| 1. |
- Ringlander, Johan, et al.
(författare)
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Hepatitis B virus particles in serum contain minus strand DNA and degraded pregenomic RNA of variable and inverse lengths
- 2024
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Ingår i: LIVER INTERNATIONAL. - 1478-3223 .- 1478-3231.
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Tidskriftsartikel (refereegranskat)abstract
- This study utilized digital PCR to quantify HBV RNA and HBV DNA within three regions of the HBV genome. Analysis of 75 serum samples from patients with chronic infection showed that HBV RNA levels were higher in core than in S and X regions (median 7.20 vs. 6.80 and 6.58 log copies/mL; p < .0001), whereas HBV DNA levels showed an inverse gradient (7.71 vs. 7.73 and 7.77 log copies/mL, p < .001). On average 80% of the nucleic acid was DNA by quantification in core. The core DNA/RNA ratio was associated with viral load and genotype. In individual patients, the relations between RNA levels in core, S and X were stable over time (n = 29; p = .006). The results suggest that pregenomic RNA is completely reverse transcribed to minus DNA in approximate to 75% of the virus particles, whereas the remaining 25% contain both RNA and DNA of lengths that reflect variable progress of the polymerase.
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| 2. |
- Larsson, Simon B., et al.
(författare)
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HBsAg quantification for identification of liver disease in chronic hepatitis B virus carriers
- 2014
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Ingår i: Liver International. - : Wiley. - 1478-3223. ; 34:7
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Tidskriftsartikel (refereegranskat)abstract
- Background & Aims: Quantification of hepatitis B surface antigen (HBsAg) has been proposed as a useful diagnostic marker for clinical staging (identification of inactive carrier state) and prognosis of chronic hepatitis B virus (HBV) infection. The aim of this study was to investigate the correlation between HBsAg levels in serum and histological liver damage in patients with chronic infection. Methods: HBsAg levels in serum (by Abbott Architect) were related to HBV DNA, ALT and histological score (n = 160) and covalently closed circular DNA (cccDNA) (n = 84). Results: HBsAg levels correlated with cccDNA, serum HBV DNA, ALT and high inflammation scores (P < 0.001). Among HBeAg-negative patients, an HBsAg level below 3.0 log(10) IU/ml identified minimal liver damage (normal ALT and mild inflammation) with a predictive value of 92% (alone) or 96% (in combination with HBV DNA <4.0 log(10) copies/ml), whereas an HBsAg level above 3.5 log(10) IU/ml identified severe inflammation with a predictive value of 16% (alone) or 33% (in combination with HBV DNA >5.0 log(10) copies/ml). Conclusions: HBsAg levels reflect clinical stage and liver disease, and a combined quantification of HBsAg and HBV DNA may improve clinical staging.
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| 3. |
- Larsson, Simon B., et al.
(författare)
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Mechanisms downstream of reverse transcription reduce serum levels of HBV DNA but not of HBsAg in chronic hepatitis B virus infection
- 2015
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Ingår i: Virology Journal. - : Springer Science and Business Media LLC. - 1743-422X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Hepatitis B virus (HBV) DNA in serum of chronically infected patients declines by 3-4 log(10) units at loss of HBe antigen (HBeAg) from serum. The mechanisms behind this decline, and the much smaller decline of surface antigen (HBsAg) levels, are still not well known. The aim of this study was to get a better understanding of this process by analysing both serum and intrahepatic markers of HBV replication. Methods: Levels of HBV DNA and HBsAg in serum, and covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) and S-RNA and total intrahepatic HBV DNA (ihDNA) in liver biopsies from 84 chronically infected patients (16 positive and 68 negative for HBeAg) were analysed. Results: Lower HBV DNA levels within HBeAg-positive stage reflected lower levels of cccDNA and pgRNA with strong correlation. In HBeAg-negative patients, ihDNA levels were greater and HBV DNA levels in serum lower than expected from pgRNA levels. A lower HBV DNA/HBsAg ratio corresponded with lower pgRNA/cccDNA (p < 0.01) and higher S-RNA/cccDNA (p < 0.0001) ratios, suggesting that in HBeAg-negative patients transcription of pgRNA, but not of S-RNA, becomes suppressed. Conclusions: The marked reduction of HBV DNA in serum after loss of HBeAg appears to be due to combined reduction of cccDNA, pgRNA and yet unidentified mechanisms downstream of reverse transcription. Such mechanisms include faster clearance of circulating virus or blocked secretion of virions, the latter supported by the observed relative increase of ihDNA in HBeAg-negative patients. The smaller reduction of S-RNA than of pgRNA partly explains why HBsAg remain high in the HBeAg-negative stage, supporting the possibility of HBsAg synthesis from integrated HBV DNA.
