scholarly journals In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 97
Author(s):  
Connie Le ◽  
Reshma Sirajee ◽  
Rineke Steenbergen ◽  
Michael A. Joyce ◽  
William R. Addison ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Human Serum ◽  
Dimethyl Sulfoxide ◽  
Hepatoma Cell ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Differentiation Markers ◽  
B Virus

An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.

scholarly journals Epitope–Paratope Interaction of a Neutralizing Human Anti-Hepatitis B Virus PreS1 Antibody That Recognizes the Receptor-Binding Motif

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 754
Author(s):  
Jisu Hong ◽  
Youngjin Choi ◽  
Yoonjoo Choi ◽  
Jiwoo Lee ◽  
Hyo Jeong Hong
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Receptor Binding ◽  
Neutralizing Antibody ◽  
Hbv Infection ◽  
Binding Motif ◽  
Alanine Scanning Mutagenesis ◽  
B Virus ◽  
Complementarity Determining Regions

Hepatitis B virus (HBV) is a global health burden that causes acute and chronic hepatitis. To develop an HBV-neutralizing antibody that effectively prevents HBV infection, we previously generated a human anti-preS1 monoclonal antibody (1A8) that binds to genotypes A–D and validated its HBV-neutralizing activity in vitro. In the present study, we aimed to determine the fine epitope and paratope of 1A8 to understand the mechanism of HBV neutralization. We performed alanine-scanning mutagenesis on the preS1 (aa 19–34, genotype C) and the heavy (HCDR) and light (LCDR) chain complementarity-determining regions. The 1A8 recognized the three residues (Leu22, Gly23, and Phe25) within the highly conserved receptor-binding motif (NPLGFFP) of the preS1, while four CDR residues of 1A8 were critical in antigen binding. Structural analysis of the epitope–paratope interaction by molecular modeling revealed that Leu100 in the HCDR3, Ala50 in the HCDR2, and Tyr96 in the LCDR3 closely interacted with Leu22, Gly23, and Phe25 of the preS1. Additionally, we found that 1A8 also binds to the receptor-binding motif (NPLGFLP) of infrequently occurring HBV. The results suggest that 1A8 may broadly and effectively block HBV entry and thus have potential as a promising candidate for the prevention and treatment of HBV infection.

scholarly journals Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

1987 ◽  
Vol 6 (3) ◽  
pp. 675-680 ◽  
Author(s):  
C.M. Chang ◽  
K.S. Jeng ◽  
C.P. Hu ◽  
S.J. Lo ◽  
T.S. Su ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Line ◽  
Transient Expression ◽  
Hepatoma Cell ◽  
Hbv Dna ◽  
Hepatoma Cell Line ◽  
B Virus

scholarly journals Primary Tupaia Javanica Hepatocyte Culture as an In Vitro Model for Human Hepatitis B Virus Infection

2022 ◽  
Vol 22 (3) ◽  
pp. 203-209
Author(s):  
Kemal Fariz Kalista ◽  
Maryati Surya ◽  
Silmi Mariya ◽  
Diah Iskandriati ◽  
Irsan Hasan ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
In Vitro Model ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Primate Research ◽  
Qualitative And Quantitative ◽  
B Virus ◽  
Vitro Model

Background: Hepatitis B virus (HBV) infection is still one of the biggest health problems in the world, which could lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Treatment for HBV infection has not yet achieved a functional cure. More studies are needed to investigate human HBV (HuHBV), but the scarcity of animal models for HuHBV infection became a barrier. Recently, many studies have shown that Tupaia are suitable for the study of HuHBV. The purpose of this study was to develop a primary tupaia hepatocyte (PTH) culture from T. javanica, a species of Tupaia found in Indonesia, and to prove that HuHBV can replicate in the PTH.Method: In vitro experimental study using PTH isolated from five wild adult T. javanica in Primate Research Center, IPB University. HuHBV was taken from humans with HBsAg and HBV-DNA (+). PTH cells then were infected with HuHBV after reaching 80% confluence. Observation on PTH cells was done everyday for 20 days. Qualitative and quantitative HBsAg were measured using a CMIA while HBV-DNA and cccDNA were measured by RT-PCR.Results: A cytopathic effect was seen on day post infection (DPI)-16. HBsAg and HBV-DNA were detected from DPI-2 until DPI-18, with HBV-DNA level peaked on DPI-12. cccDNA concentration was fluctuating from DPI-2 until DPI-20 with highest level on DPI-16.Conclusion: HuHBV could infect and replicate in PTH from T. javanica can be infected with HuHBV and HuHBV can replicate in the PTH from T. javanica.

