Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Signs or symptoms of suspected preeclampsia - A retrospective national database study of prevalence, costs, and outcomes.

BACKGROUND: Most patients with signs or symptoms (s/s) of suspected preeclampsia are not diagnosed with preeclampsia. We sought to determine and compare the prevalence of s/s, pregnancy outcomes, and costs between patients with and without diagnosed preeclampsia. METHODS: This retrospective cohort study analyzed a large insurance research database. Pregnancies with s/s of preeclampsia versus a confirmed preeclampsia diagnosis were identified using International Classification of Diseases codes. S/s include hypertension, proteinuria, headache, visual symptoms, edema, abdominal pain, and nausea/vomiting. Pregnancies were classed as 1) s/s of preeclampsia without a confirmed preeclampsia diagnosis (suspicion only), 2) s/s with a confirmed diagnosis (preeclampsia with suspicion), 3) diagnosed preeclampsia without s/s recorded (preeclampsia only), and 4) no s/s, nor preeclampsia diagnosis (control). RESULTS: Of 1,324,424 pregnancies, 29.2 % had ≥1 documented s/s of suspected preeclampsia, and 14.2 % received a preeclampsia diagnosis. Hypertension and headache were the most common s/s, leading 20.2 % and 9.2 % pregnancies developed to preeclampsia diagnosis, respectively. Preeclampsia, with or without suspicion, had the highest rates of hypertension-related severe maternal morbidity (HR [95 % CI]: 3.0 [2.7, 3.2] and 3.6 [3.3, 4.0], respectively) versus controls. A similar trend was seen in neonatal outcomes such as preterm delivery and low birth weight. Cases in which preeclampsia was suspected but not confirmed had the highest average total maternal care costs ($6096 [95 % CI: 602, 6170] over control). CONCLUSION: There is a high prevalence but poor selectivity of traditional s/s of preeclampsia, highlighting a clinical need for improved screening method and cost-effectiveness disease management.

Oxygen-dependent histone lysine demethylase 4 restricts hepatitis B virus replication.

Mammalian cells have evolved strategies to regulate gene expression when oxygen is limited. Hypoxia-inducible factors (HIF) are the major transcriptional regulators of host gene expression. We previously reported that HIFs bind and activate hepatitis B virus (HBV) DNA transcription under low oxygen conditions; however, the global cellular response to low oxygen is mediated by a family of oxygenases that work in concert with HIFs. Recent studies have identified a role for chromatin modifiers in sensing cellular oxygen and orchestrating transcriptional responses, but their role in the HBV life cycle is as yet undefined. We demonstrated that histone lysine demethylase 4 (KDM4) can restrict HBV, and pharmacological or oxygen-mediated inhibition of the demethylase increases viral RNAs derived from both episomal and integrated copies of the viral genome. Sequencing studies demonstrated that KDM4 is a major regulator of the hepatic transcriptome, which defines hepatocellular permissivity to HBV infection. We propose a model where HBV exploits cellular oxygen sensors to replicate and persist in the liver. Understanding oxygen-dependent pathways that regulate HBV infection will facilitate the development of physiologically relevant cell-based models that support efficient HBV replication.

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression.

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. Our knowledge of the host pathways that define susceptibility to infection and disease severity are limited. Hypoxia inducible factors (HIFs) define metabolic responses to low oxygen and regulate inflammatory responses in the lower respiratory tract. We demonstrate a role for HIFs to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors reduce the expression of the RSV entry receptor nucleolin and inhibit viral cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for this respiratory pathogen.

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter to transcribe pre-genomic RNA. Application of a probe-enriched long-read sequencing method to map the HBV transcriptome showed an increased abundance of all viral RNAs under low oxygen or hypoxic conditions. Importantly, the hypoxic-associated increase in HBV transcripts was dependent on N6-methyladenosine (m6A) modifications and an m6A DRACH motif in the 5' stem loop of pre-genomic RNA defined transcript half-life under hypoxic conditions. Given the essential role of m6A modifications in the viral transcriptome we assessed the oxygen-dependent expression of RNA demethylases and bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2 cell line showed increased ALKBH5 gene expression under hypoxic conditions. Silencing the demethylase reduced the levels of HBV pre-genomic RNA and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

Molecular components of the circadian clock regulate HIV-1 replication.

Human immunodeficiency virus 1 (HIV-1) causes major health burdens worldwide and still lacks curative therapies and vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organism's response to its environment and invading pathogens. Peripheral viral loads of HIV-1 infected patients show diurnal variation; however, the underlying mechanisms remain unknown. Here, we demonstrate a role for the cell-intrinsic clock to regulate rhythmic HIV-1 replication in circadian-synchronized systems. Silencing the circadian activator Bmal1 abolishes this phenotype, and we observe BMAL1 binding to the HIV-1 promoter. Importantly, we show differential binding of the nuclear receptors REV-ERB and ROR to the HIV-long terminal repeat at different circadian times, demonstrating a dynamic interplay in time-of-day regulation of HIV-1 transcription. Bioinformatic analysis shows circadian regulation of host factors that control HIV-1 replication, providing an additional mechanism for rhythmic viral replication. This study increases our understanding of the circadian regulation of HIV-1, which can ultimately inform new therapies.

