Background & Aims

Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system.

Methods

We performed studies with gt3 HEV cell culture–produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay.

Results

We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture–produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients.

Conclusions

We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.

中文翻译：

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戊型肝炎病毒的生命周期和3种形式的ORF2衣壳蛋白的鉴定

背景与目标

戊型肝炎病毒（HEV）感染是全世界急性肝炎的主要原因。大约有20亿人居住在HEV流行地区，并且有感染的风险。HEV基因组编码3种蛋白质，包括ORF2衣壳蛋白。缺乏有效的病毒培养系统阻碍了对HEV生命周期的详细分析。

方法

我们对gt3 HEV细胞培养产生的颗粒以及患者血液和粪便样本进行了研究。将样品在碘克沙醇梯度和缓冲液上分级。感染性测定是在体外和人肝嵌合小鼠中进行的。通过生化和蛋白质组学方法分析蛋白质。通过透射电子显微镜分析感染性颗粒。HEV抗原水平通过万丹酶联免疫吸附测定法进行测量。

结果

我们开发了一种有效的细胞培养系统，并分离了在体外和体内具有传染性的HEV颗粒。使用透射电子显微镜，我们定义了HEV细胞培养物产生的微粒以及患者血清和粪便样本中的微粒的超微结构。我们还确定了与感染性颗粒相关的ORF2衣壳蛋白的精确序列。在培养的细胞和患者样品中，HEV产生3种形式的ORF2衣壳蛋白：感染性/细胞内ORF2（ORF2i），糖基化ORF2（ORF2g）和裂解的ORF2（ORF2c）。ORF2i蛋白与感染性颗粒相关，而ORF2g和ORF2c蛋白是大量分泌的糖蛋白，与感染性颗粒无关。ORF2g和ORF2c是患者血清中检测到的最丰富的抗原。

结论

我们开发了细胞培养系统并鉴定了HEV颗粒；我们鉴定了3种ORF2衣壳蛋白（ORF2i，ORF2g和ORFc）。这些发现将增进我们对HEV生命周期的了解并改善诊断。