scholarly journals Hepatitis B Virus DNA Integration Occurs Early in the Viral Life Cycle in anIn VitroInfection Model via Sodium Taurocholate Cotransporting Polypeptide-Dependent Uptake of Enveloped Virus Particles

2018 ◽  
Vol 92 (11) ◽  
pp. e02007-17 ◽  
Author(s):  
Thomas Tu ◽  
Magdalena A. Budzinska ◽  
Florian W. R. Vondran ◽  
Nicholas A. Shackel ◽  
Stephan Urban
Keyword(s):  
Hepatitis B Virus ◽  
Liver Cancer ◽  
Hepatitis B ◽  
Chronic Infection ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Dna Integration ◽  
B Virus ◽  
Hbv Dna Integration

ABSTRACTChronic infection by hepatitis B virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (covalently closed circular DNA [cccDNA]), integration of HBV DNA into the host cell genome is regularly observed in the liver in infected patients. While reported as a prooncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well understood, chiefly due to the lack ofin vitroinfection models that have detectable integration events. In this study, we have established anin vitrosystem in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10,000 cells, with the most consistent detection in Huh7-NTCP cells. The integration rate remained stable between 3 and 9 days postinfection. HBV DNA integration was efficiently blocked by treatment with a 200 nM concentration of the HBV entry inhibitor Myrcludex B, but not with 10 μM tenofovir, 100 U of interferon alpha, or a 1 μM concentration of the capsid assembly inhibitor GLS4. This suggests that integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent ofde novoHBV genome replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established anin vitrosystem to interrogate the mechanisms of HBV DNA integration.IMPORTANCEHepatitis B virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of theHepadnaviridaefamily, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs are not clear. In this study, we have developed and characterized anin vitrosystem to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we showed that integration occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation.

scholarly journals Hepatitis B virus (HBV) DNA integration in patients with occult HBV infection and hepatocellular carcinoma

2015 ◽  
Vol 35 (10) ◽  
pp. 2311-2317 ◽  
Author(s):  
Carlo Saitta ◽  
Gianluca Tripodi ◽  
Adalberto Barbera ◽  
Antonio Bertuccio ◽  
Antonina Smedile ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Occult Hbv Infection ◽  
Dna Integration ◽  
Occult Hbv ◽  
B Virus ◽  
Hbv Dna Integration

scholarly journals Hepatitis B Virus DNA Integration: In Vitro Models for Investigating Viral Pathogenesis and Persistence

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 180
Author(s):  
Thomas Tu ◽  
Henrik Zhang ◽  
Stephan Urban
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
In Vitro Models ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Dna Integration ◽  
B Virus ◽  
Chronic Hbv ◽  
Globally Distributed

Hepatitis B virus (HBV) is a globally-distributed pathogen and is a major cause of liver disease. HBV (or closely-related animal hepadnaviruses) can integrate into the host genome, but (unlike retroviruses) this integrated form is replication-defective. The specific role(s) of the integrated HBV DNA has been a long-standing topic of debate. Novel in vitro models of HBV infection combined with sensitive molecular assays now enable researchers to investigate this under-characterised phenomenon with greater ease and precision. This review covers the contributions these systems have made to understanding how HBV DNA integration induces liver cancer and facilitates viral persistence. We summarise the current findings into a working model of chronic HBV infection and discuss the clinical implications of this hypothetical framework on the upcoming therapeutic strategies used to curb HBV-associated pathogenesis.

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 Two-Year Assessment of Entecavir Resistance in Lamivudine-Refractory Hepatitis B Virus Patients Reveals Different Clinical Outcomes Depending on the Resistance Substitutions Present

2006 ◽  
Vol 51 (3) ◽  
pp. 902-911 ◽  
Author(s):  
Daniel J. Tenney ◽  
Ronald E. Rose ◽  
Carl J. Baldick ◽  
Steven M. Levine ◽  
Kevin A. Pokornowski ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Clinical Outcomes ◽  
Chronic Infection ◽  
Hbv Dna ◽  
Wild Type ◽  
Pcr Assay ◽  
B Virus

ABSTRACT Entecavir (ETV) is a deoxyguanosine analog approved for use for the treatment of chronic infection with wild-type and lamivudine-resistant (LVDr) hepatitis B virus (HBV). In LVD-refractory patients, 1.0 mg ETV suppressed HBV DNA levels to below the level of detection by PCR (<300 copies/ml) in 21% and 34% of patients by Weeks 48 and 96, respectively. Prior studies showed that virologic rebound due to ETV resistance (ETVr) required preexisting LVDr HBV reverse transcriptase substitutions M204V and L180M plus additional changes at T184, S202, or M250. To monitor for resistance, available isolates from 192 ETV-treated patients were sequenced, with phenotyping performed for all isolates with all emerging substitutions, in addition to isolates from all patients experiencing virologic rebounds. The T184, S202, or M250 substitution was found in LVDr HBV at baseline in 6% of patients and emerged in isolates from another 11/187 (6%) and 12/151 (8%) ETV-treated patients by Weeks 48 and 96, respectively. However, use of a more sensitive PCR assay detected many of the emerging changes at baseline, suggesting that they originated during LVD therapy. Only a subset of the changes in ETVr isolates altered their susceptibilities, and virtually all isolates were significantly replication impaired in vitro. Consequently, only 2/187 (1%) patients experienced ETVr rebounds in year 1, with an additional 14/151 (9%) patients experiencing ETVr rebounds in year 2. Isolates from all 16 patients with rebounds were LVDr and harbored the T184 and/or S202 change. Seventeen other novel substitutions emerged during ETV therapy, but none reduced the susceptibility to ETV or resulted in a rebound. In summary, ETV was effective in LVD-refractory patients, with resistant sequences arising from a subset of patients harboring preexisting LVDr/ETVr variants and with approximately half of the patients experiencing a virologic rebound.

