PANDAA for universal, pan-lineage molecular detection of filoviruses to enable rapid epidemic response.

埃博拉病毒[EBOV]和马尔堡病毒[MARV]是引起人类严重出血热的丝状病毒， 灵长类动物，并被列为世卫组织研发蓝图中优先关注的紧迫病原体。基因组 EBOV和MARV属的序列差异> 55%，并且在每个属内， 阈值>23%，因此通过基于金标准qPCR的方法的检测受到显著的 丝状病毒之间的遗传变异性。EBOV和MARV的临床诊断是困难的，因为早期阶段（即，表示“前 出血性），表现为与一系列发热性疾病相关的非特异性症状， 受影响地区的其他传染性病原体，例如，疟疾、黄热病、登革热、克里米亚-刚果 出血性（CCHFV）或拉沙热。为了隔离受感染的个体，在EBOV或 MARV爆发只能通过实施快速准确的诊断， 性能Aldatu率先使用了PANDAA技术，该技术可以实现基于探针的qPCR， 通过同时适应和扩增不同的模板来检测高度可变的基因组区域。熊猫 独特地减轻了遗传多态性的存在，以允许通过 荧光探针。因此，PANDAA使能的qPCR是通用检测病原体的理想解决方案， 显著菌株、谱系和/或亚型序列多样性。Aldatu定位独特，可提供快速的泛- 一种基于丝状病毒qPCR的检测方法，可用于EBOV的快速、灵敏的分子诊断和鉴别 与现有诊断相比，MARV具有上级性能，并且不会受到基因变化的影响 新的病毒变种PANDAA已成功应用于15个以上的亚型独立检测 耐药突变（DRM）的艾滋病毒的目标。最近，我们开发了第一个拉沙热的泛谱系检测方法， 病毒（LASV），另一种世卫组织优先病原体，具有高爆发潜力。我们建议利用独特的 PANDAA开发用于丝状病毒检测的快速，灵敏的分子诊断方法的能力，以及第一个 EBOV和MARV的泛种覆盖，通过以下具体目标：（1）发展泛种覆盖， 丝状病毒PANDAA检测，利用成熟的技术和专有的PANDAA试剂设计;（2）分析 验证，包括确认物种包容性和高特异性;（3）PANDAA-丝状病毒的多重检测 与现有的LASV和CCHFV检测试剂盒进行比较，以产生病毒性出血热（VHF）检测试剂盒，包括 PANDAA-丝状病毒和PANDAA-VHF检测试剂的热稳定性，以满足诊断要求 （4）热稳定PANDAA-丝状病毒和PANDAA-VHF检测试剂盒的GMP生产， (5)使用代表各种流通物种和地理区域的样本进行分析验证， 在疾病预防控制中心和世卫组织附属伙伴机构的参考实验室对检测试剂盒进行现场评价。作为第一个泛- 丝状病毒检测，我们将为所有地区提供快速，标准化的检测选项，可以使用预 中心实验室现有的qPCR设备，从根本上改善诊断工作流程和流行病准备。

Ebolavirus [EBOV] and Marburgvirus [MARV] are filoviruses that cause severe hemorrhagic fevers in humans and primates and are listed among the urgently concerning pathogens prioritized in the WHO R&D Blueprint. Genomic sequences of the EBOV and MARV genera differ by >55%, and within each genera the species sequence diversity threshold is >23% thus detection by gold standard qPCR-based methods has been encumbered by significant genetic variability between filoviruses. Clinical diagnosis of EBOV and MARV is difficult as the early stages (i.e., pre- hemorrhagic) present with non-specific symptoms associated with a range of febrile illnesses that are displayed by other infectious agents endemic to affected areas e.g., malaria, yellow fever, dengue fever, Crimean-Congo hemorrhagic (CCHFV) or Lassa fevers. In order to isolate infected individuals, effective control during an EBOV or MARV outbreak can only be achieved by implementing rapid and accurate diagnostics with consistently reliable performance. Aldatu has pioneered the use of PANDAA technology, which enables probe-based qPCR for target detection in highly variable genomic regions by simultaneously adapting and amplifying diverse templates. PANDAA uniquely mitigates the presence of genetic polymorphisms to allow otherwise divergent templates to be detected by fluorescent probes. As such, PANDAA-enabled qPCR is an ideal solution for universal detection of pathogens with significant strain, lineage, and/or sub-type sequence diversity. Aldatu is uniquely positioned to deliver a rapid pan- filovirus qPCR-based assay that is rapid, sensitive molecular diagnostic for the detection and differentiation of EBOV and MARV with superior performance compared to existing diagnostics and won’t be affected by genetic changes in new viral variants. PANDAA has been successfully applied to subtype-independent detection of more than fifteen drug resistance mutation (DRM) targets in HIV. Recently, we developed the first pan-lineage assay for Lassa fever virus (LASV), another WHO priority pathogen with high outbreak potential. We propose to leverage the unique capabilities of PANDAA to develop a rapid, sensitive molecular diagnostic assay for filovirus detection, and the first with pan-species coverage of EBOV and MARV, through the following specific aims: (1) development of a pan- filovirus PANDAA assay, leveraging proven techniques and proprietary PANDAA reagent design; (2) analytical validation including confirmation of species inclusivity and high specificity; (3) multiplexing of the PANDAA-Filovirus assay with existing assays for LASV and CCHFV to produce a viral hemorrhagic fever (VHF) panel, including the thermostabilization of the both PANDAA-Filovirus and PANDAA-VHF assays, to meet the requirements of diagnostics targeted to LMICs; (4) GMP manufacturing of the thermostabilized PANDAA-Filovirus and PANDAA-VHF assays and (5) assay validation using samples representing a broad variety of circulating species and geographies, with multi- site evaluations of the test kits at reference labs at CDC- and WHO-affiliated partner institutions. As the first pan- filovirus assay, we will provide a rapid, standardized testing option for all regions that can be deployed using pre- existing qPCR equipment in central labs to radically improve the diagnostic workflow and epidemic preparedness.