Impact of the host immune response on the development of equine herpesvirus myeloencephalopathy in horses.

Herpesviruses establish a well-adapted balance with their host's immune system. Despite this co-evolutionary balance, infections can lead to severe disease including neurological disorders in their natural host. In horses, equine herpesvirus 1 (EHV-1) causes respiratory disease, abortions, neonatal foal death and myeloencephalopathy (EHM) in ~10 % of acute infections worldwide. Many aspects of EHM pathogenesis and protection from EHM are still poorly understood. However, it has been shown that the incidence of EHM increases to >70 % in female horses >20 years of age. In this study we used old mares as an experimental equine EHV-1 model of EHM to identify host-specific factors contributing to EHM. Following experimental infection with the neuropathogenic strain EHV-1 Ab4, old mares and yearling horses were studied for 21 days post-infection. Nasal viral shedding and cell-associated viremia were assessed by quantitative PCR. Cytokine/chemokine responses were evaluated in nasal secretions and cerebrospinal fluid (CSF) by Luminex assay and in whole blood by quantitative real-time PCR. EHV-1-specific IgG sub-isotype responses were measured by ELISA. All young horses developed respiratory disease and a bi-phasic fever post-infection, but only 1/9 horses exhibited ataxia. In contrast, respiratory disease was absent in old mares, but all old mares developed EHM that resulted in euthanasia in 6/9 old mares. Old mares also presented significantly decreased nasal viral shedding but higher viremia coinciding with a single fever peak at the onset of viremia. According to clinical disease manifestation, horses were sorted into an EHM group (nine old horses and one young horse) and a non-EHM group (eight young horses) for assessment of host immune responses. Non-EHM horses showed an early upregulation of IFN-α (nasal secretions), IRF7/IRF9, IL-1ß, CXCL10 and TBET (blood) in addition to an IFN-γ upregulation during viremia (blood). In contrast, IFN-α levels in nasal secretions of EHM horses were low and peak levels of IRF7, IRF9, CXCL10 and TGF-ß (blood) coincided with viremia. Moreover, EHM horses showed significantly higher IL-10 levels in nasal secretions, peripheral blood mononuclear cells and CSF and higher serum IgG3/5 antibody titres compared to non-EHM horses. These results suggest that protection from EHM depends on timely induction of type 1 IFN and upregulation cytokines and chemokines that are representative of cellular immunity. In contrast, induction of regulatory or TH-2 type immunity appeared to correlate with an increased risk for EHM. It is likely that future vaccine development for protection from EHM must target shifting this 'at-risk' immunophenotype.

Viral infection and allergy - What equine immune responses can tell us about disease severity and protection.

Horses have many naturally occurring diseases that mimic similar conditions in humans. The ability to conduct environmentally controlled experiments and induced disease studies in a genetically diverse host makes the horse a valuable intermediate model between mouse studies and human clinical trials. This review highlights important similarities in the immune landscape between horses and humans using current research on two equine diseases as examples. First, equine herpesvirus type 1 (EHV-1) infection initiates a series of innate inflammatory signals at its mucosal entry site in the upper respiratory tract. These inflammatory markers are highly synchronized and predictable between individuals during viral respiratory infection and ultimately lead to adaptive immune induction and protection. The timing of early inflammatory signals, followed by specific adaptive immune markers correlating with immunity and protection, allow accurate outbreak tracking and also provide a foundation for understanding the importance of local mucosal immunity during other viral respiratory infections. Second, rare peripheral blood immune cells that promote allergic inflammation can be analyzed during Culicoides hypersensitivity, a naturally occurring type I IgE-mediated allergic disease of horses. Rare immune cells, such as IgE-binding monocytes or basophils, can be studied repeatedly in the horse model to unravel their larger mechanistic role in inflammation during allergic and other inflammatory diseases. We conclude with a survey of all other common equine inflammatory conditions. Together, this review serves as a reference and rationale for the horse as a non-rodent model for immunological research.

Identification of Host Factors Associated with the Development of Equine Herpesvirus Myeloencephalopathy by Transcriptomic Analysis of Peripheral Blood Mononuclear Cells from Horses.