scholarly journals Na+-Taurocholate Co-Transporting Polypeptide (NTCP) in Livers, Function, Expression Regulation, and Potential in Hepatitis B Treatment

Livers ◽  
2021 ◽  
Vol 1 (4) ◽  
pp. 236-249
Author(s):  
Xiaoyu Zhao ◽  
Waqas Iqbal ◽  
Pingnan Sun ◽  
Xiaoling Zhou
Keyword(s):  
Hepatitis B ◽  
Hepatoma Cell ◽  
Sodium Taurocholate ◽  
Hbv Infection ◽  
Viral Receptor ◽  
Hepatitis D ◽  
Hepatoma Cell Lines ◽  
Chronic Hepatitis B Virus ◽  
Hepatitis B Treatment

Chronic hepatitis B virus (HBV) infection has become one of the leading causes of liver cirrhosis and hepatocellular carcinoma globally. The discovery of sodium taurocholate co-transporting polypeptide (NTCP), a solute carrier, as a key receptor for HBV and hepatitis D virus (HDV) has opened new avenues for HBV treatment. Additionally, it has led researchers to generate hepatoma cell lines (including HepG2-NTCP and Huh-7-NTCP) susceptible to HBV infection in vitro, hence, paving the way to develop and efficiently screen new and novel anti-HBV drugs. This review summarizes the history, function and critical findings regarding NTCP as a viral receptor for HBV/HDV, and it also discusses recently developed drugs targeting NTCP.

Over IFI16 expression increased inflammation in Hepatitis B Virus-Associated Glomerulonephritis by Regulating Caspase-1 and IL-1 ß

2020 ◽  
Author(s):  
zhaoqing zeng ◽  
yuyang li ◽  
jinhong yu ◽  
jing liu ◽  
shijun chen ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Line ◽  
Renal Damage ◽  
Hbv Infection ◽  
Inflammatory Factors ◽  
Inflammasome Activation ◽  
Caspase 1 ◽  
B Virus

Abstract Aims & background: IFI16 plays an important role in innate immunity against invasive microbial infection by sensing double-stranded DNA viruses due to caspase-1-dependent inflammasome activation and subsequent maturation and secretion of IL-1β. However, the role of IFI16 in regulating the immune response to viruses in vivo and in vitro, especially in sensing hepatitis B virus (HBV), has not been examined. We hypothesized that the expression of IFI16 increases corresponding to HBV-mediated inflammation in patients with hepatitis B virus associated glomerulonephritis (HBV-GN), a condition which activates inflammatory mechanisms and causes renal damage. To test this hypothesis, we therefore analyzed the expression of IFI16 and inflammatory factors in HBV-GN tissues and cell lines relative to the inflammatory response to HBV infection. Methods: A total 75 patients with chronic nephritis(CN) including 50 with HBV-GN and 25 with chronic glomerulonephritis (CCN) involved in this study. Each CN patient received renal biopsy, and immunohistochemistry(IHC) was used to detect the expression of IFI16 and inflammatory factors Caspase-1 and IL-1β in the biopsy specimens. Following IFI16 was transfected in HBV-infected and HBV-uninfected human glomerular mesangial (HGM) cell line and HEK-293T cell line, expression of Caspase-1 and IL-1β were detected by Western blot and qRT- PCR. Results: IFI16 expression in HBV-GN biopsies (80.0%) was significantly higher than in CGN (24.0%) and positively correlated with caspase-1 and IL-1𝛽 expression in HBV-GN. In vitro, over expression IFI16 increased caspase-1 and IL-1𝛽 expression in HBV -infected HGM and HEK-293T. Conclusions: The elevation of IFI16 during HBV infection or replication may contribute to renal damage due to inflammation, thus providing a putative therapeutic target and a new avenue for researching the pathogenesis of HBV-GN.

