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诊断试验来区分感染 地理区域的致病性虫媒病毒，如巴西，那里有包括寨卡病毒在内的多种黄病毒 登革热病毒(ZIKV)、登革热病毒(DENV1-4)、西尼罗河病毒(WNV)和黄热病(YFV)四种血清型，以及 基孔肯雅病毒(CHIKV)、马亚罗病毒(MAYV)(均为甲型病毒)和奥罗波什病毒(奥罗夫病毒)(a 本雅氏病毒)，是地方性的。然而，针对不同黄病毒的抗体的交叉反应性主要表现为 挑战，即使中和试验也不能确认个体中特定的黄病毒感染 有过黄病毒感染经历的人。 拟议研究的目标是开发高度敏感和特异的血清诊断试验，以 区分致病虫媒病毒的感染。中心假设是核聚变环路的组合 不同黄病毒的(FL)突变VLP和非结构蛋白1(NS1)蛋白及重组蛋白 CHIKV、MAYV和OROV复合格式的VLP可以区分不同的虫媒病毒感染。 第一个目标是开发和验证多重免疫球蛋白和免疫球蛋白微球免疫分析(Mias)，基于 重组蛋白用于区分虫媒病毒感染。我们将使用重组的NS1蛋白7 黄病毒、CHIKV、MAYV和OROV的包膜(E)2蛋白和核衣壳(N)蛋白用于多重免疫球蛋白 和IgM MIA使用Luminex 200，并用15组恢复期血清/血浆进行检测 确认感染了虫媒病毒。第二个目标是开发和验证基于Ig G和Ig M的多重MIA 以区分虫媒病毒感染。我们将使用7种黄病毒的纯化和FL突变的VLP和VLP CHIKV、MAYV和OROV，并用15组血清/血浆进行检测，与目标1相同。第三个目的是比较 免疫球蛋白M和免疫球蛋白MIA与现有血清诊断方法联合检测虫媒病毒 在巴西有多种虫媒病毒传播的东北部州巴伊亚州的血清阳性率。对于血清学诊断， 我们将检测急性发热病患者的血清样本，并在4个研究的门诊进行随访。 巴伊亚的几个地点(萨尔瓦多、费拉·德桑塔纳、伊塔库纳和坎波·福尔摩索)。对于血清阳性率研究，我们将 在4个研究地点的社区登记和测试家庭成员。 这项拟议的研究具有创新性，因为它使用了两种有希望的抗原(NS1蛋白和FL突变的VLP)来 克服黄病毒对七种黄病毒的交叉反应，而不是传统的基于E蛋白的测试，此外 CHIKV、MAYV和OROV两种多重格式，以区分巴西的虫媒病毒感染。它会 有助于详细了解巴伊亚10种虫媒病毒感染的流行病学。成功者 多重平台的应用可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.