scholarly journals Epigenetic heterogeneity after de novo assembly of native full-length Hepatitis B Virus genomes

2020 ◽  
Author(s):  
Chloe Goldsmith ◽  
Damien Cohen ◽  
Anaëlle Dubois ◽  
Maria-Guadalupe Martinez ◽  
Kilian Petitjean ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
De Novo ◽  
Expression Patterns ◽  
Cellular Transformation ◽  
Hbv Dna ◽  
World Health ◽  
Nanopore Sequencing ◽  
Increased Risk ◽  
B Virus

AbstractHepatitis B Virus (HBV) is a 3.2KB DNA virus that causes acute and chronic hepatitis. HBV infection is a world health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). Methylation of HBV DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues to why certain infections are cleared, or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. With cas9 guided RNPs to specifically target the HBV genome, we enriched in HBV DNA from Primary Human Hepatocytes (PHH) infected with different HBV genotypes, as well as enriching in HBV from infected patient liver tissue; followed by sequencing with Oxford Nanopore Technologies MinION. Detection of 5mCpG by Nanopore sequencing was benchmarked with Bisulfite-quantitative Methyl Specific PCR (BS-qMSP). 5mCpG levels in HBV determined by BS-qMSP and Nanopore sequencing were highly correlated. Our Nanopore sequencing approach achieved a coverage of ∼2000x of HBV depending on infection efficacy, sufficient coverage to perform a de novo assembly and detect small fluctuations in HBV methylation, providing the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences. Moreover, by capturing entire HBV genomes, we explored the epigenetic heterogeneity of HBV in infected patients and identified 4 epigenetically distinct clusters based on methylation profiles. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species and will serve as a valuable tool for infectious disease monitoring.

scholarly journals Cas9-targeted nanopore sequencing reveals epigenetic heterogeneity after de novo assembly of native full-length hepatitis B virus genomes

2021 ◽  
Vol 7 (5) ◽  
Author(s):  
Chloe Goldsmith ◽  
Damien Cohen ◽  
Anaëlle Dubois ◽  
Maria Guadalupe Martinez ◽  
Kilian Petitjean ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
De Novo ◽  
Expression Patterns ◽  
Cellular Transformation ◽  
Hbv Dna ◽  
Nanopore Sequencing ◽  
Infected People ◽  
Increased Risk ◽  
B Virus

Hepatitis B virus (HBV) contains a 3.2 kb DNA genome and causes acute and chronic hepatitis. HBV infection is a global health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). Methylation of HBV DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues as to why certain infections are cleared or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. With Cas9-guided RNPs to specifically target the HBV genome, we enriched in HBV DNA from primary human hepatocytes (PHHs) infected with different HBV genotypes, as well as enriching in HBV from infected patient liver tissue, followed by sequencing with Oxford Nanopore Technologies MinION. Detection of 5mCpG by nanopore sequencing was benchmarked with bisulfite-quantitative methyl-specific qPCR (BS-qMSP). The 5mCpG levels in HBV determined by BS-qMSP and nanopore sequencing were highly correlated. Our nanopore sequencing approach achieved a coverage of ~2000× of HBV depending on infection efficiency, sufficient coverage to perform a de novo assembly and detect small fluctuations in HBV methylation, providing the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences. Moreover, by capturing entire HBV genomes, we explored the epigenetic heterogeneity of HBV in infected patients and identified four epigenetically distinct clusters based on methylation profiles. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species and will serve as a valuable tool for infectious disease monitoring.

scholarly journals Severe de novo Hepatitis B Recovered from Late-Onset Liver Insufficiency with Prolonged Ascites and Hypoalbuminemia due to Hepatitis B Virus Genotype Bj with Precore Mutation

2016 ◽  
Vol 10 (3) ◽  
pp. 553-559 ◽  
Author(s):  
Akira Sato ◽  
Toshiya Ishii ◽  
Fumiaki Sano ◽  
Takayuki Yamada ◽  
Hideaki Takahashi ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
De Novo ◽  
Liver Volume ◽  
B Cell Lymphoma ◽  
Hbv Dna ◽  
Hepatocellular Damage ◽  
Precore Mutation ◽  
B Virus ◽  
De Novo Hepatitis B

