■裂谷热静脉病毒(RVFV)是一种人畜共患病原体,可引起牲畜和人类的裂谷热(RVF)。目前,没有许可的人类疫苗或抗病毒药物来控制RVF。尽管多种动物和人类都容易感染RVFV,影响易感性的宿主因素尚不清楚。
■为了确定RVFV复制所必需的宿主因子或基因,我们在人A549细胞中进行了CRISPR-Cas9敲除筛选。然后,我们使用siRNA介导的基因敲除和CRISPR-Cas9介导的基因敲除研究来验证推定的基因。分别。通过测量细胞内病毒RNA积累来评估候选基因在病毒复制周期中的作用。和通过噬斑测定或TCID50测定的病毒滴度。
■我们鉴定了大约900个可能参与RVFV感染和复制的基因。使用siRNA介导的敲减对六个基因对病毒复制的影响的进一步评估发现,沉默两个基因(WDR7和LRP1)显着损害了RVFV复制。为了进一步分析,我们专注于WDR7基因,因为LRP1在RVFV复制中的作用之前已经有详细描述.产生敲除A549细胞系,并用于剖析WRD7对RVFV和另一种布尼亚病毒的影响,LaCrosse脑炎病毒(LACV)。我们观察到WDR7敲除细胞对胞内RVFVRNA水平和病毒滴度的显著影响。在细胞内RNA水平,当与受较早复制阶段(12h)影响的LACV相比时,WRD7在其复制周期的较晚阶段(24h)影响RVFV复制。
■总之,我们已经确定WDR7是两个相关布尼亚病毒复制的必需宿主因子,RVFV和LACV。未来的研究将研究WDR7促进尼伯病毒复制的机制作用。
■裂谷热静脉病毒是一种高后果病原体,感染多种动物物种和人类。目前,目前尚无可用于治疗人类裂谷热和防止裂谷热病毒侵入非流行国家的控制措施.RVFV在流行国家对动物和人类健康构成重大威胁。RVFV复制取决于主机的机器来完成其复制周期。因此,控制病毒复制的一种方法是破坏病毒和对复制重要的宿主蛋白之间的相互作用。在这项研究中,我们确定了一个宿主因素,WDR7基因,这对于RVFV复制至关重要。这种宿主因子的鉴定很重要,因为它可能导致抗病毒策略的发展,以控制人和动物的裂谷热。
UNASSIGNED: Rift Valley fever phlebovirus (
RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to
RVFV infection, host factors affecting susceptibility are not well understood.
UNASSIGNED: To identify the host factors or genes essential for
RVFV replication, we conducted a CRISPR-Cas9 knock-out screen in human A549 cells. We then validated the putative genes using siRNA-mediated knockdowns and CRISPR-Cas9-mediated knockout studies, respectively. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers by plaque assay or TCID50 assay.
UNASSIGNED: We identified approximately 900 genes with potential involvement in
RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knockdowns found that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of LRP1 in RVFV replication was previously described in detail. Knock-out A549 cell lines were generated and used to dissect the effect of WRD7 on RVFV and another bunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knock-out cells on both intracellular
RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24h) when compared to LACV which was affected an earlier replication phase (12h).
UNASSIGNED: In summary, we have identified WDR7 as an essential host factor for the replication of two relevant bunyaviruses, RVFV and LACV. Future studies will investigate the mechanistic role by which WDR7 facilitates Phlebovirus replication.