Development of a novel BSL2 system for high-throughput analysis of hantavirus entry glycoproteins

汉坦病毒分别是美洲和欧亚大陆汉坦病毒心肺综合征（HCPS）和肾综合征出血热（HFRS）的病原体。这些病毒通常由啮齿动物宿主传播给人类。然而，南美安第斯山脉汉他病毒的多起人传人事件引起了对这些致命病毒的重大公共卫生关注，其病死率高达40%。没有FDA批准的汉坦病毒疫苗和疗法存在。汉坦病毒的细胞侵入和感染是由其病毒粒子表面Gn/Gc糖蛋白介导的，其也是保护性免疫应答的主要靶点。然而，我们的了解Gn/Gc在汉他病毒的入境和抗原性的分子决定因素仍然有限，至少部分地，由于生物安全3级（BSL 3）遏制汉他病毒的研究和缺乏反向遗传学系统的一般要求。为了解决这些限制，已经开发了多种BSL 2假病毒系统。然而，目前的BSL 2系统由于其低效的基于质粒的拯救而受到所产生病毒的可扩展性差和多样性有限的限制，这使得它们与进入糖蛋白的生物学和功能的更全面分析不相容。在这里，我们建议通过开发一种新的BSL 2系统来修复汉坦病毒领域的这一主要缺点，该系统允许对Gn/Gc在病毒进入和抗原性中的作用进行全面的反向和正向遗传分析。

Hantaviruses are the causative agents of hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) in Americas and Eurasia, respectively. These viruses are usually transmitted from their rodent reservoirs to human. However, multiple incidents of person-to-person transmission of a South American Andes hantavirus raises significant public health concern about these deadly viruses with up to 40% case fatality rates. No FDA-approved hantavirus vaccines and therapies exist. Cellular entry and infection of hantaviruses is mediated by its virion surface Gn/Gc glycoproteins, which are also the main target of protective immune responses. However, our understanding of the molecular determinants of Gn/Gc in hantavirus entry and antigenicity remains limited, at least partly, due to the general requirement of Biosafety level-3 (BSL3) containment for hantavirus research and the lack of a reverse genetics system. To address these limitations, multiple BSL2 pseudovirus systems have been developed. However, current BSL2 systems are limited by poor scalability and limited diversity of the generated viruses due to their inefficient plasmid-based rescue, which makes them incompatible with a more comprehensive analysis of the biology and function of the entry glycoproteins. Here, we propose to fix this major shortcoming of the hantavirus field by developing a novel BSL2 system that allows comprehensive reverse as well as forward genetic analysis of Gn/Gc’s role in virus entry and antigenicity.