Deep sequencing of hepatitis B virus basal core promoter and precore mutants in HBeAg-positive chronic hepatitis B patients

Yan, Linlin; Zhang, Henghui; Ma, Hui; Liu, Di; Li, Wei; Kang, Yulin; Yang, Ruifeng; Wang, Jianghua; He, Gaixia; Xie, Xingwang; Wang, Hao; Wei, Lai; Lü, Zeng; Shao, Qixiang; Chen, Hongsong

doi:10.1038/srep17950

Cited by 20 publications

(16 citation statements)

References 34 publications

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“…Other studies, however, have reported inconclusive results regarding the effect of BCP mutations on HBV replication . These heterogenic results may be explained by testing patients with different disease phases, genotype distributions, and frequency of BCP mutants . It is yet unclear which mechanisms would explain lower HBV‐DNA and HBV RNA level in presence of BCP mutations, but the key may be in HBV RNA packaging.…”

Section: Discussionmentioning

confidence: 99%

Host and viral factors associated with serum hepatitis B virus RNA levels among patients in need for treatment

et al. 2018

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Hepatitis B virus (HBV) RNA in serum is a novel biomarker for intrahepatic HBV replication and treatment response. For its proper use, it is essential to identify factors influencing serum HBV RNA level. Using a rapid amplification of complimentary DNA (cDNA) ends (RACE) PCR technique (lower limit of detection [LLD], 800 copies/mL [c/mL]), serum HBV RNA levels were measured in samples of 488 untreated individuals with chronic HBV infection who were eligible to treatment according to currently used recommendations. We explored the association of serum levels of HBV RNA with patient‐ and virus‐associated factors. HBV genotype distribution was 21/10/20/46/3% for A/B/C/D/other. Mean HBV RNA serum level was 5.9 (1.6) log10 c/mL (hepatitis B e antigen [HBeAg]‐positive chronic hepatitis B [CHB], 6.5 [1.2] log c/mL; HBeAg‐negative CHB, 4.1 [1.2] log c/mL; P < 0.001). By multivariable linear regression, factors associated with lower HBV RNA level were HBeAg negativity (β = –0.69; P < 0.001), HBV genotypes A (β = –0.13; P = 0.002), B (β = –0.07; P = 0.049), and C (β = –0.61; P < 0.001) in comparison to D, and presence of HBV basal core promoter mutation either alone (β = –0.14; P = 0.001) or in combination with precore mutation (β = –0.22; P < 0.001). Higher serum alanine aminotransferase (ALT) was associated with higher HBV RNA (β = 0.23; P < 0.001). HBV RNA correlated strongly with HBV DNA (HBeAg‐pos, r = 0.72; P < 0.001; HBeAg‐neg, r = 0.78; P < 0.001) and moderately with quantitative hepatitis B surface antigen (qHBsAg; HBeAg‐pos, r = 0.54; P < 0.001; HBeAg‐neg, r = 0.19; P = 0.04) and quantitative hepatitis B surface antigen (qHBeAg; r = 0.41; P < 0.001). Conclusion: In this multiethnic cohort of 488 untreated individuals with CHB, factors associated with serum HBV RNA level were HBeAg status, serum ALT, HBV genotype, and presence of basal core promotor mutations. For the future use of serum HBV RNA as a clinical marker, it seems mandatory to take these factors into consideration. (Hepatology 2018).

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Section: Discussionmentioning

confidence: 99%

Host and viral factors associated with serum hepatitis B virus RNA levels among patients in need for treatment

et al. 2018

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“…Protons released during nucleotide incorporation are detected using an ion sensor, which can measure slight shifts in pH ( Merriman et al, 2012 , Reuter et al, 2015 ). Rapid sequencing runs make these sequencers particularly useful for the targeted detection of viruses in clinical samples like HIV Archer et al, 2012 , Chang et al, 2013 , Gibson et al, 2014 ), hepatitis B virus ( Yan et al, 2015 ), HCV ( Gaspareto et al, 2016 , Marascio et al, 2016 ), and the rapid genome sequencing of several viruses including the toscana virus ( Nougairede et al, 2013 ), polyomavirus ( Anthony et al, 2013 ), porcine reproductive and respiratory syndrome virus ( Kvisgaard et al, 2013 ), orthoreovirus ( Steyer et al, 2013 ), bluetongue virus ( Lorusso et al, 2014 ), rotavirus ( Ndze et al, 2014 ; Nyaga et al, 2014 ), influenza virus ( Van den Hoecke et al, 2015 ) etc. Although, some studies used this technology to study viromes in skin ( Bzhalava et al, 2013 ), ticks ( Tokarz et al, 2014 ; Xia et al, 2015 ), gut virome in piglets ( Karlsson et al, 2016 ) and seals ( Kluge et al, 2016 ), this platform is not the ideal choice for virome study in human clinical samples due to lower outputs.…”

