Telomeric repeats in the commercial SB-1 vaccine facilitate viral integration and contribute to vaccine efficacy.

Marek's disease virus (MDV) integrates its genome into the telomeres of host chromosomes and causes fatal lymphomas in chickens. This integration is facilitated by telomeric repeat sequences (TMRs) at the ends of the viral genome, and is crucial for MDV-induced lymphomagenesis. The SB-1 vaccine virus is commonly used in commercial bivalent vaccines against MDV and also contains TMRs at its ends. Here, we demonstrate that SB-1 efficiently integrates its genome into the chromosomes of latently infected T cells. Deletion of the TMRs from the SB-1 genome did not affect virus replication, but severely impaired virus integration and genome maintenance in latently infected T cells and in chickens. Strikingly, the reduced integration and maintenance of latent SB-1 significantly impaired vaccine protection. Taken together, our data revealed that the TMRs facilitate SB-1 integration and that integration and/or maintenance of the latent viral genome is critical for vaccine protection.

Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes deadly lymphomas in chickens. In chickens, up to 50% of all peripheral T cells are gamma delta (γδ) T cells. Until now, their role in MDV pathogenesis and tumor formation remains poorly understood. To investigate the role of γδ T cells in MDV pathogenesis, we infected recently generated γδ T cell knockout chickens with very virulent MDV. Strikingly, disease and tumor incidence were highly increased in the absence of γδ T cells, indicating that γδ T cells play an important role in the immune response against MDV. In the absence of γδ T cells, virus replication was drastically increased in the thymus and spleen, which are potential sites of T cell transformation. Taken together, our data provide the first evidence that γδ T cells play an important role in the pathogenesis and tumor formation of this highly oncogenic herpesvirus.IMPORTANCEGamma delta (γδ) T cells are the most abundant T cells in chickens, but their role in fighting pathogens remains poorly understood. Marek's disease virus (MDV) is an important veterinary pathogen, that causes one of the most frequent cancers in animals and is used as a model for virus-induced tumor formation. Our study revealed that γδ T cells play a crucial role in combating MDV, as disease and tumor incidence drastically increased in the absence of these cells. γδ T cells restricted virus replication in the key lymphoid organs, thereby decreasing the likelihood of causing tumors and disease. This study provides novel insights into the role of γδ T cells in the pathogenesis of this highly oncogenic virus.

Mucus-Inspired Self-Healing Hydrogels: A Protective Barrier for Cells against Viral Infection.

Mucus is a dynamic biological hydrogel, composed primarily of the glycoprotein mucin, exhibits unique biophysical properties and forms a barrier protecting cells against a broad-spectrum of viruses. Here, this work develops a polyglycerol sulfate-based dendronized mucin-inspired copolymer (MICP-1) with ≈10% repeating units of activated disulfide as cross-linking sites. Cryo-electron microscopy (Cryo-EM) analysis of MICP-1 reveals an elongated single-chain fiber morphology. MICP-1 shows potential inhibitory activity against many viruses such as herpes simplex virus 1 (HSV-1) and SARS-CoV-2 (including variants such as Delta and Omicron). MICP-1 produces hydrogels with viscoelastic properties similar to healthy human sputum and with tuneable microstructures using linear and branched polyethylene glycol-thiol (PEG-thiol) as cross-linkers. Single particle tracking microrheology, electron paramagnetic resonance (EPR) and cryo-scanning electron microscopy (Cryo-SEM) are used to characterize the network structures. The synthesized hydrogels exhibit self-healing properties, along with viscoelastic properties that are tuneable through reduction. A transwell assay is used to investigate the hydrogel's protective properties against viral infection against HSV-1. Live-cell microscopy confirms that these hydrogels can protect underlying cells from infection by trapping the virus, due to both network morphology and anionic multivalent effects. Overall, this novel mucin-inspired copolymer generates mucus-mimetic hydrogels on a multi-gram scale. These hydrogels can be used as models for disulfide-rich airway mucus research, and as biomaterials.

Impact of viral telomeric repeat sequences on herpesvirus vector vaccine integration and persistence.

