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.

Long-term performance of show-jumping horses and relationship with severity of ataxia and complications associated with myeloencephalopathy caused by equine herpes virus-1.

BACKGROUND: Equine herpesvirus myeloencephalopathy (EHM) has severe impact on the sport horse population. OBJECTIVE: Study the influence of EHM on the likelihood of affected horses to return to their previous performance and investigate the association of clinical variables with prognosis. ANIMALS: Twenty-six horses positive for equine herpesvirus type 1 (EHV-1) were admitted to a veterinary teaching hospital (VTH) during a natural EHM outbreak at an international jumping event. METHODS: Data collected from the VTH, the International Equestrian Federation, and surveys completed by the riders and horse owners were retrospectively analyzed. RESULTS: Horses affected by EHM had 68% chance of returning to exercise, and 52.9% were able to achieve their preoutbreak performance level. Horses with an ataxia grade at admission ≥4/5 had an increased fatality rate (P < .05) and 10% chance of reaching their preoutbreak performance level. None of the horses with both vascular and urinary complications returned to their previous performance level. Finally, horses vaccinated against EHV-1 and those with urinary complications had a 71.4% and 43.7% fatality rate, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Horses affected by EHM were able to return to their previous performance levels, but certain clinical variables were negatively associated with postoutbreak performance. Ataxia grade upon admission and the development of systemic signs of vasculitis and urinary complications were potential poor prognostic indicators in sport horses. Variables linked to fatality included prior vaccination against EHV-1, ataxia grade upon admission, and the development of urinary complications.

Hyperoside inhibits EHV-8 infection via alleviating oxidative stress and IFN production through activating JNK/Keap1/Nrf2/HO-1 signaling pathways.

Equine herpesvirus type 8 (EHV-8) causes abortion and respiratory disease in horses and donkeys, leading to serious economic losses in the global equine industry. Currently, there is no effective vaccine or drug against EHV-8 infection, underscoring the need for a novel antiviral drug to prevent EHV-8-induced latent infection and decrease the pathogenicity of this virus. The present study demonstrated that hyperoside can exert antiviral effects against EHV-8 infection in RK-13 (rabbit kidney cells), MDBK (Madin-Darby bovine kidney), and NBL-6 cells (E. Derm cells). Mechanistic investigations revealed that hyperoside induces heme oxygenase-1 expression by activating the c-Jun N-terminal kinase/nuclear factor erythroid-2-related factor 2/Kelch-like ECH-associated protein 1 axis, alleviating oxidative stress and triggering a downstream antiviral interferon response. Accordingly, hyperoside inhibits EHV-8 infection. Meanwhile, hyperoside can also mitigate EHV-8-induced injury in the lungs of infected mice. These results indicate that hyperoside may serve as a novel antiviral agent against EHV-8 infection.IMPORTANCEHyperoside has been reported to suppress viral infections, including herpesvirus, hepatitis B virus, infectious bronchitis virus, and severe acute respiratory syndrome coronavirus 2 infection. However, its mechanism of action against equine herpesvirus type 8 (EHV-8) is currently unknown. Here, we demonstrated that hyperoside significantly inhibits EHV-8 adsorption and internalization in susceptible cells. This process induces HO-1 expression via c-Jun N-terminal kinase/nuclear factor erythroid-2-related factor 2/Kelch-like ECH-associated protein 1 axis activation, alleviating oxidative stress and triggering an antiviral interferon response. These findings indicate that hyperoside could be very effective as a drug against EHV-8.

Modulation of Equid Herpesvirus-1 Replication Dynamics In Vitro Using CRISPR/Cas9-Assisted Genome Editing.

