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Alexander Koenig
Alexander Koenig (Alexander Koenig) 저자 이메일 보기
조회 352  인쇄하기 주소복사 트위터 공유 페이스북 공유 
Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells
열기 Authors and Affiliations


Background & Aims
Hepatitis B virus (HBV) spreads through the infected liver paralleled by secretion 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.

Selection of an HBV-susceptible sodium-taurocholate cotransporting polypeptide-overexpressing HepG2 cell clone (HepG2-NTCPsec+) producing high titers of infectious progeny virus. Secreted HBV progeny was characterized by native gel electrophoresis and electron microscopy. Comparative RNA-seq transcriptomics to quantify the expression of host proviral and restriction factors. Evaluation of viral spread routes using HBV entry- or replication inhibitors; visualization of viral cell-to-cell spread in reporter cells, and nearest neighbor infection determination. Analysis of amplification kinetics of HBV genotypes B-D from 7 patients or cell culture for 8 weeks of infection.

Infected HepG2-NTCPsec+ secreted high levels of LHBs-enveloped infectious HBV progeny with typical appearance in EM. RNA-seq transcriptomics revealed that HBV does not induce significant gene expression changes in HepG2-NTCPsec+, however, transcription factors favoring HBV amplification were stronger expressed than in less permissive HepG2-NTCPsec-. Upon inoculation with HBV-containing patient sera, rates of infected cells increased from initially 10% 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 virus source. Viral spread and amplification were abolished by entry and replication inhibitors; viral rebound was observed after inhibitor discontinuation.

The novel HepG2-NTCPsec+ cells efficiently support the complete HBV lifecycle, 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 unsuitable 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 sensitivity of the patient’s HBV strain to the chosen antiviral drugs.

Keywords : complete HBV lifecycle; HepG2-NTCP; patient-derived HBV; drug sensitivity; kinetics of antigen, virion secretion, cccDNA accumulation; HBV doubling time

- 형식: Research article
- 게재일: 2019년 05월 (BRIC 등록일 2019-05-10)
- 연구진: 국내(교신)+국외 연구진태극기
- 분야: Medicine
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