De novo hepatitis B is associated with a high risk of hepatic failure often resulting in fatal fulminant hepatitis even when nucleotide analogues are administered. A 77-year-old female developed de novo hepatitis B after R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone) treatment for diffuse large B-cell lymphoma. Hepatitis B virus (HBV) isolated from the patient was of genotype Bj, with a precore mutation (G1896A) exhibiting an extremely high viral load at the onset of hepatitis. She showed markedly high levels of transaminase with mild jaundice on admission and rapid decrease of prothrombin activity after admission. Although acute liver failure was averted by the administration of entecavir and corticosteroid pulse therapy, liver volume decreased to 860 ml, and marked hypoalbuminemia accompanying massive ascites occurred 2 months after the onset of hepatitis and persisted for 3 months with high levels of HBV DNA and mild abnormal alanine aminotransferase levels. Frequent infusions of albumin solution, nutrition support, and alleviation therapy showed limited effect. However, overall improvement along with HBV DNA reduction was observed after increasing the dose of entecavir and completion of prednisolone that was administered with a minimum dose for adrenal insufficiency. An immediate and sufficient suppression of virus replication with potent antiviral therapy is critical, particularly in patients infected with HBV precore mutation (G1896A) and/or Bj genotype, which may have a high viral replication and direct hepatocellular damage.

scholarly journals Is it Safe for a Mother Infected with Hepatitis B Virus to Breastfeed Her Baby?

2012 ◽  
Vol 35 (1) ◽  
pp. 20-25
Author(s):  
ASM Nawshad Uddin Ahmed ◽  
Md Mahbubul Hoque
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Perinatal Transmission ◽  
Hepatitis B Vaccine ◽  
Hbv Infection ◽  
World Health ◽  
Increased Risk ◽  
Major Mode ◽  
B Virus ◽  
The World

One third of the world’s population has been infected by the hepatitis B virus (HBV), causing an enormous burden of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Hepatitis B virus is transmitted through contact with blood and blood products, by sexual contact, through close contact between children (horizontal transmission), or by perinatal transmission from a carrier mother to her baby. In Asia, perinatal transmission is the major mode of transmission and those who become infected perinatally with HBV are most likely to develop chronic infection. The question of whether breastfeeding by HBV-positive mothers is an additional mechanism by which infants may acquire HBV infection, has been asked for many years. Although small amounts of hepatitis B surface antigen (HBsAg) have been detected in some samples of breast milk, there is no evidence that breastfeeding by HBV-carrier mothers increase the risk of mother-to-child transmission of HBV. Infants born to known hepatitis B positive women should receive hepatitis B immune globulin (HBIG) and hepatitis B vaccine, effectively eliminating any theoretical risk of transmission through breastfeeding. However, neither screening of pregnant women for HBV infection nor use of HBIG is feasible in most developing countries. Routine immunization of infants with hepatitis B vaccine is therefore recommended by the World Health Organization. Bangladesh has already included hepatitis B vaccine as part of routine childhood immunization in EPI program since 2003. Also the risk must be balanced against the increased risk of morbidity and mortality due to malnutrition and diarrheal or other infectious diseases associated with replacement feeding. Malnutrition is responsible, directly or indirectly, for 6.5 million under 5 deaths annually. Thus, even where HBV infection is highly endemic and immunization against HBV is not available, breastfeeding remains the recommended method of feeding. DOI: http://dx.doi.org/10.3329/bjch.v35i1.10369 BJCH 2011; 35(1): 20-25

scholarly journals Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Angela M. Lam ◽  
Christine Espiritu ◽  
Robert Vogel ◽  
Suping Ren ◽  
Vincent Lau ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Activity ◽  
Viral Replication ◽  
De Novo ◽  
Hbv Dna ◽  
Cross Resistance ◽  
Capsid Assembly ◽  
B Virus

ABSTRACT NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo. The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.

scholarly journals Attitude and Vaccination Status of Healthcare Workers against Hepatitis B Infection in a Teaching Hospital, Ethiopia

Scientifica ◽  
2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Mohammed Akibu ◽  
Sodere Nurgi ◽  
Mesfin Tadese ◽  
Wendwesen Dibekulu Tsega
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
General Hospital ◽  
Health Professionals ◽  
Healthcare Workers ◽  
Vaccination Status ◽  
World Health ◽  
Medical College ◽  
Increased Risk ◽  
B Virus

