Multiplex Serodiagnostic Assays for Pathogenic Arboviruses in Brazil

巴西致病性虫媒病毒的多重血清诊断检测

• 批准号: 10406273
• 负责人: Wei-Kung Wang
• 金额: $ 15.65万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-05 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406273
• 关键词: Acute; Age; Alphavirus; Ambulatory Care Facilities; Antibodies; Antigens; Arbovirus Infections; Arboviruses; Binding; Biological Assay; Brazil; Chikungunya virus; Communities; Country; Data; Dengue Virus; Diagnosis; Employment; Enrollment; Enzyme-Linked Immunosorbent Assay; Epidemiology; FDA Emergency Use Authorization; Fever; Flavivirus; Flavivirus Infections; Geographic Locations; Household; IgG3; Immunoassay; Immunoglobulin G; Immunoglobulin M; Individual; Infection; Mayaro virus; Microspheres; Mutate; Mutation; Neutralization Tests; Nonstructural Protein; Nucleocapsid Proteins; Oropouche virus; Orthobunyavirus; Pathogenicity; Patients; Phase; Plasma; Proteins; Publications; Recombinant Proteins; Recombinants; Research; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Sensitivity and Specificity; Serology; Serology test; Seroprevalences; Serotyping; Serum; Site; Testing; Vaccination; Vaccines; Viral Proteins; Virus Diseases; Virus-like particle; West Nile virus; Yellow Fever; ZIKV infection; Zika Virus; antibody detection; base; cross reactivity; env Gene Products; experience; follow-up; implementation facilitation; innovation; member; recombinant virus; serosurveillance; transmission process

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.

抽象的 对高度敏感和特定的血清诊断测试的关键需求存在，以区分通过 地理区域（例如巴西）的病原病毒，包括寨卡病毒在内的多个黄病毒 （ZIKV），登革热病毒的四种血清型（DENV1-4），西尼罗河病毒（WNV）和黄热病（YFV）以及 Chikungunya病毒（chikv），玛莎罗病毒（Mayv）（α病毒）和Oropouche病毒（OROV）（A Bunyavirus），是地方性的。但是，针对不同黄病毒的抗体的交叉反应性 挑战，即使中和测试也无法确认个体中特定的黄病毒感染 他们以前经历过黄病毒感染。 拟议的研究的目的是开发高度敏感和特定的血清诊断测试 通过致病性弧病毒区分感染。中心假设是融合循环的组合 （FL） - 突出的VLP和非结构蛋白1（NS1）蛋白质的蛋白质和重组蛋白 以及以多重格式的Chikv，Mayv和Orov的VLP可以区分不同的Arbovirus感染。 第一个目的是基于基于基于 重组蛋白以区分arbovirus感染。我们将采用7个重组NS1蛋白 黄病毒，包络（E）2蛋白和核素（N）蛋白（N）蛋白质的chikv，Mayv和Orov用于多重IgG 使用Luminex 200和IgM MIA和IgM MIA，并用15个个体的15个康复阶段血清/等离子体进行测试 带有确认的arbovirus感染。第二个目的是开发和验证基于多路复用IgG和IgM MIAS 在VLP上以区分arbovirus感染。我们将使用7个黄病毒和VLP的纯化和fl的VLP Chikv，Mayv和Orov的作品，并用AIM 1中的15个血清/血浆测试。第三个目的是比较 具有当前可用的血清诊断测定的多重IgM MIA和IgG MIAS，以确定Arbovirus 巴伊亚州的血清阳性，这是一个东北州，在巴西有多次arbovirus透射。对于血清诊断， 我们将测试来自急性发热疾病患者的血清样品及其在4个研究的门诊诊所的随访 位于巴伊亚的地点（Salvador，Feira de Santana，Itabuna和Campo Formoso）。对于血清阳性研究，我们将 在四个研究地点的社区注册和测试家庭成员。 拟议的研究具有创新性，因为它采用了两种有前途的抗原（NS1蛋白和FL-Mutated VLP） 克服七个黄病毒的黄病毒交叉反应性，而不是传统的基于E蛋白质的测试，加上 Chikv，Mayv和Orov的两种多重格式，以区分巴西的Arbovirus感染。会 有助于详细了解巴伊亚的10种arbovirus感染的流行病学。成功 多路复用平台的使用可以用作塞子诊断和血清监视的新范式 多个Arbovirus共同循环的国家。最重要的是，这项研究将促进 在流行地区的藻类病毒疫苗的实施，特别是ZIKV，DENV和CHIKV疫苗。