Equine Herpesvirus Type Research Articles

Bacterial Toxins from Staphylococcus aureus and Bordetella bronchiseptica Predispose the Horse's Respiratory Tract to Equine Herpesvirus Type 1 Infection.

Respiratory disease in horses is caused by a multifactorial complex of infectious agents and environmental factors. An important pathogen in horses is equine herpesvirus type 1 (EHV-1). During co-evolution with this ancient alphaherpesvirus, the horse’s respiratory tract has developed multiple antiviral barriers. However, these barriers can become compromised by environmental threats. Pollens and mycotoxins enhance mucosal susceptibility to EHV-1 by interrupting cell junctions, allowing the virus to reach its basolateral receptor. Whether bacterial toxins also play a role in this impairment has not been studied yet. Here, we evaluated the role of α-hemolysin (Hla) and adenylate cyclase (ACT), toxins derived from the facultative pathogenic bacterium Staphylococcus aureus (S. aureus) and the primary pathogen Bordetella bronchiseptica (B. bronchiseptica), respectively. Equine respiratory mucosal explants were cultured at an air–liquid interface and pretreated with these toxins, prior to EHV-1 inoculation. Morphological analysis of hematoxylin–eosin (HE)-stained sections of the explants revealed a decreased epithelial thickness upon treatment with both toxins. Additionally, the Hla toxin induced detachment of epithelial cells and a partial loss of cilia. These morphological changes were correlated with increased EHV-1 replication in the epithelium, as assessed by immunofluorescent stainings and confocal microscopy. In view of these results, we argue that the ACT and Hla toxins increase the susceptibility of the epithelium to EHV-1 by disrupting the epithelial barrier function. In conclusion, this study is the first to report that bacterial exotoxins increase the horse’s sensitivity to EHV-1 infection. Therefore, we propose that horses suffering from infection by S. aureus or B. bronchiseptica may be more susceptible to EHV-1 infection.

Detection of Equid Herpesvirus Type1 from Arabian Horses with Different Clinical Presentations between 2016-2019 in Egypt

Detection of Equid Herpesvirus Type1 from Arabian Horses with Different Clinical Presentations between 2016-2019 in Egypt

Major Equine Herpesvirus ‐ 1 epizootic in Europe: Identification of a marker for epidemiological surveillance

Background: Equine herpesvirus type-1 (EHV-1) is an important threat to the equine industry, as illustrated by the ongoing outbreak of neurological disease that was initially reported during a large equestrian event in Valencia, Spain in 2021. Horses returning from this event to their training yards have contributed to the spread of the virus to nine other European countries and to Qatar. Objectives: To design a “tracking” marker in order to specifically follow the dissemination of the Valencia strain, in EHV-1 infected horses with no known epidemiological link with the Valencia outbreak. Study design: Strain isolation and genome sequences’ comparison. Methods: 67 nasopharyngeal swab samples from horses stationed in Valencia were analysed by EHV-1 qPCR and a rapid A/G/C2254 (ORF30) typing test. Positive samples were used for strain isolation in vitro, and Multilocus Sequence Typing (MLST). Results: 19/67 (28%) samples were EHV-1 positive (all 19 typed as A2254). Two strains (FR/Valencia1/2021 and FR/Valencia2/2021) were successfully isolated and characterised by MLST as belonging to clade 10. Analysis of the MLST ORF sequences revealed a mutation at position 713 of the ORF11 (A713G) in FR/Valencia1/2021 and FR/Valencia2/2021, when compared with reference strains Ab4 and V592. This A713G mutation was not present in 104 ORF11 sequences obtained from Genbank (strains from the UK, USA, China, Australia, Belgium, New-Zealand, Japan or India), or 131 and 14 ORF11 sequences from strains isolated in Ireland or France, respectively. This marker allowed subsequent confirmation of suspicious epidemiological links in EHV-1 cases. Main limitation: The limited number of ORF11 sequences available in some countries. Conclusions: Although the existence of this mutation in other field strains cannot be excluded, its absence in the 249 ORF11 sequences analysed, suggests that this SNP constitutes an interesting epidemiological marker to identify horses infected with the EHV-1 strain which was associated with the outbreak in Valencia.

Protective immunity against equine herpesvirus type 1 is associated with antibody responses to the vaccine candidate Ab4ΔORF2

Protective immunity against equine herpesvirus type 1 is associated with antibody responses to the vaccine candidate Ab4ΔORF2

Decreased virus‐neutralising antibodies against equine herpesvirus type 1 in nasal secretions of horses after 12‐hour transportation

Decreased virus‐neutralising antibodies against equine herpesvirus type 1 in nasal secretions of horses after 12‐hour transportation

Equine Herpesvirus Type 4 (EHV-4) Outbreak in Germany: Virological, Serological, and Molecular Investigations.

