Infectious Triggers of Cytokine Storm Syndromes: Herpes Virus Family (Non-EBV).

The herpesviruses are the most common infectious agents associated with both primary and secondary cytokine storm syndromes (CSS). While Epstein-Barr Virus (EBV) is most frequently reported in association with CSS, cytomegalovirus (CMV) and many other herpesviruses (e.g., herpes simplex virus, varicella zoster virus, and human herpesviruses 6 and 8) are clearly associated with CSS in children and adults. Immunocompromised hosts, whether due to primary immunodeficiency or secondary immune compromise (e.g., solid organ or stem cell transplantation), appear to be at particularly increased risk of herpesvirus-associated CSS. In this chapter, the association of the non-EBV herpesviruses with CSS will be discussed, including predisposing factors and treatment considerations.

Spatial kinetics and immune control of murine cytomegalovirus infection in the salivary glands.

Human cytomegalovirus (HCMV) is the most common congenital infection. Several HCMV vaccines are in development, but none have yet been approved. An understanding of the kinetics of CMV replication and transmission may inform the rational design of vaccines to prevent this infection. The salivary glands (SG) are an important site of sustained CMV replication following primary infection and during viral reactivation from latency. As such, the strength of the immune response in the SG likely influences viral dissemination within and between hosts. To study the relationship between the immune response and viral replication in the SG, and viral dissemination from the SG to other tissues, mice were infected with low doses of murine CMV (MCMV). Following intra-SG inoculation, we characterized the viral and immunological dynamics in the SG, blood, and spleen, and identified organ-specific immune correlates of protection. Using these data, we constructed compartmental mathematical models of MCMV infection. Model fitting to data and analysis indicate the importance of cellular immune responses in different organs and point to a threshold of infection within the SG necessary for the establishment and spread of infection.

Causality between herpes virus infections and allograft dysfunction after tissue and organ transplantation: a two-sample bidirectional Mendelian randomization study.

Background: Observational studies have suggested that herpes virus infections increase the risk of allograft dysfunction after tissue and organ transplantation, but it is still unclear whether this association is causal. The aim of this study was to assess the causal relationship between four herpes virus infections and allograft dysfunction. Methods: We used two-sample bidirectional Mendelian randomization (MR) to investigate the causality between four herpes virus infections - cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV) and varicella zoster virus (VZV) - and allograft dysfunction after tissue and organ transplantation. Based on summary data extracted from genome-wide association studies (GWAS), we chose eligible single nucleotide polymorphisms (SNPs) as instrumental variables. The Inverse variance weighted (IVW) method was used as the main analysis method, supplemented by Weighted median and MR-Egger analyses. The MR-PRESSO test, MR-Egger intercept test, heterogeneity test, leave-one-out analysis and funnel plot were used to analyze the sensitivity of MR results. Results: We found EBV early antigen-D (EA-D) antibody levels and shingles were the only two variables associated with an increased risk of allograft dysfunction. No evidence of allograft dysfunction increasing the risk of the four herpes virus infections was observed. Sensitivity analyses confirmed the robustness of our results. Conclusions: Our results suggest that EBV and VZV are involved in graft rejection or dysfunction. However, the relationship between CMV and HSV infections and allograft dysfunction remains unclear and requires further clarification.

Late-life Attenuation of Cytomegalovirus-mediated CD8 T Cell Memory Inflation: Shrinking of the Cytomegalovirus Latency Niche.

CMV drives the accumulation of virus-specific, highly differentiated CD8 memory T cells (memory inflation [MI]). In mice, MI was shown to directly correlate with the CMV infection dose, yet the CMV-associated CD8 MI plateaus over time. It is unclear how MI is regulated with aging. We infected young mice with 102, 104, and 106 PFU of murine CMV and confirmed that MI magnitude was directly proportional to the infectious dose, reaching a setpoint by midlife. By old age, MI subsided, most prominently in mice infected with 106 PFU, and reached statistical parity between groups in 26-mo-old mice. This corresponded to an age-related loss in lymphatic endothelial cells in lymph nodes, recently shown to be sufficient to drive MI in mice. We propose that MI size and persistence over the lifespan is controlled by the size of the lymphatic endothelial cell niche, whose shrinking leads to reduced MI with aging.

The EEHV1A gH/gL complex elicits humoral and cell-mediated immune responses in mice.