(1) Background: equid alphaherpesvirus-1 (EHV-1) is a highly contagious viral pathogen prevalent in most horse populations worldwide. Genome-editing technologies such as CRISPR/Cas9 have become powerful tools for precise RNA-guided genome modifications; (2) Methods: we designed single guide RNAs (sgRNA) to target three essential (ORF30, ORF31, and ORF7) and one non-essential (ORF74) EHV-1 genes and determine their effect on viral replication dynamics in vitro; (3) Results: we demonstrated that sgRNAs targeting essential lytic genes reduced EHV-1 replication, whereas those targeting ORF74 had a negligible effect. The sgRNAs targeting ORF30 showed the strongest effect on the suppression of EHV-1 replication, with a reduction in viral genomic copy numbers and infectious progeny virus output. Next-generation sequencing identified variants with deletions in the specific cleavage site of selective sgRNAs. Moreover, we evaluated the combination between different sgRNAs and found that the dual combination of sgRNAs targeting ORF30 and ORF7 significantly suppressed viral replication to lower levels compared to the use of a single sgRNA, suggesting a synergic effect; (4) Conclusion: data demonstrate that sgRNA-guided CRISPR/Cas9 can be used to inhibit EHV-1 replication in vitro, indicating that this programmable technique can be used to develop a novel, safe, and efficacious therapeutic and prophylactic approach against EHV-1.

Updated ACVIM consensus statement on equine herpesvirus-1.

Equine herpesvirus-1 (EHV-1) is a highly prevalent and frequently pathogenic infection of equids. The most serious clinical consequences of infection are abortion and equine herpesvirus myeloencephalopathy (EHM). The previous consensus statement was published in 2009 and considered pathogenesis, strain variation, epidemiology, diagnostic testing, vaccination, outbreak prevention and control, and treatment. A recent survey of American College of Veterinary Internal Medicine large animal diplomates identified the need for a revision to this original consensus statement. This updated consensus statement is underpinned by 4 systematic reviews that addressed key questions concerning vaccination, pharmaceutical treatment, pathogenesis, and diagnostic testing. Evidence for successful vaccination against, or effective treatment of EHV-1 infection was limited, and improvements in experimental design and reporting of results are needed in future studies of this important disease. This consensus statement also updates the topics considered previously in 2009.

Seroprevalence and associated risk factors of equine herpesvirus type-1/-4 in selected districts of Northwest Amhara, Ethiopia.

This study aimed to estimate the prevalence, determine the distribution, and identify the epidemiological risk factors of EHV-1/-4 infections in selected districts of Northwest Amhara Region. 460 serum samples were collected from equines using multistage cluster sampling technique, and a competitive Enzyme-linked immunosorbent assay (cELISA) was performed. Various risk factors for the occurrence of EHV-1/-4 were considered. Statistical analysis was performed using R version 4.3.1. 65.9% (303) equids were tested positive for antibodies against EHV-1/-4. Based on district, the highest prevalence was recorded in Wogera (86.1%), while the lowest was in Debark (47.4%). There was a significant difference (p <0.05; 95% CI: 1.1067993-3.682843) in the prevalence of EHV-1/-4 among species and donkeys are 2.019 times more likely to get an EHV infection than horses. The prevalence of EHV-1/-4 was highest in equids with the age of 3-8 years and lowest in < 3 years, and the difference was statistically significant (p <0.05; 95% CI: 1.9812042-6.771820). Statistically significant variation (p <0.05; 95% CI: 1.1173822-2.684013) was also observed between sex of equids in which females had 1.73 times higher chance to get EHV infection than males. Higher prevalence was found in lactating equids (81.6%), followed by pregnant equids (74.6%), and dry equids (66.4%). Generally, this study indicated a high and wide distribution of EHV-1/-4 infection in the study area, which needs due attention. Devising strategies to prevent and minimize the spread and occurrence of the infection is crucial.

Identification of neuropathogenic Varicellovirus equidalpha1 as a potential cause of respiratory disease outbreaks among horses in North Xinjiang, China, from 2021-2023.

BACKGROUND: Varicellovirus equidalpha1 (formerly Equid alphaherpesvirus 1, EqAHV-1) is among the most important viruses responsible for respiratory disease outbreaks among horses throughout the world. No reports to date have detailed the association between EqAHV-1 and respiratory disease among horses in China. This study described one such outbreak among a population of horses in north Xinjiang that occurred from April 2021 - May 2023. RESULTS: qPCR revealed that EqAHV-1 was detectable in all samples and this virus was identified as a possible source of respiratory disease, although a limited subset of these samples were also positive for EqAHV-2, EqAHV-4, and EqAHV-5. In total, three EqAHV-1 strains responsible for causing respiratory illness in horses were isolated successfully, and full-length ORF33 sequence comparisonsand phylogenetic analyses indicated that these isolates may have originated from EqAHV-1 strains detected in Yili horse abortions. ORF30 sequence data additionally suggested that these strains were neuropathic, as evidenced by the presence of a guanine residue at nucleotide position 2254 corresponding to the aspartic acid present at position 752 in the DNA polymerase encoded by this virus. CONCLUSION: This study is the first report of an outbreak of respiratory disease among horses in China caused by EqAHV-1. ORF30 sequence characterization revealed that these EqAHV-1 strains harbored a neuropathogenic genotype. Given the detection of this virus in horses suffering from respiratory disease, concern is warranted with respect to this neuropathogenic EqAHV-1 outbreak.