Equine herpesvirus-1 is the cause of respiratory disease, abortion, and equine herpesvirus myeloencephalopathy (EHM) in horses worldwide. EHM affects as many as 14% of infected horses and a cell-associated viremia is thought to be central for EHM pathogenesis. While EHM is infrequent in younger horses, up to 70% of aged horses develop EHM. The aging immune system likely contributes to EHM pathogenesis; however, little is known about the host factors associated with clinical EHM. Here, we used the "old mare model" to induce EHM following EHV-1 infection. Peripheral blood mononuclear cells (PBMCs) of horses prior to infection and during viremia were collected and RNA sequencing with differential gene expression was used to compare the transcriptome of horses that did (EHM group) and did not (non-EHM group) develop clinical EHM. Interestingly, horses exhibiting EHM did not show respiratory disease, while non-EHM horses showed significant respiratory disease starting on day 2 post infection. Multiple immune pathways differed in EHM horses in response to EHV-1. These included an upregulation of IL-6 gene expression, a dysregulation of T-cell activation through AP-1 and responses skewed towards a T-helper 2 phenotype. Further, a dysregulation of coagulation and an upregulation of elements in the progesterone response were observed in EHM horses.

Equine herpesvirus 1 elicits a strong pro-inflammatory response in the brain of mice.

Equine herpesvirus type 1 (EHV-1) is an emerging pathogen that causes encephalomyelitis in horses and non-equid species. Several aspects of the immune response in the central nervous system (CNS), mainly regarding the role of inflammatory mediators during EHV-1 encephalitis, remain unknown. Moreover, understanding the mechanisms underlying extensive neuropathology induced by viruses would be helpful to establish therapeutic strategies. Therefore, we aimed to evaluate some aspects of the innate immune response during highly neurovirulent EHV-1 infection. C57BL/6 mice infected intranasally with A4/72 and A9/92 EHV-1 strains developed a fulminant neurological disease at 3 days post-inoculation with high viral titres in the brain. These mice developed severe encephalitis with infiltration of monocytes and CD8+ T cells to the brain. The inflammatory infiltrate followed the detection of the chemokines CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL9 and CXCL-10 in the brain. Notably, the levels of CCL3, CCL4, CCL5 and CXCL9 were higher in A4/72-infected mice, which presented higher numbers of inflammatory cells within the CNS. Pro-inflammatory cytokines, such as interleukins (ILs) IL-1α, IL-1ß, IL-6, IL-12ß, and tumour necrosis factor (TNF), were also detected in the CNS, and Toll-like receptor (TLR) TLR2, TLR3 and TLR9 genes were also upregulated within the brain of EHV-1-infected mice. However, no expression of interferon-γ (IFN-γ) and IL-12α, which are important for controlling the replication of other herpesviruses, was detected in EHV-1-infected mice. The results show that the activated innate immune mechanisms could not prevent EHV-1 replication within the CNS, but most likely contributed to the extensive neuropathology. The mouse model of viral encephalitis proposed here will also be useful to study the mechanisms underlying extensive neuropathology.

Prevention of respiratory infections with alpha- and gamma-herpesviruses in weanling foals by using a modified live intra-nasal equine influenza vaccine.

This study aimed to determine if the administration of a modified live equine influenza virus vaccine (FluAvert) to foals would positively impact their health and reduce colonization of their upper airways with equine herpesviruses (EHV) during the weaning period. A single dose of FluAvert was given to 20 healthy foals 7 days prior to being weaned; 20 healthy foals served as unvaccinated controls. Nasal secretions and blood were collected before vaccination, the day of weaning, and weekly thereafter for 3 weeks. Nasal secretions were tested by quantitative polymerase chain reaction (qPCR) for EHV-1, -2, -4 and -5. Whole blood was analyzed for a complete blood cell count and fibrinogen concentration. Physical assessments were made daily. The use of FluAvert was associated with a better clinical outcome. However, the equine influenza virus (EIV) vaccine did not influence selected hematological parameters and kinetics of herpesviruses. The clinical benefit observed in vaccinates may explain the perception that the EIV vaccine induces cross-protection against respiratory agents.