Elephant endotheliotropic herpesvirus (EEHV) causes lethal hemorrhagic disease (HD) in Asian and African elephants. Although rapid detection of viremia and supportive treatments may improve survival rates, an effective vaccine would mitigate the devastating effects of this virus. In elephants, chronic infection with EEHV leads to adaptive immunity against glycoproteins gB and gH/gL, the core entry machinery for most herpesviruses. We previously evaluated two EEHV gB vaccines in mice but not a gH/gL vaccine. Here, we found that inoculation of mice with an adjuvanted EEHV gH/gL subunit vaccine induced a significant antibody response that was similar to the response observed in elephants chronically infected with EEHV. Moreover, the gH/gL heterodimer elicited polyfunctional T cells with a Th1 phenotype but no detectable Th2 response. These results suggest that gH/gL, possibly in combination with gB, may be suitable immunogens for a vaccine comprising herpesvirus glycoproteins that are known to mediate cell entry and infection.

Transcriptomic analysis reveals bovine herpesvirus 1 infection regulates innate immune response resulted in restricted viral replication in neuronal cells.

BACKGROUND: Bovine herpesvirus 1 (BoHV-1) is a major pathogen that affects the global bovine population, primarily inducing respiratory and reproductive disorders. Its ability to establish latent infections in neuronal cells and to reactivate under certain conditions poses a continual threat to uninfected hosts. In this study, we aimed to analyze the replication characteristics of BoHV-1 in neuronal cells, as well as the effects of viral replication on host cell immunity and physiology. METHODS: Using the Neuro-2a neuronal-origin cell line as a model, we explored the dynamics of BoHV-1 replication and analyzed differential gene expression profiles post-BoHV-1 infection using high-throughput RNA sequencing. RESULTS: BoHV-1 demonstrated restricted replication in Neuro-2a cells. BoHV-1 induced apoptotic pathways and enhanced the transcription of interferon-stimulated genes and interferon regulatory factors while suppressing the complement cascade in Neuro-2a cells. CONCLUSIONS: Different from BoHV-1 infection in other non-highly differentiated somatic cells result in viral dominance, BoHV-1 regulated the innate immune response in neuronal cells formed a "virus-nerve cell" relative equilibrium state, which may account for the restricted replication of BoHV-1 in neuronal cells, leading to a latent infection. These findings provide a foundation for further research into the mechanism underlying BoHV-1-induced latent infection in nerve cells.

Unraveling clonal CD8 T cell expansion and identification of essential factors in γ-herpesvirus-induced lymphomagenesis.

Alcelaphine gammaherpesvirus 1 (AlHV-1) asymptomatically persists in its natural host, the wildebeest. However, cross-species transmission to cattle results in the induction of an acute and lethal peripheral T cell lymphoma-like disease (PTCL), named malignant catarrhal fever (MCF). Our previous findings demonstrated an essential role for viral genome maintenance in infected CD8+ T lymphocytes but the exact mechanism(s) leading to lymphoproliferation and MCF remained unknown. To decipher how AlHV-1 dysregulates T lymphocytes, we first examined the global phenotypic changes in circulating CD8+ T cells after experimental infection of calves. T cell receptor repertoire together with transcriptomics and epigenomics analyses demonstrated an oligoclonal expansion of infected CD8+ T cells displaying effector and exhaustion gene signatures, including GZMA, GNLY, PD-1, and TOX2 expression. Then, among viral genes expressed in infected CD8+ T cells, we uncovered A10 that encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. Impaired A10 expression did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF. Furthermore, A10 was phosphorylated in T lymphocytes in vitro and affected T cell signaling. Finally, while AlHV-1 mutants expressing mutated forms of A10 devoid of ITAM or SH3 motifs (or both) were able to induce MCF, a recombinant virus expressing a mutated form of A10 unable to phosphorylate its tyrosine residues resulted in the lack of MCF and protected against a wild-type virus challenge. Thus, we could characterize the nature of this γ-herpesvirus-induced PTCL-like disease and identify an essential mechanism explaining its development.

Restriction factors regulating human herpesvirus infections.

Herpesviruses are DNA viruses and the cause of diseases ranging from mild skin conditions to severe brain diseases. Mammalian antiviral host defense comprises an array of mechanisms, including restriction factors (RFs), which block specific steps in viral replication cycles. In recent years, knowledge of RFs that contribute to controlling herpesvirus infections has expanded significantly, along with a new understanding of viral evasion mechanisms and disease pathogenesis. By integrating findings from human genetics, murine models, and cellular studies, this review provides a current view of RF control of herpesvirus infections. We also explore the regulation of RF expression, discuss the roles of RFs in diseases, and point towards their growing potential as candidate therapeutic targets.

Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1.

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored.

Development of a live attenuated vaccine candidate for equid alphaherpesvirus 1 control: a step towards efficient protection.

