Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, 6010 Katz Centre for Health Research, University of Alberta, Edmonton, AB T6G 2E1, Canada.
Viruses. 2021 Jan 12;13(1):97. doi: 10.3390/v13010097.
An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.
据估计，全球有 20 亿人感染了乙型肝炎病毒 (HBV)。尽管 HBV 在体内具有很高的传染性，但缺乏易于体外培养的系统阻碍了对 HBV 的研究。在肝癌细胞中过表达牛磺胆酸钠共转运多肽 (NTCP) 胆汁酸转运蛋白可提高感染效率。我们在此报告一种不需要二甲亚砜 (DMSO) 即可进行 HBV 感染的肝癌细胞培养系统。我们在 Huh7.5 细胞中过表达了 NTCP，并允许这些细胞在补充有人类血清 (HS) 的培养基中分化，而不是胎牛血清 (FBS)。我们表明，人血清培养可增强 Huh7.5-NTCP 细胞中的 HBV 感染，例如，在 HS 培养物中，与 FBS 培养物相比，HBV pgRNA 水平增加了多达 200 倍，与 FBS+DMSO 培养物相比增加了 19 倍。人血清培养可增加 Huh7.5-NTCP 细胞中肝细胞分化标志物的水平，如白蛋白分泌，与原代人肝细胞中的水平相当。在人血清中培养诱导的 NTCP 的 N-糖基化可能有助于病毒进入。我们的研究证明了 Huh7.5-NTCP 细胞在没有使用潜在毒性的 DMSO 的情况下进行体外 HBV 感染。
