Cierniak Filip, Ulrich Rainer G, Groschup Martin H, Eiden Martin
Institute of Novel and Emerging Infectious Diseases (INNT), Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany.
Partner Site Hamburg-Lübeck-Borstel-Riems, German Centre for Infection Research (DZIF), 17493 Greifswald-Insel Riems, Germany.
Pathogens. 2022 Mar 15;11(3):355. doi: 10.3390/pathogens11030355.
Zoonotic hepatitis E virus (HEV) infection is an emerging cause of acute viral hepatitis in developed countries. Known reservoirs of zoonotic genotype 3 (HEV-3) are mainly pigs and wild boar, and to a lesser extent rabbits and deer. Rabbit hepatitis E virus (HEV-3ra) is prevalent in rabbits worldwide and represents a particular risk for zoonotic infection. Current understanding of the molecular mechanisms of HEV pathogenesis is incomplete, particularly due to the limited availability of efficient and reliable cell culture systems. In order to identify genomic regions responsible for HEV propagation in cell culture, we developed a modular chimeric reporter replicon system based on cell culture-adapted (Kernow-C1/p6 and 47832mc) and rabbit-derived HEV strains. Replication in HepG2 cells was monitored on the basis of a luciferase reporter gene that was inserted in place of the open reading frame (ORF) 2 of the HEV genome. Luciferase activity of rabbit HEV-derived replicons was significantly lower than that of Kernow-C1/p6 and 47832mc replicons. Serial exchanges of defined ORF1 segments within the Kernow-C1/p6 replicon backbone indicated that HEV replication in HepG2 cells is not determined by a single domain but rather by an interplay of longer segments of the ORF1-derived nonstructural polyprotein. This implies that a specific combination of viral factors is required for efficient HEV propagation in cell culture.
人畜共患戊型肝炎病毒（HEV）感染是发达国家急性病毒性肝炎的一个新出现的病因。已知人畜共患3型基因型（HEV-3）的储存宿主主要是猪和野猪，在较小程度上还有兔子和鹿。兔戊型肝炎病毒（HEV-3ra）在全球范围内的兔子中普遍存在，是一种人畜共患感染的特殊风险因素。目前对HEV发病机制的分子机制的理解尚不完整，特别是由于高效可靠的细胞培养系统有限。为了确定负责HEV在细胞培养中传播的基因组区域，我们基于适应细胞培养的（Kernow-C1/p6和47832mc）以及兔源HEV毒株开发了一种模块化嵌合报告复制子系统。基于插入到HEV基因组开放阅读框（ORF）2位置的荧光素酶报告基因监测其在HepG2细胞中的复制情况。兔源HEV复制子的荧光素酶活性显著低于Kernow-C1/p6和47832mc复制子。在Kernow-C1/p6复制子骨架内对特定ORF1片段进行系列交换表明，HEV在HepG2细胞中的复制不是由单个结构域决定的，而是由ORF1衍生的非结构多蛋白的较长片段之间的相互作用决定的。这意味着在细胞培养中高效的HEV传播需要病毒因子的特定组合。
