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

汉坦病毒在美洲和欧亚大陆分别是引起汉坦病毒心肺综合征(HCPS)和肾综合征出血热(HFRS)的病原体。这些病毒通常从它们的啮齿动物宿主传播给人类。然而，南美安第斯汉坦病毒在人与人之间传播的多起事件引起了人们对这些致命病毒的重大公共卫生关切，病死率高达40%。目前尚不存在FDA批准的汉坦病毒疫苗和疗法。汉坦病毒的细胞侵入和感染是由其病毒颗粒表面的Gn/GC糖蛋白介导的，这些糖蛋白也是保护性免疫反应的主要靶点。然而，我们对汉坦病毒进入和抗原性中Gn/GC的分子决定因素的了解仍然有限，至少部分原因是由于生物安全等级3(BSL3)遏制汉坦病毒研究的一般要求，以及缺乏反向遗传学系统。为了解决这些限制，已经开发了多种BSL2伪病毒系统。然而，目前的BSL2系统受到可扩展性差和产生的病毒多样性有限的限制，这是因为它们基于低效的基于质粒的救援，这使得它们与对进入糖蛋白的生物学和功能的更全面的分析不相容。在这里，我们建议通过开发一种新的BSL2系统来解决汉坦病毒领域的这一主要缺陷，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.