Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA.

Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.

Association between variants in the interferon lambda 4 locus and substitutions in the hepatitis C virus non-structural protein 5A.

BACKGROUND & AIMS: Single nucleotide polymorphisms within the interferon lambda 4 (IFNL4) locus are strongly associated with spontaneous clearance of hepatitis C virus (HCV) infection and early viral response to interferon therapy. Interaction between host genotype and amino acid substitutions might also influence the risk of antiviral resistance in interferon-free direct acting antiviral (DAA) therapies. METHODS: The relationship between IFNL4 genotype and HCV substitutions was analyzed in 929 patients with chronic HCV genotype 1b infection. Ultra-deep sequencing and quasispecies reconstruction was performed on the N-terminal region of NS5A in 57 patients. RESULTS: IFNL4 genotype was strongly associated with HCV NS5A Y93 and core protein substitutions, and the number and diversity of predicted quasispecies was marginally greater in IFNL4 TT/TT patients compared to TT/ΔG, ΔG/ΔG patients. RNA secondary structure prediction of the NS5A region suggests that variable sites are more likely to occupy unpaired, high entropy positions. CONCLUSIONS: HCV infection is proposed to induce a more efficient antiviral response in individuals with the IFNL4 TT/TT genotype that results either in viral clearance or selection for viral adaptations. The association between IFNL4 TT/TT genotype and Y93 substitutions may impact the risk of antiviral resistance in NS5A inhibitors in DAA therapy.

Clinical Features of Tachycardia-induced Cardiomyopathy in Patients with Atrial Fibrillation.

Objective Atrial fibrillation (AF) is the most common cause of tachycardia-induced cardiomyopathy (TIC). However, which patients with AF are prone to developing TIC remains unclear. In this study, we investigated the clinical features of AF patients with TIC. Methods This single-center study included 722 patients with AF (average age, 63.1±10.2 years old; 191 women) who underwent radiofrequency catheter ablation. We defined TIC as an initial left ventricular ejection fraction (LVEF) of <40% and a >20% recovery of the LVEF after successful AF ablation and compared the clinical characteristics between the TIC and control groups. Results The proportions of type 2 diabetes (30.5% vs. 14.7%), renal dysfunction (34.2% vs. 23.8%), hypertension (67.1% vs. 54.8%), and persistent AF (62.2% vs. 32.2%) were significantly higher in the TIC group (n=82) than in the control group (n=640). The atrioventricular nodal effective refractory period (AVNERP) (303±72 ms vs. 332±86 ms; p=0.017) was significantly shorter in the TIC group than in the control group. A multivariable analysis found that persistent AF [odds ratio (OR), 3.19; 95% confidence interval (CI), 1.94-5.24], renal dysfunction (OR, 1.87; 95% CI, 1.06-3.32), and type 2 diabetes (OR, 2.30; 95% CI, 1.31-4.05) were significantly associated with TIC. Conclusion Comorbid renal dysfunction and type 2 diabetes were clinical features of AF patients with TIC. Persistent AF, and short AVNERP may be involved in the development of TIC.

Decreased plasma cell-free mitochondrial DNA may be a new biomarker of tachycardia-induced cardiomyopathy in patients with atrial fibrillation.

