Detection and Characterization of Flavivirus and Alphavirus Infections in Acute Suspected Arboviral Cases or Neurological Disease, Central Department, Paraguay

项目摘要 黄病毒和甲型病毒是两种最常见的虫媒病毒病原体，占 每年有数亿人感染，在过去20年里反复流行。总的来说，这些大型 属包括100多种潜在的人类病原体。有症状的黄病毒感染(例如 登革热、寨卡病毒、西尼罗河病毒)和甲型病毒(如基孔肯雅、马亚罗)存在非特异性 表现并主要导致两种重叠的急性临床综合征：伴有或 没有发烧和脑膜脑炎等神经系统疾病。然而，这是昂贵和不切实际的 对患者进行不止几种常见病原体的测试，研究方案通常集中在一个临床上 综合症。这些因素使虫媒病毒感染的检测、监测和控制工作复杂化，以及 计算准确的发病率估计。这项计划的研究将利用新的分子方法。 用于泛属检测和新的多重血清学方法，以同时筛查针对 八种虫媒病毒。该提案的目的是评估新的分子和血清学方法用于检测 黄病毒和甲型病毒感染的发病率、血清阳性率和多样性的特征 有症状的病人。研究将在亚松森和巴拉圭中央部门进行， 最近多种虫媒病毒的传播已被记录在案。我们的中心假设是1) 这些新方法将增加在有症状的患者中检测虫媒病毒的广度，2)证据 暴露在多种虫媒病毒下将是常见的，3)广泛的异型抗体反应将保护 对抗严重的疾病。这项研究将首先确定患者中黄病毒和甲病毒感染的特征。 患有急性疑似虫媒病毒病或急性神经系统疾病(脑膜炎、脑炎、格林-巴利综合症 综合症)。泛黄病毒和泛甲病毒的检测将使用一种新的分子检测方法。病毒式传播 符合研究条件的患者将描述流行病学、临床表现和患者结局。 定义。然后将确定与虫媒病毒有关的有症状和严重疾病的发生率。 出现时的抗体。将进行多重血清学检测，以检测针对五种病毒的IgM和Ig G 黄病毒和三种甲型病毒。将计算血清阳性率，并计算有症状和 严重疾病将根据感染前对异型病毒抗体的存在进行评估。 最后，将使用CRISPR/Cas9直接从临床样本中获得全基因组病毒序列 耗尽系统，以分析病毒系统发育和识别新的/罕见的病毒株。这项研究的目的是 是改进最常见的人类虫媒病毒家族的实验室方法，并识别有风险的患者 用于有症状或严重的虫媒病毒病。预计这项工作将产生重要的积极影响 因为改进的监测方法和患者风险分层将导致虫媒病毒增加 临床管理和暴发控制的检测和有针对性的干预。

PROJECT ABSTRACT Flaviviruses and alphaviruses are the two most common genera of arboviral pathogens, accounting for hundreds of millions of infections annually and repeated epidemics over the past 20 years. In total, these large genera include over a hundred potential human pathogens. Symptomatic infections with the flaviviruses (e.g. dengue, Zika, West Nile) and alphaviruses (e.g. chikungunya, Mayaro) present with non-specific manifestations and predominantly result in two overlapping acute clinical syndromes: a systemic illness with or without fever and a neurological illness such as meningoencephalitis. However, it is costly and impractical to test patients for more than a few common pathogens, and research protocols typically focus on one clinical syndrome. These factors complicate the detection of arboviral infections, surveillance and control efforts, and the calculation of accurate incidence estimates. Research in this proposal will utilize novel molecular methods for pan-genus detection and new multiplex serological methods to simultaneously screen for antibodies against eight arboviruses. The objective of the proposal is to evaluate new molecular and serological methods for the characterization of the incidence, seroprevalence and diversity of flavivirus and alphavirus infections among symptomatic patients. Research will be performed in Asunción and the Central Department of Paraguay, where recent transmission of multiple arboviruses has been documented. Our central hypotheses are that 1) these new methods will increase the breadth of arbovirus detection among symptomatic patients, 2) evidence of exposure to multiple arboviruses will be common, and 3) broad, heterotypic antibody responses will protect against severe disease. This research will first characterize flavivirus and alphavirus infections among patients with an acute suspected arboviral illness or acute neurological disease (meningitis, encephalitis, Guillain-Barré syndrome). Pan-flavivirus and pan-alphavirus detection will be performed with a novel molecular assay. Viral epidemiology, clinical findings, and patient outcomes will be described among patients who meet the study definitions. The incidence of symptomatic and severe disease will then be determined in relation to arboviral antibodies at presentation. Multiplex serological testing will be performed to detect IgM and IgG against five flaviviruses and three alphaviruses. Seroprevalence will be calculated, and the incidence of symptomatic and severe disease will be evaluated in relation to the presence of pre-infection antibodies to heterotypic viruses. Finally, whole-genome viral sequences will be obtained directly from clinical samples using a CRISPR/CAS9 depletion system to analyze viral phylogenetics and identify new/rare viral strains. The purpose of this research is to improve laboratory methods for the most common human arbovirus families and identify patients at risk for symptomatic or severe arboviral illnesses. This work is expected to have an important positive impact because improved surveillance methods and patient risk stratification will result in increased arbovirus detection and targeted interventions for clinical management and outbreak control.