Marek's disease virus (MDV) vaccines were the first vaccines that protected against cancer. The avirulent turkey herpesvirus (HVT) was widely employed and protected billions of chickens from a deadly MDV infection. It is also among the most common vaccine vectors providing protection against a plethora of pathogens. HVT establishes latency in T-cells, allowing the vaccine virus to persist in the host for life. Intriguingly, the HVT genome contains telomeric repeat arrays (TMRs) at both ends; however, their role in the HVT life cycle remains elusive. We have previously shown that similar TMRs in the MDV genome facilitate its integration into host telomeres, which ensures efficient maintenance of the virus genome during latency and tumorigenesis. In this study, we investigated the role of the TMRs in HVT genome integration, latency, and reactivation in vitro and in vivo. Additionally, we examined HVT infection of feather follicles. We generated an HVT mutant lacking both TMRs (vΔTMR) that efficiently replicated in cell culture. We could demonstrate that wild type HVT integrates at the ends of chromosomes containing the telomeres in T-cells, while integration was severely impaired in the absence of the TMRs. To assess the role of TMRs in vivo, we infected one-day-old chickens with HVT or vΔTMR. vΔTMR loads were significantly reduced in the blood and hardly any virus was transported to the feather follicle epithelium where the virus is commonly shed. Strikingly, latency in the spleen and reactivation of the virus were severely impaired in the absence of the TMRs, indicating that the TMRs are crucial for the establishment of latency and reactivation of HVT. Our findings revealed that the TMRs facilitate integration of the HVT genome into host chromosomes, which ensures efficient persistence in the host, reactivation, and transport of the virus to the skin.

Unraveling the chicken T cell repertoire with enhanced genome annotation.

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in ß and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/ß/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the ß and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.

An Antiherpesviral Host-Directed Strategy Based on CDK7 Covalently Binding Drugs: Target-Selective, Picomolar-Dose, Cross-Virus Reactivity.

The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors.

Comparison of methods for the detection of porcine cytomegalovirus/roseolovirus in relation to biosafety monitoring of xenotransplantation products.

BACKGROUND: The porcine cytomegalovirus, a porcine roseolovirus (PCMV/PRV), is widely distributed in pig populations. It has been shown that PCMV/PRV was transmitted by pig xenotransplants to non-human primates, and significantly reduced the survival time of the recipient. PCMV/PRV was also transmitted during the first transplantation of a pig heart into a human patient. PCMV/PRV establishes a lifelong persistent infection (latency) in the host, is difficult to detect in this stage, and consequential poses a threat to future clinical xenotransplantations. Therefore, sensitive and specific methods and goal-oriented strategies how, when, and where to test should be used for screening donor pigs. METHODS: In this study we compared experimentally the PCMV/PRV detection methods including PCR-based (real-time PCR, nested PCR) and immunological methods (Western blot assay, ELISA) recently published by Halecker et al. (Sci. Rep. 2022;12(1):21545) and Fischer et al. (Xenotransplantation 2023:e12803). We also compared the antigens used for antibody detection (a recombinant protein and synthetic peptides corresponding to a conserved region of the glycoprotein B, gB). RESULTS: The published methods can be used for screening donor pigs, with the results being similar. The antigens used for the detection of PCMV/PRV-specific antibodies are almost identical and give comparable results. Overall, the optimal diagnostic tests, the samples used for testing and the time of sampling play a crucial role in preventing the transmission of PCMV/PRV during xenotransplantation. CONCLUSION: Sensitive methods are available to screen donor pigs for PCMV/PRV, but a rational application of a combination of PCR-based and immunological methods as well as rational detection strategies are important for the detection of the virus during latency.

Viral and cellular telomerase RNAs possess host-specific anti-apoptotic functions.

Human telomerase RNA (hTR) is overexpressed in many cancers and protects T cells from apoptosis in a telomerase-independent manner. The most prevalent cancer in the animal kingdom is caused by the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV encodes a viral telomerase RNA (vTR) that plays a crucial role in MDV-induced tumorigenesis and shares all four conserved functional domains with hTR. In this study, we assessed whether hTR drives tumor formation in this natural model of herpesvirus-induced tumorigenesis. Therefore, we replaced vTR with hTR in the genome of a highly oncogenic MDV. Furthermore, we investigated the anti-apoptotic activity of vTR, hTR, and their counterpart in the chicken [chicken telomerase RNA (cTR)]. hTR was efficiently expressed and did not alter replication of the recombinant virus. Despite its conserved structure, hTR did not complement the loss of vTR in virus-induced tumorigenesis. Strikingly, hTR did not inhibit apoptosis in chicken cells, but efficiently inhibited apoptosis in human cells. Inverse host restriction has been observed for vTR and cTR in human cells. Our data revealed that vTR, cTR, and hTR possess conserved but host-specific anti-apoptotic functions that likely contribute to MDV-induced tumorigenesis. IMPORTANCE hTR is overexpressed in many cancers and used as a cancer biomarker. However, the contribution of hTR to tumorigenesis remains elusive. In this study, we assessed the tumor-promoting properties of hTR using a natural virus/host model of herpesvirus-induced tumorigenesis. This avian herpesvirus encodes a telomerase RNA subunit (vTR) that plays a crucial role in viral tumorigenesis and shares all conserved functional domains with hTR. Our data revealed that vTR and cellular TRs of humans and chickens possess host-specific anti-apoptotic functions. This provides important translational insights into therapeutic strategies, as inhibition of apoptosis is crucial for tumorigenesis.