Equid alphaherpesvirus 1 (EqAHV1) is a viral pathogen known to cause respiratory disease, neurologic syndromes, and abortion storms in horses. Currently, there are no vaccines that provide complete protection against EqAHV1. Marker vaccines and the differentiation of infected and vaccinated animals (DIVA) strategy are effective for preventing and controlling outbreaks but have not been used for the prevention of EqAHV1 infection. Glycoprotein 2 (gp2), located on the envelope of viruses (EqAHV1), exhibits high antigenicity and functions as a molecular marker for DIVA. In this study, a series of EqAHV1 mutants with deletion of gp2 along with other virulence genes (TK, UL24/TK, gI/gE) were engineered. The mutant viruses were studied in vitro and then in an in vivo experiment using Golden Syrian hamsters to assess the extent of viral attenuation and the immune response elicited by the mutant viruses in comparison to the wild-type (WT) virus. Compared with the WT strain, the YM2019 Δgp2, ΔTK/gp2, and ΔUL24/TK/gp2 strains exhibited reduced growth in RK-13 cells, while the ΔgI/gE/gp2 strain exhibited significantly impaired proliferation. The YM2019 Δgp2 strain induced clinical signs and mortality in hamsters. In contrast, the YM2019 ΔTK/gp2 and ΔUL24/TK/gp2 variants displayed diminished pathogenicity, causing no observable clinical signs or fatalities. Immunization with nasal vaccines containing YM2019 ΔTK/gp2 and ΔUL24/TK/gp2 elicited a robust immune response in hamsters. In particular, compared with the vaccine containing the ΔTK/gp2 strain, the vaccine containing the ΔUL24/TK/gp2 strain demonstrated enhanced immune protection upon challenge with the WT virus. Furthermore, an ELISA for gp2 was established and refined to accurately differentiate between infected and vaccinated animals. These results confirm that the ΔUL24/TK/gp2 strain is a safe and effective live attenuated vaccine candidate for controlling EqAHV1 infection.

Long-lived central memory γδ T cells confer protection against murine cytomegalovirus reinfection.

The involvement of γδ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. γδ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αß T cell deficient mice in order to analyze the memory potential of γδ T cells. As for CMV-specific αß T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ γδ TEM that principally localized in infected organ vasculature. Typifying T cell memory, γδ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking γδ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed γδ TCM cells, but not γδ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two γδ T cell memory subsets which also revealed their similarity to classically adaptive αß CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM γδ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches.

Cytokine Dynamics and Herpesvirus Interactions in Pediatric Liver and Kidney Transplant Recipients: The Distinct Behavior of HCMV, HHV6, HHV7 and EBV.

Pediatric solid organ transplant (SOT) recipients face a challenging balance between immunosuppression and graft rejection. While Epstein-Barr Virus (EBV) and cytomegalovirus (HCMV) are known contributors to post-transplant lymphoproliferative disease and graft rejection, respectively, the roles of herpesvirus 6 and 7 (HHV6 and HHV7) and the impact of these herpesviruses on cytokine levels remain unclear, leading to gaps in clinical practice. In this associative study, we measured 17 cytokines using a Bio-Plex assay in a meticulously curated plasma sample pool (N = 158) from pediatric kidney and liver transplant recipients over a one-year follow-up period. The samples included virus-negative and virus-positive cases, either individually or in combination, along with episodes of graft rejection. We observed that the elevation of IL-4, IL-8, and IL-10 correlated with graft rejection. These cytokines were elevated in samples where HCMV or HHV6 were detected alone or where EBV and HHV7 were co-detected. Interestingly, latent EBV, when detected independently, exhibited an immunomodulatory effect by downregulating cytokine levels. However, in co-detection scenarios with ß-herpesviruses, EBV transitioned to a lytic state, also associating with heightened cytokinemia and graft rejection. These findings highlight the complex interactions between the immune response and herpesviruses in transplant recipients. The study advocates for enhanced monitoring of not only EBV and HCMV but also HHV6 and HHV7, providing valuable insights for improved risk assessment and targeted interventions in pediatric SOT recipients.

Inflammation and Macrophage Loss Mark Increased Susceptibility in a Genetic Model of Acute Viral Infection-Induced Tissue Damage.

