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Journal of Clinical Virology : the... Aug 2024HDV antibody testing is recommended for universal screening and as the first line in an HDV double reflex testing strategy for effectively identifying patients with...
BACKGROUND
HDV antibody testing is recommended for universal screening and as the first line in an HDV double reflex testing strategy for effectively identifying patients with active infection for therapeutic treatments.
OBJECTIVE
The aim of this study is to evaluate the performance of a newly developed ARCHITECT HDV Total Ig (ARCHITECT HDV Ig) prototype assay.
STUDY DESIGN
Performance characteristics were determined for the ARCHITECT HDV Ig and a reference test, LIAISON XL Anti-HDV using a well-characterized specimen panel, comprising HDV RNA positive (n = 62) and negative (n = 70) samples, and healthy US blood donors.
RESULTS
Healthy US blood donors (n=200) showed 99.5% (199/200, 95%CI=97.65-99.98) specificity with ARCHITECT HDV Ig and 98.5 % (197/200, 95 %CI = 96.10-99.64) with LIAISON Anti-HDV. Among known HDV RNA positive samples, ARCHITECT HDV Ig detected 59/62 demonstrating 95.2 % sensitivity while LIAISON Anti-HDV sensitivity was 90.3 % (56/62). Among 101 HBV positive samples, 70 were reactive in the ARCHITECT test, 59 of which tested positive for HDV RNA for a positive predictive value (PPV) for the presence of HDV RNA was 84.3 %. For LIAISON Anti-HDV, 79 specimens were reactive and 56 contained HDV RNA: PPV for HDV RNA was 70.9 %. Among 70 HDV RNA negative samples, 39 were HBV positive. ARCHITECT HDV Ig negative predictive value (NPV) was 71.8 % and LIAISON Anti-HDV NPV was 41 % for the HBV positive group, respectively.
CONCLUSION
When compared to the LIASON Anti-HDV test, the ARCHITECT HDV Ig assay demonstrated enhanced sensitivity and specificity and better NPV and PPV values for HDV RNA status. The ARCHITECT HDV Ig assay represents a promising tool for universal screening of all HBsAg-positive persons.
Topics: Humans; Sensitivity and Specificity; Hepatitis D; Hepatitis Delta Virus; Hepatitis Antibodies; High-Throughput Screening Assays; Serologic Tests; Automation, Laboratory; Blood Donors
PubMed: 38781633
DOI: 10.1016/j.jcv.2024.105689 -
The Journal of General Virology May 2024Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our... (Review)
Review
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.
Topics: Hepatitis B virus; Humans; Virus Replication; Hepatitis Delta Virus; Hepatitis B; Molecular Biology; Japan; Hepatitis D; Host-Pathogen Interactions
PubMed: 38757942
DOI: 10.1099/jgv.0.001978 -
Journal For Immunotherapy of Cancer May 2024Cancer-intrinsic type I interferon (IFN-I) production triggered by radiotherapy (RT) is mainly dependent on cytosolic double-stranded DNA (dsDNA)-mediated cGAS/STING...
BACKGROUND
Cancer-intrinsic type I interferon (IFN-I) production triggered by radiotherapy (RT) is mainly dependent on cytosolic double-stranded DNA (dsDNA)-mediated cGAS/STING signaling and increases cancer immunogenicity and enhances the antitumor immune response to increase therapeutic efficacy. However, cGAS/STING deficiency in colorectal cancer (CRC) may suppress the RT-induced antitumor immunity. Therefore, we aimed to evaluate the importance of the dsRNA-mediated antitumor immune response induced by RT in patients with CRC.
METHODS
Cytosolic dsRNA level and its sensors were evaluated via cell-based assays (co-culture assay, confocal microscopy, pharmacological inhibition and immunofluorescent staining) and in vivo experiments. Biopsies and surgical tissues from patients with CRC who received preoperative chemoradiotherapy (neoCRT) were collected for multiplex cytokine assays, immunohistochemical analysis and SNP genotyping. We also generated a cancer-specific adenovirus-associated virus (AAV)-IFNβ1 construct to evaluate its therapeutic efficacy in combination with RT, and the immune profiles were analyzed by flow cytometry and RNA-seq.
