动物疱疹病毒的致病机制

Simple Summary Equine herpesvirus 8 (EHV-8) is an important pathogen primarily affecting the horse and donkey industry, but there is little information about the pathogenicity and immune response of EHV-8 in a mouse model. We aim to investigate the pathogenicity and immune response in the lung during EHV-8 infection in BALB/c mice. The results showed that EHV-8 could effectively replicate and elicits a strong proinflammatory response in the lung tissues of a mouse model. The mouse model of viral respiratory disease proposed here will also be useful for studying the underlying mechanisms of the pathology of respiration. Abstract Equine herpesvirus type 8 (EHV-8), associated with abortion and severe respiratory disease in donkeys and horses, causes significant economic losses in the global equine industry. However, the pathogenicity of EHV-8 is still unknown. Mice are widely used as an animal model to evaluate virus replication and virulence. The present study aimed to evaluate the pathogenicity of the EHV-8 SDLC66 strain in BALB/c mice. Mice were used to test for infection-associated parameters (such as clinical signs, body weights, virus replication in tissues, viremia, and cytokines) and sacrificed at 0, 2, 4, and 6 days post-infection (dpi). The mice inoculated with EHV-8 exhibited lethargy, dyspnea signs, loss in body weight, and viremia. EHV-8 was detected in the liver, spleen, brain, and lung by PCR at 4 dpi and 6 dpi, effectively replicating these tissues detected by TCID50 at 6 dpi. Proinflammatory cytokines, including IL-6, IL-1β, and TNF-α, were significantly increased at the 4 dpi and 6 dpi in the lung than in the control group. However, IFN-γ was only increased at 6 dpi in the EHV-8-infected group. These data showed that EHV-8 could enter the lungs of mice and cause respiratory disease in the mouse model, which helps reveal the pathogenicity of EHV-8.

Equine herpesvirus-9 (EHV-9), equine herpesvirus-1 (EHV-1) and zebra-borne EHV-1 are members of the family Herpesviridae and cause encephalitis and rhinopneumonitis in a range of animal species. The aim of this study was to characterize and compare the rhinopneumonitis induced by experimental intranasal inoculation of groups of hamsters with EHV-9, EHV-1 strain Ab4p or zebra-borne EHV-1 viruses. Animals inoculated with EHV-9 had earlier and more severe neurological and respiratory signs than those inoculated with EHV-1 strain Ab4p or zebra-borne EHV-1. At 4-5 days post inoculation (dpi), hamsters inoculated with EHV-9 had significantly increased expression of open reading fame (ORF) 30, the viral gene encoding the DNA polymerase, in lung tissue. ORF 30 expression at these time points was higher in the hamsters infected with EHV-9 than in those inoculated with the other two viruses. Severe, mild or very mild rhinitis was seen in animals inoculated with EHV-1 strain Ab4p, EHV-9 and zebra-borne EHV-1, respectively. Viral antigen was detected in olfactory receptor neurons, inflammatory cells and desquamated epithelial cells in animals in all groups until 5 dpi. Tracheitis was also seen in all three virus-infected groups with viral antigen detected in tracheal epithelium. Inoculated hamsters developed interstitial pneumonia of increasing severity over the course of the experiment. Bronchopneumonia and vasculitis were also seen in all three infected groups. These results confirm that, in addition to their neurotropism, EHV-9 and zebra-borne EHV-1 are pneumotropic viruses. EHV-1 strain Ab4p caused more severe upper respiratory tract disease, but no significant differences were detected in the severity of pneumonia induced by each virus.

Significance Marek’s disease virus (MDV) infects chickens and causes the most frequent clinically diagnosed cancer in the animal kingdom, and it is used as a small-animal model for virus-induced tumor formation. Until now, B cells were thought to play a central role in MDV pathogenesis. We disproved this dogma using knockout (KO) chickens that lack mature and peripheral B cells. We demonstrated that B cells are dispensable for virus replication, virus spread, and tumor formation. In the absence of B cells, T cells facilitate efficient virus replication and are subsequently transformed, resulting in deadly lymphomas. Our data pioneer the use of KO chickens in infectious disease research and expand the knowledge on the life cycle of this highly oncogenic virus. Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4+ T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4+ and CD8+ T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

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Chelonia mydas (green turtles) are being threatened worldwide by fibropapillomatosis (FP), which has seriously affected their survival. The presence of FP on the body surface and visceral organs of green turtles found dead was confirmed, causing obstruction of the gastrointestinal tract, changes in foraging behavior, and reduction of visceral functions. The etiology of FP has not yet been elucidated, and previous research generally considers that the occurrence of FP is related to the chelonid alphaherpesvirus 5 (ChHV5), associated with low animal immunity, and also with marine environmental factors, such as poor water quality and eutrophication. However, there is no evaluation on the induction of FP pathogenesis associated with the green turtle. In this study, we evaluated blood samples from green turtles with and without FP using de novo transcriptome assembly. Results indicated that 3,090 differentially expressed genes (DEGs) (p < 0.05) were identified, including 1,357 upregulated genes and 1,733 downregulated genes in turtles with or without FP. We observed that DEGs, which are significantly upregulated, are found in cancer development, namely, MAPK1IP1L and APAF1. Furthermore, the infected green turtle indicated that the greater number of DEGs was contributed by the NOD-like receptor signaling pathway, which can be activated through an endocytosis of the viral particle by the immune system cells, and the Wnt signaling pathway, which is believed to have played a role in FP tumorigenesis. We validated the more upregulated/downregulated DEGs in cancer development and immunization, and DEGs such as LEF1, BTRC, and FOSL1 participating in the NOD-like receptor signaling pathway, as well as ERBIN, TRAF6, and NFKB1 in the Wnt signaling pathway, using real-time quantitative polymerase chain reaction (RT-qPCR). Altogether, this study provided some genes as potential markers during FP infection and a further evidence of FP in endangered green turtles in Taiwan.

CD30-positive germinal center (GC)-derived B cell lymphomas are frequently linked to Epstein–Barr Virus (EBV) infection. However, a suitable animal model for the investigation of the interplay between γ-herpesvirus and host cells in B cell pathogenesis is currently lacking. Here, we present a novel in vivo model enabling the analysis of genetically modified viruses in combination with genetically modified GC B cells. As a murine γ-herpesvirus, we used MHV-68 closely mirroring the biology of EBV. Our key finding was that Cre-mediated recombination can be successfully induced by an MHV-68 infection in GC B cells from Cγ1-Cre mice allowing for deletion or activation of loxP-flanked cellular genes. The implementation of PrimeFlow RNA assay for MHV-68 demonstrated the enrichment of MHV-68 in GC and isotype-switched B cells. As illustrations of virus and cellular modifications, we inserted the EBV gene LMP2A into the MHV-68 genome and induced constitutively active CD30-signaling in GC B cells through MHV-68 infections, respectively. While the LMP2A-expressing MHV-68 behaved similarly to wildtype MHV-68, virally induced constitutively active CD30-signaling in GC B cells led to the expansion of a pre-plasmablastic population. The findings underscore the potential of our novel tools to address crucial questions about the interaction between herpesviral infections and deregulated cellular gene-expression in future studies.

A signature trait of neurotropic α-herpesviruses (α-HV) is their ability to establish stable non-productive infections of peripheral neurons termed latency. This specialized gene expression program is the foundation of an evolutionarily successful strategy to ensure lifelong persistence in the host. Various physiological stresses can induce reactivation in a subset of latently-infected neurons allowing a new cycle of viral productive cycle gene expression and synthesis of infectious virus. Recurring reactivation events ensure transmission of the virus to new hosts and contributes to pathogenesis. Efforts to define the molecular basis of α-HV latency and reactivation have been notoriously difficult because the neurons harboring latent virus in humans and in experimentally infected live-animal models, are rare and largely inaccessible to study. Increasingly, researchers are turning to cultured neuron infection models as simpler experimental platforms from which to explore latency and reactivation at the molecular level. In this review, I reflect on the strengths and weaknesses of existing neuronal models and briefly summarize the important mechanistic insights these models have provided. I also discuss areas where prioritization will help to ensure continued progress and integration.

Meningoencephalitis by Herpesvirus type 5 (BoHV-5) in cattle has some features that are similar to those of herpetic encephalitis in humans and other animal species. Human Herpesvirus 3 (commonly known as Varicella-zoster virus 1), herpes simplex viruses (HSV), and equid Herpesvirus 1 (EHV-1) induce an intense inflammatory, vascular and cellular response. In spite of the many reports describing the histological lesions associated with natural and experimental infections, the immunopathological mechanisms for the development of neurological disorder have not been established. A total of twenty calf brains were selected from the Veterinary School, University of São Paulo State, Araçatuba, Brazil, after confirmation of BoHV-5 infection by virus isolation as well as by a molecular approach. The first part of the study characterized the microscopic lesions associated with the brain areas in the central nervous system (CNS) that tested positive in a viral US9 gene hybridization assay. The frontal cortex (Fc), parietal cortex (Pc), thalamus (T) and mesencephalon (M) were studied. Secondly, distinct pathogenesis mechanisms that take place in acute cases were investigated by an immunohistochemistry assay. This study found the frontal cortex to be the main region where intense oxidative stress phenomena (AOP-1) and synaptic protein expression (SNAP-25) were closely related to inflammatory cuffs, satellitosis and gliosis, which represent the most frequently observed neurological lesions. Moreover, MMP-9 expression was shown to be localized in the leptomeninges, in the parenchyma and around mononuclear infiltrates (p < 0.0001). These data open a new perspective in understanding the role of the AOP-1, MMP-9 and SNAP-25 proteins in mediating BoHV-5 pathogenesis and the strategies of host-virus interaction in order to invade de CNS.

