Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells
Authors: Alexander König, Jaewon Yang, Eunji Jo, Kyu Ho Paul Park, Hyun Kim, Thoa Thi Than, Xiyong Song, Xiaoxuan Qi, Xinghong Dai, Soonju Park, David Shum, Wang-Shick Ryu, Jung-Hee Kim, Seung Kew Yoon, Jun Yong Park, Sang Hoon Ahn, Kwang-Hyub Han, Wolfram Hubert Gerlich, Marc Peter Windisch
Journal: Journal of Hepatology (2019)

Highlights•Cell culture system that mimicks complete HBV life cycle from entry to egress.•Efficient in vitro infection with crude HBV patient sera.•Up to 50- and 1,300-fold net amplification of patient- and cell culture-derived input HBV in the supernatant.•Polyethylene glycol-independent HBV spread to adjacent cells, forming infected cell clusters.•Evaluation of patient- and cell culture-derived HBV amplification w/wo antivirals over 8 weeks.Graphical abstractAbstractBackground & AimsAs 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.MethodsAn 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.ResultsInfected 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.ConclusionsThe 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 summaryCurrently 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.