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- Prakash, Kasthuri
(författare)
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Hepatitis B virus RNA in serum and liver tissue - quantification using digital PCR
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hepatitis B virus (HBV) infection is a global health issue that is responsible for approximately 900,000 deaths each year, by inducing liver cirrhosis and hepatocellular carcinoma (HCC). A few markers are used to classify HBV infection and monitor treatment efficacy, including HBV DNA, surface antigen (HBsAg) and e antigen (HBeAg) in serum as well as HBV DNA and RNA in liver tissue. The recent discovery of the receptor NTCP facilitates in vitro studies of HBV. The aims of this thesis were (I) to characterize a new marker of HBV infection, HBV RNA in serum (II) to investigate in vitro the neutralizing effect of HBV encoded subviral (HBsAg) particles (III) to develop and apply a new method to discriminate viral and integrated DNA in liver tissue (IV) to analyze focal differences within the liver of HBV and hepatitis D virus (HDV) and (V) to explore HBV RNA profile in liver biopsies by digital PCR. High levels of serum HBV RNA was found in the majority of 95 patient samples utilized in this study. This RNA was of full genome length, appeared in fractionation together with HBV DNA. Sequencing data supported that HBV RNA in serum represents virus-like particles with failing reverse transcription of the pregenomic RNA (pgRNA). The role of subviral particles (SVP) during HBV infection was explored in HepG2-NTCP cell line. The results support that SVP functions as a decoy to neutralize antibodies synthesized by the host. A novel droplet digital PCR (ddPCR) method was developed and applied on 70 liver biopsies to quantify circular and linear HBV DNA, in order to estimate the amount of integrated HBV DNA in the human genome. A complimentary study on the same material was performed to obtain an RNA profile using ddPCR to amplify six target regions. Together, these results indicate that integrated DNA represents the majority of intrahepatic HBV DNA in late stages of infection and is responsible for maintaining high HBsAg levels in serum. The results also suggest that reduced transcription of pgRNA via a novel mechanism may contribute to low HBV replication in HBeAg-negative phase. ddPCR analysis of a range of HBV markers was used to study focal differences in infection in 15-30 pieces of liver explant tissue from six patients with HBV or HDV induced cirrhosis. Large differences in focality was observed especially in patients with low degree of viral replication or with HDV coinfection and the results also support expression of S RNA from integrated HBV DNA. HDV infection was less focal with presence of high HDV RNA levels in the absence of HBV. In summary, this thesis compilation contributes to better understanding of HBV serum and tissue markers and their relationship to replication and integration.
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| 3. |
- Prakash, Kasthuri, et al.
(författare)
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Hepatitis B Virus RNA Profiles in Liver Biopsies by Digital Polymerase Chain Reaction
- 2020
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Ingår i: Hepatology Communications. - : Ovid Technologies (Wolters Kluwer Health). - 2471-254X. ; 4:7, s. 973-982
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Tidskriftsartikel (refereegranskat)abstract
- Replication of hepatitis B virus (HBV) originates from covalently closed circular DNA (cccDNA) and involves reverse transcription of pregenomic RNA (pgRNA), which is also called core RNA and encodes the capsid protein. The RNA coding for hepatitis B surface antigen (HBsAg) in the envelope of viral or subviral particles is produced from cccDNA or from HBV DNA integrated into the host genome. Because only cccDNA can generate the core and the 3 ' redundancy regions of HBV RNA, we aimed to clarify to what extent such HBV integrations are expressed by quantifying the different HBV RNA species in liver tissue. Digital droplet polymerase chain reaction (ddPCR) was employed to quantify six HBV RNA targets in 76 liver biopsies from patients with chronic infection, comprising 14 who were hepatitis B e antigen (HBeAg) positive and 62 who were HBeAg negative. In patients who were HBeAg negative, HBV RNA from the S RNA region was >1.6 log(10) units higher than in the core and 3 ' redundancy regions (P < 0.0001), indicating that >90% of S RNA was integration derived. HBeAg-negative samples showed 10 times lower levels of pgRNA (5 ' core) compared with core RNA (3 ' part of core; P < 0.0001), suggesting that a large proportion of core RNA might have a downstream shift of the transcription starting point. In multiple regression analysis, HBV DNA levels in serum were most strongly dependent on pgRNA. Conclusion: In patients who were HBeAg negative, integration-derived S RNA seemed to predominate and a large proportion of the core RNA lacked the 5 ' part. Because this part comprises the down-regulator of transcription 1 sequences, which are necessary for virus production (plus strand translocation), the finding might help to explain the low level of HBV DNA in serum that frequently is observed in patients with chronic HBV infection who are HBeAg negative.