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Herpesvirus-encoded microRNAs (miRNAs) have been discovered in infected cells; however, lack of a suitable animal model has hampered functional analyses of viral miRNAs in vivo. Marek’s disease virus (MDV) (Gallid alphaherpesvirus 2, GaHV-2) genome contains 14 miRNA precursors, which encode 26 mature miRNAs, grouped into three clusters. In this study, the role of MDV-encoded cluster 3 miRNAs, also known as mdv1-miR-M8-M10, in pathogenesis was evaluated in chickens, the natural host of MDV. Our results show that deletion of cluster 3 miRNAs did not affect virus replication and plaque size in cell culture, but increased early cytolytic replication of MDV in chickens. We also observed that deletion of cluster 3 miRNAs resulted in significantly higher virus reactivation from peripheral blood lymphocytes. In addition, pathogenesis studies showed that deletion of cluster 3 miRNAs resulted in more severe atrophy of lymphoid organs and reduced mean death time, but did not affect the incidence of MDV-associated visceral tumors. We confirmed these results by generating a cluster 3 miRNA revertant virus in which the parental MDV phenotype was restored. To the best of our knowledge, our study provides the first evidence that MDV cluster 3 miRNAs play an important role in modulating MDV pathogenesis.

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Primary infection and pathogenesis of equine herpesvirus type 1 (EHV-1) require an intricate interaction of virus with the mucosal epithelium, mononuclear cells and the vascular endothelium. Studies on EHV-1 have been facilitated by the development of different in vitro models that recapitulate the in vivo tissue complexity. The available in vitro assays can be categorized into (i) models mimicking the epithelium-peripheral blood mononuclear cell (PBMC) interaction, which include ex vivo mucosal (nasal and vaginal) explants and equine respiratory epithelial cells (EREC) cultures; and (ii) PBMC-endothelium mimicking models, including flow chamber and contact assays. These in vitro models have proven their worth in attempts to recapitulate the in vivo architecture and complexity, produce data relevant to natural host infection, and reduce animal use due to in vivo experiments. Although horse models are still needed for certain experiments, e.g., EHV-1 myeloencephalopathy or vaccination studies, available in vitro models can be used to obtain highly valuable data on virus-host tissue interactions. Microfluidic based 3D culture system (e.g., horse-on-a-chip) could be a potential upgraded version of these in vitro models for future research.

KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA. ABSTRACT The latency-associated nuclear antigen from Kaposi's sarcoma-associated herpesvirus (KSHV), kLANA, and its homolog from the murid herpesvirus 4 (MuHV-4), mLANA, are essential for viral latency. kLANA is nearly four times the size of mLANA, mainly due to an extensive central repeat region that is absent in mLANA. Both proteins harbor a C-terminal DNA binding domain (DBD). The DBD binds the terminal repeat (TR) DNA sequences of the viral genome to mediate persistence. Despite structural conservation, the kLANA and mLANA DBDs differ in sequence and mode of oligomerization. kLANA DBD oligomers are flexible and bent, while mLANA DBD oligomers bind DNA in a rigid, linear conformation. We previously reported that kLANA and mLANA acted reciprocally on TR sequences. Furthermore, a MuHV-4 expressing kLANA instead of mLANA (v-kLANA) established latency in mice, albeit at a lower magnitude than the wild-type (WT) virus. Here, we asked if kLANA can accommodate the mLANA DBD and generated a fusion protein which contains kLANA but with the mLANA C-terminal region in place of that of kLANA. We report a recombinant MuHV-4 (v-KM) encoding this LANA fusion protein instead of mLANA. The fusion protein was expressed in lytic infection in vitro and assembled nuclear LANA dots in infected splenocytes. Results demonstrated that kLANA functionally accommodated mLANA's mode of DNA binding, allowing MuHV-4 chimeric virus to establish latency in vivo. Notably, v-KM established latency in germinal center B cells more efficiently than did v-kLANA, although levels were reduced compared to WT MuHV-4. IMPORTANCE KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency in vivo more efficiently than MuHV-4 expressing full-length kLANA.

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae subfamily, causes significant economic losses to the cattle industry worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an essential role in the viral entry into permissive cells and possibly cooperates with other envelope glycoproteins. The herpesvirus gD induces a protective immune response against diseases in cattle or animal models. Mapping epitopes on gD will facilitate the understanding of the BoHV-1 pathogenesis and development of alternative vaccines against various diseases associated with the virus. In this study, a monoclonal antibody (MAb), designated as 3C1, was generated using naive BoHV-1 in vaccination of mice, demonstrating that 3C1 was specific to gD and represents a neutralizing activity against BoHV-1 infection in Madin-Darby bovine kidney cells. Panels of overlapping gD recombinant proteins with glutathione S-transferase tag were prepared to define the epitope recognized by 3C1. The data demonstrated that the N-terminus of gD 23APRVTVYVD31 was recognized by 3C1. Furthermore, the 26VTVYVD31 motif was the minimal amino acid sequence for the recognition. The epitope identified in this study is highly conserved among the typical strains of BoHV-1 and BoHV-5, suggesting that this epitope may be useful in the diagnosis of diseases. In addition, the defined region on gD of BoHV-1 might be essential in viral entry upon comparison with the prototype virus in herpes simplex virus (Alphaherpesvirinae). The data will elucidate the roles of gD of BoHV-1 in viral entry and pathogenesis and its potential application for the development of vaccine candidates and diagnostic techniques based on the conserved epitopes on gD or in combination with those of other herpesvirus glycoproteins.

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Adaptive immune responses to primary Kaposi sarcoma-associated herpesvirus (KSHV) infection are poorly defined. To develop better small-animal models for understanding KSHV pathogenesis and immunity, we previously generated a chimeric virus in which the KSHV latency-associated nuclear antigen (kLANA), a conserved multifunctional protein critical for viral latency, was exchanged for the LANA homolog in murine gammaherpesvirus 68 (MHV68). Despite comparable levels of latent infection between WT and KLKI MHV68, kLANA directly repressed MHV68 lytic replication and reactivation. We therefore hypothesized that suppression of lytic replication by kLANA dampens adaptive immune responses. To test this, mice were infected with equivalent doses of either WT or KLKI MHV68 and adaptive immune responses were evaluated over time. B and T cell activation was starkly reduced following KLKI MHV68 infection, despite a potent virus-specific effector CD8+ T cell response against both viruses. These phenotypes were independent of inoculating dose, as high dose infection with KLKI MHV68 still showed reduced adaptive immune cell activation. In contrast, infection of Ifnar1-/-mice, which support enhanced KLKI MHV68 lytic replication, led to potent adaptive cellular and humoral immune activation by both WT and KLKI viruses, suggesting that the level of viral replication, and not simply amount of virus present, is a major driver of adaptive immunity during GHV infection. Collectively, these data support the hypothesis that kLANA-mediated suppression of lytic replication facilitates immune evasion by holding viral replication below a threshold for potent induction of adaptive immunity. IMPORTANCE KSHV is a gammaherpesvirus that establishes lifelong, chronic infections in humans and increases the risk of virus-associated cancers. Currently, there is little information on how primary KSHV infection influences adaptive immune development in healthy individuals. Rodent models, such as murine gammaherpesvirus 68 (MHV68), provide a valuable laboratory system for studying gammaherpesvirus pathogenesis in vivo. In this study, we report that infection with a previously characterized chimeric KSHV-MHV68 virus expressing KSHV LANA represses lytic viral replication and elicits weak antiviral adaptive immune responses following primary infection, despite efficient latency establishment. Using this chimeric MHV68 virus, we demonstrate that lytic viral amplification must breach a threshold to trigger a potent virus-specific adaptive immune response. We propose that KSHV, through LANA, evades detection by repressing lytic viral replication to remain “below the radar” of adaptive immune defenses during host colonization.

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Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) can both establish latency in the trigeminal ganglion. Non-neural sites of latency have been described for BoHV-1 but not for BoHV-5. The aim of this study was to determine whether peripheral blood leukocytes and tonsils are targets for BoHV-5 infection and to establish whether all stages of that virus's infectious cycle can occur in those cell types. Comparisons with BoHV-1 infection of these tissues were also made in order to better understand the pathogenesis of both viruses. BoHV-1 and BoHV-5 were isolated from tonsils of acutely-infected calves. BoHV-5 was also isolated from a tonsil homogenate after dexamethasone-induced reactivation. During latency, infectious virus was recovered from a tonsil explant of one BoHV-5-infected calf. The genomes of BoHV-5 and BoHV-1 were detected in tonsils from acutely-infected calves although were not detected in tonsils from latently-infected calves or from calves treated with dexamethasone. Virus DNA was intermittently detected in leukocytes. The study has shown that BoHV-5 can establish latency in bovine tonsils and peripheral white blood cells, and that it can be reactivated from latently-infected tonsils, which might contribute to viral transmission. The titres of BoHV-1 and BoHV-5 in tonsils were similar, suggesting that replication at this site is a common feature for both viruses.

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Upper respiratory tract infections with Equid Herpesvirus 1 (EHV-1) typically result in a peripheral blood mononuclear cell-associated viremia, which can lead to vasculopathy in the central nervous system. Primary EHV-1 infection also likely establishes latency in trigeminal ganglia (TG) via retrograde axonal transport and in respiratory tract-associated lymphatic tissue. However, latency establishment and reactivation are poorly understood. To characterize the pathogenesis of EHV-1 latency establishment and maintenance, two separate groups of yearling horses were experimentally infected intranasally with EHV-1, strain Ab4, and euthanized 30 days post infection (dpi), (n = 9) and 70 dpi (n = 6). During necropsy, TG, sympathetic trunk (ST), retropharyngeal and mesenteric lymph nodes (RLn, MesLn) and kidney samples were collected. Viral DNA was detected by quantitative PCR (qPCR) in TG, ST, RLn, and MesLn samples in horses 30 and 70 dpi. The number of positive TG, RLn and MesLn samples was reduced when comparing horses 30 and 70 dpi and the viral copy number in TG and RLn significantly declined from 30 to 70 dpi. EHV-1 late gene glycoprotein B reverse transcriptase PCR and IHC results for viral protein were consistently negative, thus lytic replication was excluded in the present study. Mild inflammation could be detected in all neural tissue samples and inflammatory infiltrates mainly consisted of CD3+ T-lymphocytes (T-cells), frequently localized in close proximity to neuronal cell bodies. To identify latently infected cell types, in situ hybridization (ISH, RNAScope®) detecting viral DNA was used on selected qPCR- positive neural tissue sections. In ganglia 30 dpi, EHV-1 ISH signal was located in the neurons of TG and ST, but also in non-neuronal support or interstitial cells surrounding the neuron. In contrast, distinct EHV-1 signal could only be observed in neurons of TG 70 dpi. Overall, detection of latent EHV-1 in abdominal tissue samples and non-neuronal cell localization suggests, that EHV-1 uses T-cells during viremia as alternative route toward latency locations in addition to retrograde neuronal transport. We therefore hypothesize that EHV-1 follows the same latency pathways as its close relative human pathogen Varicella Zoster Virus.

