Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.

中文翻译：

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戊型肝炎病毒的强劲感染和人类肝细胞中的转录反应。

戊型肝炎病毒（HEV）是人类中戊型肝炎的病原体，也是全球范围内急性病毒性肝炎的主要原因。该病毒被归类为Hepeviridae家族的Orthohepevirus A属成员。由于缺乏用于HEV感染的可靠的细胞培养模型，因此对病毒生命周期的分析，有效抗病毒药和疫苗的开发受到严重限制。在这项研究中，我们建立了基于HEV基因型3 p6（Kernow C-1）和具有不同培养基条件的人肝癌细胞系HepG2和HepG2 / C3A的协议，以产生带有病毒的细胞内HEV细胞培养衍生颗粒（HEVcc）滴度在105至106 FFU / mL之间。病毒滴度可以通过在RNA依赖的RNA聚合酶中具有突变的HEV变体进一步增强。这些HEVcc颗粒具有密度梯度特征，可以实现亚基因组报告基因HEV复制子的反式互补。此外，体外产生的细胞内来源的颗粒在肝脏人源化小鼠中具有传染性，在血清和粪便中可检测到高RNA拷贝数。可以观察到使用所开发的方案有效感染人和猪原代肝细胞，并被利巴韦林抑制。最后，HEV感染的原代人肝细胞的RNA测序研究证明了时间结构的转录防御反应。总之，这种强大的HEV感染细胞培养模型为研究病毒与宿主之间的相互作用提供了强大的工具，应有助于发现这种重要的人畜共患病原体的抗病毒药物。