Background. World Health Organization and Centers for Disease Control and Prevention recommend all health professionals to get vaccinated against hepatitis B virus before they start the clinical attachments during their stay in the medical school. However, only 18–39% of healthcare workers in low- and middle-income countries received the vaccine. Therefore, this study aims to determine the attitude and vaccination status of health professionals working at Adama General Hospital and Medical College.Methods. An institution-based cross-sectional study was conducted from December 2016 to February 2017 with 403 health professionals working at Adama General Hospital and Medical College. Data were collected using self-administered questionnaire distributed at the participant’s work unit and analyzed using SPSS version 20. Multiple logistic regression analysis was conducted to identify factors that affect the complete vaccination status andpvalue < 0.05 was considered statistically significant.Result. The prevalence of complete vaccination against hepatitis B virus was 25.6%. The most frequently mentioned reasons for not being vaccinated were high cost of the vaccine (41%) and unavailability of the vaccine (36%). More than three-fourths (77.8%) of study participants strongly agreed that hepatitis B is a major public health threat and there was tendency among participants to believe that their profession will put them at increased risk of acquiring the disease (strongly agreed: 75.9%). Attending infection-prevention training [AOR = 2.3; 95% CI, 1.24–6.31], history of exposure to risky behavior [AOR = 5.5; 95% CI, 2.86–9.29], and long years of work experience [AOR = 3.1; 95% CI, 1.98–5.24] were statistically significant with complete vaccination status.Conclusion. Only one-quarter of health professionals received the recommended full dose of the vaccine. Sustained hepatitis B vaccination programs for healthcare workers need to be established by collaboration of different stakeholders to optimize health professionals’ safety against this contagious infection.

Frequency and load of hepatitis B virus DNA in first-time blood donors with antibodies to hepatitis B core antigen

Blood ◽  
2002 ◽  
Vol 100 (7) ◽  
pp. 2637-2641 ◽  
Author(s):  
Holger Hennig ◽  
Ines Puchta ◽  
Jürgen Luhm ◽  
Peter Schlenke ◽  
Siegfried Goerg ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Blood Donors ◽  
Hbv Dna ◽  
World Health ◽  
Core Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Blood Donations ◽  
B Virus ◽  
First Time

The objective of this study was to determine the frequency and load of hepatitis B virus (HBV) DNA in anti-HBc–positive first-time blood donors; it was designed to contribute to determining whether anti-HBc screening of blood donations might reduce the residual risk of posttransfusion HBV infection. A total of 14 251 first-time blood donors were tested for anti-HBc using a microparticle enzyme immunoassay; positive results were confirmed by a second enzyme-linked immunosorbent assay (ELISA). For the detection of HBV DNA from plasma samples, we developed a novel and highly sensitive real-time polymerase chain reaction (PCR) assay. The 95% detection limit of the method amounted to 27.8 IU/mL, consistent with the World Health Organization (WHO) international standard for HBV DNA. A total of 216 blood donors (1.52%) tested anti-HBc–positive in both tests, and 205 of them (16 HBsAg+, 189 HBsAg−) were tested for HBV DNA. In 14 (87.5%) of the HBsAg-positive blood donors, HBV DNA was repeatedly detected, and in 3 (1.59%) of the HBsAg-negative donors, HBV DNA was also found repeatedly. In the 3 HBV DNA–positive, HBsAg-negative cases, anti-HBe and anti-HBs (> 100 IU/L) were also detectable. HBV DNA in HBsAg-negative as well as HBsAg-positive samples was seen at a low level. Thus, HBV DNA is sometimes found in HBsAg-negative, anti-HBc–positive, and anti-HBs–positive donors. Retrospective studies on regular blood donors and recipients are necessary to determine the infection rate due to those donations. Routine anti-HBc screening of blood donations could probably prevent some transfusion-transmitted HBV infections.

scholarly journals Complete Genome Sequence of the WHO International Standard for Hepatitis B Virus DNA

2017 ◽  
Vol 5 (7) ◽  
Author(s):  
Adrian Jenkins ◽  
Rehan Minhas ◽  
Clare Morris ◽  
Neil Berry
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Hbv Dna ◽  
Nucleic Acid Amplification ◽  
World Health ◽  
International Standard ◽  
B Virus

ABSTRACT The World Health Organization (WHO) international standard (IS) for hepatitis B virus (HBV) DNA for use in nucleic acid amplification assays was characterized by determining the complete genome sequence, which was assigned genotype A. This information will aid the design, development, and evaluation of HBV DNA amplification assays.

scholarly journals Possible role of tocopherols in the modulation of host microRNA with potential antiviral activity in patients with hepatitis B virus-related persistent infection: a systematic review

2014 ◽  
Vol 112 (11) ◽  
pp. 1751-1768 ◽  
Author(s):  
S. Fiorino ◽  
L. Bacchi-Reggiani ◽  
S. Sabbatani ◽  
F. Grizzi ◽  
L. di Tommaso ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Activity ◽  
Hbv Infection ◽  
Cochrane Library ◽  
Viral Pathogen ◽  
Increased Risk ◽  
B Virus ◽  
Chronic Hbv