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Feline herpesvirus 1 (FeHV-1) is an alphaherpesvirus that causes feline viral rhinotracheitis, an important viral disease of cats on a worldwide basis. Acute FeHV-1 infection is associated with both upper respiratory and ocular signs. Following the acute phase of the disease lifelong latency is established, primarily in sensory neuronal cells. As is the case with human herpes simplex viruses, latency reactivation can result in recrudescence, which can manifest itself in the form of serious ocular lesions. FeHV-1 infection in cats is a natural host model that is useful for the identification of viral virulence genes that play a role in replication at the mucosal portals of entry or are mediators of the establishment, maintenance, or reactivation of latency. It is also a model system for defining innate and adaptive immunity mechanisms and for immunization strategies that can lead to better protection against this and other alphaherpesvirus infections.

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Alphaherpesviruses are widespread in the human population, and include herpes simplex virus 1 (HSV-1) and 2, and varicella zoster virus (VZV). These viral pathogens cause epithelial lesions, and then infect the nervous system to cause lifelong latency, reactivation, and spread. A related veterinary herpesvirus, pseudorabies (PRV), causes similar disease in livestock that result in significant economic losses. Vaccines developed for VZV and PRV serve as useful models for the development of an HSV-1 vaccine. We present full genome sequence comparisons of the PRV vaccine strain Bartha, and two virulent PRV isolates, Kaplan and Becker. These genome sequences were determined by high-throughput sequencing and assembly, and present new insights into the attenuation of a mammalian alphaherpesvirus vaccine strain. We find many previously unknown coding differences between PRV Bartha and the virulent strains, including changes to the fusion proteins gH and gB, and over forty other viral proteins. Inter-strain variation in PRV protein sequences is much closer to levels previously observed for HSV-1 than for the highly stable VZV proteome. Almost 20% of the PRV genome contains tandem short sequence repeats (SSRs), a class of nucleic acids motifs whose length-variation has been associated with changes in DNA binding site efficiency, transcriptional regulation, and protein interactions. We find SSRs throughout the herpesvirus family, and provide the first global characterization of SSRs in viruses, both within and between strains. We find SSR length variation between different isolates of PRV and HSV-1, which may provide a new mechanism for phenotypic variation between strains. Finally, we detected a small number of polymorphic bases within each plaque-purified PRV strain, and we characterize the effect of passage and plaque-purification on these polymorphisms. These data add to growing evidence that even plaque-purified stocks of stable DNA viruses exhibit limited sequence heterogeneity, which likely seeds future strain evolution.

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BoHV-1, a neurotropic herpesvirus, establishes, maintains, and reactivates from latency in neurons. BoHV-1 DNA is also detected in pharyngeal tonsil (PT) from latently infected calves. ABSTRACT Bovine herpesvirus 1 (BoHV-1), an important pathogen of cattle, establishes lifelong latency in sensory neurons within trigeminal ganglia (TG) after acute infection. The BoHV-1 latency-reactivation cycle, like other alphaherpesvirinae subfamily members, is essential for viral persistence and transmission. Notably, cells within pharyngeal tonsil (PT) also support a quiescent or latent BoHV-1 infection. The synthetic corticosteroid dexamethasone, which mimics the effects of stress, consistently induces BoHV-1 reactivation from latency allowing early stages of viral reactivation to be examined in the natural host. Based on previous studies, we hypothesized that stress-induced cellular factors trigger expression of key viral transcriptional regulatory genes. To explore this hypothesis, RNA-sequencing studies compared viral gene expression in PT during early stages of dexamethasone-induced reactivation from latency. Strikingly, RNA encoding infected cell protein 4 (bICP4), which is translated into an essential viral transcriptional regulatory protein, was detected 30 min after dexamethasone treatment. Ninety minutes after dexamethasone treatment bICP4 and, to a lesser extent, bICP0 RNA were detected in PT. All lytic cycle viral transcripts were detected within 3 h after dexamethasone treatment. Surprisingly, the latency related (LR) gene, the only viral gene abundantly expressed in latently infected TG neurons, was not detected in PT during latency. In TG neurons, bICP0 and the viral tegument protein VP16 are expressed before bICP4 during reactivation, suggesting distinct viral regulatory genes mediate reactivation from latency in PT versus TG neurons. Finally, these studies confirm PT is a biologically relevant site for BoHV-1 latency, reactivation from latency, and virus transmission. IMPORTANCE BoHV-1, a neurotropic herpesvirus, establishes, maintains, and reactivates from latency in neurons. BoHV-1 DNA is also detected in pharyngeal tonsil (PT) from latently infected calves. RNA-sequencing studies revealed the viral infected cell protein 4 (bICP4) RNA was expressed in PT of latently infected calves within 30 min after dexamethasone was used to initiate reactivation. As expected, bICP4 RNA was not detected during latency. All lytic cycle viral genes were expressed within 3 h after dexamethasone treatment. Conversely, bICP0 and the viral tegument protein VP16 are expressed prior to bICP4 in trigeminal ganglionic neurons during reactivation. The viral latency related gene, which is abundantly expressed in latently infected neurons, was not abundantly expressed in PT during latency. These studies provide new evidence PT is a biologically relevant site for BoHV-1 latency and reactivation. Finally, we predict other alphaherpesvirinae subfamily members utilize PT as a site for latency and reactivation.

Neurotropic α-herpesvirinae subfamily members, herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BoHV-1), are important viral pathogens in their respective hosts. Following acute infection on mucosal surfaces, these viruses establish life-long latency in neurons within trigeminal ganglia (TG) and central nervous system. Chronic or acute stress (physiological or psychological) increases the frequency of reactivation from latency, which leads to virus shedding, virus transmission, and recurrent disease. While stress impairs immune responses and inflammatory signaling cascades, we predict stressful stimuli directly stimulate viral gene expression and productive infection during early stages of reactivation from latency. For example, BoHV-1 and HSV-1 productive infection is impaired by glucocorticoid receptor (GR) antagonists but is stimulated by the synthetic corticosteroid dexamethasone. Promoters that drive expression of key viral transcriptional regulatory proteins are cooperatively stimulated by GR and specific Krüppel like transcription factors (KLF) induced during stress induced reactivation from latency. The BoHV-1 immediate early transcription unit 1 promoter and contains two GR response elements (GRE) that are essential for cooperative transactivation by GR and KLF15. Conversely, the HSV-1 infected cell protein 0 (ICP0) and ICP4 promoter as well as the BoHV-1 ICP0 early promoter lack consensus GREs: however, these promoters are cooperatively transactivated by GR and KLF4 or KLF15. Hence, growing evidence suggests GR and stress-induced transcription factors directly stimulate viral gene expression and productive infection during early stages of reactivation from latency. We predict the immune inhibitory effects of stress enhance virus spread at late stages during reactivation from latency.

Steroid hormone fluctuations are predicted to enhance or initiate bovine herpesvirus 1 (BoHV-1) replication and virus spread in cattle. For example, stress increases the incidence of BoHV-1 reactivation from latency in cattle, and the synthetic corticosteroid dexamethasone consistently induces reactivation from latency. ABSTRACT Acute infection of the ocular, oral, or nasal cavity by bovine herpesvirus 1 (BoHV-1) culminates in lifelong latency in sensory neurons within trigeminal ganglia. The BoHV-1 latency reactivation cycle, including calves latently infected with commercially available modified live vaccines, can lead to reproductive complications, including abortions. Recent studies demonstrated progesterone stimulated BoHV-1 productive infection and sporadically induced reactivation from latency in male rabbits. The progesterone receptor (PR) and progesterone transactivate the immediate early transcription unit 1 (IEtu1) promoter and the infected cell protein 0 (bICP0) early promoter. These viral promoters drive expression of two viral transcriptional regulatory proteins (bICP0 and bICP4) that are crucial for productive infection. Based on these observations, we hypothesize that progesterone induces reactivation in a subset of calves latently infected with BoHV-1. These studies demonstrated progesterone was less efficient than dexamethasone at initiating reactivation from latency in female calves. Notably, heat stress correlated with enhancing the ability of progesterone to induce reactivation from latency. Previous studies demonstrated that heat stress activates the glucocorticoid receptor (GR), which suggested GR activation augments progesterone-mediated reactivation from latency. Additional studies revealed GR and PR cooperatively stimulated productive infection and synergistically transactivated the IEtu1 promoter when cultures were treated with dexamethasone. Mutating one or both GR binding sites in the IEtu1 promoter blocked transactivation. Collectively, these studies indicated that progesterone intermittently triggered reactivation from latency, and heat stress augmented reactivation from reactivation. Finally, these studies suggest progesterone enhances virus spread in tissues and cells where PR is abundantly expressed. IMPORTANCE Steroid hormone fluctuations are predicted to enhance or initiate bovine herpesvirus 1 (BoHV-1) replication and virus spread in cattle. For example, stress increases the incidence of BoHV-1 reactivation from latency in cattle, and the synthetic corticosteroid dexamethasone consistently induces reactivation from latency. The glucocorticoid receptor (GR) and dexamethasone stimulate key viral regulatory promoters and productive infection, in part because the viral genome contains numerous consensus GR-responsive elements (GREs). The progesterone receptor (PR) and GR belong to the type I nuclear hormone receptor family. PR and progesterone specifically bind to and transactivate viral promoters that contain GREs and stimulate BoHV-1 productive infection. Although progesterone did not induce reactivation from latency in female calves as efficiently as dexamethasone, heat stress enhanced progesterone-mediated reactivation from latency. Consequently, we predict that low levels of stressful stimuli can cooperate with progesterone to induce reactivation from latency or promote virus spread.