RESULTS
Our studies revealed that RT stimulates the autonomous release of dsRNA from cancer cells to activate TLR3-mediated IFN-I signatures to facilitate antitumor immune responses. Patients harboring a dysfunctional TLR3 variant had reduced serum levels of IFN-I-related cytokines and intratumoral CD8 immune cells and shorter disease-free survival following neoCRT treatment. The engineered cancer-targeted construct AAV-IFNβ1 significantly improved the response to RT, leading to systematic eradication of distant tumors and prolonged survival in defective TLR3 preclinical models.
CONCLUSION
Our results support that increasing cancer-intrinsic IFNβ1 expression is an immunotherapeutic strategy that enhances the RT-induced antitumor immune response in locally patients with advanced CRC with dysfunctional TLR3.
Topics: Humans; Colorectal Neoplasms; RNA, Double-Stranded; Interferon-beta; Mice; Animals; Interferon Type I; Signal Transduction; Female; Male
PubMed: 38749537
DOI: 10.1136/jitc-2023-008515 -
Molecular Cell Jun 2024The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral...
The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.
Topics: Nucleotidyltransferases; Humans; Membrane Proteins; Signal Transduction; Micronuclei, Chromosome-Defective; Nucleotides, Cyclic; Phosphorylation; Chromosome Segregation; DNA Replication; Protein Serine-Threonine Kinases; Interferon Regulatory Factor-3; Immunity, Innate; DNA Damage; HEK293 Cells; Animals; Radiation, Ionizing; HeLa Cells
PubMed: 38749421
DOI: 10.1016/j.molcel.2024.04.017 -
Emerging Microbes & Infections Dec 2024Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we...
Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we developed a novel LASV reverse genetics system which, to our knowledge, is the first to study the complete LASV life cycle under BSL-2 conditions. Viral particles can be produced efficiently when LASV minigenomic RNA harbouring minimal viral -elements and reporter genes is transfected into a helper cell line stably expressing viral NP, GP, Z and L proteins. The resulting defective virions, named LASVmg, can propagate only in the helper cell line, providing a BSL-2 model to study the complete LASV life cycle. Using this model, we found that a previously reported cellular receptor α-dystroglycan is dispensable for LASVmg infection. Furthermore, we showed that ribavirin can inhibit LASVmg infection by inducing viral mutations. This new BSL-2 system should facilitate studying the LASV life cycle and screening antivirals.
Topics: Lassa virus; Reverse Genetics; Humans; Animals; Antiviral Agents; Chlorocebus aethiops; Cell Line; Virus Replication; Lassa Fever; Ribavirin; Vero Cells; Containment of Biohazards; Genome, Viral; Virion
PubMed: 38747061
DOI: 10.1080/22221751.2024.2356149 -
Research Square May 2024Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver miniaturized dystrophin (micro-dystrophin or µDys), which...
Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver miniaturized dystrophin (micro-dystrophin or µDys), which does not provide full protection for striated muscles as it lacks many important functional domains within full-length (FL) dystrophin. Here we develop a triple vector system to deliver FL-dystrophin into skeletal and cardiac muscles. We rationally split FL-dystrophin into three fragments (N, M, and C) linked to two orthogonal pairs of split intein, allowing efficient, unidirectional assembly of FL-dystrophin. The three fragments packaged in myotropic AAV (MyoAAV4A) restore FL-dystrophin expression in both skeletal and cardiac muscles in male mice. Dystrophin-glycoprotein complex components are also restored in the sarcolemma of dystrophic muscles. MyoAAV4A-delivered FL-dystrophin significantly improves muscle histopathology, contractility, and overall strength comparable to µDys, but unlike µDys, it also restores defective ERK signaling in heart. The FL-dystrophin gene therapy therefore promises to offer superior protection for DMD.
PubMed: 38746161
DOI: 10.21203/rs.3.rs-3867299/v1 -
Methods in Molecular Biology (Clifton,... 2024Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid...
Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid molecules of positive sense antigenome and negative sense genome. They possess perfectly complementary ends allowing the formation of extremely stable double-stranded RNA panhandle structures. The presence of the 3'-terminal promoter allows replication of these molecules by the viral polymerase. They thereby negatively interfere with replication of standard genomes. In addition, the double-stranded RNA stem structures are highly immunostimulatory and activate antiviral cell-intrinsic innate immune responses. Thus, copy-back defective interfering RNAs severely affect the virulence and pathogenesis of morbillivirus stocks. We describe two biochemical methods to analyze copy-back defective interfering RNAs in virus-infected samples, or purified viral RNA. First, we present our Northern blotting protocol that allows accurate size determination of defective interfering RNA molecules and estimation of the relative contamination level of virus preparations. Second, we describe a PCR approach to amplify defective interfering RNAs specifically, which allows detailed sequence analysis.
Topics: RNA, Viral; Morbillivirus; Animals; Blotting, Northern; Virus Replication; Polymerase Chain Reaction; RNA, Small Interfering; Genome, Viral; RNA, Double-Stranded; Humans
PubMed: 38743363
DOI: 10.1007/978-1-0716-3870-5_6 -
Methods in Molecular Biology (Clifton,... 2024RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource...
RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource competition or by interferon stimulation are known as defective interfering (DI) genomes. DI genomes can be successfully packaged into virus-like-particles referred to as defective interfering particles (DIPs). Such DIPs can sustainably coexist with the full-length virus particles and have been shown to negatively impact virus replication in vitro and in vivo. Here, we describe a method to generate a clonal DI genome population by reverse genetics. This method is applicable to other RNA viruses and will enable assessment of DIPs for their antiviral properties.
Topics: Reverse Genetics; Defective Viruses; Animals; Virus Replication; Genome, Viral; Morbillivirus; Humans; Virion; Vero Cells; Chlorocebus aethiops; RNA, Viral
PubMed: 38743362
DOI: 10.1007/978-1-0716-3870-5_5 -
PLoS Pathogens May 2024Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely...
Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNα/βR KO and wild type mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.
Topics: Animals; Mice; Hepatocytes; Receptor-Interacting Protein Serine-Threonine Kinases; Cytokines; Hepatitis Delta Virus; Hepatitis D; Cell Death; Mice, Inbred C57BL; Apoptosis; Mice, Knockout; Humans; Tumor Necrosis Factor-alpha; Disease Models, Animal
PubMed: 38739648
DOI: 10.1371/journal.ppat.1011749 -
The Journal of Dermatological Treatment Dec 2024Dystrophic epidermolysis bullosa (DEB), a rare genetic skin disease caused by loss-of-function mutations in , the gene encoding type VII collagen (COL7), is... (Review)
Review
BACKGROUND/PURPOSE
Dystrophic epidermolysis bullosa (DEB), a rare genetic skin disease caused by loss-of-function mutations in , the gene encoding type VII collagen (COL7), is characterized by skin blistering, scarring, and extracutaneous manifestations that markedly reduce patient quality-of-life. Beremagene geperpavec-svdt ('B-VEC') is a gene therapy employing a non-integrating, replication-defective herpes simplex virus type 1 (HSV-1)-based vector encoding two copies of full-length human to restore COL7 protein after topical administration to DEB wounds. B-VEC was approved in the United States in 2023 as the first topical gene therapy and the first approved treatment for DEB. However, few providers have experience with use of this gene therapy.
METHODS
Data was obtained through literature review and the experience of providers who participated in the B-VEC clinical study or initiated treatment after B-VEC approval.
RESULTS
This review discusses the burden of disease, describes the clinical trial outcomes of B-VEC, and provides physician and patient/caregiver recommendations as a practical guide for the real-world use of B-VEC, which can be administered in-office or at the patient's home.
CONCLUSIONS
By continuing to optimize the practical aspects of B-VEC administration, the focus will continue to shift to patient-centric considerations and improved patient outcomes.
Topics: Humans; Genetic Therapy; Epidermolysis Bullosa Dystrophica; Collagen Type VII; Genetic Vectors; Herpesvirus 1, Human; Treatment Outcome; Quality of Life
PubMed: 38724041
DOI: 10.1080/09546634.2024.2350232