scholarly journals Comparison of Anti-Hepatitis B Virus Activities of Lamivudine and Clevudine by a Quantitative Assay

2003 ◽  
Vol 47 (1) ◽  
pp. 324-336 ◽  
Author(s):  
Ayman M. Abdelhamed ◽  
Colleen M. Kelley ◽  
Thomas G. Miller ◽  
Phillip A. Furman ◽  
Edward E. Cable ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Treatment ◽  
Hbv Dna ◽  
Hbv Infection ◽  
Extracellular Dna ◽  
Quantitative Assay ◽  
Hbv Replication ◽  
B Virus

ABSTRACT In this study, we used a quantitative assay to measure the concentration-dependent effects of antivirals on extracellular hepatitis B virus (HBV) DNA as well as on different cytoplasmic and nuclear forms of HBV DNA that participate in HBV replication. HBV recombinant baculovirus, which efficiently delivers the HBV genome to HepG2 cells, was used for this study because (i) antivirals can be administered prior to initiation of HBV infection or after HBV infection and (ii) sufficiently high HBV replication levels are achieved that HBV covalently closed circular (CCC) DNA can be easily detected and individual HBV DNA species can be quantitatively analyzed separately from total HBV DNA. The results showed that the levels of HBV replicative intermediate and extracellular DNA decreased in a concentration-dependent fashion following antiviral treatment. The 50% effective concentration (EC50) and EC90 values and the Hill slopes differed for the different HBV DNA species analyzed. The data clearly indicated that (i) nuclear HBV DNAs are more resistant to antiviral therapy than cytoplasmic or extracellular HBV DNAs and (ii) nuclear HBV CCC DNA is more resistant than the nuclear relaxed circular form. This report presents the first in vitro comparison of the effects of two antivirals administered prior to initiation of HBV infection and the first thorough in vitro quantitative study of concentration-dependent antiviral effects on HBV CCC DNA.

Hepatitis B virus (HBV) DNA integration is not driven by viral proteins

2019 ◽  
Author(s):  
T Tu ◽  
B Zehnder ◽  
M Levy ◽  
G Micali ◽  
L Tran ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Proteins ◽  
Hbv Dna ◽  
Dna Integration ◽  
B Virus ◽  
Hbv Dna Integration

scholarly journals Hepatitis B Virus DNA Integration, Chronic Infections and Hepatocellular Carcinoma

2021 ◽  
Vol 9 (8) ◽  
pp. 1787
Author(s):  
Maria Bousali ◽  
George Papatheodoridis ◽  
Dimitrios Paraskevis ◽  
Timokratis Karamitros
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Molecular Mechanisms ◽  
Horizontal Transmission ◽  
Acute Infection ◽  
Hbv Dna ◽  
Clinical Aspects ◽  
Dna Integration ◽  
B Virus ◽  
Hbv Dna Integration

Hepatitis B Virus (HBV) is an Old World virus with a high mutation rate, which puts its origins in Africa alongside the origins of Homo sapiens, and is a member of the Hepadnaviridae family that is characterized by a unique viral replication cycle. It targets human hepatocytes and can lead to chronic HBV infection either after acute infection via horizontal transmission usually during infancy or childhood or via maternal–fetal transmission. HBV has been found in ~85% of HBV-related Hepatocellular Carcinomas (HCC), and it can integrate the whole or part of its genome into the host genomic DNA. The molecular mechanisms involved in the HBV DNA integration is not yet clear; thus, multiple models have been described with respect to either the relaxed-circular DNA (rcDNA) or the double-stranded linear DNA (dslDNA) of HBV. Various genes have been found to be affected by HBV DNA integration, including cell-proliferation-related genes, oncogenes and long non-coding RNA genes (lincRNAs). The present review summarizes the advances in the research of HBV DNA integration, focusing on the evolutionary and molecular side of the integration events along with the arising clinical aspects in the light of WHO’s commitment to eliminate HBV and viral hepatitis by 2030.

scholarly journals Cellular Genomic Sites of Hepatitis B Virus DNA Integration

Genes ◽  
2018 ◽  
Vol 9 (7) ◽  
pp. 365 ◽  
Author(s):  
Magdalena A. Budzinska ◽  
Nicholas A. Shackel ◽  
Stephan Urban ◽  
Thomas Tu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Insertional Mutagenesis ◽  
Current Knowledge ◽  
Hbv Dna ◽  
Dna Integration ◽  
Open Questions ◽  
Cellular Genes ◽  
B Virus ◽  
Hbv Dna Integration

Infection with the Hepatitis B Virus (HBV) is one of the strongest risk-factors for liver cancer (hepatocellular carcinoma, HCC). One of the reported drivers of HCC is the integration of HBV DNA into the host cell genome, which may induce pro-carcinogenic pathways. These reported pathways include: induction of chromosomal instability; generation of insertional mutagenesis in key cancer-associated genes; transcription of downstream cancer-associated cellular genes; and/or formation of a persistent source of viral protein expression (particularly HBV surface and X proteins). The contribution of each of these specific mechanisms towards carcinogenesis is currently unclear. Here, we review the current knowledge of specific sites of HBV DNA integration into the host genome, which sheds light on these mechanisms. We give an overview of previously-used methods to detect HBV DNA integration and the enrichment of integration events in specific functional and structural cellular genomic sites. Finally, we posit a theoretical model of HBV DNA integration during disease progression and highlight open questions in the field.

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