M.R2k/b mice are identical to the MA/My parent strain aside from a 5.58-Mb C57L-derived region on chromosome 17 (Cmv5s) that causes increased susceptibility to acute murine CMV (MCMV) infection and the development of significant spleen tissue damage. Spleen pathology begins at the marginal zone (MZ), apparent by 2 d postinfection (dpi), and progresses throughout the red pulp by 4 dpi. To better understand how M.R2k/b mice respond to infection and how Cmv5s contributes to tissue damage in the spleen, we assessed the regulation of myeloid cells and inflammation during acute MCMV infection in MA/My and M.R2k/b mice. We found that Cmv5s drove increased neutrophil accumulation and cell death at the MZ, which corresponded with evidence of localized oxidative stress and increased overall spleen IL-6 and TGF-ß1 early during infection. Further assessment of MCMV infection dynamics at the early MZ revealed infected SIGNR1+ MZ macrophages as the first apparent cell type lost during infection in these mice and the likely target of early neutrophil recruitment. Spleen macrophages were also identified as the mediators of differential spleen IL-6 and TGF-ß1 between MA/My and M.R2k/b mice. Interrogation of MCMV progression past 2 dpi revealed substantial M.R2k/b F480+ red pulp macrophage loss along with buildup of oxidative stress and MZ macrophage debris that was not neutrophil dependent. Together we identify Cmv5s-driven macrophage loss and inflammation during acute MCMV infection corresponding with the spatial and temporal development of spleen tissue damage.

KSHV infection of B cells primes protective T cell responses in humanized mice.

Kaposi sarcoma associated herpesvirus (KSHV) is associated with around 1% of all human tumors, including the B cell malignancy primary effusion lymphoma (PEL), in which co-infection with the Epstein Barr virus (EBV) can almost always be found in malignant cells. Here, we demonstrate that KSHV/EBV co-infection of mice with reconstituted human immune systems (humanized mice) leads to IgM responses against both latent and lytic KSHV antigens, and expansion of central and effector memory CD4+ and CD8+ T cells. Among these, KSHV/EBV dual-infection allows for the priming of CD8+ T cells that are specific for the lytic KSHV antigen K6 and able to kill KSHV/EBV infected B cells. This suggests that K6 may represent a vaccine antigen for the control of KSHV and its associated pathologies in high seroprevalence regions, such as Sub-Saharan Africa.

Transcriptomic profiling of thymic dysregulation and viral tropism after neonatal roseolovirus infection.

Introduction: Herpesviruses, including the roseoloviruses, have been linked to autoimmune disease. The ubiquitous and chronic nature of these infections have made it difficult to establish a causal relationship between acute infection and subsequent development of autoimmunity. We have shown that murine roseolovirus (MRV), which is highly related to human roseoloviruses, induces thymic atrophy and disruption of central tolerance after neonatal infection. Moreover, neonatal MRV infection results in development of autoimmunity in adult mice, long after resolution of acute infection. This suggests that MRV induces durable immune dysregulation. Methods: In the current studies, we utilized single-cell RNA sequencing (scRNAseq) to study the tropism of MRV in the thymus and determine cellular processes in the thymus that were disrupted by neonatal MRV infection. We then utilized tropism data to establish a cell culture system. Results: Herein, we describe how MRV alters the thymic transcriptome during acute neonatal infection. We found that MRV infection resulted in major shifts in inflammatory, differentiation and cell cycle pathways in the infected thymus. We also observed shifts in the relative number of specific cell populations. Moreover, utilizing expression of late viral transcripts as a proxy of viral replication, we identified the cellular tropism of MRV in the thymus. This approach demonstrated that double negative, double positive, and CD4 single positive thymocytes, as well as medullary thymic epithelial cells were infected by MRV in vivo. Finally, by applying pseudotime analysis to viral transcripts, which we refer to as "pseudokinetics," we identified viral gene transcription patterns associated with specific cell types and infection status. We utilized this information to establish the first cell culture systems susceptible to MRV infection in vitro. Conclusion: Our research provides the first complete picture of roseolovirus tropism in the thymus after neonatal infection. Additionally, we identified major transcriptomic alterations in cell populations in the thymus during acute neonatal MRV infection. These studies offer important insight into the early events that occur after neonatal MRV infection that disrupt central tolerance and promote autoimmune disease.

Evaluation of Bispecific T-Cell Engagers Targeting Murine Cytomegalovirus.

Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups.

Transcriptional and functional remodeling of lung-resident T cells and macrophages by Simian varicella virus infection.