Prévention des infections respiratoires causées par les alpha- et gamma-herpesvirus chez les poulains au sevrage en utilisant un vaccin vivant modifié intra-nasal contre l'influenza. La présente étude visait à déterminer si l'administration d'un vaccin vivant modifié du virus de l'influenza (FluAvert) à des poulains affecterait positivement leur santé et réduirait la colonisation de leurs voies respiratoires supérieures par les herpesvirus équins (EHV) durant la période de sevrage. Une dose unique de FluAvert fut administrée à 20 poulains en santé 7 jours avant le sevrage; 20 poulains en santé ont servi de témoins non-vaccinés. Des sécrétions nasales et du sang furent prélevés avant la vaccination, le jour du sevrage, et de manière hebdomadaire pour les trois semaines suivantes. Les sécrétions nasales furent testées par réaction d'amplification en chaîne par la polymérase quantitative (qPCR) pour EHV-1, -2, -4 et -5. Le sang entier fut analysé pour un dénombrement complet des cellules sanguines et la concentration de fibrinogène. Des examens physiques étaient réalisés quotidiennement. L'utilisation de FluAvert fut associée avec une meilleure issue clinique. Toutefois, le vaccin contre le virus de l'influenza équin (EIV) n'influença pas des paramètres hématologiques sélectionnés et la cinétique des herpesvirus. Les bienfaits cliniques observés chez les chevaux vaccinés pourraient expliquer la perception que le vaccin EIV induit une protection croisée contre des agents infectieux respiratoires.(Traduit par Dr Serge Messier).

Vaccination of foals with a modified live, equid herpesvirus-1 gM deletion mutant (RacHΔgM) confers partial protection against infection.

Equid herpesvirus-1 (EHV-1) causes respiratory and neurological disease and late gestation abortion in pregnant mares. Current vaccines contain either inactivated or live EHV-1, but fail to provide complete clinical or virological protection, namely prevention of nasopharyngeal shedding and cell-associated viraemia. Thus, the development of novel products, such as modified live virus (MLV) vaccines which stimulate virus-specific, humoral and cell mediated immune responses more effectively remains a priority. Two groups of weaned foals (n = 6 each group) were used in a longitudinal, prospective, experimental study to evaluate immune responses elicited by two vaccinations with a glycoprotein M (gM) deletion mutant of EHV-1 (RacHdeltagM). Following two concurrent intranasal and intramuscular inoculations six weeks apart, vaccinated (8.4 ±â€¯0.2 months old) and control foals (6.2 ±â€¯0.4 months) were challenge infected intranasally with EHV-1 Ab4/8 four weeks after the second vaccination and clinical signs and virological replication measured. Vaccination caused no adverse events, but did stimulate significantly higher complement fixing and virus neutralizing antibodies in serum compared with control foals at either equivalent or pre-vaccination time points. Virus-specific nasopharyngeal antibody levels and cytotoxic T lymphocyte responses were not significantly different between the groups. Following challenge infection, these immune responses were associated with a reduction in clinical signs and virological replication in the vaccinated foals, including a reduction in duration and magnitude of pyrexia, nasopharyngeal shedding and cell-associated viraemia. We conclude that the RacHΔgM MLV primed EHV-1-specific humoral immune responses in weaned foals. However, complete virological protection by vaccination against EHV-1 requires further research.

Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells.

Equid herpesvirus 1 (EHV-1) is a viral pathogen of horse populations worldwide spread by the respiratory route and is known for causing outbreaks of neurologic syndromes and abortion storms. Previously, we demonstrated that an EHV-1 strain of the neuropathogenic genotype, T953, downregulates the beta interferon (IFN-ß) response in vitro in equine endothelial cells (EECs) at 12 h postinfection (hpi). In the present study, we explored the molecular correlates of this inhibition as clues toward an understanding of the mechanism. Data from our study revealed that EHV-1 infection of EECs significantly reduced both Toll-like receptor 3 (TLR3) and TLR4 mRNA expression at 6 hpi and 12 hpi. While EHV-1 was able to significantly reduce IRF9 mRNA at both 6 hpi and 12 hpi, the virus significantly reduced IFN regulatory factor 7 (IRF7) mRNA only at 12 hpi. EHV-1 did not alter the cellular level of Janus-activated kinase 1 (JAK1) at any time point. However, EHV-1 reduced the cellular level of expression of tyrosine kinase 2 (TYK2) at 12 hpi. Downstream of JAK1-TYK2 signaling, EHV-1 blocked the phosphorylation and activation of signal transducer and activator of transcription 2 (STAT2) when coincubated with exogenous IFN, at 12 hpi, although not at 3 or 6 hpi. Immunofluorescence staining revealed that the virus prevented the nuclear translocation of STAT2 molecules, confirming the virus-mediated inhibition of STAT2 activation. The pattern of suppression of phosphorylation of STAT2 by EHV-1 implicated viral late gene expression. These data help illuminate how EHV-1 strategically inhibits the host innate immune defense by limiting steps required for type I IFN sensitization and induction.IMPORTANCE To date, no commercial vaccine label has a claim to be fully protective against the diseases caused by equid herpesvirus 1 (EHV-1), especially the neurologic form. The interferon (IFN) system, of which type I IFN is of great importance, still remains a viable immunotherapeutic option against EHV-1 infection. The type I IFN system has been exploited successfully to treat other viral infections, such as chronic hepatitis B and C in humans. The current state of research on how EHV-1 interferes with the protective effect of type I IFN has indicated transient induction of type I IFN production followed by a rapid shutdown in vitro in equine endothelial cells (EECs). The significance of our study is the identification of certain steps in the type I IFN signaling pathway targeted for inhibition by EHV-1. Understanding this pathogen-host relationship is essential for the long-term goal of developing effective immunotherapy against EHV-1.

