Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Berlin, Germany.
Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, United States of America.
PLoS Negl Trop Dis. 2020 Jan 21;14(1):e0007952. doi: 10.1371/journal.pntd.0007952. eCollection 2020 Jan.
The significance of the integral membrane protein Niemann-Pick C1 (NPC1) in the ebolavirus entry process has been determined using various cell lines derived from humans, non-human primates and fruit bats. Fruit bats have long been purported as the potential reservoir host for ebolaviruses, however several studies provide evidence that Mops condylurus, an insectivorous microbat, is also an ebolavirus reservoir. NPC1 receptor expression in the context of ebolavirus replication in microbat cells remains unstudied. In order to study Ebola virus (EBOV) cellular entry and replication in M. condylurus, we derived primary and immortalized cell cultures from 12 different organs. The NPC1 receptor expression was characterized by confocal microscopy and flow cytometry comparing the expression levels of M. condylurus primary and immortalized cells, HeLa cells, human embryonic kidney cells and cells from a European microbat species. EBOV replication kinetics was studied for four representative cell cultures using qRT-PCR. The aim was to elucidate the suitability of primary and immortalized cells from different tissues for studying NPC1 receptor expression levels and their potential influence on EBOV replication. The NPC1 receptor expression level in M. condylurus primary cells differed depending on the organ they were derived from and was for most cell types significantly lower than in human cell lines. Immortalized cells showed for most cell types higher expression levels than their corresponding primary cells. Concluding from our infection experiments with EBOV we suggest a potential correlation between NPC1 receptor expression level and virus replication rate in vitro.
Niemann-Pick C1（NPC1）整合膜蛋白在埃博拉病毒进入过程中的重要性已通过源自人类、非人类灵长类动物和果蝠的各种细胞系来确定。果蝠长期以来一直被认为是埃博拉病毒的潜在储存宿主，但有几项研究提供的证据表明，食虫性的小蝙蝠 Mops condylurus 也是埃博拉病毒的储存宿主。NPC1 受体在小蝙蝠细胞中埃博拉病毒复制的情况下的表达尚未得到研究。为了研究 M. condylurus 中的埃博拉病毒（EBOV）细胞进入和复制，我们从 12 种不同的器官中衍生出原代和永生化细胞培养物。通过共聚焦显微镜和流式细胞术比较 M. condylurus 原代和永生化细胞、HeLa 细胞、人胚肾细胞和来自欧洲小蝙蝠物种的细胞的表达水平来表征 NPC1 受体的表达。使用 qRT-PCR 研究了四种代表性细胞培养物中的 EBOV 复制动力学。目的是阐明不同组织的原代和永生化细胞在研究 NPC1 受体表达水平及其对 EBOV 复制的潜在影响方面的适用性。M. condylurus 原代细胞中的 NPC1 受体表达水平取决于它们来自的器官，并且在大多数细胞类型中明显低于人类细胞系。永生化细胞在大多数细胞类型中表现出比其相应的原代细胞更高的表达水平。根据我们用 EBOV 进行的感染实验，我们建议 NPC1 受体表达水平与体外病毒复制率之间存在潜在的相关性。