Cyprinid herpesvirus 2 (CyHV-2, species Cyprinid herpesvirus 2) causes severe mortality in ornamental goldfish, crucian carp (Carassius auratus), and gibel carp (Carassius gibelio). It has been shown that the genomic DNA of CyHV-2 could be detected in subclinical fish, which implied that CyHV-2 could establish persistent infection. In this study, the latency of CyHV-2 was investigated in the survival fish after primary infection. CyHV-2 genomic DNA was detected in multiple tissues of acute infection samples; however, detection of CyHV-2 DNA was significantly reduced in fish recovered from the primary infection on day 300 postinfection. No active viral gene transcription, such as DNA polymerase and ORF99, was detected in recovered fish. Following temperature stress, an increase of CyHV-2 DNA copy numbers and gene transcription were observed in tissues examined, which suggests that CyHV-2 was reactivated under stress. In addition, a cell line (GCBLat1) derived from the brain tissue from CyHV-2-exposed fish harbored CyHV-2 genome but did not produce infectious virions under normal culture conditions. However, CyHV-2 replication and viral gene transcription occurred when GCBLat1 cells were treated with trichostatin A (TSA) or phorbol 12-myristate 13-acetate (TPA). It suggests CyHV-2 can remain latent in vitro and can reactivate under stress condition.

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Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, can cause abortions in pregnant cows. Progesterone maintains pregnancy and promotes spermiogenesis and testosterone biosynthesis in males: furthermore, progesterone is a neuro-steroid. Recent published studies demonstrated progesterone stimulated the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, and two glucocorticoid receptor response elements within the promoter were required for progesterone mediated transactivation. In this study, we tested whether progesterone induces reactivation from latency in rabbits. As expected, the synthetic corticosteroid dexamethasone consistently induced reactivation from latency in males and females. While progesterone induced reactivation from latency in approximately one-half of male rabbits, virus shedding was sporadic compared to dexamethasone and less efficient in female rabbits. Progesterone significantly increased productive infection in rabbit skin cells, which correlated with stimulating reactivation. These studies suggest progesterone promotes BoHV-1 spread in cattle, in part, by increasing the frequency of reactivation from latency.

Alpha herpesvirus infections (α-HVs) are widespread, affecting more than 70% of the adult human population. Typically, the infections start in the mucosal epithelia, from which the viral particles invade the axons of the peripheral nervous system. In the nuclei of the peripheral ganglia, α-HVs establish a lifelong latency and eventually undergo multiple reactivation cycles. Upon reactivation, viral progeny can move into the nerves, back out toward the periphery where they entered the organism, or they can move toward the central nervous system (CNS). This latency–reactivation cycle is remarkably well controlled by the intricate actions of the intrinsic and innate immune responses of the host, and finely counteracted by the viral proteins in an effort to co-exist in the population. If this yin-yang- or Nash-equilibrium-like balance state is broken due to immune suppression or genetic mutations in the host response factors particularly in the CNS, or the presence of other pathogenic stimuli, α-HV reactivations might lead to life-threatening pathologies. In this review, we will summarize the molecular virus–host interactions starting from mucosal epithelia infections leading to the establishment of latency in the PNS and to possible CNS invasion by α-HVs, highlighting the pathologies associated with uncontrolled virus replication in the NS.

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A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses.

Herpesviruses are a group of DNA viruses that infect both humans and animals. Alphaherpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasion in vivo. Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1, and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alphaherpesvirus neuroinvasion and the interaction between virus and host during PRV infection. ABSTRACT Following its entry into cells, pseudorabies virus (PRV) utilizes microtubules to deliver its nucleocapsid to the nucleus. Previous studies have shown that PRV VP1/2 is an effector of dynein-mediated capsid transport. However, the mechanism of PRV for recruiting microtubule motor proteins for successful neuroinvasion and neurovirulence is not well understood. Here, we provide evidence that PRV pUL21 is an inner tegument protein. We tested its interaction with the cytoplasmic light chains using a bimolecular fluorescence complementation (BiFC) assay and observed that PRV pUL21 interacts with Roadblock-1. This interaction was confirmed by coimmunoprecipitation (co-IP) assays. We also determined the efficiency of retrograde and anterograde axonal transport of PRV strains in explanted neurons using a microfluidic chamber system and investigated pUL21’s contribution to PRV neuroinvasion in vivo. Further data showed that the carboxyl terminus of pUL21 is essential for its interaction with Roadblock-1, and this domain contributes to PRV retrograde axonal transport in vitro and in vivo. Our findings suggest that the carboxyl terminus of pUL21 contributes to PRV neuroinvasion. IMPORTANCE Herpesviruses are a group of DNA viruses that infect both humans and animals. Alphaherpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasion in vivo. Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1, and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alphaherpesvirus neuroinvasion and the interaction between virus and host during PRV infection.

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The Alphaherpesvirinae include the neurotropic pathogens herpes simplex virus and varicella zoster virus of humans and pseudorabies virus of swine. These viruses establish lifelong latency in the nuclei of peripheral ganglia, but utilize the peripheral tissues those neurons innervate for productive replication, spread, and transmission. Delivery of virions from replicative pools to the sites of latency requires microtubule-directed retrograde axonal transport from the nerve terminus to the cell body of the sensory neuron. As a corollary, during reactivation newly assembled virions must travel along axonal microtubules in the anterograde direction to return to the nerve terminus and infect peripheral tissues, completing the cycle. Neurotropic alphaherpesviruses can therefore exploit neuronal microtubules and motors for long distance axonal transport, and alternate between periods of sustained plus end- and minus end-directed motion at different stages of their infectious cycle. This review summarizes our current understanding of the molecular details by which this is achieved.

BackgroundpUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited.ResultsWe verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays.ConclusionsThe DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21.

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The lytic-latent balance is a major viral persistence strategy and obstacle to curing viral diseases, yet its mechanisms remain poorly understood. Following lytic infection in non-neuronal cells, herpes simplex virus (HSV) establishes latency specifically in neurons and is reactivated by stresses. Here we identify forkhead box (FOX) transcription factors that can strongly activate or repress replication of HSV-1 and other alphaherpesviruses, and show that neurons express activating Fox (e.g., Foxf) genes poorly but repressive Fox (Foxk) genes abundantly while non-neuronal or stressed neuronal cells exhibit higher expression of activating Fox genes. Remarkably, knockdown of Foxk1 or overexpression of activating Fox genes induces reactivation from latency in male mouse neuronal culture and in vivo. Of note, FOX proteins bind the viral genome globally and nonsequence-specifically and interact with epigenetic cofactors for gene regulation. FOXF1 interacts with CBP and P300 to acetylate and open viral chromatin. FOXK1 works with SIN3A, a cofactor of histone deacetylation, and MAX to suppress HSV-1 and antagonize activating FOX proteins. Therefore, the viral lytic-latent balance is controlled by the relative abundance of counteracting host transcription factors that recruit different epigenetic regulators to the viral genome.

To date, no herpesvirus has been shown to latently persist in fibroblastic cells. Here, we show that murine cytomegalovirus, a β-herpesvirus, persists for the long term and across organs in PDGFRα-positive fibroblastic cells, with similar or higher genome loads than in the previously known sites of murine cytomegalovirus latency. Whereas murine cytomegalovirus gene transcription in PDGFRα-positive fibroblastic cells is almost completely silenced at 5 months post-infection, these cells give rise to reactivated virus ex vivo, arguing that they support latent murine cytomegalovirus infection. Notably, PDGFRα-positive fibroblastic cells also support productive virus replication during primary murine cytomegalovirus infection. Mechanistically, Stat1-deficiency promotes lytic infection but abolishes latent persistence of murine cytomegalovirus in PDGFRα-positive fibroblastic cells in vivo. In sum, fibroblastic cells have a dual role as a site of lytic murine cytomegalovirus replication and a reservoir of latent murine cytomegalovirus in vivo and STAT1 is required for murine cytomegalovirus latent persistence in vivo.

Targeting key antiviral proteins of the host immune system is a common viral strategy. However, little is known about whether viruses employ more refined immune evasion tactics in the few polyploid animal hosts that exist. Here, we report that ORF56 of Carassius auratus herpesvirus (CaHV) diminishes IFN production by degrading MAVS-B, but not MAVS-A, via the autophagy pathway in amphitriploid (AAABBB) gibel carp (Carassius gibelio) with two triploid sets of chromosomes. Screening assays demonstrated that ORF56 suppresses IFN promoter activity and promotes CaHV replication. ORF56 specifically binds to and degrades gibel carp MAVS-B, a process that can be rescued by autophagy inhibitors. Moreover, co-expression of ORF56 and MAVS-B induces pronounced autophagic flux. Interestingly, although the selective autophagy receptors OPTN-A and OPTN-B both interact with ORF56, only OPTN-B is involved in ORF56 mediated degradation of MAVS-B. In summary, during CaHV infection of polyploid gibel carp, the viral protein ORF56 does not indiscriminately target both MAVS isoforms but selectively degrades MAVS-B through the autophagy pathway. These findings reveal a sophisticated viral immune evasion mechanism adapted for polyploid hosts.

