Investigation of norovirus replication in a human cell line
Investigation of norovirus replication in a human cell line
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DOI：
10.1007/s00705-005-0720-9
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发表时间：
2006-07-01
影响因子：
2.7
通讯作者：
Takeda, N.
中科院分区：
文献类型：
--
作者：
Katayama, K.;Hansman, G. S.;Takeda, N.
诺如病毒属于诺如病毒属，属于杯状病毒科。NOV是胃肠炎暴发的主要原因，但由于缺乏细胞培养系统或实用的动物模型，除了小鼠NOV外，对NOV分子特征及其复制策略的了解一直受到阻碍。为了阐明新城疫病毒基因组的转录和复制，从T7 RNA聚合酶启动子下游克隆了人新城疫病毒II组株的全基因组，并利用T7痘苗病毒表达系统在人胚胎肾293T/17细胞中进行了表达。在感染细胞中发现了一条7.6kb的负链RNA、一条7.6kb的正链基因组RNA和一条2.6kb的正链亚基因组样RNA。而Western blotting未检测到重组衣壳蛋白(RVP1)和rVP2。当含有VP1和VP2基因的构建体与全长构建体共转染时，观察到浮力密度为1.271 g/cm(3)的病毒样颗粒(VLP)的表达。我们还观察到直径为20-80 nm的圆形颗粒，浮力密度为1.318 g/cm(3)。我们的结果表明，NOV RNA被整合到较重的颗粒中。然而，需要进一步的研究来调查这些颗粒的抗原性，并确定它们是否代表未发育的VLP。
Noroviruses (NoVs) belong to the genus Norovirus and are members of the family Caliciviridae. NoVs are the dominant cause of outbreaks of gastroenteritis, but progress in understanding the molecular characteristics of NoV and its replication strategies have been hampered by the lack of a cell culture system or a practical animal model, except for murine NoVs. To elucidate the transcription and replication of the NoV genome, a complete genome of a human NoV genogroup II strain was cloned downstream of a T7 RNA polymerase promoter and expressed in human embryonic kidney (HEK) 293T/17 cells using a T7 vaccinia virus expression system. Bands for a 7.6-kb negative-strand RNA, a 7.6-kb positive-strand genomic RNA, and a 2.6-kb positive-strand subgenomic-like RNA were found in the infected cells. However, recombinant capsid protein (rVP1) and rVP2 were not detected by Western blotting. When a construct containing VP1 and VP2 genes was co-transfected with a full-length construct, the expression of virus-like particles (VLPs) with a buoyant density of 1.271 g/cm(3) was observed. We also observed round particles, 20 to 80 nm in diameter, with a buoyant density of 1.318 g/cm(3). Our results indicated that NoV RNA was incorporated into the heavier particles. However, further studies are needed to investigate the antigenicity of these particles and to determine if they represent undeveloped VLPs.