Construction of an Infectious Cdna Clone of Hepatitis E Virus Strain Je03-1760f That Can Propagate Efficiently in Cultured Cells
Construction of an Infectious Cdna Clone of Hepatitis E Virus Strain Je03-1760f That Can Propagate Efficiently in Cultured Cells
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戊型肝炎病毒株 Je03-1760f 感染性 cdna 克隆的构建，可在培养细胞中高效传播
DOI：
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发表时间：
2008
期刊：
影响因子：
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通讯作者：
Hiroaki Okamoto
中科院分区：
文献类型：
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作者：
Kentaro Yamada;Masaharu Takahashi;Yu Hoshino;Hideyuki Takahashi;K. Ichiyama;Toshinori Tanaka;Hiroaki Okamoto Correspondence;Hiroaki Okamoto
从粪便标本中获得了戊型肝炎病毒(HEV)JE03-1760F株全长感染性克隆(pJE03-1760F/wt)。PJE03-1760F/wt体外转录本转入PLC/PRF/5细胞后，培养上清液中病毒RNA水平在第6天开始升高(P.T.)免疫荧光法检测第5、7、11、15天表达ORF2蛋白的细胞数增加，表明HEV感染在细胞培养中扩散，将培养上清液中的病毒接种到PLC/PRF/5或A549细胞中，其生长速度与粪源病毒相当，接种后30天达106拷贝·ml~(-1)。我们的HEV反向遗传学系统可用于强大的细胞培养系统，这将有助于阐明HEV复制的机制和HEV蛋白的功能作用。戊型肝炎病毒(HEV)导致人类急性和暴发型戊型肝炎。基因1和基因2的HEV作为水传播的流行病导致了戊型肝炎的暴发，基因3和基因4在最有可能是人畜共患病的E型肝炎的零星病例中被发现(Okamoto，2007)。HEV被归类为HepeVirus属的唯一成员(Emerson等，2004a)，属于Hepeviridae科。戊型肝炎病毒是一种无包膜病毒，其基因组是单链、阳性的核糖核酸，是有帽和多腺化的(Kabrane-Lazizi等人，1999年；等人，1991年)。基因组全长约7.2kb，包含3个开放阅读框，即ORF1、ORF2和ORF3(等，1991年)。ORF1编码非结构蛋白，包括RNA解旋酶和依赖于RNA的RNA聚合酶(Agrawal等人，2001；Koning等人，1992；Magden等人，2001)。ORF2和ORF3蛋白是从单个亚基因组RNA翻译而来的(Graff等人，2006年；Huang等人，2007年)。ORF2蛋白是病毒衣壳蛋白，由660个氨基酸组成。ORF3蛋白是一种小蛋白(113-114氨基酸)，对动物的病毒感染性是必不可少的(2007)。然而，HEV的复制机制和蛋白功能尚不完全清楚。但目前还没有开发出一种高效的细胞培养系统。几个研究小组已经建立了HEV的感染性cDNA克隆，观察到这些克隆可以在非人类灵长类动物或猪身上复制(Emerson等人。Panda et…
A full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 hepatitis E virus (HEV) (strain JE03-1760F) obtained from a faecal specimen was constructed in this study. Upon transfection of the capped in vitro transcripts of pJE03-1760F/wt into PLC/PRF/5 cells, the viral RNA levels in the culture supernatant started to increase on day 6 post-transfection (p.t.) and reached 10 7 copies ml " 1 on day 28 p.t. Detection of increasing numbers of cells with ORF2 protein expression by immunofluorescence assay at 5, 7, 11 and 15 days p.t. indicated the spread of HEV infection in cell culture. When the cDNA-derived virus in culture supernatant was inoculated into PLC/PRF/5 or A549 cells, it grew as efficiently as the faeces-derived virus in both cells, reaching 10 6 copies ml " 1 at 30 days post-inoculation. Our reverse genetics system for HEV that is usable in a robust cell-culture system will be useful for elucidation of the mechanism of HEV replication and functional roles of HEV proteins. Hepatitis E virus (HEV) causes acute and fulminant hepatitis E in humans. Genotype 1 and 2 HEVs have caused outbreaks of hepatitis E as water-borne epidemics, and genotype 3 and 4 HEVs were found in sporadic cases of hepatitis E, which were most likely zoonotic in origin (Okamoto, 2007). HEV is classified as the sole member of the genus Hepevirus (Emerson et al., 2004a) in the family Hepeviridae. HEV is a non-enveloped virus and its genome is a single-stranded, positive-sense RNA, which is capped and polyadenylated (Kabrane-Lazizi et al., 1999; Tam et al., 1991). The genome is approximately 7.2 kb and contains three open reading frames (ORFs), ORF1, ORF2 and ORF3 (Tam et al., 1991). The ORF1 encodes non-structural proteins including RNA helicase and RNA-dependent RNA polymerase (Agrawal et al., 2001; Koonin et al., 1992; Magden et al., 2001). ORF2 and ORF3 proteins are translated from a single subgenomic RNA (Graff et al., 2006; Huang et al., 2007). The ORF2 protein is the viral capsid protein, consisting of 660 aa. The ORF3 protein is a small protein (113–114 aa) that is essential for viral infectivity in animals 2007). However, the replication mechanism and protein functions of HEV are not fully understood. but an efficient cell culture system has not been developed. Several research groups have established infectious cDNA clones of HEV, which were observed to replicate in non-human primates or pigs (Emerson et al. Panda et …
影响因子：
9.4
作者：
Takahashi, Masaharu;Tanaka, Toshinori;Okamoto, Hiroaki
通讯作者：
Okamoto, Hiroaki
影响因子：
2.7
作者：
Takahashi, Masaharu;Yamada, Kentaro;Okamoto, Hiroaki
通讯作者：
Okamoto, Hiroaki