者中有很大一部分是无症状的， 因此，非侵入性POC平台将非常有益于在感染者中筛查普通人群， 监控病毒传播。第三，VLEAD平台可以适于检测其他 感兴趣的病原体，有可能产生更多的社会影响。

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.