OBJECTIVES: To determine cell-free mitochondrial DNA (mt-cfDNA) levels in tachycardia-induced cardiomyopathy (TIC) and non-TIC among atrial fibrillation (AF) cases. BACKGROUNDS: TIC is a reversible cardiomyopathy resulting from tachyarrhythmias, such as AF. The exact cause of TIC is not fully understood, but mitochondrial dysfunction has been reported in a variety of cardiomyopathies and may be involved in TIC as well. AF is recognized to be associated with systemic inflammation, and studies have shown that in patients with AF have elevated levels of mt-cfDNA increased, and this increase is linked to systemic inflammation. METHODS: We enrolled 67 patients with TIC (TIC group) and 671 patients without TIC (non-TIC group), who underwent catheter ablation for AF at our hospital between November 2009 and September 2016 and did not meet the exclusion criteria. We performed quantitative PCR analysis of plasma mt-cfDNA and nuclear-cfDNA and compared clinical factors and these measurements between the two groups. RESULTS: Levels of mt-cfDNA were significantly lower in the TIC group than in the non-TIC group (1110.01 vs. 1918.71 copies/µg plasma, P = 0.027), while levels of nuclear-cfDNA were comparable between these two groups. In particular, mt-cfDNA (P = 0.0003, odds ratio [OR] 2.54), non-paroxysmal AF (P < 0.0001, OR 3.07), and diabetes mellitus (P = 0.006, OR 2.36) were identified as independent factors associated with TIC. CONCLUSION: There are lower mt-cfDNA in TIC, and decreased plasma levels of circulating mt-cfDNA may be a new biomarker and involve in related mechanisms for AF associated TIC. CONDENSED ABSTRACT: Tachycardia-induced cardiomyopathy (TIC) is a reversible cardiomyopathy caused by tachyarrhythmias, such as atrial fibrillation (AF) tachycardia. The pathogenesis of TIC remains incompletely understood, and there is currently no method to predict its development in patients. In this study, we show that cell-free mitochondrial DNA (mt-cfDNA) levels were significantly lower in the TIC group than in the non-TIC group. Persistent AF, coexisting diabetes mellitus, and decreased mt-cfDNA levels were independently associated with TIC. Decreased mt-cfDNA levels may serve as a novel biomarker for predicting TIC in patients with AF.

Circulating microRNA-22 correlates with microRNA-122 and represents viral replication and liver injury in patients with chronic hepatitis B.

Hepatitis B virus (HBV) infection is associated with increased expression of microRNA-122. Serum microRNA-122 and microRNA-22 levels were analyzed in 198 patients with chronic HBV who underwent liver biopsy and were compared with quantitative measurements of HBsAg, HBeAg, HBV DNA, and other clinical and histological findings. Levels of serum microRNA-122 and microRNA-22 were determined by reverse transcription-TaqMan PCR. Serum levels of microRNA-122 and microRNA-22 were correlated (R(2) = 0.576; P < 0.001), and both were elevated in chronic HBV patients. Significant linear correlations were found between microRNA-122 or microRNA-22 and HBsAg levels (R(2) = 0.824, P < 0.001 and R(2) = 0.394, P < 0.001, respectively) and ALT levels (R(2) = 0.498, P < 0.001 and R(2) = 0.528, P < 0.001, respectively). MicroRNA-122 levels were also correlated with HBV DNA titers (R(2) = 0.694, P < 0.001 and R(2) = 0.421, P < 0.001). Levels of these microRNAs were significantly higher in HBeAg-positive patients compared to HBeAg-negative patients (P < 0.001 and P < 0.001). MicroRNA-122 levels were also lower in patients with advanced liver fibrosis (P < 0.001) and lower inflammatory activity (P < 0.025). These results suggest that serum micro-RNA levels are significantly associated with multiple aspects of HBV infection. The biological meaning of the correlation between microRNA-122 and HBsAg and should be investigated further.

Plasma MicroRNAs as noninvasive diagnostic biomarkers in patients with Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) can be diagnosed by a type 1 BrS tracing in a 12-lead electrocardiogram (ECG). However, there are daily variations in the ECGs of BrS patients, which presents a challenge when diagnosing BrS. Although many susceptibility genes have been identified, the SCN5A gene is reportedly the main causative gene of BrS. However, most patients do not have an evidence of genetic predisposition to develop BrS. In addition, the diagnosis and risk stratification for ventricular fibrillation (VF) in patients with BrS presents some problems. Meanwhile, circulating micro RNAs (miRNAs) have drawn increased attention as potential biomarkers of various diseases. We hypothesize that circulating miRNAs may be potential diagnostic biomarkers for BrS. METHODS: We enrolled 70 Japanese BrS patients and 34 controls for the screening cohort. A total of 2,555 miRNA sequences were detected using the 3D-Gene miRNAs labeling kit and 3D-Gene Human miRNAs Oligo Chip. We compared the expression of the miRNAs between the BrS patients and the controls. We validated whether the miRNA were significantly up- or downregulated in the screening cohort using RT-PCR. We also enrolled 72 Japanese BrS patients and 56 controls to replicate these miRNAs. RESULTS: Eight miRNAs (hsa-miR-223-3p, hsa-miR-22-3p, hsa-miR-221-3p, hsa-miR-4485-5p, hsa-miR-550a-5p, hsa-miR-423-3p, hsa-miR-23a-3p, and hsa-miR-30d-5p) were downregulated, and one miRNA (hsa-miR-873-3p) was upregulated by more than 3-fold in BrS patients. The multivariate logistic regression analysis determined that hsa-miR-423-3p, hsa-miR-223-3p, and hsa-miR-23a-3p were independently associated with BrS (P < 0.0001). The AUC based on cross validation was 0.871 with a sensitivity and specificity of 83.5% and 81.1%, respectively. CONCLUSIONS: The plasma miRNAs are potential noninvasive biomarkers of BrS, and the constructed logistic model was useful for discriminating BrS.

