Chaudhry Yasmin, Skinner Michael A, Goodfellow Ian G
Calicivirus Research Group, Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
Vaccine Vector Group, Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
J Gen Virol. 2007 Aug;88(Pt 8):2091-2100. doi: 10.1099/vir.0.82940-0.
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.
尽管人类诺如病毒感染造成了巨大的疾病负担，但针对这些病毒的高效组织培养系统仍然难以实现。鼠诺如病毒（MNV）是研究诺如病毒生物学的理想替代物，因为该病毒能在组织培养中高效复制，且有低成本的动物模型可供使用。在本报告中，描述了一种用于MNV的反向遗传学系统，首次利用表达T7 RNA聚合酶的禽痘病毒（FWPV）重组体在组织培养中拯救出基因明确的MNV。这些研究表明，对杯状病毒科其他成员已证明成功的方法未能成功拯救出MNV。这是因为我们观察到痘苗病毒感染对MNV复制有负面影响。相比之下，FWPV感染没有有害影响，并能从先前用MNV cDNA构建体转染的细胞中拯救出有感染性的MNV。这些研究还表明，3'-末端核苷酸的性质对高效拯救病毒至关重要，并且在3'末端包含丁型肝炎病毒核酶可以提高病毒拯救的效率。该系统现在能够拯救基因明确的诺如病毒，并将有助于分析基因变异对诺如病毒发病机制的影响。
