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Cyprinid herpesvirus is a causative agent of a destructive disease in common and koi carp ( Cyprinus carpio ), which leads to substantial global financial losses in aquaculture industries. Among the strains of C. herpesvirus , C. herpesvirus 1 (CyHV-1) and C. herpesvirus 3 (CyHV-3) are known as highly pathogenic to carp fishes in Europe, Asia, and Africa. To date, no effective vaccine has been developed to combat these viruses. This study aimed to develop unique multi-epitope subunit vaccines targeting the CyHV-1 and CyHV-3 using a reverse vaccinology approach. The study began with a comprehensive literature review to identify the most critical proteins, which were then subjected to in silico analyses to predict highly antigenic epitopes. These analyses involved assessing antigenicity, transmembrane topology screening, allergenecity, toxicity, and molecular docking approaches. We constructed two multi-epitope-based vaccines incorporating a suitable adjuvant and appropriate linkers. It revealed that both the vaccines are non-toxic and immunogenic. The tertiary structures of the vaccine proteins were generated, refined, and validated to ensure their suitability. The binding affinity between the vaccine constructs and TLR3 and TLR5 receptors were assessed by molecular docking studies. Molecular dynamics simulations indicated that vaccine construct V1 exhibited greater stability with both TLR3 and TLR5 based on RMSD analysis. Hydrogen bond analysis revealed a stronger binding affinity between the vaccine constructs and TLR5 compared to TLR3. Furthermore, MM-PBSA analysis suggested that both vaccine constructs exhibited a better affinity for TLR5. Considering all aspects, the results suggest that in silico development of CyHV vaccines incorporating multiple epitopes holds promise for management of diseases caused by CyHV-1 and CyHV-3. However, further in vivo trials are highly recommended to validate the efficacies of these vaccines.

Herpesviruses establish latent infections, and most reactivate frequently, resulting in symptoms and virus shedding in healthy individuals. In immunocompromised patients, reactivating virus can cause severe disease. Persistent EBV has been associated with several malignancies in both immunocompromised and nonimmunocompromised persons. Reactivation and shedding occur with most herpesviruses, despite potent virus-specific antibodies and T cell immunity as measured in the blood. The licensure of therapeutic vaccines to reduce zoster indicates that effective therapeutic vaccines for other herpesviruses should be feasible. However, varicella-zoster virus is different from other human herpesviruses in that it is generally only shed during varicella and zoster. Unlike prophylactic vaccines, in which the correlate of immunity is antibody function, T cell immunity is the correlate of immunity for the only effective therapeutic herpesvirus vaccine-zoster vaccine. While most studies of therapeutic vaccines have measured immunity in the blood, cellular immunity at the site of reactivation is likely critical for an effective therapeutic vaccine for certain viruses. This Review summarizes the status of therapeutic vaccines for herpes simplex virus, cytomegalovirus, and Epstein-Barr virus and proposes approaches for future development.

We report the results of the world’s first trial of a vaccine against elephant endotheliotropic herpesvirus (EEHV) in elephants. EEHV-induced haemorrhagic disease is a major threat to juvenile Asian elephants. A vaccine preventing severe disease and death would support conservation efforts for this endangered species. We developed a heterologous, recombinant modified vaccinia virus Ankara prime and adjuvanted protein boost vaccine, containing regulatory protein EE2 and major capsid protein. Vaccine design targeted Th1 and cytotoxic T cell responses, crucial for herpesvirus immunity. In a proof-of-concept trial, safety and immunogenicity were tested in adult elephants. A modified interferon-γ release (IFNG) point-of-care vaccine-specific whole blood assay was established to avoid sample transport-related loss of immune readouts and determine T cell responses by RT-qPCR first. Subsequently, RNA sequencing was utilised to investigate transcriptomic changes post-vaccination. No adverse reactions were observed following heterologous vaccination. IFNG responses to candidate antigens were detected against the pre-existing latent immunity in adult elephants. Over-representation analysis revealed induction of T cell-associated pathways. Thus, we show that the vaccine has a favourable safety profile and stimulates EEHV-specific T cell-biased immune responses, warranting further evaluation. Elephant endotheliotropic herpesvirus (EEHV) haemorrhagic disease is a major threat for juvenile Asian elephants. This study reports a proof-of-concept trial that evaluates the safety and immunogenicity of a vaccine against EEHV.

