Management Department of Biosafety and Laboratory Animal, National Institute of Infectious Diseases, Tokyo 162-0052, Japan;
Department of Virology II, National Institute of Infectious Diseases, Tokyo 208-0011, Japan;
Proc Natl Acad Sci U S A. 2020 Dec 15;117(50):32078-32085. doi: 10.1073/pnas.2007310117. Epub 2020 Nov 30.
Human sapoviruses (HuSaVs) cause acute gastroenteritis similar to human noroviruses. Although HuSaVs were discovered four decades ago, no HuSaV has been grown in vitro, which has significantly impeded the understanding of viral biology and the development of antiviral strategies. In this study, we identified two susceptible human cell lines, that originated from testis and duodenum, that support HuSaV replication and found that replication requires bile acids. HuSaVs replicated more efficiently in the duodenum cell line, and viral RNA levels increased up to ∼6 log-fold. We also detected double-stranded RNA, viral nonstructural and structural proteins in the cell cultures, and intact HuSaV particles. We confirmed the infectivity of progeny viruses released into the cell culture supernatants by passaging. These results indicate the successful growth of HuSaVs in vitro. Additionally, we determined the minimum infectious dose and tested the sensitivities of HuSaV GI.1 and GII.3 to heat and ultraviolet treatments. This system is inexpensive, scalable, and reproducible in different laboratories, and can be used to investigate mechanisms of HuSaV replication and to evaluate antivirals and/or disinfection methods for HuSaVs.
人类杯状病毒（HuSaVs）可引起类似于人类诺如病毒的急性胃肠炎。尽管 HuSaVs 是在四十年前被发现的，但至今仍未能在体外培养，这极大地阻碍了对病毒生物学的理解和抗病毒策略的发展。在本研究中，我们鉴定了两种易感性人源细胞系，它们分别来源于睾丸和十二指肠，可支持 HuSaV 复制，并且发现复制需要胆汁酸。HuSaVs 在十二指肠细胞系中复制效率更高，病毒 RNA 水平增加了约 6 个对数级。我们还在细胞培养物中检测到双链 RNA、病毒非结构和结构蛋白以及完整的 HuSaV 颗粒。我们通过传代证实了释放到细胞培养上清液中的子代病毒的感染性。这些结果表明 HuSaVs 已成功在体外生长。此外，我们确定了最小感染剂量，并测试了 HuSaV GI.1 和 GII.3 对热和紫外线处理的敏感性。该系统价格低廉、可扩展且在不同实验室中具有可重复性，可用于研究 HuSaV 复制的机制，并评估针对 HuSaVs 的抗病毒药物和/或消毒方法。