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Many acute viral infections can be controlled by vaccination; however, vaccinating against persistent infections remains problematic. Herpesviruses are a classic example. Here, we discuss their immune control, particularly that of gamma-herpesviruses, relating the animal model provided by murid herpesvirus-4 (MuHV-4) to human infections. The following points emerge: (i) CD8(+) T-cell evasion by herpesviruses confers a prominent role in host defence on CD4(+) T cells. CD4(+) T cells inhibit MuHV-4 lytic gene expression via gamma-interferon (IFN-gamma). By reducing the lytic secretion of immune evasion proteins, they may also help CD8(+) T cells to control virus-driven lymphoproliferation in mixed lytic/latent lesions. Similarly, CD4(+) T cells specific for Epstein-Barr virus lytic antigens could improve the impact of adoptively transferred, latent antigen-specific CD8(+) T cells. (ii) In general, viral immune evasion necessitates multiple host effectors for optimal control. Thus, subunit vaccines, which tend to prime single effectors, have proved less successful than attenuated virus mutants, which prime multiple effectors. Latency-deficient mutants could make safe and effective gamma-herpesvirus vaccines. (iii) The antibody response to MuHV-4 infection helps to prevent disease but is suboptimal for neutralization. Vaccinating virus carriers with virion fusion complex components improves their neutralization titres. Reducing the infectivity of herpesvirus carriers in this way could be a useful adjunct to vaccinating naive individuals with attenuated mutants.

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Herpesviruses are highly prevalent viruses associated with numerous pathologies both in animal and human populations. Until now, most of the strategies used to prevent or to cure these infections have been unsuccessful because these viruses have developed numerous immune evasion mechanisms. Therefore, a better understanding of their complex lifecycle is needed. In particular, while the genome of numerous herpesviruses has been sequenced, the exact composition of virions remains unknown for most of them. Mass spectrometry has recently emerged as a central method and has permitted fundamental discoveries in virology. Here, we review mass spectrometry-based approaches that have recently allowed a better understanding of the composition of the herpesvirus virion. In particular, we describe strategies commonly used for proper sample preparation and fractionation to allow protein localization inside the particle but also to avoid contamination by nonstructural proteins. A collection of other important data regarding post-translational modifications or the relative abundance of structural proteins is also described. This review also discusses the poorly studied importance of host proteins in herpesvirus structural proteins and the necessity to develop a quantitative workflow to better understand the dynamics of the structural proteome. In the future, we hope that this collaborative effort will assist in the development of new strategies to fight these infections.

Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which have evolved to be transmitted in presence of specific immune response. Identifying their mode of transmission and their mechanisms of immune evasion is therefore essential to develop prophylactic and therapeutic strategies against these infections. As the known human gammaherpesviruses, Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus are host-specific and lack a convenient in vivo infection model; related animal gammaherpesviruses, such as murine gammaherpesvirus-68 (MHV-68), are commonly used as general models of gammaherpesvirus infections in vivo. To date, it has however never been possible to monitor viral excretion or virus transmission of MHV-68 in laboratory mice population. In this study, we have used MHV-68 associated with global luciferase imaging to investigate potential excretion sites of this virus in laboratory mice. This allowed us to identify a genital excretion site of MHV-68 following intranasal infection and latency establishment in female mice. This excretion occurred at the external border of the vagina and was dependent on the presence of estrogens. However, MHV-68 vaginal excretion was not associated with vertical transmission to the litter or with horizontal transmission to female mice. In contrast, we observed efficient virus transmission to naïve males after sexual contact. In vivo imaging allowed us to show that MHV-68 firstly replicated in penis epithelium and corpus cavernosum before spreading to draining lymph nodes and spleen. All together, those results revealed the first experimental transmission model for MHV-68 in laboratory mice. In the future, this model could help us to better understand the biology of gammaherpesviruses and could also allow the development of strategies that could prevent the spread of these viruses in natural populations.

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Animal and bacterial cells sense and defend against viral infections using evolutionarily conserved antiviral signaling pathways. Here, we show that viruses overcome host signaling using mechanisms of immune evasion that are directly shared across the eukaryotic and prokaryotic kingdoms of life. Structures of animal poxvirus proteins that inhibit host cGAS-STING signaling demonstrate architectural and catalytic active-site homology shared with bacteriophage Acb1 proteins, which inactivate CBASS anti-phage defense. In bacteria, phage Acb1 proteins are viral enzymes that degrade host cyclic nucleotide immune signals. Structural comparisons of poxvirus protein-2'3'-cGAMP and phage Acb1-3'3'-cGAMP complexes reveal a universal mechanism of host nucleotide immune signal degradation and explain kingdom-specific additions that enable viral adaptation. Chimeric bacteriophages confirm that animal poxvirus proteins are sufficient to evade immune signaling in bacteria. Our findings identify a mechanism of immune evasion conserved between animal and bacterial viruses and define shared rules that explain host-virus interactions across multiple kingdoms of life.

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The cellular DNA sensor cGMP-AMP synthase (cGAS) detects cytosolic viral DNA via the stimulator of interferon genes (STING) to initiate innate antiviral response. Herpesviruses are known to target key immune signaling pathways to persist in an immune-competent host. Marek’s disease virus (MDV), a highly pathogenic and oncogenic herpesvirus of chickens, can antagonize host innate immune responses to achieve persistent infection. With a functional screen, we identified five MDV proteins that blocked beta interferon (IFN-β) induction downstream of the cGAS-STING pathway. Specifically, the MDV major oncoprotein Meq impeded the recruitment of TANK-binding kinase 1 and IFN regulatory factor 7 (IRF7) to the STING complex, thereby inhibiting IRF7 activation and IFN-β induction. Meq overexpression markedly reduced antiviral responses stimulated by cytosolic DNA, whereas knockdown of Meq heightened MDV-triggered induction of IFN-β and downstream antiviral genes. Moreover, Meq-deficient MDV induced more IFN-β production than wild-type MDV. Meq-deficient MDV also triggered a more robust CD8+ T cell response than wild-type MDV. As such, the Meq-deficient MDV was highly attenuated in replication and lymphoma induction compared to wild-type MDV. Taken together, these results revealed that MDV evades the cGAS-STING DNA sensing pathway, which underpins the efficient replication and oncogenesis. These findings improve our understanding of the virus-host interaction in MDV-induced lymphoma and may contribute to the development of novel vaccines against MDV infection.

Among various pathogens, viruses pose significant threats to the livestock and poultry industry, resulting in substantial annual costs due to production losses and vaccination. The MHC-I presentation pathway is a crucial surveillance mechanism for preventing viral infections. Consequently, many viruses have evolved sophisticated strategies to inhibit the presentation of viral peptides by MHC-I to CD8+ T-cells, thereby evading the immune system. Understanding the mechanisms that suppress the MHC-I pathway and identifying specific binding peptides are essential for comprehending viral immune evasion and developing effective animal vaccines. This review summarizes the viral strategies for evading immune recognition, including the inhibition of MHC-I molecules synthesis, degradation, transport, and assembly, which affect MHC-I surface expression during viral infections. We also present evidence that MHC-I surface expression is frequently lost during numerous viral infections in livestock and poultry and offer new insights into the underlying mechanisms through which viruses inactivate the MHC-I antigen presentation pathway. Collectively, these advanced findings on viral evasion from the MHC-I pathway could inform the development of more effectives strategies to restore immunological control over viral infections and improve vaccines for the livestock and poultry industry.

ABSTRACT Viruses are important and lethal pathogens that hamper aquatic animals. The result of the battle between host and virus would determine the occurrence of diseases. The host will fight against virus infection with various responses such as innate immunity, adaptive immunity, apoptosis, and so on. On the other hand, the virus also develops numerous strategies such as immune evasion to antagonize host antiviral responses. Here, We review the research advances on virus mediated immune evasions to host responses containing interferon response, NF‐&kgr;B signaling, apoptosis, and adaptive response, which are executed by viral genes, proteins, and miRNAs from different aquatic animal viruses including Alloherpesviridae, Iridoviridae, Nimaviridae, Birnaviridae, Reoviridae, and Rhabdoviridae. Thus, it will facilitate the understanding of aquatic animal virus mediated immune evasion and potentially benefit the development of novel antiviral applications. HighlightsItemized are host antiviral responses in aquatic animals including fish and shrimp.Summed up are aquatic animal virus and viral components mediated evasions.Outlined are three main immune evasion strategies of aquatic animal virus.

Establishment of latent infection within specific tissues in the host is a common biological feature of the herpesviruses. In the case of bovine herpesvirus 2 (BoHV-2), latency is established in neuronal tissues, while bovine herpesvirus 4 (BoHV-4) and ovine herpesvirus 2 (OvHV-2) latent virus targets on cells of the monocytic lineage. This study was conducted in quest of BoHV-2, BoHV-4 and OvHV-2 DNA in two hundred trigeminal ganglia (TG) specimens, derived from one hundred clinically healthy cattle, majority of them naturally infected with bovine herpesvirus 1 (BoHV-1) and bovine herpesvirus 5 (BoHV-5). Total DNA extracted from ganglia was analyzed by polymerase chain reaction (PCR) designed to amplify part of the genes coding for BoHV-2, and BoHV-4 glycoprotein B and, for OvHV-2, the gene coding for phosphoribosylformylglycinamidine synthase-like protein. BoHV-2 DNA was detected in TG samples of two (2%) and BoHV-4 DNA in nine (9%) of the animals, whereas OvHV-2 DNA could not be detected in any of the TG DNA. The two animals in which BoHV-2 DNA was identified were also co-infected with BoHV-1 and BoHV-5. Within the nine animals in which BoHV-4 DNA was detected, six were also co-infected with BoHV-1 and BoHV-5. This report provides for the first time evidence that viral DNA from BoHV-2 and BoHV-4 can be occasionally detected in TG of naturally infected cattle. Likewise, in this report we provided for the first time evidence that the co-infection of cattle with three distinct bovine herpesviruses might be a naturally occurring phenomenon.

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs 467YTSL470 and 563YTVV566, which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport.