GJA1 gene polymorphism is a genetic predictor of recurrence after pulmonary vein isolation in patients with paroxysmal atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) and recurrence of AF after pulmonary vein isolation (PVI) have been linked to sinus node dysfunction. OBJECTIVE: The purpose of this study was to investigate the association between the heart rate-associated single nucleotide polymorphisms (SNPs) identified in genome-wide association studies and recurrence of AF after PVI. METHODS: In this study, patients with paroxysmal AF who underwent initial PVI, including 522 patients for screening and 172 patients for replication, were recruited and 21 heart rate-associated SNPs identified in genome-wide association studies were genotyped. The association between these SNPs and the recurrence of AF was investigated. RESULTS: Throughout the follow-up period of 21 ± 12 months, 119 patients with paroxysmal AF (22.8%) exhibited AF recurrences in the screening set. The rate of AF recurrence was significantly associated with the minor allele C of the gap junction alpha-1 protein (GJA1) rs1015451 (additive model: odds ratio 2.07; P = 9.32 × 10-7), but not with other SNPs. This association was confirmed in the replication set (allelic model: odds ratio 1.81; P = 2.70 × 10-2). Multivariate analysis revealed that the recurrence of AF after AF ablation was independently related to the GJA1 SNP rs1015451 additive model, duration of AF >1 year, AF from non-pulmonary vein foci, and thicker interventricular septum. CONCLUSION: The GJA1 SNP rs1015451, coding for a gap junction protein (connexin-43), may be considered a novel genetic marker for AF recurrence after PVI.

HBx protein is indispensable for development of viraemia in human hepatocyte chimeric mice.

The non-structural X protein, HBx, of hepatitis B virus (HBV) is assumed to play an important role in HBV replication. Woodchuck hepatitis virus X protein is indispensable for virus replication, but the duck hepatitis B virus X protein is not. In this study, we investigated whether the HBx protein is indispensable for HBV replication in vivo using human hepatocyte chimeric mice. HBx-deficient (HBx-def) HBV was generated in HepG2 cells by transfection with an overlength HBV genome. Human hepatocyte chimeric mice were infected with HBx-def HBV with or without hepatic HBx expression by hydrodynamic injection of HBx expression plasmids. Serum virus levels and HBV sequences were determined with mice sera. The generated HBx-def HBV peaked in the sucrose density gradient at points equivalent to the generated HBV wild type and the virus in a patient's serum. HBx-def HBV-injected mice developed measurable viraemia only in continuously HBx-expressed liver. HBV DNA in the mouse serum increased up to 9 log(10) copies ml(-1) and the viraemia persisted for more than 2 months. Strikingly, all revertant viruses had nucleotide substitutions that enabled the virus to produce the HBx protein. It was concluded that the HBx protein is indispensable for HBV replication and could be a target for antiviral therapy.

Publisher Correction: Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Regulation of the Hepatitis B virus replication and gene expression by the multi-functional protein TARDBP.

Hepatitis B virus (HBV) infects the liver and is a key risk factor for hepatocellular carcinoma. Identification of host factors that support viral replication is important to understand mechanisms of viral replication and to develop new therapeutic strategies. We identified TARDBP as a host factor that regulates HBV. Silencing or knocking out the protein in HBV infected cells severely impaired the production of viral replicative intermediates, mRNAs, proteins, and virions, whereas ectopic expression of TARDBP rescued production of these products. Mechanistically, we found that the protein binds to the HBV core promoter, as shown by chromatin precipitation as well as mutagenesis and protein-DNA interaction assays. Using LC-MS/MS analysis, we also found that TARDBP binds to a number of other proteins known to support the HBV life cycle, including NPM1, PARP1, Hsp90, HNRNPC, SFPQ, PTBP1, HNRNPK, and PUF60. Interestingly, given its key role as a regulator of RNA splicing, we found that TARDBP has an inhibitory role on pregenomic RNA splicing, which might help the virus to export its non-canonical RNAs from the nucleus without being subjected to unwanted splicing, even though mRNA nuclear export is normally closely tied to RNA splicing. Taken together, our results demonstrate that TARDBP is involved in multiple steps of HBV replication via binding to both HBV DNA and RNA. The protein's broad interactome suggests that TARDBP may function as part of a RNA-binding scaffold involved in HBV replication and that the interaction between these proteins might be a target for development of anti-HBV drugs.

