Recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA polymerase.
Recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA polymerase.
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DOI：
10.1099/vir.0.82940-0
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发表时间：
2007-08
期刊：
影响因子：
--
通讯作者：
Goodfellow IG
中科院分区：
文献类型：
--
作者：
Chaudhry Y;Skinner MA;Goodfellow IG
尽管人类诺如病毒感染造成了显著的疾病负担，但这些病毒的有效组织培养系统仍然难以捉摸。鼠诺如病毒（MNV）是研究诺如病毒生物学的理想替代物，因为病毒在组织培养中有效复制，并且容易获得低成本的动物模型。本文报道了一种MNV的反向遗传学系统，首次利用表达T7 RNA聚合酶的鸡痘病毒（FWPV）重组体在组织培养中回收遗传上确定的MNV。这些研究表明，已经证明对杯状病毒科的其他成员成功的方法未能导致MNV的回收。这是由于我们观察到牛痘病毒感染对MNV复制有负面影响。相反，FWPV感染没有有害影响，并允许从先前用MNV cDNA构建体转染的细胞中回收感染性MNV。这些研究还表明，3′端核苷酸的性质对于有效的病毒回收至关重要，并且在3′端包含丁型肝炎病毒核酶可以提高病毒回收的效率。该系统现在允许回收遗传上确定的诺如病毒，并将有助于分析遗传变异对诺如病毒发病机制的影响。
Despite the significant disease burden caused by human norovirus infection, an efficient tissue-culture system for these viruses remains elusive. Murine norovirus (MNV) is an ideal surrogate for the study of norovirus biology, as the virus replicates efficiently in tissue culture and a low-cost animal model is readily available. In this report, a reverse-genetics system for MNV is described, using a fowlpox virus (FWPV) recombinant expressing T7 RNA polymerase to recover genetically defined MNV in tissue culture for the first time. These studies demonstrated that approaches that have proved successful for other members of the family Caliciviridae failed to lead to recovery of MNV. This was due to our observation that vaccinia virus infection had a negative effect on MNV replication. In contrast, FWPV infection had no deleterious effect and allowed the recovery of infectious MNV from cells previously transfected with MNV cDNA constructs. These studies also indicated that the nature of the 3′-terminal nucleotide is critical for efficient virus recovery and that inclusion of a hepatitis delta virus ribozyme at the 3′ end can increase the efficiency with which virus is recovered. This system now allows the recovery of genetically defined noroviruses and will facilitate the analysis of the effects of genetic variation on norovirus pathogenesis.
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