BACKGROUND After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. METHODS In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. RESULTS We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. CONCLUSIONS The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

中文翻译：

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深入分析咽病毒的复制周期。

背景技术在分离棘阿米巴多噬菌丝拟病毒（APMV）之后，已经加强了对新的巨型病毒的研究和搜索。大多数巨型病毒都与棘阿米巴属（Acanthamoeba）的自由生活的变形虫相关。但是，在Vermamoeba vermiformis中也分离出了其他巨型病毒，例如短小病毒，柯姆巴病毒和原疱病毒。这些研究极大地扩展了我们对巨型病毒的多样性，结构，基因组学和进化的知识。到现在为止，只有一种Orpheovirus分离株，其生命周期的许多方面仍有待阐明。方法在本研究中，我们通过透射和扫描电子显微镜，光学显微镜和IF分析对原核病毒的复制周期和颗粒进行了深入的表征。结果我们通过光学和中频显微镜观察到，在咽病毒感染过程中，V。vermiformis细胞的形态发生了变化，以及感染后12 h（hpi）的运动性增强。通过透射电子显微镜分析病毒工厂的形成和病毒颗粒的形态发生，揭示线粒体和膜募集进入电子透明病毒工厂及其周围。膜交通抑制剂（布雷菲德菌素A）对颗粒形态产生负面影响。在颗粒形态发生过程中观察到的第一个结构是月牙形体，其延伸并被内部内容物填充，直到形成多层成熟颗粒。我们还观察到了具有不同形状和尺寸的缺陷颗粒的形成。病毒学检测表明，病毒在12 hpi时通过胞吞作用从宿主中释放出来，这与上清液中颗粒计数的增加有关。结论本文介绍的结果有助于更好地了解正痘病毒复制周期中的生物学，结构和重要步骤。