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| 4. |
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| 5. |
- Malmström, Sebastian, 1976, et al.
(författare)
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Genotype impact on long-term virological outcome of chronic hepatitis B virus infection
- 2012
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Ingår i: Journal of Clinical Virology. - : Elsevier BV. - 1386-6532. ; 54:4, s. 321-326
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Tidskriftsartikel (refereegranskat)abstract
- Background: The importance of hepatitis B virus (HBV) genotype on the clinical course of chronic HBV infection is not yet clarified. Objectives: To investigate genotype impact on long- term virological outcome of chronic HBV infection. Study design: HBsAg, HBeAg, ALT and HBV DNA levels were determined after a median of 9.2 years of follow-up of 124 adults with chronic HBV infection, of whom 33 were HBeAg-positive at inclusion. Results: HBV DNA levels decreased significantly in patients carrying genotype A (n = 28), B (n = 21) or D (n = 63), but not in those with genotype C infection (n = 12). Loss of HBeAg was seen in 44% (4/9) of patients with genotype C, as compared with 92% (22/24) with non-C genotypes. Loss of HBsAg was seen in 36% (10/28) patients with genotype A, 5% (1/21) with B, 0% (0/12) with C, and 11% (7/63) with genotype D. Conclusions: HBV DNA levels decreased over time in patients infected with genotypes A, B or D. However, highly active genotype C or D infection often remained highly active, implying a risk for progressive liver damage. (c) 2012 Elsevier B.V. All rights reserved.
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| 6. |
- Malmström, Sebastian, 1976, et al.
(författare)
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Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact
- 2012
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 7:7
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Tidskriftsartikel (refereegranskat)abstract
- Background: Quantification of hepatitis B virus (HBV) DNA and surface antigen (HBsAg) serum levels have become increasingly important for the assessment of clinical stage and response to treatment for chronic hepatitis B. Effective immune clearance results in reduction of viremia by 4-5 log units and HBsAg levels by 2 log, but these processes are not well understood. Thus, it is uncertain to what extent mechanisms that inhibit transcription of the pregenomic RNA (pgRNA), an RNA intermediate, contribute to suppression of viremia. Likewise, it is unclear if transcriptional regulation is important for the excessive production of surface antigen (HBsAg) that is a hallmark of HBV infection. Methods: HBV RNA and cccDNA were quantified in 19 liver biopsies from patients with chronic HBV infection, as well as in transfected Huh7.5 cells and in PLC/PRF/5 cells carrying integrated HBV genome. Results: Patients negative for HBeAg had 2.15 log lower levels of cccDNA in liver tissue, 4.84 log lower serum levels of HBV DNA and 1.45 log lower serum levels of HBsAg, than HBeAg-positive patients. The pgRNA in liver tissue correlated strongly with cccDNA (R-2 = 0.87, p<0.0001) and HBV DNA levels in serum (R-2 = 0.81, p<0.0001), whereas S-RNA correlated strongly with cccDNA (R-2 = 0.65, p<0.0001) and HBsAg levels (R-2 = 0.57, p = 0.0003). The S-RNA/pgRNA ratio was higher in HBeAg-negative patients (ratio 40 vs. 3, p = 0.01) and in PLC/PRF/5 cells, and was in transfected Huh7.5 cells not influenced by mutations in the HBV core promoter. Conclusion: The reduction of viremia that is observed after loss of HBeAg was mainly explained by reduced cccDNA load in the liver, whereas the contribution of down-regulation of pgRNA transcription was relatively small. Enhanced transcription of S-RNA does not explain excessive production of HBsAg.
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| 7. |
- Malmström, Sebastian, 1976, et al.