Equine herpesvirus type 4 (EHV-4) is enzootic in equine populations throughout the world. A large outbreak of EHV-4 respiratory infection occurred at a Standardbred horse-breeding farm in northern Germany in 2017. Respiratory illness was observed in a group of in-housed foals and mares, which subsequently resulted in disease outbreak. Out of 84 horses in the stud, 76 were tested and 41 horses were affected, including 20 foals, 10 stallions, and 11 mares. Virological investigations revealed the involvement of EHV-4 in all cases of respiratory illness, as confirmed by virus isolation, qPCR, and/or serological follow-up using virus neutralization test and peptide-specific ELISA. Among infected mares, 73% (8 out of 11) and their corresponding foals shed the virus at the same time. EHV-4 was successfully isolated from four animals (including one stallion and three foals), and molecular studies revealed a different restriction fragment length polymorphism (RFLP) profile in all four isolates. We determined the complete 144 kbp genome sequence of EHV-4 isolated from infected horses by next-generation sequencing and de novo assembly. Hence, EHV-4 is genetically stable in nature, different RFLP profiles, and genome sequences of the isolates, suggesting the involvement of more than one animal as a source of infection due to either true infection or reactivation from a latent state. In addition, epidemiological investigation revealed that stress caused by seasonal changes, management practices, routine equestrian activities, and exercises contributed as a multifactorial causation for disease outbreak. This study shows the importance of implementing stress alleviating measures and management practices in breeding farms in order to avoid immunosuppression and occurrence of disease.

Recombinant Glycoprotein B of Equine herpesvirus Type 1 Elicits Protective Immune Response against Challenge in BALB/c Mouse Model

Background: Equine herpesvirus type 1 (EHV-1) is the most important viral pathogen of equines, causing respiratory illness, abortion, neonatal foal mortality and neurologic disorders. Large numbers of commercial EHV-1 vaccines are available to protect equines from the disease, but they provide only partial protection. Despite immunization with inactivated and modified live virus vaccine, mares show abortions. Present study was aimed to investigate the immunogenicity and protective efficacy of EHV-1 recombinant glycoprotein B (rgB) and gB expressing plasmid DNA against EHV-1 infection in BALB/c mice model.Methods: About 3-4 weeks old 225 female BALB/c mice were selected for the comparative study of immunization followed by challenged with EHV-1/India/Tohana/96-2 strain virus in 5 different groups of 45 animals each.Result: Following immunization, rgB vaccinated mice showed optimal stimulation of EHV-1 gB specific cell mediated and humoral mediated immunity (HMI and CMI). The gB expressing plasmid DNA vaccinated mice developed only CMI while inactivated whole virus vaccinated mice had only HMI. Upon EHV-1 challenge, all infected mice displayed variable levels of clinical signs with changes in body weight, however, vaccinated mice showed very rapid recovery with optimal protection. Positive control group mice showed severe pulmonary lesions along with persistence virus infection till 5 days post challenge (dpc) whereas vaccinated mice had less pulmonary lesion only up to 3dpc. Minimal lung lesions and early virus clearance was observed in the rgB immunized mice in comparison to the gB plasmid DNA and inactivated EHV-1 vaccine immunized mice. It has been concluded that immunization with rgB elicits optimum protective immune response against EHV-1 infection in mice model. The rgB could be a potential vaccine candidate against EHV-1 infection in equine in the future.

Decreased Virus-Neutralizing Antibodies Against Equine Herpesvirus type 1 In Nasal Secretions of Horses After 12-hour Transportation

This study evaluated the effects of 12-hour transportation on immune responses to equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Possible replication of EHV-1 and EHV-4 was monitored by real-time PCR of nasal swabs and peripheral blood mononuclear cells (PBMCs), and changes in systemic and mucosal antibodies were investigated. Six healthy Thoroughbreds with transport experience were transported in commercial trucks, repeating the same three-hour route four times. Blood samples for cortisol measurement were taken before departure and every three hours. Nasal swabs, PBMCs, nasal wash and serum samples were collected before departure, at unloading, two and six days after arrival. Cortisol concentration increased significantly after three and six hours of transport (P < 0.05), confirming acute transport stress. However, no evidence of viral replication or lytic infection was observed, and serum virus neutralization (VN) titers for EHV-1 and EHV-4 were unchanged, except for one horse that showed a four-fold decrease in titer against EHV-1 after transportation. Urea and total IgA concentration in nasal washes increased significantly after transportation (P < 0.05), while total IgA/protein ratio was unchanged. A transient, ≥4-fold decrease in VN titers for EHV-1 in nasal wash concentrates was observed in four out of six horses after transportation (geometric mean titer declined from 202 to 57, P < 0.05), suggesting suppression of VN capacity in the nasal mucosa may contribute to susceptibility to EHV-1 after transportation. VN antibodies against EHV-4 in nasal secretion were not detected at any timepoint.