Equine herpesvirus 1 infection orchestrates the expression of chemokines in equine respiratory epithelial cells.

The ancestral equine herpesvirus 1 (EHV1), closely related to human herpes viruses, exploits leukocytes to reach its target organs, accordingly evading the immune surveillance system. Circulating EHV1 strains can be divided into abortigenic/neurovirulent, causing reproductive/neurological disorders. Neurovirulent EHV1 more efficiently recruits monocytic CD172a+ cells to the upper respiratory tract (URT), while abortigenic EHV1 tempers monocyte migration. Whether similar results could be expected for T lymphocytes is not known. Therefore, we questioned whether differences in T cell recruitment could be associated with variations in cell tropism between both EHV1 phenotypes, and which viral proteins might be involved. The expression of CXCL9 and CXCL10 was evaluated in abortigenic/neurovirulent EHV1-inoculated primary respiratory epithelial cells (ERECs). The bioactivity of chemokines was tested with a functional migration assay. Replication of neurovirulent EHV1 in the URT resulted in an enhanced expression/bioactivity of CXCL9 and CXCL10, compared to abortigenic EHV1. Interestingly, deletion of glycoprotein 2 resulted in an increased recruitment of both monocytic CD172a+ cells and T lymphocytes to the corresponding EREC supernatants. Our data reveal a novel function of EHV1-gp2, tempering leukocyte migration to the URT, further indicating a sophisticated virus-mediated orchestration of leukocyte recruitment to the URT.

An Equine Herpesvirus Type 1 (EHV-1) Ab4 Open Reading Frame 2 Deletion Mutant Provides Immunity and Protection from EHV-1 Infection and Disease.

Equine herpesvirus type 1 (EHV-1) outbreaks continue to occur despite widely used vaccination. Therefore, development of EHV-1 vaccines providing improved immunity and protection is ongoing. Here, an open reading frame 2 deletion mutant of the neuropathogenic EHV-1 strain Ab4 (Ab4ΔORF2) was tested as a vaccine candidate. Three groups of horses (n = 8 each) were infected intranasally with Ab4ΔORF2 or the parent Ab4 virus or were kept as noninfected controls. Horses infected with Ab4ΔORF2 had reduced fever and nasal virus shedding compared to those infected with Ab4 but mounted similar adaptive immunity dominated by antibody responses. Nine months after the initial infection, all horses were challenged intranasally with Ab4. Previously noninfected horses (control/Ab4) displayed clinical signs, shed large amounts of virus, and developed cell-associated viremia. In contrast, 5/8 or 3/8 horses previously infected with Ab4ΔORF2 or Ab4, respectively, were fully protected from challenge infection as indicated by the absence of fever, clinical disease, nasal virus shedding, and viremia. All of these outcomes were significantly reduced in the remaining, partially protected 3/8 (Ab4ΔORF2/Ab4) and 5/8 (Ab4/Ab4) horses. Protected horses had EHV-1-specific IgG4/7 antibodies prior to challenge infection, and intranasal antibodies increased rapidly postchallenge. Intranasal inflammatory markers were not detectable in protected horses but quickly increased in control/Ab4 horses during the first week after infection. Overall, our data suggest that preexisting nasal IgG4/7 antibodies neutralize EHV-1, prevent viral entry, and thereby protect from disease, viral shedding, and cell-associated viremia. In conclusion, improved protection from challenge infection emphasizes further evaluation of Ab4ΔORF2 as a vaccine candidate.IMPORTANCE Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to preexisting local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies and lack of nasal type I interferon and chemokine induction. These host immune parameters may constitute markers of protection against EHV-1 and be utilized as indicators for improved vaccine development and informed vaccination strategies.