Hepatitis B virus (HBV) infection represents a serious global health problem and persistent HBV infection is associated with an increased risk of cirrhosis, hepatocellular carcinoma and liver failure. Recently, the study of the role of microRNA (miRNA) in the pathogenesis of HBV has gained considerable interest as well as new treatments against this pathogen have been approved. A few studies have investigated the antiviral activity of vitamin E (VE) in chronic HBV carriers. Herein, we review the possible role of tocopherols in the modulation of host miRNA with potential anti-HBV activity. A systematic research of the scientific literature was performed by searching the MEDLINE, Cochrane Library and EMBASE databases. The keywords used were ‘HBV therapy’, ‘HBV treatment’, ‘VE antiviral effects’, ‘tocopherol antiviral activity’, ‘miRNA antiviral activity’ and ‘VE microRNA’. Reports describing the role of miRNA in the regulation of HBV life cycle,in vitroandin vivoavailable studies reporting the effects of VE on miRNA expression profiles and epigenetic networks, and clinical trials reporting the use of VE in patients with HBV-related chronic hepatitis were identified and examined. Based on the clinical results obtained in VE-treated chronic HBV carriers, we provide a reliable hypothesis for the possible role of this vitamin in the modulation of host miRNA profiles perturbed by this viral pathogen and in the regulation of some cellular miRNA with a suggested potential anti-HBV activity. This approach may contribute to the improvement of our understanding of pathogenetic mechanisms involved in HBV infection and increase the possibility of its management and treatment.

272 Use of LioCyx-M, autologous hepatitis B virus (HBV)-Specific T cell receptor (TCR) T-cells, in advanced HBV-related hepatocellular carcinoma (HCC)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A297-A297
Author(s):  
Fu-Sheng Wang ◽  
Fanping Meng ◽  
Jiehua Jin ◽  
Yuanyuan Li ◽  
Regina Wanju Wong ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Hepatitis B Virus ◽  
T Cell ◽  
Hepatitis B ◽  
T Cell Receptor ◽  
Dose Level ◽  
Cell Receptor ◽  
Hbv Dna ◽  
B Virus

BackgroundWe have demonstrated the ability of Hepatitis-B-virus (HBV)-specific T cell receptor (TCR) bioengineered T cells to recognize and lyse Hepatocellular carcinoma (HCC) cells expressing HBV antigens derived from HBV-DNA integration in patients with liver transplant.1 LioCyx-M is an immunotherapeutic product composing of autologous T cells transiently modified with in-vitro transcribed mRNA encoding HBV-specific TCR. The transient TCR expression makes LioCyx -M amenable to a dose escalating posology.MethodsThe primary endpoint of this phase 1 trial is to assess the safety and tolerability of LioCyx-M in patients with advanced HBV-HCC without curative treatment options. Eligible patients were diagnosed with Barcelona clinic liver cancer stage B or C HCC (Child-Pugh < 7 points), receiving >1 year antiviral treatment prior to enrollment. These patients had matching HLA class I genotypes which present HBV encoded antigen. Peripheral blood was collected from each patient prior to each dose for LioCyx-M manufacturing. Patients received 4 escalating doses of 1×104 cells/kg, 1×105 cells/kg, 1×106 cells/kg, 5×106 cells/kg bodyweight (BW) in the first treatment cycle, each intravenously administered weekly. Patients underwent 1-month safety assessment post the 4th infusion, according to Common Terminology NCI CTCAE Version 4.0.3. If there were no dose associated toxicities, patients were eligible to continue administration of LioCyx-M at dose of 5 × 106 cells/kg BW weekly. Tumor response per RECIST 1.1 criteria and survival time were assessed.ResultsAt data cutoff (30 April 2020), eight patients were enrolled, with a median age of 53 (range: 49 - 67). These patients received a median number of 6 (range: 4 - 12) infusions of LioCyx-M. 1 patient developed Grade 3 elevations in alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST) and bilirubin after receiving LioCyx-M at dose level of 1×105 cells/kg BW. Another patient had Grade 1 transient fever after receiving LioCyx-M at dose level 5×106 cells/kg BW in the 4th, 5th and 6th infusions. No treatment-related adverse events (trAEs) such as cytokine release syndrome or neurotoxicity were observed. No fatal trAEs were observed. The median time to progression was 1.9 months (range: 0.2 - 9.5 months). The median overall survival was 34 months (range: 3 - 38.2 months).ConclusionsThe encouraging clinical outcome and tolerable safety highlight the good benefit-risk profile of LioCyx-M. Therefore, further exploration of efficacy of LioCyx-M treatment for advanced HBV-HCC is warranted in a Phase 2 proof-of-concept clinical study.AcknowledgementsFunding: Lion TCR.Trial RegistrationNCT03899415Ethics ApprovalThe study was approved by Fifth Medical Center of Chinese PLA General Hospital’s Ethics Board, approval number R2016185DI010.ReferenceTan AT, Yang N, Lee Krishnamoorthy T, et al. Use of Expression Profiles of HBV-DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy. Gastroenterology 2019;156(6):1862–1876.e9.

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