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.
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