Department of Infection and Immunity, Jichi Medical University School of Medicine, Shimotsuke-Shi, Tochigi, Japan.
Rev Med Virol. 2011 Jan;21(1):18-31. doi: 10.1002/rmv.678.
Attempts have been made to propagate hepatitis E virus (HEV) in primary hepatocyte culture and various other cultured cells. However, the replication ability of HEV recovered from culture media remains extremely low. Recently, efficient culture systems have been established in PLC/PRF/5 (hepatocellular carcinoma) and A549 (lung cancer) cell lines for HEV strains of genotypes 3 and 4 in our laboratory. They originated in fecal extracts from patients containing HEV RNA in extremely high-titers (10(7) copies/ml), and named the JE03-1760F (genotype 3) and HE-JF5/15F (genotype 4) strains, respectively. HEV RNA in culture supernatants reached 10(8) copies/ml in titer, and were transmitted successively through many passages. An infectious HEV cDNA clone (pJE03-1760F/wt) was constructed that has replication activity comparable to that of the wild-type JE03-1760F in feces. The ORF3 protein is indispensable for shedding HEV particles from cells in the reverse genetics system. HEV recovered from culture media, as well as circulating HEV, possess ORF3 proteins on the surface and are covered with cellular membranes, and therefore, ORF2 epitopes are buried in these particles. In contrast, HEV excreted into feces are naked nucleocapsids without a lipid layer or surface expression of the ORF3 protein. HEV in sera of patients with acute hepatitis E can infect and replicate in PLC/PRF/5 and A549 cells, with efficiency comparable to the circulating HEV RNA levels. High-efficiency cell culture systems for infectious viruses, thus developed, are expected to open up a new era and resolve many mysteries in the epidemiology, molecular biology, and treatment of HEV.
已尝试在原代肝细胞培养物和各种其他培养细胞中繁殖戊型肝炎病毒 (HEV)。然而，从培养物中回收的 HEV 的复制能力仍然极低。最近，我们实验室在 PLC/PRF/5（肝癌）和 A549（肺癌）细胞系中建立了高效的培养系统，用于 3 型和 4 型 HEV 株。它们源自粪便提取物，其中含有 HEV RNA 的极高滴度（10(7) 拷贝/ml），分别命名为 JE03-1760F（基因型 3）和 HE-JF5/15F（基因型 4）株。培养上清液中的 HEV RNA 滴度达到 10(8) 拷贝/ml，并成功传代多次。构建了具有复制活性的感染性 HEV cDNA 克隆（pJE03-1760F/wt），其活性与粪便中野生型 JE03-1760F 相当。在反向遗传学系统中，ORF3 蛋白对于从细胞中释放 HEV 颗粒是必不可少的。从培养物中回收的 HEV 以及循环中的 HEV 在表面都具有 ORF3 蛋白，并被细胞膜覆盖，因此 ORF2 表位被隐藏在这些颗粒中。相比之下，分泌到粪便中的 HEV 是没有脂质层或表面表达 ORF3 蛋白的裸露核衣壳。急性戊型肝炎患者血清中的 HEV 可以感染和复制 PLC/PRF/5 和 A549 细胞，其效率与循环 HEV RNA 水平相当。因此，开发出的高效细胞培养系统有望开创一个新时代，解决 HEV 流行病学、分子生物学和治疗方面的许多谜团。