An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome.

Hepatitis B virus (HBV) is one of the smallest human DNA viruses and its 3.2 Kb genome encodes multiple overlapping open reading frames, making its viral transcriptome challenging to dissect. Previous studies have combined quantitative PCR and Next Generation Sequencing to identify viral transcripts and splice junctions, however the fragmentation and selective amplification used in short read sequencing precludes the resolution of full length RNAs. Our study coupled an oligonucleotide enrichment protocol with state-of-the-art long read sequencing (PacBio) to identify the repertoire of HBV RNAs. This methodology provides sequencing libraries where up to 25 % of reads are of viral origin and enable the identification of canonical (unspliced), non-canonical (spliced) and chimeric viral-human transcripts. Sequencing RNA isolated from de novo HBV infected cells or those transfected with 1.3 × overlength HBV genomes allowed us to assess the viral transcriptome and to annotate 5' truncations and polyadenylation profiles. The two HBV model systems showed an excellent agreement in the pattern of major viral RNAs, however differences were noted in the abundance of spliced transcripts. Viral-host chimeric transcripts were identified and more commonly found in the transfected cells. Enrichment capture and PacBio sequencing allows the assignment of canonical and non-canonical HBV RNAs using an open-source analysis pipeline that enables the accurate mapping of the HBV transcriptome.

Project20: maternity care mechanisms that improve access and engagement for women with social risk factors in the UK - a mixed-methods, realist evaluation.

OBJECTIVES: To evaluate how women access and engage with different models of maternity care, whether specialist models improve access and engagement for women with social risk factors, and if so, how? DESIGN: Realist evaluation. SETTING: Two UK maternity service providers. PARTICIPANTS: Women accessing maternity services in 2019 (n=1020). METHODS: Prospective observational cohort with multinomial regression analysis to compare measures of access and engagement between models and place of antenatal care. Realist informed, longitudinal interviews with women accessing specialist models of care were analysed to identify mechanisms. MAIN OUTCOME MEASURES: Measures of access and engagement, healthcare-seeking experiences. RESULTS: The number of social risk factors women were experiencing increased with deprivation score, with the most deprived more likely to receive a specialist model that provided continuity of care. Women attending hospital-based antenatal care were more likely to access maternity care late (risk ratio (RR) 2.51, 95% CI 1.33 to 4.70), less likely to have the recommended number of antenatal appointments (RR 0.61, 95% CI 0.38 to 0.99) and more likely to have over 15 appointments (RR 4.90, 95% CI 2.50 to 9.61) compared with community-based care. Women accessing standard care (RR 0.02, 95% CI 0.00 to 0.11) and black women (RR 0.02, 95% CI 0.00 to 0.11) were less likely to have appointments with a known healthcare professional compared with the specialist model. Qualitative data revealed mechanisms for improved access and engagement including self-referral, relational continuity with a small team of midwives, flexibility and situating services within deprived community settings. CONCLUSION: Inequalities in access and engagement with maternity care appears to have been mitigated by the community-based specialist model that provided continuity of care. The findings enabled the refinement of a realist programme theory to inform those developing maternity services in line with current policy.

Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model.

Understanding the host pathways that define susceptibility to Severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in the Syrian hamster model. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.

Inflammatory Gene Expression Associates with Hepatitis B Virus cccDNA- but Not Integrant-Derived Transcripts in HBeAg Negative Disease.

Chronic hepatitis B virus (HBV) infection is a global health problem that presents as a spectrum of liver disease, reflecting an interplay between the virus and the host immune system. HBV genomes exist as episomal covalently closed circular DNA (cccDNA) or chromosomal integrants. The relative contribution of these genomes to the viral transcriptome in chronic hepatitis B (CHB) is not well-understood. We developed a qPCR method to estimate the abundance of HBV cccDNA- and integrant-derived viral transcripts and applied this to a cohort of patients diagnosed with CHB in the HBe antigen negative phase of disease. We noted a variable pattern of HBV transcripts from both DNA templates, with preS1/S2 mRNAs predominating and a significant association between increasing age and the expression of integrant-derived mRNAs, but not with inflammatory status. In contrast, cccDNA-derived transcripts were associated with markers of liver inflammation. Analysis of the inflammatory hepatic transcriptome identified 24 genes significantly associated with cccDNA transcriptional activity. Our study uncovers an immune gene signature that associates with HBV cccDNA transcription and increases our understanding of viral persistence.

Cost effectiveness of the sFlt1/PlGF ratio test as an adjunct to the current practice of evaluating suspected preeclampsia in the United States.