Introduction: Varicella zoster virus (VZV) causes varicella and can reactivate as herpes zoster, and both diseases present a significant burden worldwide. However, the mechanisms by which VZV establishes latency in the sensory ganglia and disseminates to these sites remain unclear. Methods: We combined a single-cell sequencing approach and a well-established rhesus macaque experimental model using Simian varicella virus (SVV), which recapitulates the VZV infection in humans, to define the acute immune response to SVV in the lung as well as compare the transcriptome of infected and bystander lung-resident T cells and macrophages. Results and discussion: Our analysis showed a decrease in the frequency of alveolar macrophages concomitant with an increase in that of infiltrating macrophages expressing antiviral genes as well as proliferating T cells, effector CD8 T cells, and T cells expressing granzyme A (GZMA) shortly after infection. Moreover, infected T cells harbored higher numbers of viral transcripts compared to infected macrophages. Furthermore, genes associated with cellular metabolism (glycolysis and oxidative phosphorylation) showed differential expression in infected cells, suggesting adaptations to support viral replication. Overall, these data suggest that SVV infection remodels the transcriptome of bystander and infected lung-resident T cells and macrophages.

Construction and in vitro characterisation of virus-vectored immunocontraceptive candidates derived from felid alphaherpesvirus 1.

There is a pressing need for effective feral cat management globally due to overabundant feline populations, disease transmission and their destructive impact on biodiversity. Virus-vectored immunocontraception (VVIC) is an attractive method for cat population management. Virus-vectored immunocontraceptives could be self-disseminating through horizontal transmission of the VVIC in feral cat populations, or they may be modified to act as non-transmissible vaccine-type immunocontraceptives for delivery to individual cats. These later constructs may be particularly attractive for use in owned (pet) cats and stray cats but could also be used for feral cats that are caught, vaccinated, and released. Here, we report the construction of three felid alphaherpesvirus 1 (FHV-1) derived immunocontraceptive candidates containing genes that encode for feline zona pellucida subunit 3 (ZP3) and gonadotropin-releasing hormone (GnRH). Two of the vaccine candidates were engineered to include disruptions to the thymidine kinase viral virulence gene to reduce the ability of the vaccines to be horizontally transmitted. Analysis of in vitro growth characteristics and protein expression are reported, and their potential for use as a population management tool for cats is discussed.

Equine herpesvirus type 1 (EHV-1) replication at the upper respiratory entry site is inhibited by neutralizing EHV-1-specific IgG1 and IgG4/7 mucosal antibodies.

Equine herpesvirus type 1 (EHV-1) is a contagious respiratory pathogen that infects the mucosa of the upper respiratory tract (URT). Mucosal immune responses at the URT provide the first line of defense against EHV-1 and are crucial for orchestrating immunity. To define host-pathogen interactions, we characterized B-cell responses, antibody isotype functions, and EHV-1 replication of susceptible (non-immune) and clinically protected (immune) horses after experimental EHV-1 infection. Nasal secretion and nasal wash samples were collected and used for the isolation of DNA, RNA, and mucosal antibodies. Shedding of infectious virus, EHV-1 copy numbers, viral RNA expression, and host B-cell activation in the URT were compared based on host immune status. Mucosal EHV-1-specific antibody responses were associated with EHV-1 shedding and viral RNA transcription. Finally, mucosal immunoglobulin G (IgG) and IgA isotypes were purified and tested for neutralizing capabilities. IgG1 and IgG4/7 neutralized EHV-1, while IgG3/5, IgG6, and IgA did not. Immune horses secreted high amounts of mucosal EHV-1-specific IgG4/7 antibodies and quickly upregulated B-cell pathway genes, while EHV-1 was undetected by virus isolation and PCR. RNA transcription analysis reinforced incomplete viral replication in immune horses. In contrast, complete viral replication with high viral copy numbers and shedding of infectious viruses was characteristic for non-immune horses, together with low or absent EHV-1-specific neutralizing antibodies during viral replication. These data confirm that pre-existing mucosal IgG1 and IgG4/7 and rapid B-cell activation upon EHV-1 infection are essential for virus neutralization, regulation of viral replication, and mucosal immunity against EHV-1.IMPORTANCEEquine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion storms, and neurologic outbreaks known as equine herpes myeloencephalopathy (EHM). EHV-1 is transmitted with respiratory secretions by nose-to-nose contact or via fomites. The virus initially infects the epithelium of the upper respiratory tract (URT). Host-pathogen interactions and mucosal immunity at the viral entry site provide the first line of defense against the EHV-1. Robust mucosal immunity can be essential in protecting against EHV-1 and to reduce EHM outbreaks. It has previously been shown that immune horses do not establish cell-associated viremia, the prerequisite for EHM. Here, we demonstrate how mucosal antibodies can prevent the replication of EHV-1 at the epithelium of the URT and, thereby, the progression of the virus to the peripheral blood. The findings improve the mechanistic understanding of mucosal immunity against EHV-1 and can support the development of enhanced diagnostic tools, vaccines against EHM, and the management of EHV-1 outbreaks.