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.

Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.

In vitro infection of primary chicken lymphocytes with Marek's disease virus.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects immune cells and causes a deadly lymphoproliferative disease in chickens. Cytokines and monoclonal antibodies promote the survival of chicken lymphocytes in vitro. Here, we describe protocols for the isolation, maintenance, and efficient MDV infection of primary chicken lymphocytes and lymphocyte cell lines. This facilitates the investigation of key aspects of the MDV life cycle in the primary target cells of viral replication, latency, genome integration, and reactivation. For complete details on the use and execution of this protocol, please refer to Schermuly et al.,1 Bertzbach et al. (2019),2 and You et al.3 For a comprehensive background on MDV, please see Osterrieder et al.4 and Bertzbach et al. (2020).5.

Mucin-Inspired Single-Chain Polymer (MIP) Fibers as Potent SARS-CoV-2 Inhibitors.

Mucins are the key component of the defensive mucus barrier. They are extended fibers of very high molecular weight with diverse biological functions depending strongly on their specific structural parameters. Here, we present a mucin-inspired nanostructure, produced via a synthetic methodology to prepare methacrylate-based dendronized polysulfates (MIP-1) on a multi gram-scale with high molecular weight (MW=450 kDa) and thiol end-functionalized mucin-inspired polymer (MIP) via RAFT polymerization. Cryo-electron tomography (Cryo-ET) analysis of MIP-1 confirmed a mucin-mimetic wormlike single-chain fiber structure (length=144±59 nm) in aqueous solution. This biocompatible fiber showed promising activity against SARS-CoV-2 and its mutant strain, with a remarkable low half maximal (IC50 ) inhibitory concentration (IC50 =10.0 nM). Additionally, we investigate the impact of fiber length on SARS-CoV-2 inhibition by testing other functional polymers (MIPs) of varying fiber lengths.

Genomic investigations of unexplained acute hepatitis in children.

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

A Special Issue on Marek's Disease Virus-The Editors' View.

Marek's disease virus (MDV), an Alphaherpesvirus belonging to the genus Mardivirus, causes T cell lymphomas in chickens and remains one of the greatest threats to poultry production worldwide [...].

Excision of Integrated Human Herpesvirus 6A Genomes Using CRISPR/Cas9 Technology.

Human herpesviruses 6A and 6B are betaherpesviruses that can integrate their genomes into the telomeres of latently infected cells. Integration can also occur in germ cells, resulting in individuals who harbor the integrated virus in every cell of their body and can pass it on to their offspring. This condition is termed inherited chromosomally integrated HHV-6 (iciHHV-6) and affects about 1% of the human population. The integrated HHV-6A/B genome can reactivate in iciHHV-6 patients and in rare cases can also cause severe diseases including encephalitis and graft-versus-host disease. Until now, it has remained impossible to prevent virus reactivation or remove the integrated virus genome. Therefore, we developed a system that allows the removal of HHV-6A from the host telomeres using the CRISPR/Cas9 system. We used specific guide RNAs (gRNAs) targeting the direct repeat region at the ends of the viral genome to remove the virus from latently infected cells generated in vitro and iciHHV-6A patient cells. Fluorescence-activated cell sorting (FACS), quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH) analyses revealed that the virus genome was efficiently excised and lost in most cells. Efficient excision was achieved with both constitutive and transient expression of Cas9. In addition, reverse transcription-qPCR (RT-qPCR) revealed that the virus genome did not reactivate upon excision. Taken together, our data show that our CRISPR/Cas9 approach allows efficient removal of the integrated virus genome from host telomeres. IMPORTANCE Human herpesvirus 6 (HHV-6) infects almost all humans and integrates into the telomeres of latently infected cells to persist in the host for life. In addition, HHV-6 can also integrate into the telomeres of germ cells, which results in about 80 million individuals worldwide who carry the virus in every cell of their body and can pass it on to their offspring. In this study, we develop the first system that allows excision of the integrated HHV-6 genome from host telomeres using CRISPR/Cas9 technology. Our data revealed that the integrated HHV-6 genome can be efficiently removed from the telomeres of latently infected cells and cells of patients harboring the virus in their germ line. Virus removal could be achieved with both stable and transient Cas9 expression, without inducing viral reactivation.

