Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells
Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells
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DOI：
10.1016/j.jhep.2019.04.010
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发表时间：
2019-08-01
影响因子：
25.7
通讯作者：
Windisch, Marc Peter
中科院分区：
文献类型：
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作者：
Konig, Alexander;Yang, Jaewon;Windisch, Marc Peter
背景与目的：乙型肝炎病毒（HBV）通过受感染的肝脏传播，同时分泌到血液中。hbv易感的体外感染模型不能有效地扩增病毒子代或支持细胞间传播。我们试图建立一种从临床标本中扩增感染性HBV的细胞培养系统。方法：选择hbv易感的牛磺胆酸钠共转运多肽过表达HepG2细胞克隆（HepG2- ntcpsec +）产生高滴度的感染性后代。用天然凝胶电泳和电子显微镜对分泌的HBV子代进行鉴定。比较RNA-seq转录组学用于量化宿主原病毒和限制性因子的表达。使用HBV进入或复制抑制剂、报告细胞中病毒细胞间传播的可视化和最近邻居感染测定来评估病毒传播途径。分析HBV基因型B-D的扩增动力学。结果：HepG2-NTCPsec+感染后高水平分泌HBV表面大蛋白包膜的感染性HBV子代，电镜下表现典型。RNA-seq转录组学显示HBV在HepG2-NTCPsec+中没有引起显著的基因表达变化，然而，有利于HBV扩增的转录因子比在不太允许的HepG2-NTCPsec-中表达得更强烈。用含有hbv的患者血清接种后，由于病毒向邻近细胞传播，感染细胞的比例从最初的10%增加到70%，并且病毒子代和抗原被有效分泌。根据病毒来源的不同，HepG2-NTCPsec+支持高达1300倍的HBV基因组净扩增。病毒的扩散和扩增被进入和复制抑制剂所抑制；停药后观察到病毒反弹。结论：新型HepG2-NTCPsec+细胞有效支持来自患者或细胞培养的HBV的完整生命周期、长期病毒传播和扩增，与HBV感染患者的相关特征相似。摘要：目前可用的实验室系统无法重现乙型肝炎病毒（HBV）通过受感染的肝脏传播并释放到血液中的动态。我们开发了一种缓慢分裂的肝源细胞系，该细胞系在接种患者或细胞培养源HBV后增殖感染性病毒颗粒。这种新的感染模型可以通过提前测量患者HBV毒株对特定抗病毒药物的敏感性来改善治疗。(C) 2019欧洲肝脏研究协会。Elsevier B.V.出版
Background & Aims: As hepatitis B virus (HBV) spreads through the infected liver it is simultaneously secreted into the blood. HBV-susceptible in vitro infection models do not efficiently amplify viral progeny or support cell-to-cell spread. We sought to establish a cell culture system for the amplification of infectious HBV from clinical specimens.Methods: An HBV-susceptible sodium-taurocholate cotransporting polypeptide-overexpressing HepG2 cell clone (HepG2-NTCPsec+) producing high titers of infectious progeny was selected. Secreted HBV progeny were characterized by native gel electrophoresis and electron microscopy. Comparative RNA-seq transcriptomics was performed to quantify the expression of host proviral and restriction factors. Viral spread routes were evaluated using HBV entry- or replication inhibitors, visualization of viral cell-to-cell spread in reporter cells, and nearest neighbor infection determination. Amplification kinetics of HBV genotypes B-D were analyzed.Results: Infected HepG2-NTCPsec+ secreted high levels of large HBV surface protein-enveloped infectious HBV progeny with typical appearance under electron microscopy. RNA-seq transcriptomics revealed that HBV does not induce significant gene expression changes in HepG2-NTCPsec+, however, transcription factors favoring HBV amplification were more strongly expressed than in less permissive HepG2-NTCPsec-. Upon inoculation with HBV-containing patient sera, rates of infected cells increased from 10% initially to 70% by viral spread to adjacent cells, and viral progeny and antigens were efficiently secreted. HepG2-NTCPsec+ supported up to 1,300-fold net amplification of HBV genomes depending on the source of virus. Viral spread and amplification were abolished by entry and replication inhibitors; viral rebound was observed after inhibitor discontinuation.Conclusions: The novel HepG2-NTCPsec+ cells efficiently support the complete HBV life cycle, long-term viral spread and amplification of HBV derived from patients or cell culture, resembling relevant features of HBV-infected patients.Lay summary: Currently available laboratory systems are unable to reproduce the dynamics of hepatitis B virus (HBV) spread through the infected liver and release into the blood. We developed a slowly dividing liver-derived cell line which multiplies infectious viral particles upon inoculation with patient- or cell culture-derived HBV. This new infection model can improve therapy by measuring, in advance, the sensitivity of a patient's HBV strain to specific antiviral drugs. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V.