Intranasal IgG4/7 antibody responses protect horses against equid herpesvirus-1 (EHV-1) infection including nasal virus shedding and cell-associated viremia.

Equid herpesvirus-1 (EHV-1) outbreaks continue despite widely used vaccination. We demonstrated previously that an ORF1/ORF71 gene deletion mutant of the EHV-1 strain Ab4 (Ab4ΔORF1/71) is less virulent than its parent Ab4 virus. Here, we describe the Ab4 challenge infection evaluating protection induced by the Ab4ΔORF1/71 vaccine candidate. Susceptible control horses developed respiratory disease, fever, nasal shedding, and viremia. Full protection after challenge infection was observed in 5/5 previously Ab4 infected horses and 3/5 Ab4ΔORF1/71 horses. Two Ab4ΔORF1/71 horses developed short-lasting viremia and/or virus shedding. Protective immunity in the respiratory tract was characterized by pre-existing EHV-1-specific IgG4/7 antibodies, the absence of IFN-α secretion and rapidly increasing IgG4/7 upon challenge infection. Pre-existing systemic EHV-1-specific IgG4/7 highly correlated with protection. T-cell immunity was overall low. In conclusion, protective immunity against EHV-1 infection including prevention of viremia was associated with robust systemic and intranasal IgG4/7 antibodies suggesting immediate virus neutralization at the local site.

TLR-5 agonist Salmonella abortus equi flagellin FliC enhances FliC-gD-based DNA vaccination against equine herpesvirus 1 infection.

Equine herpesvirus 1 (EHV-1) induces serious respiratory infections, viral abortion, neurological signs, and neonatal mortality in horses. Despite the use of vaccines, EHV-1 infection also causes a high annual economic burden to the equine industry. The poor immunogenicity of and protection conferred by EHV-1 vaccines are the major factors responsible for the spread of EHV-1 infection. The present study examined the immunogenicity of a novel DNA vaccine co-expressing FliC, a flagellin protein, in Salmonella abortus equi and the gD protein of EHV-1. Mice and horses were immunized intramuscularly with the vaccine, and mice were challenged with EHV-1. Immunofluorescence and western blotting revealed that FliC and gD can be efficiently expressed in cells. This novel vaccine significantly increased gD-specific antibody and interferon gamma (IFN-γ) levels in immunized mice and horses. Compared with controls, the viral load and morbidity were markedly reduced in FliC-gD-immunized mice after they were challenged with EHV-1. Furthermore, the immunogenicity of FliC-gD in a natural host was tested. Our results indicate that vaccinated mice and horses exhibit increased humoral and improved cellular immune responses.

Equine Herpesvirus 1 Bridles T Lymphocytes To Reach Its Target Organs.