The 14-3-3 molecular scaffolds promote type I interferon (IFN) responses by stabilizing the interaction of RIG-I with the TRIM25 ligase. Viruses have evolved unique strategies to halt this cellular response to support their replication and spread. Here, we report that the ubiquitin deconjugase (DUB) encoded in the N-terminus of the Epstein-Barr virus (EBV) large tegument protein BPLF1 harnesses 14-3-3 molecules to promote TRIM25 autoubiquitination and sequestration of the ligase into inactive protein aggregates. Catalytically inactive BPLF1 induced K48-linked autoubiquitination and degradation of TRIM25 while the ligase was mono- or di-ubiquitinated in the presence of the active viral enzyme and formed cytosolic aggregates decorated by the autophagy receptor p62/SQSTM1. Aggregate formation and the inhibition of IFN response were abolished by mutations of solvent exposed residues in helix-2 of BPLF1 that prevented binding to 14-3-3 while preserving both catalytic activity and binding to TRIM25. 14-3-3 interacted with the Coiled-Coil (CC) domain of TRIM25 in in vitro pulldown, while BPLF1 interacted with both the CC and B-box domains, suggesting that 14-3-3 positions BPLF1 at the ends of the CC dimer, close to known autoubiquitination sites. Our findings provide a molecular understanding of the mechanism by which a viral deubiquitinase inhibits the IFN response and emphasize the role of 14-3-3 proteins in modulating antiviral defenses.

MicroRNAs (miRNAs) are small non-coding RNAs important in gene regulation. They are able to regulate mRNA translation through base-pair complementarity. Cellular miRNAs have been involved in the regulation of nearly all cellular pathways, and their deregulation has been associated with several diseases such as cancer. Given the importance of microRNAs to cell homeostasis, it is no surprise that viruses have evolved to take advantage of this cellular pathway. Viruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. Moreover, viral inhibition of key proteins from the microRNA pathway and important changes in cellular microRNA pool have been reported upon viral infection. In addition, viruses have developed multiple mechanisms to avoid being targeted by cellular microRNAs. This complex interaction between host and viruses to control the microRNA pathway usually favors viral infection and persistence by either reducing immune detection, avoiding apoptosis, promoting cell growth, or promoting lytic or latent infection. One of the best examples of this virus-host-microRNA interplay emanates from members of the Herperviridae family, namely the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), human herpesvirus 8 (HHV-8), and the Epstein–Barr virus (EBV). In this review, we will focus on the general functions of microRNAs and the interactions between herpesviruses, human hosts, and microRNAs and will delve into the related mechanisms that contribute to infection and pathogenesis.

Human herpesviruses (HHV) cause a variety of clinically relevant conditions upon primary infection of typically young and immunocompetent hosts. Both primary infection and reactivation after latency can lead to more severe disease, such as encephalitis, congenital defects and cancer. Infections with HHV are also associated with cardiovascular and neurodegenerative disease. However, most of the associations are based on retrospective case-control analyses and well-powered prospective cohort studies are needed for assessing temporality and causality. To enable comprehensive investigations of HHV-related disease etiology in large prospective population-based cohort studies, we developed HHV Multiplex Serology. This methodology represents a low-cost, high-throughput technology that allows simultaneous measurement of specific antibodies against five HHV species: Herpes simplex viruses 1 and 2, Varicella zoster virus, Epstein-Barr virus, and Cytomegalovirus. The newly developed HHV species-specific ('Monoplex') assays were validated against established gold-standard reference assays. The specificity and sensitivity of the HHV species-specific Monoplex Serology assays ranged from 92.3% to 100.0% (median 97.4%) and 91.8% to 98.7% (median 96.6%), respectively. Concordance with reference assays was very high with kappa values ranging from 0.86 to 0.96 (median kappa 0.93). Multiplexing the Monoplex Serology assays resulted in no loss of performance and allows simultaneous detection of antibodies against the 5 HHV species in a high-throughput manner.

Key Points Herpesviruses are a large family of DNA viruses, eight of which can cause diseases in humans, particularly in children and immunocompromised individuals. All herpesviruses have the capacity to cause lytic infection in permissive cells and to establish latent or recurrent infections in other cell types. The innate immune system detects infections using germline-encoded pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are membrane-bound PRRs that detect microorganisms in extracellular and endosomal locations. TLR2, TLR3 and TLR9 are well-described sensors of herpesvirus infections. In the cytoplasm, active innate immune surveillance takes place and is mediated by nucleic acid sensors. Herpesvirus infections are sensed by both RNA and DNA sensing systems, and recent reports suggest a particularly important role for the cytosolic DNA-sensing AIM2-like receptor (ALR) family in intracellular detection of herpesviruses. Given the relatively slow replication cycle of herpesviruses and the establishment of life-long infections, these viruses are highly dependent on efficient immune evasion strategies. It is now known that herpesviruses evade all classes of PRRs (including ALRs), as well as the downstream signalling machinery. Innate immune defence against herpesviruses is highly dependent on the type I interferon system and natural killer cells. Model studies in mice and genetic data from humans have revealed essential roles for both TLRs and intracellular DNA sensors in mounting protective immune responses against herpesviruses. Here the authors describe how multiple pattern recognition receptors, at the host cell surface, in endosomes and in the cytoplasm, are involved in detecting herpesviruses. How do they each contribute to immune defence against the virus, and how does the virus evade this detection and persist in the host? Advances in innate immunity over the past decade have revealed distinct classes of pattern recognition receptors (PRRs) that detect pathogens at the cell surface and in intracellular compartments. This has shed light on how herpesviruses, which are large disease-causing DNA viruses that replicate in the nucleus, are initially recognized during cellular infection. Surprisingly, this involves multiple PRRs both on the cell surface and within endosomes and the cytosol. In this article we describe recent advances in our understanding of innate detection of herpesviruses, how this innate detection translates into anti-herpesvirus host defence, and how the viruses seek to evade this innate detection to establish persistent infections.