Impact of Host Telomere Length on HHV-6 Integration.

Human herpesvirus 6A and 6B are two closely related viruses that infect almost all humans. In contrast to most herpesviruses, HHV-6A/B can integrate their genomes into the telomeres during the infection process. Both viruses can also integrate in germ cells and subsequently be inherited in children. How HHV-6A/B integrate into host telomeres and the consequences of this remain a subject of active research. Here, we developed a method to measure telomere length by quantitative fluorescence in situ hybridization, confocal microscopy, and computational processing. This method was validated using a panel of HeLa cells having short or long telomeres. These cell lines were infected with HHV-6A, revealing that the virus could efficiently integrate into telomeres independent of their length. Furthermore, we assessed the telomere lengths after HHV-6A integration and found that the virus-containing telomeres display a variety of lengths, suggesting that either telomere length is restored after integration or telomeres are not shortened by integration. Our results highlight new aspects of HHV-6A/B biology and the role of telomere length on virus integration.

In vivo imaging reveals novel replication sites of a highly oncogenic avian herpesvirus in chickens.

In vivo bioluminescence imaging facilitates the non-invasive visualization of biological processes in living animals. This system has been used to track virus infections mostly in mice and ferrets; however, until now this approach has not been applied to pathogens in avian species. To visualize the infection of an important avian pathogen, we generated Marek's disease virus (MDV) recombinants expressing firefly luciferase during lytic replication. Upon characterization of the recombinant viruses in vitro, chickens were infected and the infection visualized in live animals over the course of 14 days. The luminescence signal was consistent with the known spatiotemporal kinetics of infection and the life cycle of MDV, and correlated well with the viral load measured by qPCR. Intriguingly, this in vivo bioimaging approach revealed two novel sites of MDV replication, the beak and the skin of the feet covered in scales. Feet skin infection was confirmed using a complementary fluorescence bioimaging approach with MDV recombinants expressing mRFP or GFP. Infection was detected in the intermediate epidermal layers of the feet skin that was also shown to produce infectious virus, regardless of the animals' age at and the route of infection. Taken together, this study highlights the value of in vivo whole body bioimaging in avian species by identifying previously overlooked sites of replication and shedding of MDV in the chicken host.

The Diverse Major Histocompatibility Complex Haplotypes of a Common Commercial Chicken Line and Their Effect on Marek's Disease Virus Pathogenesis and Tumorigenesis.

The major histocompatibility complex (MHC) is crucial for appropriate immune responses against invading pathogens. Chickens possess a single predominantly-expressed class I molecule with strong associations between disease resistance and MHC haplotype. For Marek's disease virus (MDV) infections of chickens, the MHC haplotype is one of the major determinants of genetic resistance and susceptibility. VALO specific pathogen free (SPF) chickens are widely used in biomedical research and vaccine production. While valuable findings originate from MDV infections of VALO SPF chickens, their MHC haplotypes and associated disease resistance remained elusive. In this study, we used several typing systems to show that VALO SPF chickens possess MHC haplotypes that include B9, B9:02, B15, B19 and B21 at various frequencies. Moreover, we associate the MHC haplotypes to MDV-induced disease and lymphoma formation and found that B15 homozygotes had the lowest tumor incidence while B21 homozygotes had the lowest number of organs with tumors. Finally, we found transmission at variable levels to all contact birds except B15/B21 heterozygotes. These data have immediate implications for the use of VALO SPF chickens and eggs in the life sciences and add another piece to the puzzle of the chicken MHC complex and its role in infections with this oncogenic herpesvirus.

Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA.

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic-latent switch and viral non-coding RNAs3,4. Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30-p53-DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.