The development of a multiplexed Soluble Phage Array (SPAr) for the detection of zoonotic pathogens

血清监测是监测动物种群疾病的一种有价值的方法，以促进有效的干预和遏制策略。有人建议，对人畜共患疾病进行有效的血清监测可以为人类人群中可能出现的人畜共患病病原体提供一种早期预警系统。然而，到目前为止，血清监测在某种程度上受到单一病原体焦点的限制，缺乏区分非常密切相关的病原体的能力。因此，需要一种负担得起的、可获得的、具有非常高特异性的多重方法来监测广泛的既有和新出现的病原体。噬菌体展示多肽库的巨大多样性与下一代测序(NGS)在被称为下一代噬菌体展示(NGPD)的过程中的筛选能力相结合，可用于发现由抗体对疾病的反应识别的大量多肽模拟表位。我们建议，一个富含多个感染特异性模拟表位的噬菌体多肽亚库本身可以被用作诊断特定病原体感染的检测‘抗原’；我们称之为可溶性噬菌体阵列(SPAR)的检测格式。这项测试将通过对与抗体结合的噬菌体进行NGS分析，来测量血清样本中抗体对感染特异性噬菌体展示模拟表位的识别。我们假设，NGPD可以用来开发SPAR分析，可以准确地识别人畜共患病原体的感染，并且这些检测可以以高精度和重复性进行多重检测。这项概念验证研究将集中在四种影响绵羊或马的紧急人畜共患病病毒上。这些病毒是绵羊的卢平病(LIV)、壁虱传播的脑炎(TBEV)和裂谷热(RVFV)病毒，以及马的西尼罗河病毒(WNV)。所选病毒代表黄病毒属(Liv、TBEV和WNV)和蛇毒(RVFV)。这些病毒经过精心挑选，以代表对新出现的人畜共患病进行血清监测的复杂性，以证明SPAR检测的潜力。与现有的血清监测方法相比，多重SPAR分析将具有相当大的优势。它将不限于单一病原体的方法，并将有能力区分关系非常密切的病原体，这一特性对于制定有效的人畜共患病病原体控制战略至关重要。

Serosurveillance is a valuable approach to disease monitoring in animal populations to facilitate effective intervention and containment strategies. It has been suggested that effective serosurveillance of zoonotic diseases could provide an early-warning system for the potential emergence of a zoonotic pathogen in human populations. However, to date, serosurveillance is somewhat limited by a single-pathogen focus and lacks the ability to distinguish between very closely related pathogens. Therefore, there is a need for an affordable, accessible, multiplexed approach with very high specificity to monitor for a wide range of established and emerging pathogens. The huge diversity of phage display peptide libraries combined with the screening power of next generation sequencing (NGS) in a process termed next generation phage display (NGPD) can be applied to discover large panels of peptide mimotopes recognised by antibody responses to disease. We propose that a phage-peptide sub-library enriched to contain multiple infection-specific mimotopes can itself be used as the assay 'antigen' to diagnose infection with a particular pathogen; an assay format we have termed Soluble Phage Array (SPAr). This assay would measure recognition of the panel of infection-specific phage-displayed mimotopes by antibodies in serum samples by NGS analysis of phage that are bound by the antibodies. We hypothesise that NGPD can be used to develop SPAr assays that can accurately identify infection with zoonotic pathogens and that these can be multiplexed with high accuracy and reproducibility.This proof-of-concept study will focus on four emergent zoonotic viruses that affect sheep or horses. These are louping ill (LIV), tick-borne encephalitis (TBEV) and Rift Valley fever (RVFV) viruses in sheep, and West Nile virus (WNV) in horses. The selected viruses represent the genera Flavivirus (LIV, TBEV and WNV) and Phlebovirus (RVFV). These viruses have been carefully chosen to represent the complexity of serosurveillance of emerging viral zoonoses in order to demonstrate the potential of the SPAr assay. A multiplexed SPAr assay would have considerable advantages over existing serosurveillance approaches. It would not be limited to a single-pathogen approach and would have the ability to distinguish very closely related pathogens, a property vital to inform effective control strategies for zoonotic pathogens.