Equine herpesvirus 1 (EHV1) replicates in the respiratory epithelium and disseminates through the body via a cell-associated viremia in leukocytes, despite the presence of neutralizing antibodies. "Hijacked" leukocytes, previously identified as monocytic cells and T lymphocytes, transmit EHV1 to endothelial cells of the endometrium or central nervous system, causing reproductive (abortigenic variants) or neurological (neurological variants) disorders. In the present study, we questioned the potential route of EHV1 infection of T lymphocytes and how EHV1 misuses T lymphocytes as a vehicle to reach the endothelium of the target organs in the absence or presence of immune surveillance. Viral replication was evaluated in activated and quiescent primary T lymphocytes, and the results demonstrated increased infection of activated versus quiescent, CD4+ versus CD8+, and blood- versus lymph node-derived T cells. Moreover, primarily infected respiratory epithelial cells and circulating monocytic cells efficiently transferred virions to T lymphocytes in the presence of neutralizing antibodies. Albeit T-lymphocytes express all classes of viral proteins early in infection, the expression of viral glycoproteins on their cell surface was restricted. In addition, the release of viral progeny was hampered, resulting in the accumulation of viral nucleocapsids in the T cell nucleus. During contact of infected T lymphocytes with endothelial cells, a late viral protein(s) orchestrates T cell polarization and synapse formation, followed by anterograde dynein-mediated transport and transfer of viral progeny to the engaged cell. This represents a sophisticated but efficient immune evasion strategy to allow transfer of progeny virus from T lymphocytes to adjacent target cells. These results demonstrate that T lymphocytes are susceptible to EHV1 infection and that cell-cell contact transmits infectious virus to and from T lymphocytes.IMPORTANCE Equine herpesvirus 1 (EHV1) is an ancestral alphaherpesvirus that is related to herpes simplex virus 1 and causes respiratory, reproductive, and neurological disorders in Equidae. EHV1 is indisputably a master at exploiting leukocytes to reach its target organs, accordingly evading the host immunity. However, the role of T lymphocytes in cell-associated viremia remains poorly understood. Here we show that activated T lymphocytes efficiently become infected and support viral replication despite the presence of protective immunity. We demonstrate a restricted expression of viral proteins on the surfaces of infected T cells, which prevents immune recognition. In addition, we indicate a hampered release of progeny, which results in the accumulation of nucleocapsids in the T cell nucleus. Upon engagement with the target endothelium, late viral proteins orchestrate viral synapse formation and viral transfer to the contact cell. Our findings have significant implications for the understanding of EHV1 pathogenesis, which is essential for developing innovative therapies to prevent the devastating clinical symptoms of infection.

Abortigenic but Not Neurotropic Equine Herpes Virus 1 Modulates the Interferon Antiviral Defense.

Equine herpesvirus 1 (EHV1) is considered as a major pathogen of Equidae, causing symptoms from mild respiratory disease to late-term abortion and neurological disorders. Different EHV1 strains circulating in the field have been characterized to be of abortigenic or neurovirulent phenotype. Both variants replicate in a plaque-wise manner in the epithelium of the upper respiratory tract (URT), where the abortigenic strains induce more prominent viral plaques, compared to the neurovirulent strains. Considering the differences in replication at the URT, we hypothesized that abortigenic strains may show an increased ability to modulate the type I IFN secretion/signaling pathway, compared to strains that display the neurovirulent phenotype. Here, we analyze IFN levels induced by abortigenic and neurovirulent EHV1 using primary respiratory epithelial cells (EREC) and respiratory mucosa ex vivo explants. Similar levels of IFNα (~70 U/ml) were detected in explants inoculated with both types of EHV1 strains from 48 to 72 hpi. Second, EREC and mucosa explants were treated with recombinant equine IFNα (rEqIFNα) or Ruxolitinib (Rux), an IFN signaling inhibitor, prior to and during inoculation with abortigenic or neurovirulent EHV1. Replication of both EHV1 variants was suppressed by rEqIFNα. Further, addition of Rux increased replication in a concentration-dependent manner, indicating an IFN-susceptibility for both variants. However, in two out of three horses, at a physiological concentration of 100 U/ml of rEqIFNα, an increase in abortigenic EHV1 replication was observed compared to 10 U/ml of rEqIFNα, which was not observed for the neurovirulent strains. Moreover, in the presence of Rux, the plaque size of the abortigenic variants remained unaltered, whereas the typically smaller viral plaques induced by the neurovirulent variants became larger. Overall, our results demonstrate the importance of IFNα in the control of EHV1 replication in the URT for both abortigenic and neurovirulent variants. In addition, our findings support the speculation that abortigenic variants of EHV1 may have developed anti-IFN mechanisms that appear to be absent or less pronounced in neurovirulent EHV1 strains.

Deletion of the ORF2 gene of the neuropathogenic equine herpesvirus type 1 strain Ab4 reduces virulence while maintaining strong immunogenicity.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses' clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection. RESULTS: Infection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4ΔORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4ΔORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10-12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5-10 pi) and was similar in both infected groups, Ab4 and Ab4ΔORF2. CONCLUSIONS: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4ΔORF2 and parent Ab4 viruses are identical. Graphical abstract - Deletion of ORF2 reduces virulence of EHV-1 Ab4. Graphical summary of the main findings of this study: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa.

Comparison of protective efficacies between intranasal and intramuscular vaccination of horses with a modified live equine herpesvirus type-1 vaccine.

