Background & Aims

The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1–4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells.

Methods

Using a cell culture–adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture–adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies.

Results

HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture–adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A.

Conclusions

Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin.

中文翻译：

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在干细胞衍生的肝细胞系统中泛基因型戊型肝炎病毒复制

背景与目标

感染人类的​​4种E型肝炎病毒（HEV）基因型（1-4型基因）在地理分布，传播和发病机理上各不相同。对导致这些变异的HEV或其宿主的特性知之甚少。初级分离株在细胞培养中生长较差；大多数研究都依赖于适应癌细胞系的变异体，这些变异体可能会改变病毒生物学。我们调查了人类胚胎干细胞和诱导多能干细胞衍生的肝细胞样细胞（HLCs）中HEV的主要分离株的感染和复制。

方法

使用适应细胞培养的基因型3菌株和基因型1-4的主要分离株，我们比较了病毒复制动力学，对药物的敏感性以及HEV激活先天免疫应答的能力。我们使用定量逆转录酶聚合酶链反应，免疫荧光测定法和酶联免疫吸附测定法研究了HLC。我们使用了一种胚胎干细胞系，该细胞系可以被诱导表达CRISPR-Cas9机制来破坏肽基脯氨酰异构酶A基因，该基因编码亲环蛋白A（CYPA），据报道该蛋白抑制细胞培养适应性HEV的复制。我们进一步修饰了该品系，以在最终分化为HLC之前挽救CYPA的表达，并进行了HEV感染研究。

结果

HLC允许被非适应性HEV基因型1至4的主要分离株感染。HLC的HEV感染诱导了复制依赖性III型干扰素应答。与适应细胞培养的菌株不同，主要的HEV分离株的复制不受肽基脯氨酰异构酶A基因的破坏或CYPA抑制剂环孢菌素A暴露的影响。

结论

细胞培养适应性改变了HEV的复制能力。HLC为研究主要HEV分离株的复制提供了一种改良的，具有生理相关性且在遗传上易处理的系统。HLC可以提供一个模型，以帮助开发HEV药物，并提供一种系统来指导个性化治疗方案，特别是对于已对利巴韦林产生抗药性的慢性戊型肝炎患者。