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ABSTRACT Alpha herpesvirus (α-HV) particles enter their hosts from mucosal surfaces and efficiently maintain fast transport in peripheral nervous system (PNS) axons to establish infections in the peripheral ganglia. The path from axons to distant neuronal nuclei is challenging to dissect due to the difficulty of monitoring early events in a dispersed neuron culture model. We have established well-controlled, reproducible, and reactivateable latent infections in compartmented rodent neurons by infecting physically isolated axons with a small number of viral particles. This system not only recapitulates the physiological infection route but also facilitates independent treatment of isolated cell bodies or axons. Consequently, this system enables study not only of the stimuli that promote reactivation but also the factors that regulate the initial switch from productive to latent infection. Adeno-associated virus (AAV)-mediated expression of herpes simplex-1 (HSV-1) VP16 alone in neuronal cell bodies enabled the escape from silencing of incoming pseudorabies virus (PRV) genomes. Furthermore, the expression of HSV VP16 alone reactivated a latent PRV infection in this system. Surprisingly, the expression of PRV VP16 protein supported neither PRV escape from silencing nor reactivation. We compared transcription transactivation activity of both VP16 proteins in primary neurons by RNA sequencing and found that these homolog viral proteins produce different gene expression profiles. AAV-transduced HSV VP16 specifically induced the expression of proto-oncogenes including c-Jun and Pim2. In addition, HSV VP16 induces phosphorylation of c-Jun in neurons, and when this activity is inhibited, escape of PRV silencing is dramatically reduced. IMPORTANCE During latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection. During latency, alpha herpesvirus genomes are silenced yet retain the capacity to reactivate. Currently, host and viral protein interactions that determine the establishment of latency, induce escape from genome silencing or reactivation are not completely understood. By using a compartmented neuronal culture model of latency, we investigated the effect of the viral transcriptional activator, VP16 on pseudorabies virus (PRV) escape from genome silencing. This model recapitulates the physiological infection route and enables the study of the stimuli that regulate the initial switch from a latent to productive infection. We investigated the neuronal transcriptional activation profiles of two homolog VP16 proteins (encoded by HSV-1 or PRV) and found distinct gene activation signatures leading to diverse infection outcomes. This study contributes to understanding of how alpha herpesvirus proteins modulate neuronal gene expression leading to the initiation of a productive or a latent infection.

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External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency. ABSTRACT Neurotropic Alphaherpesvirinae subfamily members such as bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) establish and maintain lifelong latent infections in neurons. Following infection of ocular, oral, or nasal cavities, sensory neurons within trigeminal ganglia (TG) are an important site for latency. Certain external stressors can trigger reactivation from latency, in part because activation of the glucocorticoid receptor (GR) stimulates productive infection and promoters that drive expression of key viral transcriptional regulators. The Akt serine/threonine protein kinase family is linked to maintaining latency. For example, Akt3 is detected in more TG neurons during BoHV-1 latency than in reactivation and uninfected calves. Furthermore, Akt signaling correlates with maintaining HSV-1 latency in certain neuronal models of latency. Finally, an active Akt protein kinase is crucial for the ability of the HSV-1 latency-associated transcript (LAT) to inhibit apoptosis in neuronal cell lines. Consequently, we hypothesized that viral and/or cellular factors impair stress-induced transcription and reduce the incidence of reactivation triggered by low levels of stress. New studies demonstrate that Akt1 and Akt2, but not Akt3, significantly reduced GR-mediated transactivation of the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter, the HSV-1 infected cell protein 0 (ICP0) promoter, and the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Akt3, but not Akt1 or Akt2, significantly enhanced neurite formation in mouse neuroblastoma cells, which correlates with repairing damaged neurons. These studies suggest that unique biological properties of the three Akt family members promote the maintenance of latency in differentiated neurons. IMPORTANCE External stressful stimuli are known to increase the incidence of reactivation of Alphaherpesvirinae subfamily members. Activation of the glucocorticoid receptor (GR) by the synthetic corticosteroid dexamethasone (DEX) stimulates bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) reactivation. Furthermore, GR and dexamethasone stimulate productive infection and promoters that drive expression of viral transcriptional regulators. These observations lead us to predict that stress-induced transcription is impaired by factors abundantly expressed during latency. Interestingly, activation of the Akt family of serine/threonine protein kinases is linked to maintenance of latency. New studies reveal that Akt1 and Ak2, but not Akt3, impaired GR- and dexamethasone-mediated transactivation of the BoHV-1 immediate early transcription unit 1 and HSV-1 ICP0 promoters. Strikingly, Akt3, but not Akt1 or Akt2, stimulated neurite formation in mouse neuroblastoma cells, a requirement for neurogenesis. These studies provide insight into how Akt family members may promote the maintenance of lifelong latency.

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Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies. Author Summary Alpha-herpesviruses represent a group of large, enveloped DNA viruses that are capable to establish a quiescent (also called latent) but reactivatable form of infection in the peripheral nervous system of their hosts. Following reactivation of latent genomes, virus progeny are formed in the soma of neuronal cells and depend on sorting into the axon for anterograde spread of infection to mucosal sites and potentially new host. We studied two alpha-herpesviruses (the veterinary pathogen pseudorabies virus (PRV) and human herpes simplex virus 1 (HSV-1)) and found viral membrane proteins Us7, Us8, and Us9 to form a complex, which is able to recruit kinsin-3 motors. Motor recruitment facilitates axonal sorting and subsequent transport to distal egress sites. Complex formation occurs at the trans-Golgi network and mediates efficiency of axonal sorting and motility characteristics of egressing capsids. We also used an artificial kinesin-3 recruitment system, which allows controlled induction of axonal sorting and transport for virus mutants lacking Us7, Us8, and Us9. Overall, these data contribute to our understanding of anterograde alpha-herpesvirus spread and kinesin-mediated sorting of vesicular axonal cargoes.

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ABSTRACT Following reactivation of a latent alphaherpesvirus infection, viral particles are assembled in neuronal cell bodies, trafficked anterogradely within axons to nerve termini, and spread to adjacent epithelial cells. The virally encoded membrane proteins US9p and the glycoprotein heterodimer gE/gI of pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1) play critical roles in anterograde spread, likely as a tripartite gE/gI-US9p complex. Two kinesin motors, kinesin-1 and kinesin-3, are implicated in the egress of these viruses, but how gE/gI-US9p coordinates their activities is poorly understood. Here, we report that PRV, in addition to associating with the kinesin-3 motor KIF1A, recruits the neuronal kinesin-1 isoforms KIF5A and KIF5C, but not the broadly expressed isoform KIF5B, during egress from differentiated CAD neurons. Similarly, in the axons of dorsal root ganglia (DRG)-derived sensory neurons, PRV colocalized with KIF5C but not KIF5B. In differentiated CAD cells, the association of KIF1A with egressing PRV was dependent upon US9p, whereas the recruitment of KIF5 isoforms required gE/gI. Consistent with these findings, the number of PRV particles trafficking within CAD neurites and the axons of DRG neurons increased when kinesin-1 motor activity was upregulated by hyperacetylating microtubules using trichostatin A (TSA) or tubacin, and this enhanced trafficking depended upon the presence of gE/gI. We propose that, following its recruitment by US9p, KIF1A delivers PRV particles to a location where KIF5 motors are subsequently added by a gE/gI-dependent mechanism. KIF5A/C isoforms then serve to traffic viral particles along axons, resulting in characteristic recrudescent infection. IMPORTANCE Alphaherpesviruses include important human and veterinary pathogens that share a unique propensity to establish life-long latent infections in the peripheral nervous system. Upon reactivation, these viruses navigate back to body surfaces and transmit to new hosts. In this study, we demonstrate that the virus gE/gI-US9p membrane complex routes virus particles down this complex neuronal egress pathway by coordinating their association with multiple kinesin microtubule motors. Alphaherpesviruses include important human and veterinary pathogens that share a unique propensity to establish life-long latent infections in the peripheral nervous system. Upon reactivation, these viruses navigate back to body surfaces and transmit to new hosts. In this study, we demonstrate that the virus gE/gI-US9p membrane complex routes virus particles down this complex neuronal egress pathway by coordinating their association with multiple kinesin microtubule motors.

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Equine herpesvirus myeloencephalitis (EHM) remains one of the most devastating manifestations of equine herpesvirus type 1 (EHV-1) infection but our understanding of its pathogenesis remains rudimentary, partly because of a lack of adequate experimental models. EHV-1 infection of the ocular vasculature may offer an alternative model as EHV-1-induced chorioretinopathy appears to occur in a significant number of horses, and the pathogenesis of EHM and ocular EHV-1 may be similar. To investigate the potential of ocular EHV-1 as a model for EHM, and to determine the frequency of ocular EHV-1, our goal was to study: (1) Dissemination of virus following acute infection, (2) Development and frequency of ocular lesions following infection, and (3) Utility of a GFP-expressing virus for localization of the virus in vivo. Viral antigen could be detected following acute infection in ocular tissues and the central nervous system (experiment 1). Furthermore, EHV-1 infection resulted in multifocal choroidal lesions in 90% (experiment 2) and 50% (experiment 3) of experimentally infected horses, however ocular lesions did not appear in vivo until between 3 weeks and 3 months post-infection. Taken together, the timing of the appearance of lesions and their ophthalmoscopic features suggest that their pathogenesis may involve ischemic injury to the chorioretina following viremic delivery of virus to the eye, mirroring the vascular events that result in EHM. In summary, we show that the frequency of ocular EHV-1 is 50-90% following experimental infection making this model attractive for testing future vaccines or therapeutics in an immunologically relevant age group.