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- Prakash, Kasthuri, et al.
(författare)
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High serum levels of pregenomic RNA reflect frequently failing reverse transcription in hepatitis B virus particles
- 2018
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Ingår i: Virology Journal. - : Springer Science and Business Media LLC. - 1743-422X. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Hepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA (pgRNA), which is reverse transcribed during replication. Particles containing HBV RNA are present in serum of infected individuals, and quantification of this HBV RNA could be clinically useful. Methods: In a retrospective study of 95 patients with chronic HBV infection, we characterised HBV RNA in serum in terms of concentration, particle association and sequence. HBV RNA was detected by real-time PCR at levels almost as high as HBV DNA. Results: The HBV RNA was protected from RNase and it was found in particles of similar density as particles containing HBV DNA after fractionation on a Nycodenz gradient. Sequencing the epsilon region of the RNA did not reveal mutations that would preclude its binding to the viral polymerase before encapsidation. Specific quantification of precore RNA and pgRNA by digital PCR showed almost seven times lower ratio of precore RNA/pgRNA in serum than in liver tissue, which corresponds to poorer encapsidation of this RNA as compared with pgRNA. The serum ratio between HBV DNA and HBV RNA was higher in genotype D as compared with other genotypes. Conclusions: The results suggest that HBV RNA in serum is present in viral particles with failing reverse transcription activity, which are produced at almost as high rates as viral particles containing DNA. The results encourage further studies of the mechanisms by which these particles are produced, the impact of genotype, and the potential clinical utility of quantifying HBV RNA in serum.
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- Ringlander, Johan, et al.
(författare)
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Deep sequencing of hepatitis B virus using Ion Torrent fusion primer method
- 2022
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934. ; 299
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Tidskriftsartikel (refereegranskat)abstract
- Background: Hepatitis B virus (HBV) infection is worldwide a major cause of liver cirrhosis and hepatocellular carcinoma. Thousands of years ago, several HBV genotypes (A-I) evolved and have, as a result of human migration, become globally disseminated. Sequencing of HBV is used for genotyping, and investigation of out -breaks or of antiviral resistance. The present study describes a simplified deep sequencing of the whole HBV genome. Methods: Sequencing by Ion Torrent was evaluated and its performance compared with Sanger sequencing on clinical samples. Results: Amplification of overlapping segments spanning the entire HBV genome was successful at HBV DNA levels in serum as low as 100 IU/mL. The use of primers carrying adapter tags generated libraries without the need for fragmentation and ligation steps, and inclusion of barcode sequences allowed parallel analysis of multiple samples. A streamlined bioinformatic platform generated consensus sequences and superior mutation assessment as compared with Sanger sequencing, with which there was a 99.8 % average agreement. Conclusion: Deep sequencing of the whole HBV genome by using PCR primers tagged with adapters that prepare overlapping amplicons for Ion Torrent analysis was efficient and accurate.
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- Ringlander, Johan, et al.
(författare)
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Deep sequencing of liver explant transcriptomes reveals extensive expression from integrated hepatitis B virus DNA
- 2020
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Ingår i: Journal of Viral Hepatitis. - : Wiley. - 1352-0504 .- 1365-2893. ; 27:11, s. 1162-1170
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC). Integration of HBV DNA into the human genome may contribute to oncogenesis and to the production of the hepatitis B surface antigen (HBsAg). Whether integrations contribute to HBsAg levels in the blood is poorly known. Here, we characterize the HBV RNA profile of HBV integrations in liver tissue in patients with chronic HBV infection, with or without concurrent hepatitis D infection, by transcriptome deep sequencing. Transcriptomes were determined in liver tissue by deep sequencing providing 200 million reads per sample. Integration points were identified using a bioinformatic pipeline. Explanted liver tissue from five patients with end-stage liver disease caused by HBV or HBV/HDV was studied along with publicly available transcriptomes from 21 patients. Almost all HBV RNA profiles were devoid of reads in the core and the 3 ' redundancy (nt 1830-1927) regions, and contained a large number of chimeric viral/human reads. Hence, HBV transcripts from integrated HBV DNA rather than from covalently closed circular HBV DNA (cccDNA) predominated in late-stage HBV infection, in particular in cases with hepatitis D virus co-infection. The findings support the suggestion that integrated HBV DNA can be a significant source of HBsAg in humans.
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| 7. |
- Ringlander, Johan, et al.