Differences in serum microRNA profiles in hepatitis B and C virus infection.

OBJECTIVES: Patients infected with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) are at greater risk of cirrhosis and hepatocellular carcinoma. The objective of this study was to identify virus-specific serum microRNA profiles associated with liver function and disease progression. Microarray analysis of serum microRNAs was performed using the Toray 3D array system in 22 healthy subjects, 42 HBV patients, and 30 HCV patients. Selected microRNAs were then validated by qRT-PCR in 186 HBV patients, 107 HCV patients, and 22 healthy subjects. RESULTS: Microarray analysis showed up-regulation of a number of microRNAs in serum of both HBV and HCV patients. In qRT-PCR analysis, miR-122, miR-99a, miR-125b, miR-720, miR-22, and miR-1275 were up-regulated both in HBV patients relative to healthy subjects, and all except miR-1275 were up-regulated in HBeAg-positive patients relative to HBeAg-negative patients. Specific microRNAs were independently associated with different aspects of HBV infection. MiR-122 was independently associated with HBV DNA level, whereas miR-125b was independently associated with levels of HBV DNA, HBsAg, and HBeAg. MiR-22 and miR-1275 were independently associated with serum γ-glutamyl transpeptidase levels. CONCLUSIONS: Serum microRNA levels reflect differences in the etiology and stage of viral hepatitis.

Hepatitis B virus-specific miRNAs and Argonaute2 play a role in the viral life cycle.

UNLABELLED: Disease-specific serum miRNA profiles may serve as biomarkers and might reveal potential new avenues for therapy. An HBV-specific serum miRNA profile associated with HBV surface antigen (HBsAg) particles has recently been reported, and AGO2 and miRNAs have been shown to be stably associated with HBsAg in serum. We identified HBV-associated serum miRNAs using the Toray 3D array system in 10 healthy controls and 10 patients with chronic hepatitis B virus (HBV) infection. 19 selected miRNAs were then measured by quantitative RT-PCR in 248 chronic HBV patients and 22 healthy controls. MiRNA expression in serum versus liver tissue was also compared using biopsy samples. To examine the role of AGO2 during the HBV life cycle, we analyzed intracellular co-localization of AGO2 and HBV core (HBcAg) and surface (HBsAg) antigens using immunocytochemistry and proximity ligation assays in stably transfected HepG2 cells. The effect of AGO2 ablation on viral replication was assessed using siRNA. Several miRNAs, including miR-122, miR-22, and miR-99a, were up-regulated at least 1.5 fold (P<2E-08) in serum of HBV-infected patients. AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments. HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments. Conversely, HBx localized non-specifically in the nucleus and cytoplasm, and no interaction between AGO2 and HBx was detected. SiRNA ablation of AGO2 suppressed production of HBV DNA and HBs antigen in the supernatant. CONCLUSION: These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.

G to A hypermutation of TT virus.

APOBEC3 proteins function as part of innate antiviral immunity and induce G to A hypermutation in retroviruses and hepatitis B virus (HBV) genomes. Whether APOBEC3 proteins affect viruses that replicate without a reverse transcription step is unknown. TT virus (TTV), known to present in serum of healthy individuals and HBV carriers, has a single-stranded circular DNA genome and replicates without reverse transcription. In this study, we examined 67 blood samples obtained from healthy individuals and HBV carriers and observed G to A hypermutation of genomes of TTV in both healthy individuals and HBV carriers. During ALT flare-up in HBV carriers, G to A hypermutation of HBV increased, but TTV genomes significantly decreased in number and hypermutated TTV genomes became undetectable. Our results show that hypermutated TTV exist in healthy individuals and HBV carriers and that TTV genomes were susceptible to immune reaction directed to HBV by interacting with APOBEC3 proteins.