Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero
Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero
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DOI：
10.1371/journal.pone.0232274
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发表时间：
2020-04
期刊：
影响因子：
3.7
通讯作者：
Kyoko Saito;M. Fukasawa;Yoshitaka Shirasago;R. Suzuki;N. Osada;T. Yamaji;T. Wakita;E. Konishi;K. Hanada
中科院分区：
文献类型：
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作者：
Kyoko Saito;M. Fukasawa;Yoshitaka Shirasago;R. Suzuki;N. Osada;T. Yamaji;T. Wakita;E. Konishi;K. Hanada
黄病毒科（Flaviviridae）是具有正义单链RNA基因组的包膜病毒家族。它含有许多威胁人类健康的病毒，如黄病毒属的日本脑炎病毒（JEV）和黄热病病毒（YFV）以及肝炎病毒属的丙型肝炎病毒。高度容许黄病毒科病毒的细胞培养系统对于它们的分离、繁殖和诊断、理解它们的生物学以及疫苗和抗病毒剂的开发非常有用。先前，我们分离出一种人肝癌HuH-7衍生的细胞克隆Huh 7.5.1 -8，其对丙型肝炎病毒感染高度容许。在这里，我们的特点是黄病毒感染的Huh 7.5.1 -8细胞系相比，在非洲绿色猴肾衍生的Vero细胞系，这是允许的一个广泛的病毒。在感染JEV后，Huh 7.5.1 -8细胞在感染早期产生更大量的病毒颗粒，并且比Vero细胞更容易受到病毒诱导的细胞死亡的影响。当用另一种黄病毒YFV（17 D-204株）感染细胞时，获得了类似的结果。细胞和细胞外病毒RNA的定量显示，Huh7.5.1-8细胞中的高JEV产量可归因于快速的病毒复制动力学和感染早期的有效病毒释放。在噬斑测定中，Huh 7.5.1 -8细胞比Vero细胞更快地形成JEV噬斑。虽然YFV噬斑的情况并非如此，但Huh 7.5.1 -8细胞比Vero细胞产生更多数量的YFV噬斑。对编码抗病毒RNA解旋酶RIG-I的cDNA的序列分析表明，Huh 7.5.1 -8细胞不仅表达具有已知显性负义错义突变的全长RIG-I mRNA，而且还表达没有突变的变体。然而，后者的mRNA缺乏外显子5/6−12，表明细胞中RIG-I的功能丧失。Huh7.5.1-8细胞系的这些特征有助于黄病毒检测、滴定和增殖。
The Flaviviridae is a family of enveloped viruses with a positive-sense single-stranded RNA genome. It contains many viruses that threaten human health, such as Japanese encephalitis virus (JEV) and yellow fever virus (YFV) of the genus Flavivirus as well as hepatitis C virus of the genus Hepacivirus. Cell culture systems highly permissive for the Flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. Previously, we isolated a human hepatoma HuH-7-derived cell clone, Huh7.5.1–8, which is highly permissive to hepatitis C virus infection. Here, we have characterized flavivirus infection in the Huh7.5.1–8 cell line by comparing with that in the African green monkey kidney-derived Vero cell line, which is permissive for a wide spectrum of viruses. Upon infection with JEV, Huh7.5.1–8 cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than Vero cells. Similar outcomes were obtained when the cells were infected with another flavivirus, YFV (17D-204 strain). Quantification of cellular and extracellular viral RNA revealed that high JEV production in Huh7.5.1–8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. In a plaque assay, Huh7.5.1–8 cells developed JEV plaques more rapidly than Vero cells. Although this was not the case with YFV plaques, Huh7.5.1–8 cells developed higher numbers of YFV plaques than Vero cells. Sequence analysis of cDNA encoding an antiviral RNA helicase, RIG-I, showed that Huh7.5.1–8 cells expressed not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants without the mutation. However, the latter mRNAs lacked exon 5/6−12, indicating functional loss of RIG-I in the cells. These characteristics of the Huh7.5.1–8 cell line are helpful for flavivirus detection, titration, and propagation.