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.

Apparent Breed Predilection for Equid Herpesvirus-1-Associated Myeloencephalopathy (EHM) in a Multiple-Breed Herd.

Equid herpesvirus type 1 (EHV-1) causes several outbreaks of abortion and/or equid herpesvirus-associated myeloencephalopathy (EHM) worldwide each year. EHM is of great concern, as permanent neurological gait anomalies can leave a horse unfit for future use. The study assesses the risk factors associated with the occurrence of EHM. During an unmitigated outbreak, 141 adult horses/ponies of several distinct breeds were evaluated—using multiple Bayesian logistic regression calculating the odds ratios for breed, age, and sex. In total, 33 of the 141 horses showed signs of EHM. Fjord horses and warmblood horses were overrepresented among those developing EHM. The pony breeds, Welsh and Shetland ponies, were underrepresented. In addition, age and sex were not associated with the risk for EHM. The main limitation was that it was a retrospective analysis with some flaws of documentation. It can be concluded that breed was a significant risk factor for developing EHM during this outbreak.

The Pathogenesis and Immune Evasive Mechanisms of Equine Herpesvirus Type 1.

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.

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-1β, IL-6, IL-12β, 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.

Persistence of virus-neutralizing antibodies in horses inoculated with two doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals.

The antibody response in horses inoculated with 2 doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals (1 to 3 months) was evaluated with regard to the persistence of virus-neutralizing (VN) antibodies. The durations for which the geometric mean VN titers were maintained significantly higher than those before the first vaccination (P<0.05) were up to 5 months in horses that received the vaccination with a 1-month interval (n=17) and 7 months for those that received it with a 2-month (n=17) or 3-month interval (n=14 or 17). The vaccination program with the 2-month interval was the most effective in maintaining VN antibodies for a long duration with the smallest gap of antibody decline between the first and second vaccinations.

Identification of a Novel α-herpesvirus Associated with Ulcerative Stomatitis in Donkeys.

An outbreak of ulcerative stomatitis was observed in a donkey (Equus asinus) dairy herd. Similar lesions were also observed on the dams’ udders and, sporadically, in genital areas. The lesions typically resolved in 1–3 weeks. An α-herpesvirus, Varicellovirus, genetically related to equid herpesvirus type 3, was identified.

Evaluation of Colostral Immunity Against Equine Herpesvirus Type 1 (EHV-1) in Martina Franca's Foals.

Eight Martina Franca pregnant jennies were selected in order to evaluate the transfer of colostral antibodies against equine herpesvirus type 1 in their relative foals after immunization with a commercial inactivated vaccine, compared with an unvaccinated group. Samples of serum and colostrums/milk were collected from jennies and foals under study starting from 10 min before and up to 21 days after the foaling. Specific anti-EHV-1 antibody titers were evaluated by means of a serum neutralization test, and the results obtained from both groups were analyzed. The serological titers in the vaccinated jennies was significantly higher (p < 0.01). No significant differences were found in the specific time-point intervals in both groups examined (p > 0.05). The antibody titers in milk at the time of delivery and subsequent withdrawal (T0 and T1) were very high in both groups, but no significant differences were found between the two groups (p > 0.05). In the foal sera, a significant difference was found between foals in the vaccinated group compared with those in the unvaccinated group (p < 0.05). Finally, a significant correlation (p < 0.05) was observed between the antibody titers found in serum and colostrum of jennies and the foal titers in the first time-point sampling (up to 12 h after foaling). The results confirm a substantial homology in the antibody production compared with other most investigated equids, highlighting the efficacy of the vaccination against EHV-1 of the jennies to ensure the protective immunity to their foals during the first weeks after delivery.

Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs.

Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.

Efficiency of alfalfa granulated feed additive in balanced ration of Ukrainian Riding Horse

We established positive influence of granulated alfalfa feed on the growth, development intensity, and sport qualities of the race horses. We registered the increase in live weight of young horses, which consumed the full granulated alfalfa feed by 8.0-9.8%. These animals had a more pronounced constitutional breed type. Thus, at the age of 18 months, they outclassed their peers in main linear dimensions: they had higher values of thoracic development (by 4.6%), higher thoracic diameter (by 4.5%), higher body length (by 5.7%). At the age of 24 months, these advantages were 6.4, 8.2, and 6.0% respectively. We found that the colts and fillies in the experimental group at the age of 12 months outperformed the same year horses from the control group by 1.1% by the index of format. Further, at the age of 18 and 24 months, the animals of the experimental group outperformed their counterparts from the control group by 0.2 and 0.4%, respectively. In terms of breast circumference index, the advantage of the exerimentary group animals at the age of 12 months was 0.1%, at the age of 18 months - 1.7%, at the age of 24 months - 1.8%, respectively. Similar results were recorded for the compactness index, where the advantage at the age of 12 months was 1.2%, at 18 months - 1.9%, at 24 months - 2.3% respectively. The effective use of environmentally friendly feed is confirmed by the results of hematological blood tests. It was found that for the vast majority of indicators they correspond to the physiological norm. This blood composition naturally characterizes changes in horses during the whole period of research. The received hematological profile is the basic characteristic of the state of health of horses in the research enterprise.