Equine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Besides epithelial cells, CD172a+ monocytic cells become infected with EHV-1 in the respiratory mucosa and transport the virus from the apical side of the epithelium to the lamina propria en route to the lymph and blood circulation. Whether CD172a+ monocytic cells are specifically recruited to the infection sites in order to pick up virus is unknown. In our study, equine nasal mucosa explants were inoculated with EHV-1 neurological strains 03P37 and 95P105 or the non-neurological strains 97P70 and 94P247 and the migration of monocytic cells was examined by immunofluorescence. Further, the role of monokines CCL2 and CCL5 was determined and the effect of migration inhibitors rosiglitazone (RSG) or quinacrine was analyzed. It was shown that with neurological strains but not with the non-neurological strains, CD172a+ cells specifically migrated towards EHV-1 infected regions and that CCL2 and CCL5 were involved. CCL2 started to be expressed in infected epithelial cells at 24 h post-incubation (hpi) and CCL5 at 48 hpi, which corresponded with the CD172a+ migration. RSG treatment of EHV-1-inoculated equine nasal mucosa had no effect on the virus replication in the epithelium, but decreased the migration of CD172a+ cells in the lamina propria. Overall, these findings bring new insights in the early pathogenesis of EHV-1 infections, illustrate differences between neurological and non-neurological strains and show the way for EHV-1 treatment.

Equid herpesvirus 1 (EHV-1) causes respiratory disease, abortion and neurological disorders in horses. Similarly, to other alphaherpesviruses, EHV-1 is neurotropic and establishes latency in the neurons of its natural host. Despite the fact that many studies have been devoted to the pathogenesis of various clinical forms of EHV-1 infection, mechanisms of the neuronal damage are still not fully understood. The aim of this study was to define the phosphorylation status of tau protein in neuronal cell culture infected with EHV-1. Phosphorylation of tau was tested at tau-ser199/ser202, tau-ser404, tau-ser262, tau-thr181, tau-thr217 and tau-thr205 sites. We described, for the first time, that EHV-1 infection leads to the accumulation of hyperphosphorylated tau in primary murine neurons. We showed that non-neuropathogenic and neuropathogenic EHV-1 strains specifically induce hyperphosphorylation of tau-ser199/ser202, tau-ser404 and tau-thr205 during long-term infection and after a controlled activation of productive infection. Keywords: tau protein; hyperphosphorylation; equid herpesvirus 1 (EHV-1); neuronal cell culture.

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Mouse BALB/3T3-A31-1-1 (A31) cells are non-permissive to bovine herpes virus-1 (BHV-1) but permissive to pseudorabies virus (PrV). The promoter activity of the immediate early gene of BHV-1 (BICP4) was very weak when compared with that of PrV in A31 cells. Infectious BHV-1 genomic DNA co-transfected into A31 cells with plasmids expressing BICP4 and BICP0 by a strong promoter failed to yield any progeny virus. Growth of BHV-1 in non-permissible A31 cells is restricted in many phases of the growth. The fact that expression of BICP4 and/or BICP0 in A31 cells does not improve the yield of progeny virus from infectious BHV-1 genomic DNA suggests that some more growth restrictions exist beyond the expression of BHV-1 immediate early proteins.

Bovine herpesvirus-1 (BHV-1) has been used as a vector of live recombinant vaccines for cattle which express the genes of other pathogens. Because of the importance of the choice of the promoter which allows the efficient expression of the foreign genes in the BHV-1 vector, we compared the relative efficacy of various promoters integrated in the BHV-1 genome. The promoter sequences of the BHV-1 thymidine kinase (tk), gB, gC, SV40 early, and pseudorabies virus (PRV) immediate early (IE) genes were placed at the upstream of the open reading frame of the chloramphenycol acetyl transferase (CAT) gene and the promoter-CAT sequences were integrated into the tk gene of BHV-1 by homologous recombination. The promoter activity was assayed by measuring the CAT activity in the extracts of Madin Darby bovine kidney (MDBK) cells infected with the recombinant BHV-1. The PRV IE promoter was activated earlier and maintained at a higher level activity than the BHV-1 gB or gC promoters throughout the most of the growth phase of BHV-1. At the late phase, however, the activities of the BHV-1 gB and gC promoters reached the higher level. The BHV-1 tk promoter activity was low and the SV40 early promoter was hardly activated when integrated into the BHV-1 genome. promoter, recombinant BHV-1.

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Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus related to pseudorabies virus (PRV) and varicella-zoster virus (VZV). This virus is one of the major pathogens affecting horses worldwide. EHV-1 is responsible for respiratory disorders, abortion, neonatal foal death and equine herpes myeloencephalopathy (EHM). Over the last decade, EHV-1 has received growing attention due to the frequent outbreaks of abortions and/or EHM causing serious economical losses to the horse industry worldwide. To date, there are no effective antiviral drugs and current vaccines do not provide full protection against EHV-1-associated diseases. Therefore, there is an urgent need to gain a better understanding of the pathogenesis of EHV-1 in order to develop effective therapies. The main objective of this review is to provide state-of-the-art information on the pathogenesis of EHV-1. We also highlight recent findings on EHV-1 immune evasive strategies at the level of the upper respiratory tract, blood circulation and endothelium of target organs allowing the virus to disseminate undetected in the host. Finally, we discuss novel approaches for drug development based on our current knowledge of the pathogenesis of EHV-1.

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The aim of this study was to investigate the role of immediate early gene (gene63) in the pathogenesis of equine herpesvirus 1 (EHV-1) acute and latent infections in equine and murine models. EHV-1 gene63 mutant virus (g63mut) along with EHV-1 (Ab4) was used for intracerebral and intranasal infection of 3 and 17-day-old mice. Both viruses were recovered at the same frequency from tissues after infection. Two Welsh ponies were infected via the intranasal route with each of the viruses. Acute infection was monitored by virus isolation from nasal swabs and peripheral blood leukocytes. Six weeks post infection, peripheral blood leukocytes were taken from ponies and in vitro reactivation was positive for both viruses. At autopsy, both viruses were isolated by co-cultivation from bronchial and submandibular lymph nodes. These findings indicate that the mutation of EHV-1 gene63 does not play a role in the establishment and reactivation from latency.

Equine herpesvirus-1 (EHV-1) infection remains a significant problem despite the widespread use of vaccines. The inability to generate a protective immune response to EHV-1 vaccination or infection is thought to be due to immunomodulatory properties of the virus, and the ORF1 and ORF2 gene products have been hypothesized as potential candidates with immunoregulatory properties. A pony infection study was performed to define immune responses to EHV-1, and to determine if an EHV-1 ORF1/2 deletion mutant (ΔORF1/2) would have different disease and immunoregulatory effects compared to wild type EHV-1 (WT). Infection with either virus led to cytokine responses that coincided with the course of clinical disease, particularly the biphasic pyrexia, which correlates with respiratory disease and viremia, respectively. Similarly, both viruses caused suppression of proliferative T-cell responses on day 7 post infection (pi). The ΔORF1/ORF2 virus caused significantly shorter primary pyrexia and significantly reduced nasal shedding, and an attenuated decrease in PBMC IL-8 as well as increased Tbet responses compared to WT-infected ponies. In conclusion, our findings are (i) that infection of ponies with EHV-1 leads to modulation of immune responses, which are correlated with disease pathogenesis, and (ii) that the ORF1/2 genes are of importance for disease outcome and modulation of cytokine responses.

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ABSTRACT Pseudorabies virus (PRV) infection poses a significant threat to reproductive success in pregnant pigs, often leading to abortion and stillbirth. Systematic studies on the vertical transmission mechanism of PRV through the placenta remain limited. To address this research gap, we developed a PRV infection model in pregnant mice. Immunohistochemical analysis detected strong PRV-positive signals in placental trophoblasts and fetal brains, with viral loads significantly higher than those in the maternal bloodstream. These results suggest that PRV can infect the placenta via maternal circulation and subsequently be transmitted to the fetus. Further analysis revealed that PRV infection disrupts placental vascular architecture and compromises the integrity of tight junction proteins (TJPs) ZO-1 and occludin, facilitating viral entry into fetal tissues. Transcriptomic profiling of placental tissues showed activation of inflammatory responses, oxidative stress, and cell death pathways. Concurrently, genes involved in hormone biosynthesis, angiogenesis, and trophoblast proliferation and differentiation were markedly downregulated. Together, these results indicate that PRV compromises placental integrity in mice through a combination of physical barrier disruption, inflammatory microenvironment formation, and vascular dysfunction. This provides a reference for the mechanism of vertical transmission of PRV through the placenta in mice. IMPORTANCE Pseudorabies virus can be transmitted vertically by disrupting the barrier function of the placenta. Maternal viremia drives preferential PRV replication within the placenta. PRV preferentially colonizes decidual and labyrinthine placental regions. PRV can cause pregnancy failure via inflammation. Pseudorabies virus can be transmitted vertically by disrupting the barrier function of the placenta. Maternal viremia drives preferential PRV replication within the placenta. PRV preferentially colonizes decidual and labyrinthine placental regions. PRV can cause pregnancy failure via inflammation.

During human herpesvirus infection, dynamic alterations of N6-methyladenosine (m6A) modification have been extensively observed in viral and cellular transcriptomes. This modification plays a crucial role in RNA metabolism, serving as a novel regulator of gene expression alongside DNA and protein modifications. Notably, reversible changes in a single m6A modification site can impact viral replication and pathogenicity. Recent studies have reported changes in m6A modification-associated epitranscriptomes and their functional analysis during animal herpesvirus infections. This review focuses on the research progress of m6A modification on the transcriptome in both human and animal herpesvirus infections within the same family. Specifically, it examines the dynamic alterations of m6A modification-associated epitranscriptomes, the expression of m6A-machinery proteins, regulatory molecular mechanisms associated with herpesvirus infection, and potential clinical applications. By addressing the gaps in research on m6A modification in animal viruses, new insights into the regulatory molecular mechanisms of viral diseases may be uncovered. Furthermore, natural hosts infected with animal herpesvirus serve as valuable biomedical models for studying the regulation of m6A modification on viral replication and pathogenesis, thereby supporting the development of novel vaccine and drug targets.