Immune responses were compared after intranasal (IN) and intramuscular (IM) vaccination of horses with a modified live equine herpesvirus type-1 (EHV-1) vaccine, and the protective effect after EHV-1 challenge was evaluated. IN- and IM-vaccinated groups (n = 5 each) showed significant rises in serum virus-neutralizing titers with increased levels of IgGa and IgGb antibodies after the first vaccination (P < 0.05). In nasal secretions, the IN group had significantly increased levels of IgA antibodies after vaccination (P < 0.05), whereas the response of the IM group was dominated by IgGa and IgGb subclasses. After challenge infection, the numbers of pyretic horses from 1 to 4 days post-inoculation were 3/5 in the placebo (PBO) group (n = 5), 0/5 in the IN group, and 1/5 in the IM group. The IN and IM groups had significantly lower levels of virus shedding than the PBO group (P < 0.05). There were no significant between-group differences in the numbers of viremic horses each day. Notably, two horses in the IM group had no virus shedding or viremia, whereas all horses in the other group did. Both IN and IM vaccination induced systemic humoral immunity and mucosal immunity, suppressing virus replication in the nasal mucosa, and partially protected horses from pyrexia, especially early in infection. This study showed a mucosal antibody response was induced, not only by IN vaccination but also by IM vaccination.

Intramuscular Immunization of Mice with the Live-Attenuated Herpes Simplex Virus 1 Vaccine Strain VC2 Expressing Equine Herpesvirus 1 (EHV-1) Glycoprotein D Generates Anti-EHV-1 Immune Responses in Mice.

Vaccination remains the best option to combat equine herpesvirus 1 (EHV-1) infection, and several different strategies of vaccination have been investigated and developed over the past few decades. Herein, we report that the live-attenuated herpes simplex virus 1 (HSV-1) VC2 vaccine strain, which has been shown to be unable to enter into neurons and establish latency in mice, can be utilized as a vector for the heterologous expression of EHV-1 glycoprotein D (gD) and that the intramuscular immunization of mice results in strong antiviral humoral and cellular immune responses. The VC2-EHV-1-gD recombinant virus was constructed by inserting an EHV-1 gD expression cassette under the control of the cytomegalovirus immediate early promoter into the VC2 vector in place of the HSV-1 thymidine kinase (UL23) gene. The vaccines were introduced into mice through intramuscular injection. Vaccination with both the VC2-EHV-1-gD vaccine and the commercially available vaccine Vetera EHVXP 1/4 (Vetera; Boehringer Ingelheim Vetmedica) resulted in the production of neutralizing antibodies, the levels of which were significantly higher in comparison to those in VC2- and mock-vaccinated animals (P < 0.01 or P < 0.001). Analysis of EHV-1-reactive IgG subtypes demonstrated that vaccination with the VC2-EHV-1-gD vaccine stimulated robust IgG1 and IgG2a antibodies after three vaccinations (P < 0.001). Interestingly, Vetera-vaccinated mice produced significantly higher levels of IgM than mice in the other groups before and after challenge (P < 0.01 or P < 0.05). Vaccination with VC2-EHV-1-gD stimulated strong cellular immune responses, characterized by the upregulation of both interferon- and tumor necrosis factor-positive CD4+ T cells and CD8+ T cells. Overall, the data suggest that the HSV-1 VC2 vaccine strain may be used as a viral vector for the vaccination of horses as well as, potentially, for the vaccination of other economically important animals.IMPORTANCE A novel virus-vectored VC2-EHV-1-gD vaccine was constructed using the live-attenuated HSV-1 VC2 vaccine strain. This vaccine stimulated strong humoral and cellular immune responses in mice, suggesting that it could protect horses against EHV-1 infection.

Occurrence of viral diseases in donkeys (Equus asinus) in São Paulo State, Brazil / Ocorrência de enfermidades virais em asininos (Equus asinus) no estado de São Paulo, Brasil

Among the diseases that affect equines, viral diseases play an important role from a health and economic point of view, especially influenza, viral arteritis, herpes infections and vesicular stomatitis. In the Brazilian literature, there is little or no account of the occurrence of infectious diseases in donkeys. Given the importance of donkeys in different activities and the lack of information on infections that may occur in these animals, the aim of this study was to determine the frequency of anti-equine herpesvirus (EHV), anti-equine arteritis virus (EAV), anti-vesicular stomatitis, and anti-equine influenza (H3N8) antibodies in the serum of 85 donkeys bred in some regions of the state of São Paulo. We found the following antibody frequencies: 50.6% (43/85) antibodies against influenza virus subtype H3N8, 47% (40/85) anti-EHV, and 20% (17/85) anti-EAV. The donkeys were not seropositive for vesicular stomatitis. The results suggested that the agents EHV, EAV, and equine influenza subtype H3N8 circulate among donkeys in some regions of the state of São Paulo, Brazil, reinforcing the importance of establishing a routine diagnosis and epidemiological study of this species.(AU)