(författare)
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Mutation analysis of lamivudine resistant hepatitis B virus strains by TaqMan PCR.
- 2007
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Ingår i: Journal of virological methods. - : Elsevier BV. - 0166-0934. ; 143:2, s. 147-52
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis B virus infected patients on long-term lamivudine treatment are exposed to a 15-32% risk compounded annually of developing resistance mutations. Such resistance results in a progression of the liver damage caused by chronic hepatitis B, and may also impair the effect of other antivirals through cross-resistance. At present lamivudine is used frequently as monotherapy because of its relatively low price and negligible side effects. Thus, simple methods for identifying resistance mutations are required. A method based on real-time polymerase chain reaction with TaqMan chemistry is described. The method combines both primer specificity, in order to target wild type and mutant viral strains at codon 180, and a mixture of three minor groove binding probes distinguishing the YMDD wild type and the YVDD and YIDD variants at codon 204. The accuracy of the method was verified by concordance with results of direct sequencing and restriction fragment length polymorphism when examining 27 samples from five patients, in whom lamivudine resistance was known to have developed. This method is rapid, cost effective, and should prove useful for monitoring patients treated with lamivudine.
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| 8. |
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| 9. |
- Malmström, Sebastian, 1976
(författare)
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Real-time PCR studies of genotypes, mutations and replication of hepatitis B virus
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Infection with hepatitis B virus (HBV) is an important cause of liver disease and affects 350 million people worldwide, causing 600,000 deaths/year. Treatment includes interferon and nucleoside analogues (NAs) such as lamivudine, entecavir, and tenofovir. During treatment with NAs, substitutions may arise in the viral genome that confer resistance to treatment, impairing or abolishing the effect. Clinical prognosis and outcome of treatment are affected by viral genotype, and to date there are eight established (A-H) and two putative (I-J) genotypes, as well as several subgenotype strains described. Levels of viral DNA and surface antigen (HBsAg) in serum are used to monitor the course of infection and the response to treatment. It is however not clear to what extent mechanisms that inhibit transcription of the pregenomic RNA (pgRNA), contribute to suppression of viremia, which mainly occurs in parallel with loss of HBeAg from blood. Likewise, it is unclear how the excessive production of HBsAg is regulated. The aims of this thesis were to develop methods for genotyping and resistance mutation analysis, to investigate the impact of genotypes on clinical outcome, and to investigate the role of the regulation of viral transcripts for replication and HBsAg production. Two real-time PCR based assays were designed and evaluated. The first focused on amino acid positions 180 and 204 in the viral polymerase enzyme, which are important for resistance against treatment with the NA lamivudine. The second aimed to include all established genotypes in a multiplex genotyping assay for accurate and rapid analysis. It was not possible to find one single genomic segment that could be used for amplification and identification of all genotypes. Instead, we chose to target a number of segments in different parts of the genome, and for genotypes A-C two segments each were targeted, to obtain reliable accuracy. Both methods showed high accuracy and concordance with earlier methods, adding the possibility to identify mixed infections and assign relative proportions to the strains in the mixture. Genotype impact on virological outcome was investigated after 9.2 years in 124 chronically infected adults. HBV DNA levels declined in patients carrying genotype A, B, and D, among whom HBeAg loss was observed in 92%. Genotype A and D showed 36% and 11% loss of HBsAg. In contrast, viral activity and aminotransferase elevation persisted in genotype C infections. In the final study, real-time PCR was used to analyse the levels of cccDNA and viral RNA in biopsies and cell lines with focus on differences between HBeAg positive and negative stage. Patients negative for HBeAg had 2.15 log lower levels of cccDNA in liver tissue, 4.84 log lower serum levels of HBV DNA and 1.45 log lower serum levels of HBsAg, than HBeAg-positive patients. The pgRNA in liver tissue correlated strongly with cccDNA (R2=0.87) and HBV DNA levels in serum (R2=0.81). The S-RNA/pgRNA ratio was higher in HBeAg-negative patients, which may reflect specific down-regulation of pgRNA, or enhanced S-RNA production. Transcription efficiency was lower in vitro than in biopsies, and was not influenced by HBV core promoter mutations in transfected Huh7.5 cells.
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