Okamoto Hiroaki
Division of Virology, Department of Infection and Immunity, Jichi Medical University School of Medicine, 3311-1 Yakushiji, Shimotsuke-shi, Tochigi-ken 329-0498, Japan.
Uirusu. 2010 Jun;60(1):93-104. doi: 10.2222/jsv.60.93.
Early studies reported propagation of hepatitis E virus (HEV) in primary hepatocytes or several established cell lines, but replication was inefficient. Recently, using inocula comprised of fecal suspensions with high loads of HEV, originally obtained from Japanese patients who contracted domestic infection of genotype 3 HEV (the JE03-1760F strain, 2.0 x 10(7) copies/ml) or genotype 4 HEV (the HE-JF5/15F strain, 1.3 x 10(7) copies/ml), we developed an efficient cell culture system for HEV in PLC/PRF/5 and A549 cells, which yielded the highest HEV load of 10(8) copies/ml in the culture supernatant, and we successfully propagated six or more generations in serial passages of culture supernatant. In addition, we constructed a full-length infectious cDNA clone (pJE03-1760F/wt) of the JE03-1760F strain, which can replicate efficiently in PLC/PRF/5 and A549 cells. Using a derivative ORF3-deficient (delta ORF3) mutant, we demonstrated that the ORF3 protein of HEV is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which is associated with lipids. Various HEV strains in blood circulation were also propagated efficiently in PLC/PRF/5 and A549 cells. Our in vitro cell culture system can be used for propagation of a wide variety of HEV strains in feces and sera from various infected patients, allowing extended studies on viral replication specific to different HEV strains.
早期研究报道戊型肝炎病毒（HEV）可在原代肝细胞或几种已建立的细胞系中增殖，但复制效率较低。最近，我们使用最初从感染3型HEV（JE03 - 1760F株，2.0×10⁷拷贝/ml）或4型HEV（HE - JF5/15F株，1.3×10⁷拷贝/ml）的日本患者粪便悬液中获得的高病毒载量接种物，在PLC/PRF/5和A549细胞中建立了一种高效的HEV细胞培养系统，该系统在培养上清液中产生的最高HEV载量为10⁸拷贝/ml，并且我们成功地在培养上清液的连续传代中传代了六代或更多代。此外，我们构建了JE03 - 1760F株的全长感染性cDNA克隆（pJE03 - 1760F/wt），其可在PLC/PRF/5和A549细胞中高效复制。使用一种缺失ORF3的衍生突变体（ΔORF3），我们证明HEV的ORF3蛋白负责病毒粒子从感染细胞中释放，并且存在于释放的HEV颗粒表面，与脂质相关。血液循环中的各种HEV毒株也能在PLC/PRF/5和A549细胞中高效增殖。我们的体外细胞培养系统可用于增殖来自各种感染患者粪便和血清中的多种HEV毒株，从而能够对不同HEV毒株特异性的病毒复制进行深入研究。