Dentre as doenças que acometem os equídeos, as enfermidades virais assumem um papel importante do ponto de vista sanitário e econômico, especialmente a influenza, arterite viral, as infecções herpéticas e a estomatite vesicular. Na literatura nacional, existe pouco ou nenhum relato sobre a ocorrência de enfermidades infecciosas nos asininos. Tendo em vista a importância dos asininos para diferentes atividades e a falta de informações sobre as doenças que acometem esses animais, este trabalho teve como objetivo estudar a frequência de anticorpos anti-EHV, antivírus da arterite equina, anti-estomatite vesicular e anti-influenza equina (H3N8) em 85 soros de jumentos criados no estado de São Paulo. Estimou-se que 50,6% apresentavam anticorpos contra o subtipo H3N8 do vírus da influenza; 47% (40/85) apresentavam anticorpos contra o EHV e 20% apresentavam anticorpos contra o vírus da arterite. Os jumentos não foram soro reagentes contra a estomatite vesicular. Os resultados obtidos sugerem que os agentes EHV, vírus da arterite equina e influenza equina subtipo H3N8, circulam entre os jumentos do estado de São Paulo, caracterizando a importância do estabelecimento de uma rotina diagnóstica e estudos epidemiológicos na espécie.(AU)

Immunization with Attenuated Equine Herpesvirus 1 Strain KyA Induces Innate Immune Responses That Protect Mice from Lethal Challenge.

UNLABELLED: Equine herpesvirus 1 (EHV-1) is a major pathogen affecting equines worldwide. The virus causes respiratory disease, abortion, and, in some cases, neurological disease. EHV-1 strain KyA is attenuated in the mouse and equine, whereas wild-type strain RacL11 induces severe inflammation of the lung, causing infected mice to succumb at 4 to 6 days postinfection. Our previous results showed that KyA immunization protected CBA mice from pathogenic RacL11 challenge at 2 and 4 weeks postimmunization and that KyA infection elicited protective humoral and cell-mediated immune responses. To investigate the protective mechanisms of innate immune responses to KyA, KyA-immunized mice were challenged with RacL11 at various times postvaccination. KyA immunization protected mice from RacL11 challenge at 1 to 7 days postimmunization. Immunized mice lost less than 10% of their body weight and rapidly regained weight. Virus titers in the lungs of KyA-immunized mice were 1,000-fold lower at 2 days post-RacL11 challenge than virus titers in the lungs of nonimmunized mice, indicating accelerated virus clearance. Affymetrix microarray analysis revealed that gamma interferon (IFN-γ) and 16 antiviral interferon-stimulated genes (ISGs) were upregulated 3.1- to 48.2-fold at 8 h postchallenge in the lungs of RacL11-challenged mice that had been immunized with KyA. Murine IFN-γ inhibited EHV-1 infection of murine alveolar macrophages and protected mice against lethal EHV-1 challenge, suggesting that IFN-γ expression is important in mediating the protection elicited by KyA immunization. These results suggest that EHV-1 KyA may be used as a live attenuated EHV-1 vaccine as well as a prophylactic agent in horses. IMPORTANCE: Viral infection of cells initiates a signal cascade of events that ultimately attempts to limit viral replication and prevent infection through the expression of host antiviral proteins. In this study, we show that EHV-1 KyA immunization effectively protected CBA mice from pathogenic RacL11 challenge at 1 to 7 days postvaccination and increased the expression of IFN-γ and 16 antiviral interferon-stimulated genes (ISGs). The administration of IFN-γ blocked EHV-1 replication in murine alveolar macrophages and mouse lungs and protected mice from lethal challenge. To our knowledge, this is the first report of an attenuated EHV-1 vaccine that protects the animal at 1 to 7 days postimmunization by innate immune responses. Our findings suggested that IFN-γ serves as a novel prophylactic agent and may offer new strategies for the development of anti-EHV-1 agents in the equine.