Antibody Responses Against Equine Influenza Virus Induced by Concurrent and by Consecutive Use of an Inactivated Equine Influenza Virus Vaccine and a Modified Live Equine Herpesvirus Type 1 Vaccine in Thoroughbred Racehorses

An inactivated equine influenza virus (EIV) vaccine and a live equine herpesvirus type 1 (EHV-1) vaccine are usually administered concurrently to Thoroughbred racehorses in Japan. The objective of this study was to evaluate whether concurrent administration of an inactivated EIV vaccine and a live EHV-1 vaccine in Thoroughbred racehorses influences the antibody response against EIV. We compared the antibody response against EIV in horses administered both vaccines on the same day (Group A; n = 27) and the response in horses administered an inactivated EIV vaccine first and then a live EHV-1 vaccine 1–2 weeks later (Group B; n = 20). In both groups, geometric mean hemagglutination inhibition (HI) titers against A/equine/Ibaraki/1/2007 and A/equine/Yokohama/aq13/2010 increased significantly after EIV vaccination. However, the percentage of horses that showed a twofold increase or greater in HI titers against A/equine/Yokohama/aq13/2010 was significantly higher in Group B (75%) than in Group A (37%; P = .02). These results suggest that the concurrent use of an inactivated EIV vaccine and a live EHV-1 vaccine reduced the immune response against EIV to some extent, and it would be better to use these vaccines consecutively, especially for naïve horses or horses whose vaccination history is incomplete.

Characterization of high virulent strains of equine rhinopneumonitis virus

This article presents results of screening samples collected from horses during acute outbreaks of abortion and neurological disorders recorded in 2019-2020 in Russia. The signs of disease were characterized by severe hemorrhagic vasculitis. Massive hemorrhages were observed in organs of aborted fetuses and dead horses. Equine herpesvirus type one was isolated in cell culture. Testing to identify genes encoding glycoprotein B and polymerase catalytic subunit of certain viral isolates were carried out. Virus isolated from horses with signs of encephalomyelitis from the Northwest region had a high degree of phylogenetic relationship with the strain “89c25” of equine herpesvirus type one from Japan, 1989. The same virus was found in horses with signs of rhinopneumonia from the Ural region. The virus isolated after equine abortion from the North Caucasus region had a high degree of phylogenetic relationship with the strain “IT19m10” of equine herpesvirus type one from Italy, 2019. It was established that all viral isolates belong to non-neuropathogenic genotype - A2254.

Environmental persistence of equid herpesvirus type-1.

Equid herpesvirus type 1 (EHV-1) is ubiquitous in equine populations causing respiratory disease, and complications including late-term abortion and neurological disease. Eradication of EHV-1 from housing environments that typically contain unsealed wood and porous bedding materials can be challenging. However, consideration should be given to take advantage of the viral envelope's susceptibility to environmental conditions. To determine environmental persistence of EHV-1 on materials and in environmental conditions commonly found in equine facilities. We hypothesised that environmental conditions and materials would limit environmental persistence of EHV-1 in horse housing environments. Experimental study. Standard inoculum of EHV-1 strain OH03 was applied to leather, polyester-cotton fabric, two bedding materials (pinewood shavings and wheat straw) and polystyrene (plastic), and placed under three different environmental conditions (4°C, indoors and outdoors). Virus titration and quantitative PCR (qPCR) were performed at six time points between 0 and 48hours and the number of plaque-forming units (PFUs) was determined. Viable EHV-1 was recovered up to 48hours from all material-environmental condition combinations, with persistence decreasing over time. In general, outdoor environment had the greatest impact, irrespective of material tested, followed by indoor environment and 4°C. On average, wood shavings had the greatest impact on persistence, followed by leather, straw, fabric and polystyrene. The inoculum used in this study was not in a milieu consistent with nasal secretions. As such, virus particles may have been more sensitive to the materials and/or environmental conditions evaluated. Environmental factors had variable effects on environmental persistence. Although there were significant reductions in PFUs within the first 3hours, irrespective of environment-material evaluated, viable virus was still recovered at 48hours likely representing a transmission risk. Barrier precautions should be used to prevent spread of EHV-1 from unrecognised environmental reservoirs.