Multiplex Serodiagnostic Assays for Pathogenic Arboviruses in Brazil

摘要 迫切需要高灵敏度和特异性的血清学诊断测试，以通过以下方式区分感染： 病原性虫媒病毒在地理区域，如巴西，其中多种黄病毒，包括寨卡病毒， （ZIKV）、登革病毒（DENV 1 -4）、西尼罗河病毒（WNV）和黄热病（YFV）的四种血清型，以及 基孔肯雅病毒（CHIKV）、马亚罗病毒（MAYV）（均为甲病毒）和奥罗波什病毒（Oropouche）（a 布尼亚病毒）是地方性的。然而，针对不同黄病毒的抗体的交叉反应性存在主要的差异。 挑战，例如即使中和试验也不能确认个体中的特定黄病毒感染 有过黄病毒感染史的人 拟议研究的目标是开发高度敏感和特异性的血清学诊断试验， 区分病原性虫媒病毒感染。核心假设是融合环 不同黄病毒的（FL）突变VLP和非结构蛋白1（NS 1）蛋白和重组蛋白 CHIKV、MAYV和Ostromus的VLP在多重形式中可以区分不同的虫媒病毒感染。 第一个目的是开发和验证多重IgG和IgM微球免疫测定（MIA）， 重组蛋白来区分虫媒病毒感染。我们将使用重组NS 1蛋白7 用于多重IgG的黄病毒、CHIKV、MAYV和Ostrous的包膜（E）2蛋白和核衣壳（N）蛋白 使用Luminex 200和15组来自个体的恢复期血清/血浆进行检测 被证实感染虫媒病毒第二个目标是开发和验证多重IgG和IgM MIA， 来区分虫媒病毒感染。我们将使用7种黄病毒和VLP的纯化和FL突变的VLP CHIKV、MAYV和OYB的血清/血浆，并用15组血清/血浆进行测试，如目的1中所述。第三个目的是比较 多重IgM MIA和IgG MIA与目前可用的血清学诊断测定法，以确定虫媒病毒 巴西东北部一个有多种虫媒病毒传播的州巴伊亚的血清阳性率。对于血清诊断， 我们将在4个研究的门诊诊所检测急性发热性疾病患者的血清样本及其随访 在巴伊亚的几个地点（萨尔瓦多、费拉德桑塔纳、伊塔布纳和坎波福莫索）。至于血清阳性率研究，我们会 在4个研究中心的社区招募和测试家庭成员。 这项研究具有创新性，因为它采用了两种有前途的抗原（NS 1蛋白和FL突变的VLP）， 克服了黄病毒对七种黄病毒的交叉反应性，而不是传统的基于E蛋白的检测， CHIKV、MAYV和Ostrous以两种多重形式区分巴西的虫媒病毒感染。它将 有助于详细了解巴伊亚10种虫媒病毒感染的流行病学。成功 多重平台的使用可以作为血清诊断和血清监测的新范例， 多种虫媒病毒共同传播的国家。最重要的是，这项研究将促进 在流行地区实施虫媒病毒疫苗，特别是ZIKV、DENV和CHIKV疫苗。

Abstract A critical need exists for highly sensitive and specific serodiagnostic tests to discriminate infections by pathogenic arboviruses in geographic regions, such as Brazil, where multiple flaviviruses including Zika virus (ZIKV), four serotypes of dengue virus (DENV1-4), West Nile virus (WNV), and yellow fever (YFV), as well as chikungunya virus (CHIKV), Mayaro virus (MAYV) (both alphaviruses) and Oropouche virus (OROV) (a bunyavirus), are endemic. However, cross-reactivity of antibodies against different flaviviruses present major challenges, such that even neutralization tests cannot confirm a specific flavivirus infection among individuals who have experienced previous flavivirus infections. The objective of the proposed research is to develop highly sensitive and specific serodiagnostic tests to discriminate infections by pathogenic arboviruses. The central hypothesis is that a combination of fusion loop (FL)-mutated VLPs and nonstructural protein 1 (NS1) proteins of different flaviviruses and recombinant proteins and VLPs of CHIKV, MAYV and OROV in multiplex formats can distinguish different arbovirus infections. The first Aim is to develop and validate multiplex IgG and IgM microsphere immunoassays (MIAs) based on recombinant proteins to distinguish arbovirus infections. We will employ recombinant NS1 proteins of 7 flaviviruses, envelope (E) 2 protein and nucleocapsid (N) protein of CHIKV, MAYV and OROV for multiplex IgG and IgM MIAs using Luminex 200 and test with 15 panels of convalescent-phase serum/plasma from individuals with confirmed arbovirus infections. The second Aim is to develop and validate multiplex IgG and IgM MIAs based on VLPs to distinguish arbovirus infections. We will use purified and FL-mutated VLPs of 7 flaviviruses and VLPs of CHIKV, MAYV and OROV, and test with 15 panels of serum/plasma as in Aim 1. The third Aim is to compare the multiplex IgM MIAs and IgG MIAs with currently available serodiagnostic assays to determine arbovirus seroprevalence in Bahia, a northeastern state with multiple arbovirus transmissions in Brazil. For serodiagnosis, we will test serum samples from patients with acute febrile illness and their follow-up at outpatient clinics of 4 study sites (Salvador, Feira de Santana, Itabuna and Campo Formoso) in Bahia. For seroprevalence study, we will enroll and test household members at communities of the 4 study sites. The proposed study is innovative as it employs two promising antigens (NS1 protein and FL-mutated VLPs) to overcome flavivirus cross-reactivity for seven flaviviruses, as opposed to traditional E protein-based tests, plus CHIKV, MAYV and OROV in two multiplex formats to discriminate arbovirus infections in Brazil. It would contribute to a detailed understanding of the epidemiology of 10 arbovirus infections in Bahia. The successful employment of the multiplex platforms can serve as a new paradigm for serodiagnosis and serosurveillance in countries where multiple arboviruses co-circulate. Most importantly, this research will facilitate the implementation of arbovirus vaccines, in particular ZIKV, DENV and CHIKV vaccines in endemic regions.