The transmembrane and cytosolic domains of equine herpesvirus type 1 glycoprotein D determine Golgi retention by regulating vesicle formation.

Accumulating evidence underscores the pivotal role of envelope proteins in viral secondary envelopment. However, the intricate molecular mechanisms governing this phenomenon remain elusive. To shed light on these mechanisms, we investigated a Golgi-retained gD of EHV-1 (gDEHV-1), distinguishing it from its counterparts in Herpes Simplex Virus-1 (HSV-1) and Pseudorabies Virus (PRV). To unravel the specific sequences responsible for the Golgi retention phenotype, we employed a gene truncation and replacement strategy. The results suggested that Golgi retention signals in gDEHV-1 exhibiting a multi-domain character. The extracellular domain of gDEHV-1 was identified as an endoplasmic reticulum (ER)-resident domain, the transmembrane domain and cytoplasmic tail (TM-CT) of gDEHV-1 were integral in facilitating the protein's residence within the Golgi complex. Deletion or replacement of either of these dual domains consistently resulted in the mutant gDEHV-1 being retained in an ER-like structure. Moreover, (TM-CT)EHV-1 demonstrated a preference for binding to endomembranes, inducing the generation of a substantial number of vesicles, potentially originate from the Golgi complex or the ER-Golgi intermediate compartment. In conclusion, our findings provide insights into the intricate molecular mechanisms governing the Golgi retention of gDEHV-1, facilitating the comprehension of the processes underlying viral secondary envelopment.

Pharmacologic interventions for the treatment of equine herpesvirus-1 in domesticated horses: A systematic review.

BACKGROUND: Equine herpes virus type 1 (EHV-1) infection in horses is associated with upper respiratory disease, neurological disease, abortions, and neonatal death. REVIEW QUESTION: Does pharmacological therapy decrease either the incidence or severity of disease or infection caused by EHV-1 in domesticated horses? METHODS: A systematic review was preformed searching AGRICOLA, CAB Abstracts, Cochrane, PubMed, Web of Science, and WHO Global Health Index Medicus Regional Databases to identify articles published before February 15, 2021. Selection criteria were original research reports published in peer reviewed journals, and studies investigating in vivo use of therapeutic agents for prevention or treatment of EHV-1 in horses. Outcomes assessed included measures related to clinical outcomes that reflect symptomatic EHV-1 infection or virus infection. We evaluated risk of bias and performed a GRADE evaluation of the quality of evidence for interventions. RESULTS: A total of 7009 unique studies were identified, of which 9 met the inclusion criteria. Two studies evaluated valacyclovir or small interfering RNAs, and single studies evaluated the use of a Parapoxvirus ovis-based immunomodulator, human alpha interferon, an herbal supplement, a cytosine analog, and heparin. The level of evidence ranged between randomized controlled studies and observational trials. The risk of bias was moderate to high and sample sizes were small. Most studies reported either no benefit or minimal efficacy of the intervention tested. CONCLUSIONS AND CLINICAL IMPORTANCE: Our review indicates minimal or limited benefit either as a prophylactic or post-exposure treatment for any of the studied interventions in the mitigation of EHV-1-associated disease outcome.

Detection of equine herpesvirus-1 (EHV-1) in urine samples during outbreaks of equine herpesvirus myeloencephalopathy.