Herpesviruses are ubiquitous DNA viruses that can establish latency and cause a range of mild to life-threatening diseases in humans. Upon infection, herpesviruses trigger the activation of several host antiviral defense programs that play critical roles in curbing virus replication and dissemination. Recent work from many groups has integrated our understanding of TRIM (tripartite motif) proteins, a specific group of E3 ligase enzymes, as pivotal orchestrators of mammalian antiviral immunity. In this review, we summarize recent advances in the modulation of innate immune signaling by TRIM proteins during herpesvirus infection, with a focus on the detection of herpes simplex virus type 1 (HSV-1, a prototype herpesvirus) by cGAS-STING, RIG-I-like receptors, and Toll-like receptors. We also review the latest progress in understanding the intricate relationship between herpesvirus replication and TRIM protein-regulated autophagy and apoptosis. Finally, we discuss the maneuvers used by HSV-1 and other herpesviruses to overcome TRIM protein-mediated virus restriction.

IFI16 (gamma-interferon-inducible protein 16), a predominantly nuclear protein involved in transcriptional regulation, also functions as an innate immune response DNA sensor and induces the IL-1β and antiviral type-1 interferon-β (IFN-β) cytokines. We have shown that IFI16, in association with BRCA1, functions as a sequence independent nuclear sensor of episomal dsDNA genomes of KSHV, EBV and HSV-1. Recognition of these herpesvirus genomes resulted in IFI16 acetylation, BRCA1-IFI16-ASC-procaspase-1 inflammasome formation, cytoplasmic translocation, and IL-1β generation. Acetylated IFI16 also interacted with cytoplasmic STING and induced IFN-β. However, the identity of IFI16 associated nuclear proteins involved in STING activation and the mechanism is not known. Mass spectrometry of proteins precipitated by anti-IFI16 antibodies from uninfected endothelial cell nuclear lysate revealed that histone H2B interacts with IFI16. Single and double proximity ligation microscopy, immunoprecipitation, EdU-genome labeled virus infection, and chromatin immunoprecipitation studies demonstrated that H2B is associated with IFI16 and BRCA1 in the nucleus in physiological conditions. De novo KSHV and HSV-1 infection as well as latent KSHV and EBV infection induces the cytoplasmic distribution of H2B-IFI16, H2B-BRCA1 and IFI16-ASC complexes. Vaccinia virus (dsDNA) cytoplasmic replication didn't induce the redistribution of nuclear H2B-IFI16 or H2B into the cytoplasm. H2B is critical in KSHV and HSV-1 genome recognition by IFI16 during de novo infection. Viral genome sensing by IFI16-H2B-BRCA1 leads to BRCA1 dependent recruitment of p300, and acetylation of H2B and IFI16. BRCA1 knockdown or inhibition of p300 abrogated the acetylation of H2B-IFI16 or H2B. Ran-GTP protein mediated the translocation of acetylated H2B and IFI16 to the cytoplasm along with BRCA1 that is independent of IFI16-ASC inflammasome. ASC knockdown didn't affect the acetylation of H2B, its cytoplasmic transportation, and the association of STING with IFI16 and H2B during KSHV infection. Absence of H2B didn't affect IFI16-ASC association and cytoplasmic distribution and thus demonstrating that IFI16-H2B complex is independent of IFI16-ASC-procaspase-1-inflammasome complex formed during infection. The H2B-IFI16-BRCA1 complex interacted with cGAS and STING in the cytoplasm leading to TBK1 and IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production. Silencing of H2B, cGAS and STING inhibited IFN-β induction but not IL-1β secretion, and cGAMP activity is significantly reduced by H2B and IFI16 knockdown during infection. Silencing of ASC inhibited IL-1β secretion but not IFN-β secretion during de novo KSHV and HSV-1 infection. These studies identify H2B as an innate nuclear sensor mediating a novel extra chromosomal function, and reveal that two IFI16 complexes mediate KSHV and HSV-1 genome recognition responses, with recognition by the IFI16-BRCA1-H2B complex resulting in IFN-β responses and recognition by IFI16-BRCA1 resulting in inflammasome responses.

Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.