OBJECTIVE: Preeclampsia is a major obstetric disorder that can lead to severe maternal, fetal and infant outcomes. In women with suspected preeclampsia, measurement of the soluble fms-like tyrosine kinase-1 (sFlt1) and placental growth factor (PlGF) ratio has been shown to have a high negative predictive value (>97%). Our aim was to estimate the value to the US healthcare system of adopting this test into clinical practice. STUDY DESIGN: An economic model was developed for the evaluation of suspected preeclampsia from a US payer perspective using data from a US observational study of 459 women evaluated between 23 and 34.6 weeks. Test results were not available to clinicians. The model compares two strategies for managing suspected preeclampsia: standard care versus a biomarker-informed pathway utilizing the sFlt1/PlGF ratio. RESULTS: Utilization of the sFlt1/PlGF ratio test reduced the number of women admitted for suspected preeclampsia by 34-49%. Despite fewer admissions, a higher proportion of women admitted to hospital subsequently developed preeclampsia, and the proportion of women not admitted who would subsequently develop preeclampsia remained low (3.2%-6.7%). Cost savings arising from a reduction in admissions are estimated to be $1050 in the base case; varying the hospitalization cost ±25% would lead to savings in the range $771 to $1330 per patient at 2020 prices. CONCLUSION: Adopting the sFlt1/PlGF ratio test as an adjunct to clinical criteria improves the assessment of risk in women presenting with suspicion of preeclampsia and has the potential to safely reduce unnecessary admissions and save costs.

Optogenetic axon guidance in embryonic zebrafish.

Axons form the long-range connections of biological neuronal networks, which are built through the developmental process of axon guidance. Here, we describe a protocol to precisely and non-invasively control axonal growth trajectories in live zebrafish embryos using focal light activation of a photoactivatable Rac1. We outline techniques for photostimulation, time-lapse imaging, and immunohistochemistry. These approaches enable engineering of long-range axonal circuitry or repair of defective circuits in living zebrafish, despite a milieu of competing endogenous signals and repulsive barriers. For complete details on the use and execution of this protocol, please refer to Harris et al. (2020).

The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells.

The coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract via spike glycoprotein binding to angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism's response to its environment and can regulate host susceptibility to virus infection. We demonstrate that silencing the circadian regulator Bmal1 or treating lung epithelial cells with the REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry and replication. Importantly, treating infected cells with SR9009 limits SARS-CoV-2 replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced interferon-stimulated gene transcripts in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to limit SARS-CoV-2 infection. Our study highlights alternative approaches to understand and improve therapeutic targeting of SARS-CoV-2.

Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint.

Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC.

Evaluation of the Onclarity HPV assay on the high-throughput COR system.

Background: Here we compare the performance of the high-throughput BD COR System (COR) to the Viper LT System (Viper) using the BD Onclarity HPV assay.Research Design and Methods: Remnant clinical specimens, contrived specimens in SurePath (BD) and PreservCyt (Hologic) media, and prospective clinical specimens in BD Cervical Brush Diluent (CBD) were tested. Outcomes included intra-laboratory agreement of Onclarity results on COR and inter-system agreement between COR and Viper.Results: Onclarity reproducibility on COR resulted in standard deviation and correlation of variation of Ct values ranging from 0.14 to 1.98 and 0.49% to 2.15%, respectively, for contrived specimens, and 0.9-3.08 and 2.89-9.21%, respectively, for clinical specimens. In the COR and Viper clinical agreement study, OPA for Onclarity ranged from 97.1%-98.9%, depending on the collection media type. PPA values for pooled, HPV(+) specimens at low positive (C95), and moderate positive (3XC95) target concentrations were ≥95.0% and 100%, respectively; PPA values associated with HPV 16, 18, 31, 45, 33/58, 52, 35/39/68, 51, and 56/59/66, individually, ranged from 93.8%-100%.Conclusions: Onclarity performance on COR is equivalent to Viper, and is accurate and reproducible for detection of all high-risk HPV genotypes, with a throughput of 330 results from a single 8-hour shift.

The circadian clock component BMAL1 regulates SARS-CoV-2 entry and replication in lung epithelial cells.

The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via Spike glycoprotein binding angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism’s response to its environment and can regulate host susceptibility to virus infection. We demonstrate a circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with a synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced a wide spectrum of interferon stimulated genes in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to dampen SARS-CoV-2 infection. Our study suggests new approaches to understand and improve therapeutic targeting of SARS-CoV-2.

Circadian control of hepatitis B virus replication.

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide for which there are no curative therapies. The major challenge in curing infection is eradicating or silencing the covalent closed circular DNA (cccDNA) form of the viral genome. The circadian factors BMAL1/CLOCK and REV-ERB are master regulators of the liver transcriptome and yet their role in HBV replication is unknown. We establish a circadian cycling liver cell-model and demonstrate that REV-ERB directly regulates NTCP-dependent hepatitis B and delta virus particle entry. Importantly, we show that pharmacological activation of REV-ERB inhibits HBV infection in vitro and in human liver chimeric mice. We uncover a role for BMAL1 to bind HBV genomes and increase viral promoter activity. Pharmacological inhibition of BMAL1 through REV-ERB ligands reduces pre-genomic RNA and de novo particle secretion. The presence of conserved E-box motifs among members of the Hepadnaviridae family highlight an evolutionarily conserved role for BMAL1 in regulating this family of small DNA viruses.