Gilead Sciences, Inc., Foster City, California, USA.
J Virol. 2022 Sep 28;96(18):e0084922. doi: 10.1128/jvi.00849-22. Epub 2022 Aug 29.
The existing cell culture-based methods to study hepatitis B virus (HBV) have limitations and do not allow for viral long-term passage. The aim of this study was to develop a robust long-term viral passage system with optimized cell culture conditions and a viral isolate with the ability to spread and passage. An HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized primary human hepatocyte (PHH)/human fibroblast coculture system. The passaged UV-treated virus was sequenced and further characterized. In addition, a panel of mutant viruses containing different combinations of mutations observed in this virus was investigated. The clinical isolate was passaged for 20 rounds with 21 days per round in an optimized PHH/human fibroblast coculture system while subject to UV mutagenesis. This passaged UV-mutated isolate harbored four mutations: G225A (sR24K) in the S gene, A2062T in the core gene, and two mutations G1764A and C1766T (xV131I) in the basal core promoter (BCP) region. characterization of the four mutations suggested that the two BCP mutations G1764A and C1766T contributed to the increased viral replication and viral infectivity. A robust long-term HBV viral passage system has been established by passaging a UV-treated clinical isolate in an optimized PHH/fibroblast coculture system. The two BCP mutations played a key role in the virus's ability to passage. This passage system can be used for studying the entire life cycle of HBV and has the potential for drug-resistance selection upon further optimization. The existing cell culture-based methods to study HBV have limitations and do not allow for viral long-term passage. In this study, an HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized PHH/human fibroblast coculture system. This passaged UV-mutated isolate carried four mutations across the HBV genome, and characterization of the four mutations suggested that the two basal core promoter (BCP) mutations G1764A and C1766T played a key role in the virus's ability to passage. In summary, we have developed a robust long-term HBV viral passage system by passaging an UV-treated HBV genotype A clinical isolate in an optimized PHH/human fibroblast coculture system. This passage system can be used for studying the entire life cycle of HBV and has the potential for drug-resistance selection upon further optimization.
现有的基于细胞培养的乙型肝炎病毒 (HBV) 研究方法存在局限性，不允许病毒进行长期传代。本研究旨在开发一种稳健的长期病毒传代系统，优化细胞培养条件，并使用具有传播和传代能力的病毒分离株。对一株乙型肝炎病毒基因型 A 临床分离株进行多次紫外线处理，并在优化的人原代肝细胞 (PHH)/人成纤维细胞共培养系统中进行传代。对传代的紫外线处理病毒进行测序并进一步表征。此外，还研究了包含该病毒中观察到的不同组合突变的突变病毒。将临床分离株在优化的 PHH/人成纤维细胞共培养系统中进行 20 轮传代，每轮 21 天，同时进行紫外线诱变。在优化的 PHH/人成纤维细胞共培养系统中，经过紫外线诱变和 20 轮传代，该传代的紫外线突变分离株携带四个突变：S 基因中的 G225A（sR24K）、核心基因中的 A2062T 以及前核心启动子（BCP）区域中的两个突变 G1764A 和 C1766T（xV131I）。对四个突变的特征分析表明，两个 BCP 突变 G1764A 和 C1766T 有助于病毒复制和病毒感染力的增强。通过在优化的 PHH/成纤维细胞共培养系统中用紫外线处理的临床分离株进行传代，建立了一种稳健的长期 HBV 病毒传代系统。两个 BCP 突变在病毒传代能力中起关键作用。该传代系统可用于研究 HBV 的整个生命周期，并在进一步优化后具有耐药性选择的潜力。现有的基于细胞培养的乙型肝炎病毒 (HBV) 研究方法存在局限性，不允许病毒进行长期传代。本研究中，一株乙型肝炎病毒基因型 A 临床分离株经过多轮紫外线处理，并在优化的 PHH/人成纤维细胞共培养系统中进行传代。该传代的紫外线突变分离株在 HBV 基因组中携带四个突变，对四个突变的特征分析表明，两个前核心启动子 (BCP) 突变 G1764A 和 C1766T 在病毒的传代能力中起关键作用。综上所述，我们通过在优化的 PHH/人成纤维细胞共培养系统中用紫外线处理的 HBV 基因型 A 临床分离株进行传代，建立了一种稳健的长期 HBV 病毒传代系统。该传代系统可用于研究 HBV 的整个生命周期，并在进一步优化后具有耐药性选择的潜力。
