Coexistence of Hepatitis B Virus Quasispecies Enhances Viral Replication and the Ability To Induce Host Antibody and Cellular Immune Responses

Cao, Liang; Wu, Chunchen; Shi, Hui; Gong, Zuojiong; Zhang, Ejuan; Wang, Hui; Zhao, Kaitao; Liu, Shuhui; Li, Songxia; Gao, Xiuzhu; Wang, Yun; Pei, Rongjuan; Lu, Mengji; Chen, Xinwen

doi:10.1128/jvi.01123-14

Cited by 58 publications

(55 citation statements)

References 42 publications

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“…The primary antibodies were rabbit anti-HBcAg polyclonal antibody (Dako, Glostrup, Denmark), mouse anti-HBsAg monoclonal antibody (Cao L. et al, 2014), mouse anti-CD81 antibody (Santa Cruz, California, USA), and rabbit antihNTCP polyclonal antibody (Sigma: HPA042727). The secondary antibodies were Alexa Fluor-488 or Alexa Fluor-568 conjugated (Life Technologies, Waltham, Massachusetts, USA).…”

Section: Immunofluorescence Assaymentioning

confidence: 99%

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

et al. 2017

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Feasible and effective cell models for hepatitis B virus (HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol (DMSO/PEG), hNTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line (Huh7D hNTCP ) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO / 4% PEG8000, and one resistant cell line (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7D hNTCP ) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Huh7D hNTCP cells with or without DMSO treatment were characterized. Finally, the susceptibility of Huh7D hNTCP cells to HBV infection was assessed. Our results showed that Huh7D cells were resistant to 2.5% DMSO / 4% PEG8000, whereas the others were not. Huh7D hNTCP cells were established to express a high level of hNTCP compared to liver extracts, and Huh7D hNTCP cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Huh7D hNTCP cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.

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Section: Immunofluorescence Assaymentioning

confidence: 99%

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

et al. 2017

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“…At 48 h post-transfection, the cellular RNA was extracted and analysed by Northern blotting as described previously (Hao et al, 2015). At 96 h post-transfection, the viral replication intermediate DNA (Cao et al, 2014) and extracellular virion DNA (Tian et al, 2011) were extracted as described previously. The intracellular replication intermediates or extracellular virion DNA were then analysed by qPCR; the primers (2RC/CCS and 2RC/CCAS) were adopted from a previously published paper (Werle-Lapostolle et al, 2004).…”

Section: Inhibition Of Hbv By Crispr/cas9mentioning

confidence: 99%

Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome

Liu

Hao

Chen

et al. 2015

Journal of General Virology

135

103

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Hepatitis B virus (HBV) remains a global health threat as chronic HBV infection may lead to liver cirrhosis or cancer. Current antiviral therapies with nucleoside analogues can inhibit the replication of HBV, but do not disrupt the already existing HBV covalently closed circular DNA. The newly developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a powerful tool to target cellular genome DNA for gene editing. In order to investigate the possibility of using the CRISPR/Cas9 system to disrupt the HBV DNA templates, we designed eight guide RNAs (gRNAs) that targeted the conserved regions of different HBV genotypes, which could significantly inhibit HBV replication both in vitro and in vivo. Moreover, the HBV-specific gRNA/Cas9 system could inhibit the replication of HBV of different genotypes in cells, and the viral DNA was significantly reduced by a single gRNA/Cas9 system and cleared by a combination of different gRNA/Cas9 systems.

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“…Selection pressures for the virus come from (i) competition between viral variants (differences in replicative efficiency) (5) and/or (ii) host immune activity (3). It was recently documented in a Chinese study that the coexistence of the HBV variants (A1762T/G1764A/G1896A mutation and preS deletion/S deletion at a 1:4 ratio) from an HBVassociated liver failure patient could increase, in vivo and in vitro, HBV replication, and this could significantly modulate specific host immune responses (6).…”

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confidence: 99%

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

et al. 2015

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Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T-and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

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Coexistence of Hepatitis B Virus Quasispecies Enhances Viral Replication and the Ability To Induce Host Antibody and Cellular Immune Responses

Cited by 58 publications

References 42 publications

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

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