Powassan病毒(POWV)是一种蜱传播的黄病毒,以引起人类致命的神经侵袭性疾病而闻名。最近,POWV感染明显增加,强调了解病毒复制的紧迫性,发病机制,发展干预措施。值得注意的是,没有批准的POWV疫苗或疗法,将其分类为生物安全3级(BSL-3)制剂阻碍了研究。为了克服这些障碍,我们开发了一个复制子系统,一种缺乏结构蛋白的自我复制的RNA,使其在bsl-2环境下操作安全。我们构建了一个携带高斯荧光素酶(Gluc)报告基因和杀瘟素(BSD)选择标记的POWV复制子。连续的BSD选择导致获得稳定的携带POWV复制子的Huh7细胞系。我们鉴定了细胞培养适应性突变G4079A,G4944T和G6256A,导致NS2AR195K,NS3G122G,和NS3V560M,增强RNA复制。我们证明了POWV复制子系统用于高通量筛选(HTS)测定以鉴定有希望的抗POWV复制的抗病毒药物的实用性。我们进一步探索了POWV复制子系统的应用,通过用编码病毒衣壳(C)的质粒转染Huh7-POWV复制子细胞来产生单轮感染性颗粒(SRIPs),前膜(PRM),和包膜(E)蛋白,并揭示了POWV与ZIKV的独特抗原特征。总之,POWV复制子和SRIP系统代表了POWV生命周期的遗传和功能分析以及促进抗病毒药物发现的关键平台。重要性鉴于最近POWV引起的人类感染激增,生物安全3级(BSL-3)分类的病毒,迫切需要了解病毒的生命周期和制定有效的对策。为了解决这个问题,我们率先建立了POWVRNA复制子系统和基于复制子的POWVSRIP系统。重要的是,这些系统可在BSL-2实验室运行,能够对病毒生命周期进行全面调查,并促进抗病毒筛查。总之,这些有用的工具有望促进我们对POWV生命周期的理解,并加快抗病毒干预措施的发展.
Powassan virus (POWV) is a tick-borne flavivirus known for causing fatal neuroinvasive diseases in humans. Recently, there has been a noticeable increase in POWV infections, emphasizing the urgency of understanding viral replication, pathogenesis, and developing interventions. Notably, there are no approved vaccines or therapeutics for POWV, and its classification as a biosafety level-3 (BSL-3) agent hampers research. To overcome these obstacles, we developed a
replicon system, a self-replicating RNA lacking structural proteins, making it safe to operate in a BSL-2 environment. We constructed a POWV
replicon carrying the Gaussia luciferase (Gluc) reporter gene and blasticidin (BSD) selectable marker. Continuous BSD selection led to obtain a stable POWV
replicon-carrying Huh7 cell lines. We identified cell culture adaptive mutations G4079A, G4944T and G6256A, resulting in NS2AR195K, NS3G122G, and NS3V560M, enhancing RNA replication. We demonstrated the utility of the POWV
replicon system for high-throughput screening (HTS) assay to identify promising antivirals against POWV replication. We further explored the applications of the POWV replicon system, generating single-round infectious particles (SRIPs) by transfecting Huh7-POWV replicon cells with plasmids encoding viral capsid (C), premembrane (prM), and envelope (E) proteins, and revealed the distinct antigenic profiles of POWV with ZIKV. In summary, the POWV replicon and SRIP systems represent crucial platforms for genetic and functional analysis of the POWV life cycle and facilitating the discovery of antiviral drugs.IMPORTANCEIn light of the recent surge in human infections caused by POWV, a biosafety level-3 (BSL-3) classified virus, there is a pressing need to understand the viral life cycle and the development of effective countermeasures. To address this, we have pioneered the establishment of a POWV RNA
replicon system and a
replicon-based POWV SRIP system. Importantly, these systems are operable in BSL-2 laboratories, enabling comprehensive investigations into the viral life cycle and facilitating antiviral screening. In summary, these useful tools are poised to advance our understanding of the POWV life cycle and expedite the development of antiviral interventions.