scholarly journals Identification and characterization of SEC24D as a susceptibility gene for hepatitis B virus infection

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xianzhong Jiang ◽  
Bin Zhang ◽  
Junsheng Zhao ◽  
Yi Xu ◽  
Haijun Han ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Time Course ◽  
Expression Profiles ◽  
Association Studies ◽  
Susceptibility Gene ◽  
Hbv Infection ◽  
Genome Wide Association Studies ◽  
B Virus

Abstract Single nucleotide polymorphisms (SNPs) and genes associated with susceptibility to hepatitis B virus (HBV) infection that have been identified by genome-wide association studies explain only a limited portion of the known heritability, indicating more genetic variants remain to be discovered. In this study, we adopted a new research strategy to identify more susceptibility genes and variants for HBV infection. We first performed genetic association analysis of 300 sib-pairs and 3,087 case-control samples, which revealed that 36 SNPs located in 31 genes showed nominal associations with HBV infection in both samples. Of these genes, we selected SEC24D for further molecular analysis according to the following two main lines of evidence. First, a time course analysis of the expression profiles from HBV-infected primary human hepatocytes (PHH) demonstrated that SEC24D expression increased markedly as time passed after HBV infection (P = 4.0 × 10−4). Second, SNP rs76459466 in SEC24D was adversely associated with HBV risk (ORmeta = 0.82; Pmeta = 0.002), which again indicated that SEC24D represents a novel susceptibility gene for HBV infection. Moreover, SEC24D appeared to be protective against HBV infection in vitro. Consistently, we found that SEC24D expression was significantly enhanced in non-infected liver tissues (P = 0.002). We conclude that SEC24D is a novel candidate gene linked to susceptibility to HBV infection.

scholarly journals Hepatitis B Virus-Encoded MicroRNA Controls Viral Replication

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Xi Yang ◽  
Hongfeng Li ◽  
Huahui Sun ◽  
Hongxia Fan ◽  
Yaqi Hu ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Persistent Infection ◽  
Hepatoma Cell ◽  
Hbv Infection ◽  
Dependent Manner ◽  
Dna Virus ◽  
Hbv Replication ◽  
B Virus ◽  
Self Replication

ABSTRACT MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding, functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and Northern blotting. HBV-miR-3 is located at nucleotides (nt) 373 to 393 of the HBV genome and was generated from 3.5-kb, 2.4-kb, and 2.1-kb HBV in a classic miRNA biogenesis (Drosha-Dicer-dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV+ hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released into the circulation by exosomes and HBV virions, and HBV-miR-3 expression had a positive correlation with HBV titers in the sera of patients in the acute phase of HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg, and replication of HBV. HBV-miR-3 targets the unique site of the HBV 3.5-kb transcript to specifically reduce HBc protein expression, levels of pregenomic RNA (pgRNA), and HBV replication intermediate (HBV-RI) generation but does not affect the HBV DNA polymerase level, thus suppressing HBV virion production (replication). This may explain the low levels of HBV virion generation with abundant subviral particles lacking core during HBV replication, which may contribute to the development of persistent infection in patients. Taken together, our findings shed light on novel mechanisms by which HBV-encoded miRNA controls the process of self-replication by regulating HBV transcript during infection. IMPORTANCE Hepatitis B is a liver infection caused by the hepatitis B virus (HBV) that can become a long-term, chronic infection and lead to cirrhosis or liver cancer. HBV is a small DNA virus that belongs to the hepadnavirus family, with virions and subviral forms of particles that lack a core. MicroRNA (miRNA), a small (∼22-nt) noncoding RNA, was recently found to be an important regulator of gene expression. We found that HBV encodes miRNA (HBV-miR-3). More importantly, we revealed that HBV-miR-3 targets its transcripts to attenuate HBV replication. This may contribute to explaining how HBV infection leads to mild damage in liver cells and the subsequent establishment/maintenance of persistent infection. Our findings highlight a mechanism by which HBV-encoded miRNA controls the process of self-replication by regulating the virus itself during infection and might provide new biomarkers for diagnosis and treatment of hepatitis B.

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