(författare)
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Impact of ADAR-induced editing of minor viral RNA populations on replication and transmission of SARS-CoV-2
- 2022
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 119:6
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Tidskriftsartikel (refereegranskat)abstract
- Adenosine deaminases acting on RNA (ADAR) are RNA-editing enzymes that may restrict viral infection. We have utilized deep sequencing to determine adenosine to guanine (AfiG) mutations, signifying ADAR activity, in clinical samples retrieved from 93 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)- infected patients in the early phase of the COVID-19 pandemic. AfiG mutations were detected in 0.035% (median) of RNA residues and were predominantly nonsynonymous. These mutations were rarely detected in the major viral population but were abundant in minor viral populations in which AfiG was more prevalent than any other mutation (P < 0.001). The AfiG substitutions accumulated in the spike protein gene at positions corresponding to amino acids 505 to 510 in the receptor binding motif and at amino acids 650 to 655. The frequency of AfiG mutations in minor viral populations was significantly associated with low viral load (P < 0.001). We additionally analyzed AfiG mutations in 288,247 SARSCoV- 2 major (consensus) sequences representing the dominant viral population. The AfiG mutations observed in minor viral populations in the initial patient cohort were increasingly detected in European consensus sequences between March and June 2020 (P < 0.001) followed by a decline of these mutations in autumn and early winter (P < 0.001). We propose that ADAR-induced deamination of RNA is a significant source of mutated SARS-CoV-2 and hypothesize that the degree of RNA deamination may determine or reflect viral fitness and infectivity. © 2022 National Academy of Sciences. All rights reserved.
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| 8. |
- Rydell, Gustaf E, et al.
(författare)
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Abundance of non-circular intrahepatic hepatitis B virus DNA may reflect frequent integration into human DNA in chronically infected patients.
- 2022
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Ingår i: The Journal of infectious diseases. - : Oxford University Press (OUP). - 1537-6613 .- 0022-1899. ; 225:11, s. 1982-1990
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis B virus (HBV) integration has implications for cancer development and surface antigen (HBsAg) production, but methods to quantify integrations are lacking. The aim of this study was to develop a digital PCR (ddPCR) assay discriminating between circular and integrated HBV DNA, and to relate the distribution between the two forms to other HBV markers.ddPCR comprising primers spanning the typical linearization breakpoint in the HBV genome allowed for quantification of the absolute copy numbers of total and circular HBV DNA, and calculation of linear HBV DNA.Analysis of 70 liver biopsies from patients with chronic HBV infection revealed that the fraction of linear HBV DNA, which includes integrations, was higher in HBeAg-negative patients than HBeAg-positive. The ratio between HBsAg and HBV DNA levels in serum correlated with the intrahepatic proportion of linear HBV DNA. Furthermore, ddPCR experiments on serum samples and experiments with nuclease indicated the contribution of encapsidated double-stranded linear DNA and replication intermediates to be limited.The degree of integration of intrahepatic HBV DNA in the HBeAg-negative stage may be higher than previously anticipated, and integrated DNA may explain the persistence of high HBsAg serum levels in patients with low HBV DNA levels.
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| 9. |
- Rydell, Gustaf E, et al.
(författare)
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Hepatitis B surface antigen on subviral particles reduces the neutralizing effect of anti-HBs antibodies on hepatitis B viral particles in vitro
- 2017
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Ingår i: Virology. - : Elsevier BV. - 0042-6822. ; 509, s. 67-70
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Tidskriftsartikel (refereegranskat)abstract
- During hepatitis B virus (HBV) infections subviral particles (SVP) consisting mainly of hepatitis B surface antigen are present at much higher concentration than viral particles (VP) in serum. To investigate reasons for this excess of SVP production, SVP and VP were fractionated on a Nycodenz gradient and analyzed for HBV infection of HepG2-NTCP cells with and without anti-HBs antibodies. Our findings showed that SVP significantly reduced the neutralization of VP by anti-HBs, while SW had little effect on viral entry, supporting the assumption that SW serve as decoy facilitating cell-to-cell spread of HBV in the presence of neutralizing antibodies.
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| 10. |
- Rydell, Gustaf E, et al.
(författare)
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Quantification of viral RNA in multiple pieces of explant liver tissue shows distinct focal differences of hepatitis B infection.
- 2022
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Ingår i: The Journal of infectious diseases. - : Oxford University Press (OUP). - 1537-6613 .- 0022-1899. ; 226:6, s. 1036-1040
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis B virus (HBV) DNA and RNA were quantified by digital PCR assays in 20-30 tissue pieces from each of four liver explants with cirrhosis caused by HBV. The within-patient variability of HBV RNA levels between pieces was up to thousand-fold. Core RNA and S RNA levels were similar and correlated strongly when replication was high, supporting transcription from cccDNA. By contrast, enhanced expression of S RNA relative to cccDNA and core RNA in patients with medium-high or low replication supports that HBV surface antigen (HBsAg) can be expressed mainly from integrated HBV DNA in such patients.
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