Section: Evolution and Use Of Sequencing Technologies For Virus Metagmentioning

confidence: 99%

Evolution of selective-sequencing approaches for virus discovery and virome analysis

2017

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Recent advances in sequencing technologies have transformed the field of virus discovery and virome analysis. Once, mostly confined to the traditional Sanger sequencing based individual virus discovery, is now entirely replaced by high throughput sequencing (HTS) based virus metagenomics that can be used to characterize the nature and composition of entire viromes. To better harness the potential of HTS for study of viromes, sample preparation methodologies used different refinements to exclude amplification of non-viral components that can overshadow low-titer viruses. These virus-sequence enrichment approaches mostly focused on the sample preparation methods, like enzymatic digestion of non-viral nucleic acids and size exclusion of non-viral constituents by column filtration, ultrafiltration or density centrifugation. However, recently an approach of virus-sequence enrichment called virome-capture sequencing, focused on the amplification or HTS library preparation stage, was shown to increase the ability of virome characterization. This new approach has the potential to further transform the field of virus discovery and virome analysis, but its technical complexity and sequence-dependence warrants further improvements. In this review we have listed the different methods, their applications and evolution, for selective sequencing based virome analysis and also the major refinements needed to harness the full potential of HTS for virome analysis.

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“…HBV-related oncogenesis is complex and is influenced by viral characteristics such as genetic variants particularly within the X/basal core promoter (BCP)/pre-core (PC) region and integration into the host chromosomes contributing to genomic instability and hepatocarcinogenesis. Through next-generation sequencing (NGS), our group and others have demonstrated the variability of HBV within CHB carriers either with or without end-stage liver disease (cirrhosis and cancer) ( Yan et al, 2015 ; Lau et al, 2018 ; Wu et al, 2018 ). Moreover, we have shown HBV genome integration in both liver and lymphoid cells in individuals with hepatic and extrahepatic malignancy ( Lau et al, 2019 , 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Differences in HBV Replication, APOBEC3 Family Expression, and Inflammatory Cytokine Levels Between Wild-Type HBV and Pre-core (G1896A) or Basal Core Promoter (A1762T/G1764A) Mutants

et al. 2020

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Background: Chronic hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC) worldwide. HBV variants, particularly the G1896A precore (PC) and A1762T/G1764A basal core promoter (BCP) mutations, are established risk factors for cirrhosis and HCC, but the molecular biological basis is unclear. We hypothesized that these variants result in differential HBV replication, APOBEC3 family expression, and cytokine/chemokine expression. Methods: HepG2 cells were transfected with monomeric full-length containing wildtype, PC, or BCP HBV. Cells and supernatant were collected to analyze viral infection markers (i.e., HBsAg, HBeAg, HBV DNA, and RNA). Cellular APOBEC3 expression and activity was assessed by quantitative real-time (qRT)-PCR, immunoblot, differential DNA denaturation PCR, and sequencing. Cytokine/chemokines in the supernatant and in serum from 11 CHB carriers (4 non-cirrhotic; 7 cirrhotic and/or HCC) with predominantly wild-type, PC, or BCP variants were evaluated by Luminex. Results: HBeAg expression was reduced in PC and BCP variants, and higher supernatant HBV DNA and HBV RNA levels were found with A1762T/G1764A vs. G1896A mutant (p < 0.05). Increased APOBEC3G protein levels in wild-type vs. mutant were not associated with HBV covalently closed circular DNA G-to-A hypermutations. Differences in cytokine/chemokine expression in culture supernatants, especially IL-13 were observed amongst the variants analyzed. Noticeable increases of numerous

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Deep sequencing of hepatitis B virus basal core promoter and precore mutants in HBeAg-positive chronic hepatitis B patients

Cited by 20 publications

References 34 publications

Host and viral factors associated with serum hepatitis B virus RNA levels among patients in need for treatment

Host and viral factors associated with serum hepatitis B virus RNA levels among patients in need for treatment

Evolution of selective-sequencing approaches for virus discovery and virome analysis

Differences in HBV Replication, APOBEC3 Family Expression, and Inflammatory Cytokine Levels Between Wild-Type HBV and Pre-core (G1896A) or Basal Core Promoter (A1762T/G1764A) Mutants

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