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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 
Citation
 JOURNAL OF HEPATOLOGY, Vol.71(2) : 289-300, 2019 
Journal Title
JOURNAL OF HEPATOLOGY
ISSN
 0168-8278 
Issue Date
2019
Keywords
Complete HBV life cycle ; Drug sensitivity ; HBV doubling time ; HBV spread ; HepG2-NTCP ; Kinetics of antigen ; Patient-derived HBV ; Virion secretion ; cccDNA accumulation
Abstract
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.
Files in This Item:
T201903868.pdf Download
DOI
10.1016/j.jhep.2019.04.010
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 1. Journal Papers
Yonsei Authors
Park, Jun Yong(박준용) ORCID logo https://orcid.org/0000-0001-6324-2224
Ahn, Sang Hoon(안상훈) ORCID logo https://orcid.org/0000-0002-3629-4624
Han, Kwang-Hyub(한광협) ORCID logo https://orcid.org/0000-0003-3960-6539
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/173073
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