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Cell culture techniques are increasingly favored over animal models due to rising costs, time constraints, and ethical concerns regarding animal use. These techniques serve critical roles in disease modeling, drug screening, drug discovery, and toxicity analysis. Notably, cell cultures facilitate primary virus isolation, infectivity assays, biochemical studies, and vaccine production. However, viral contamination in cell cultures poses significant challenges, particularly due to the necessity for complex and sophisticated detection methods. Among the prevalent viruses, Epstein Barr virus (EBV) is ubiquitous across human populations, infecting approximately 98% of individuals. Despite its prevalence, the detection of EBV is often not considered a safety priority, as its detection methods are well-established, including PCR assays that can identify both active and latent forms of the virus. Conversely, ovine herpesvirus 2 (OvHV-2), a relative of EBV, presents a critical concern due to its ability to infect a wide range of organs and species, including over 33 animal species and nearly all domestic sheep. This makes the detection of OvHV-2 crucial for the safety of cell cultures across various species. The literature reveals a gap in the comprehensive understanding of both EBV and OvHv-2 detection in cell culture systems, highlighting an urgent need for developing robust detection methodologies specific to EBV and OvHv-2 to ensure bioprocess safety.

Cell adhesion molecules (CAMs) are surface ligands, usually glycoproteins, which mediate cell-to-cell adhesion. They play a critical role in maintaining tissue integrity and mediating migration of cells, and some of them also act as viral receptors. It has been known that soluble forms of the viral receptors bind to the surface glycoproteins of the viruses and neutralize them, resulting in inhibition of the viral entry into cells. Nectin-1 is one of important CAMs belonging to immunoglobulin superfamily and herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor family. Both CAMs also act as alphaherpesvirus receptor. Transgenic mice expressing the soluble form of nectin-1 or HVEM showed almost complete resistance against the alphaherpesviruses. As another CAM, sialic acid-binding immunoglobulin-like lectins (Siglecs) that recognize sialic acids are also known as an immunoglobulin superfamily member. Siglecs play an important role in the regulation of immune cell functions in infectious diseases, inflammation, neurodegeneration, autoimmune diseases and cancer. Siglec-9 is one of Siglecs and capsular polysaccharide (CPS) of group B Streptococcus (GBS) binds to Siglec-9 on neutrophils, leading to suppress host immune response and provide a survival advantage to the pathogen. In addition, Siglec-9 also binds to tumor-produced mucins such as MUC1 to lead negative immunomodulation. Transgenic mice expressing the soluble form of Siglec-9 showed significant resistance against GBS infection and remarkable suppression of MUC1 expressing tumor proliferation. This review describes recent developments in the understanding of the potency of soluble forms of CAMs in the transgenic mice and discusses potential therapeutic interventions that may alter the outcomes of certain diseases.

Guinea Pig Herpes-Like Virus (GPHLV) is a virus isolated from leukemic guinea pigs with herpes virus-like morphology described by Hsiung and Kaplow in 1969. GPHLV transformed embryonic cells from Syrian hamsters or rats, which were tumorigenic in adult animals. Herein, we present the genomic sequence of GPHLV strain LK40 as a reference for future molecular analysis. GPHLV has a broad host tropism and replicates efficiently in Guinea pig, Cat, and Green African Monkey-derived cell lines. GPHLV has a GC content of 35.45%. The genome is predicted to encode at least 75 open-reading frames (ORFs) with 84% (63 ORFs) sharing homology to human Kaposi Sarcoma Associated Herpes Virus (KSHV). Importantly, GPHLV encodes homologues of the KSHV oncogenes, vBCL2 (ORF16), vPK (ORF36), viral cyclin (v-cyclin, ORF72), the latency associated nuclear antigen (LANA, ORF73), and vGPCR (ORF74). GPHLV is a Rhadinovirus of Cavia porcellus, and we propose the formal name of Caviid gamma herpesvirus 1 (CaGHV-1). GPHLV can be a novel small animal model of Rhadinovirus pathogenesis with broad host tropism.

ABSTRACT Equine herpesvirus type 1 (EHV-1) is one of the most prevalent respiratory pathogens in horses with a high impact on animal health worldwide. Entry of the virus into epithelial cells of the upper respiratory tract and rapid local viral replication is followed by infection of local lymphoid tissues leading to cell-associated viremia and disease progression. Pre-existing mucosal immunity has previously been shown to reduce viral shedding and prevent viremia, consequently limiting severe disease manifestations. Here, nasopharyngeal transcriptomic profiling was used to identify differentially expressed genes following EHV-1 challenge in horses with different EHV-1 immune statuses. Immune horses (n = 4) did neither develop clinical disease nor viremia and did not shed virus after experimental infection, while non-immune horses (n = 4) did all the above. RNA sequencing was performed on nasopharyngeal samples pre- and 24 hours post-infection (24hpi). At 24hpi, 109 and 44 genes were upregulated in immune horses and non-immune horses, respectively, and three genes were explored in further detail. Antileukoproteinase (SLPI) gene expression increased 2.1-fold within 24 hours in immune horses in concert with protein secretion. Interferon (IFN)-induced proteins with tetratricopeptide repeats 2 (IFIT2) and 3 (IFIT3) were upregulated in non-immune horses, corresponding with nasal IFN-α secretion and viral replication. By contrast, neither IFIT expression nor IFN-α secretion was induced by EHV-1 infection of immune horses. Transcriptomic profiling offered a tool to identify, for the first time, the role of SLPI in innate immunity against EHV-1, and further emphasized the central role of the type I IFN response in the anti-viral defense of non-immune horses. IMPORTANCE Equine herpesvirus type 1 (EHV-1) remains a considerable concern in the equine industry, with yearly outbreaks resulting in morbidity, mortality, and economic losses. In addition to its importance in equine health, EHV-1 is a respiratory pathogen and an alphaherpesvirus, and it may serve as a model for other viruses with similar pathogenicity or phylogeny. Large animal models allow the collection of high-volume samples longitudinally, permitting in-depth investigation of immunological processes. This study was performed on bio-banked nasopharyngeal samples from an EHV-1 infection experiment, where clinical outcomes had previously been determined. Matched nucleic acid and protein samples throughout infection permitted longitudinal quantification of the protein or related proteins of selected differentially expressed genes detected during the transcriptomic screen. The results of this manuscript identified novel innate immune pathways of the upper respiratory tract during the first 24 hours of EHV-1 infection, offering a first look at the components of early mucosal immunity that are indicative of protection. Equine herpesvirus type 1 (EHV-1) remains a considerable concern in the equine industry, with yearly outbreaks resulting in morbidity, mortality, and economic losses. In addition to its importance in equine health, EHV-1 is a respiratory pathogen and an alphaherpesvirus, and it may serve as a model for other viruses with similar pathogenicity or phylogeny. Large animal models allow the collection of high-volume samples longitudinally, permitting in-depth investigation of immunological processes. This study was performed on bio-banked nasopharyngeal samples from an EHV-1 infection experiment, where clinical outcomes had previously been determined. Matched nucleic acid and protein samples throughout infection permitted longitudinal quantification of the protein or related proteins of selected differentially expressed genes detected during the transcriptomic screen. The results of this manuscript identified novel innate immune pathways of the upper respiratory tract during the first 24 hours of EHV-1 infection, offering a first look at the components of early mucosal immunity that are indicative of protection.

Simple Summary Equine herpesvirus type 8 (EHV-8), a member of the alphaherpesvirus subfamily, is a significant pathogen in donkeys and causes abortion and respiratory infections, which result in considerable economic losses within the equine industry. Despite its importance, limited research has been conducted on the molecular response to EHV-8 in donkeys. The present study investigated the host cell response to EHV-8 infection in rabbit kidney (RK-13) cells through transcriptomic and proteomic approaches. Our findings identified several candidate genes and proteins, along with their associated signaling pathways, involved in the cellular response to EHV-8 infection in this in vitro model. However, because RK-13 cells may not accurately replicate viral–host interactions in equine species, additional in vivo studies in horses and donkeys are necessary to achieve a more thorough understanding of viral pathogenesis in these animals.

Acipenserid herpesvirus 2 (AciHV-2) is a large double-stranded DNA virus in the family Alloherpesviridae that causes catastrophic outbreaks in young naive white sturgeon (Acipenser transmontanus) populations, with mortalities of up to 80%. Survivors of these infections are suspected to remain latently infected. The gram-positive zoonotic bacterium Streptococcus iniae is another important sturgeon pathogen that causes severe myositis and up to 50% mortality during natural outbreaks. Throughout the last decade, co-infections of AciHV-2 and S. iniae have been reported in cultured white sturgeon in California resulting in severe presentations of piscine streptococcosis. This phenomenon of herpesvirus and streptococcus co-infection appears to span multiple taxa since in humans, it is recognized that a Human herpesvirus 3 infection (VZV) is a negative prognostic indicator for pediatric Invasive Group A Streptococcal infections (IGASI). While a decrease in humoral immunity caused by VZV has been hypothesized as a potentially important factor in IGASI cases, no natural animal model exists to study this process. Moreover, no studies have investigated these reported co-infections in white sturgeon. Therefore, the goal of this study was to investigate the effects of a recent AciHV-2 infection on the outcome of a subsequent S. iniae challenge in white sturgeon fingerlings. When fish were infected with 108 colony forming units (CFU) of S. iniae intramuscularly (IM), a statistically significant decrease in survival of 41% was detected in the co-infection group compared to the S. iniae group (p-value < 0.001). This difference was not observed when fish were infected with 106 CFU of S. iniae IM. At this lower infection dose, however, a statistically significant downregulation of tnfα was observed in the spleen of fish in the co-infection group compared to the S. iniae group (p-value = 0.0098). Analysis of serum from survivors revealed a statistically significant reduction in anti-S. iniae serum IgM and serum serotransferrin in fish from the co-infection group compared to the S. iniae group (p-value = 0.0134 and p-value = 0.0183, respectively). Further studies are indicated to determine what interactions lead to the decreased production of pathogen-specific IgM, serotransferrin, and TNFα in the host.

ABSTRACT Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses. IMPORTANCE There are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets. There are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.