Background

Lack of universal replication system for hepatitis B virus with narrow host range and organ tropism has hampered to uncover the pathogenesis of HBV. Previously, we reported the essentiality of humoral milieu and its components toward HBV and hepatitis C virus survival/viability in vitro. Of these components, the precise role of enterohepatic humoral milieu such as bile acid (BA) on HBV cultivation in vitro and in vivo is unknown.

Objective

We explored whether BA, specifically taurochenodeoxycholic acid (tCDCA) would directly regulate the viral DNA and surface antigen expression of HBV in vitro, consequently rendering HBV to enter into human or murine immortalized hepatocytes, and non-hepatocytes.

Result

We found that higher concentration of taurochenodeoxycholic acid (tCDCA) is able to preserve the genomic stability of HBV in cell-free DMEM, showing higher the surface antigenicity than taurocholic acid (tCA). In line, we found that in vitro cell culture condition (100 μmol/L of tCDCA coupled with 1 × 10⁸ g e/mL HBV) would be optimal for HBV entry into target cells. Using this, human (HepG2, Huh7), and rodent (Hepa1c1c7, H4-II-E) hepatoma cell lines were infected by HBV, as evidenced by the presence of HBV biomarkers (HBsAg, and HBV DNA in culture supernatant, as well as HBcAg in cell). Further, cellular entry test revealed that HBV is able to infect 12 different non-hepatic cell lines regardless of species, and organ/tissue, consequently reproducing progeny as confirmed by HBV biomarkers. Last, reinfection test showed that the progenies of HBV from immortalized HepG2, and Hepa1c1c7 cells are able to enter into each or vice versa naïve HepG2, and Hepa1c1c7 cells with or without BA.

Conclusion

This study demonstrates that enterohepatic humoral milieu such as BA, specifically tCDCA would directly regulate HBV DNA and its surface antigen expression in vitro, consequently rendering HBV to enter into human or murine immortalized hepatocytes, and non-hepatocytes. This is the first note to render HBV permissive to human or rodent hepatic and non-hepatic cells via sole manipulation of humoral milieu, thus establishing the platform for in vitro robust replication system of HBV.

中文翻译：

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胆汁酸（BA）在乙型肝炎病毒体外细胞进入和释放中的关键作用

背景

乙型肝炎病毒宿主范围狭窄和器官嗜性缺乏通用的复制系统，阻碍了乙型肝炎病毒的发病机理的发现。以前，我们报道了体液环境及其对HBV和C型肝炎病毒存活/存活的必要性。在这些成分中，肠肝液性环境如胆汁酸（BA）在体外和体内培养HBV的确切作用尚不清楚。

目的

我们探讨了BA，特别是牛磺去氧胆酸（tCDCA）是否会在体外直接调节病毒DNA和HBV表面抗原表达，从而使HBV进入人或鼠永生的肝细胞和非肝细胞。

结果

我们发现更高浓度的牛磺去氧去氧胆酸（tCDCA）能够保留无细胞DMEM中HBV的基因组稳定性，显示出比牛磺胆酸（tCA）更高的表面抗原性。我们发现，体外细胞培养条件（tCDCA 100μmol/ L加上1×10 ⁸ Ge / mL HBV）对于HBV进入靶细胞而言是最佳的。使用这种方法，人类（HepG2，Huh7）和啮齿类动物（Hepa1c1c7，H4-II-E）肝细胞瘤细胞均被HBV感染，这可以通过培养上清液中存在HBV生物标志物（HBsAg和HBV DNA）以及细胞中的HBcAg）。此外，细胞进入测试表明，HBV能够感染12种不同的非肝细胞株，无论其种类，器官/组织如何，因此均能繁殖出后代，如HBV生物标记所证实。最后，再感染试验表明，来自永生化的HepG2和Hepa1c1c7细胞的HBV子代能够进入含有HepG2和Hepa1c1c7的每个细胞，反之亦然。

结论

这项研究表明肠肝体液环境，例如BA，特别是tCDCA，将在体外直接调节HBV DNA及其表面抗原的表达，从而使HBV进入人或鼠永生的肝细胞和非肝细胞。这是通过单独操纵体液环境使HBV对人或啮齿动物的肝细胞和非肝细胞具有容许性的第一个说明，从而为HBV体外鲁棒复制系统奠定了平台。