Elucidating the immune response of Schreiber's bats to Lloviu virus infection in vitro and in vivo

摘要 蝙蝠作为许多RNA病毒的天然宿主发挥着重要作用，这些病毒有可能引起重大的 伤害人类。在这项提案中，我们重点关注Lloviu病毒（LLOV），这是一种研究不足的丝状病毒， 在欧洲的施赖伯蝙蝠（Miniopterus schreibersii）中。尽管LLOV对人类的致病潜力 LLOV与高致病性埃博拉病毒和马尔堡病毒的密切关系引起了人们的关注 一个潜在的溢出事件可能会导致人类之间的爆发。 LLOV于2002年在西班牙首次发现Schreiber蝙蝠，然后于2016年在匈牙利再次发现。序列 西班牙和匈牙利LLOV RNA基因组的比较表明，细胞RNA编辑 脱氨酶如阿达尔和APOBEC可能在LLOV序列多样化中起作用。在本申请中， 我们建议探索宿主介导的RNA编辑是否驱动LLOV序列的分化和进化， 施赖伯的蝙蝠。 在目标1中，我们建议从地理上不同的群体中采样施赖伯蝙蝠，并获得LLOV序列 从受感染的蝙蝠中提取信息进行比较分析。我们将进一步开发基于高度敏感的 液滴RT-PCR和RNA FISH，可以确定ADAR和APOBECs的表达模式， LLOV感染的蝙蝠细胞培养物和受感染动物的血液样品中。 在目标2中，基于确定的阿达尔和APOBEC表达模式，我们将敲除选择阿达尔 和/或APOBEC基因，这些基因可能参与蝙蝠细胞系中的LLOV RNA编辑，并检查了 在连续传代实验和细胞培养中，这些基因在LLOV序列多样化和病毒适应性中起作用 感染研究。 完成这项工作后，我们将揭示宿主特异性RNA编辑器是否是LLOV的驱动程序 蝙蝠细胞的进化这项工作将有助于我们理解宿主驱动的病毒序列差异 并可能有助于评估潜在的LLOV溢出事件的风险从蝙蝠到人类通过主机驱动 病毒序列的变化。

ABSTRACT Bats play an important role as natural reservoirs of numerous RNA viruses with the potential to cause significant harm to humans. In this proposal, we focus on Lloviu virus (LLOV), an under-investigated filovirus that circulates in Schreiber’s bats (Miniopterus schreibersii) in Europe. Although the pathogenic potential of LLOV for humans is not known, the close relationship of LLOV to the highly pathogenic Ebola and Marburg viruses raises concerns that a potential spillover event could lead to an outbreak among humans. LLOV was first detected Schreiber’s bats in Spain in 2002 and then again in Hungary in 2016. Sequence comparison of the Spanish and the Hungarian LLOV RNA genomes suggests that RNA editing by cellular deaminases, such as ADAR and APOBEC, might play a role in LLOV sequence diversification. In this application, we propose to explore if host-mediated RNA editing drives LLOV sequence divergence and evolution in Schreiber’s bats. In Aim 1, we propose to sample Schreiber’s bats from geographically distinct colonies and obtain LLOV sequence information from infected bats for comparative analysis. We will further develop tools based on highly sensitive droplet RT-PCR and RNA FISH that allow to determine the expression pattern of ADARs and APOBECs in LLOV-infected bat cell culture and in blood samples from infected animals. In Aim 2, based on the determined ADAR and APOBEC expression patterns, we will knockout select ADAR and/or APOBEC genes that might be involved in LLOV RNA editing in the bat cell line and examine the role of these genes in LLOV sequence diversification and viral fitness in serial passaging experiments and cell culture infection studies. Upon completion of this work, we will have revealed whether host-specific RNA editors are the drivers of LLOV evolution in bat cells. This work will contribute to our understanding of host-driven viral sequence divergence and might help assess the risk of potential LLOV spillover events from bats to humans through host-driven changes in viral sequences.