BACKGROUND: Real-time PCR is the diagnostic technique of choice for the diagnosis and control of equine herpesvirus-1 (EHV-1) in an outbreak setting. The presence of EHV-1 in nasal swabs (NS), whole blood, brain and spinal cord samples has been extensively described; however, there are no reports on the excretion of EHV-1 in urine, its DNA detection patterns, and the role of urine in viral spread during an outbreak. OBJECTIVES: To determine the presence of EHV-1 DNA in urine during natural infection and to compare the DNA detection patterns of EHV-1 in urine, buffy coat (BC) and NS. STUDY DESIGN: Descriptive study of natural infection. METHODS: Urine and whole blood/NS samples were collected at different time points during the hospitalisation of 21 horses involved in two EHV-1 myeloencephalopathy outbreaks in 2021 and 2023 in Spain. Quantitative real-time PCR was performed to compare the viral DNA load between BC-urine samples in 2021 and NS-urine samples in 2023. Sex, age, breed, presence of neurological signs, EHV-1 vaccination status and treatment data were recorded for all horses. RESULTS: A total of 18 hospitalised horses during the 2021 and 2023 outbreaks were positive for EHV-1, and viral DNA was detected in urine samples from a total of 11 horses in both outbreaks. Compared with BC samples, DNA presence was detected in urine samples for longer duration and with slightly higher concentration; however, compared with NS, detection of EHV-1 in urine was similar in duration with lower DNA concentrations. MAIN LIMITATIONS: Limited sample size, different sampling times and protocols (BC vs. NS) in two natural infection outbreak settings. CONCLUSIONS: EHV-1 was detected in the urine from naturally infected horses. Urine should be considered as complimentary to blood and NS in diagnosis of EHV-1 infection.

HISTORIAL: PCR en tiempo real es la técnica diagnostica de preferencia para el diagnóstico y control del herpes virus equino­1 (EHV­1) en una situación de brote. La presencia de EHV­1 en torulas nasales (TN), muestras de sangre entera, cerebro, y medula espinal ha sido descrita en forma extensa; sin embargo, no hay informes de excreción de EHV­1 en orina, la detección del patrón de ADN, y el rol de la orina en la propagación vírica durante un brote. OBJETIVOS: Determinar la presencia de ADN de EHV­1 en muestras de orina durante un brote infeccioso natural y comparar los patrones de detección de ADN de EHV­1 en orina, capa leucocitaria (CL) y TN. DISEÑO DEL ESTUDIO: Estudio prospectivo en una infección natural en caballos hospitalizados. MÉTODOS: Muestras de orina y sangre entera/TN fueron recolectadas a distintos tiempos durante la hospitalización de veintiún caballos involucrados en dos brotes de mielo encefalopatía por EHV­1 en 2021 y 2023 en España. PCR a tiempo real cuantitativo fue llevado a cabo para comparar la carga de ADN viral entre muestras de CL­orina en 2021 y muestras TN­orina en 2023. Sexo, edad, raza, presencia de síntomas neurológicos, estatus de vacunación y datos de tratamiento fueron anotados para todos los caballos. RESULTADOS: Un total de diez y ocho caballos hospitalizados durante los brotes de 2021 y 2023 resultaron positivos a EHV­1, y ADN viral fue detectado en muestras de orina en un total de 11 caballos de ambos brotes. En comparación a muestras de CL, la presencia de AND fue detectado por mas largo tiempo y con una concentración ligeramente mas alta; sin embargo, en comparación a TN, la detección de EHV­1 en orina fue similar en tiempo pero demostró menor concentración de ADN. LIMITACIONES PRINCIPALES: Tamaño de muestra limitado, tiempos de muestreo diferentes, y de protocolos (CL vs. TN) en dos situaciones de brotes naturales. CONCLUSIONES: Se detecto EHV­1 en orina de caballos infectados naturalmente. La recolección, no invasive, de orina debería considerarse como un complemento a las muestras de sangre y TN en el control de caballos infectados en situaciones de brote.

Effect of dexamethasone on antibody response of horses to vaccination with a combined equine influenza virus and equine herpesvirus-1 vaccine.

BACKGROUND: Dexamethasone is routinely administered to horses but its effect on the antibody response to a commercial EIV/EHV vaccine is unclear. HYPOTHESIS: Horses receiving dexamethasone will have lower postvaccination antibody levels against EIV and EHV-1 than vaccinated controls. ANIMALS: Fifty-five healthy adult research horses. METHODS: Randomized cohort study. Control (no vaccine, group 1), vaccination only (EIV/EHV-1/EHV-4, Prestige 2, Merck Animal Health, group 2), vaccination and concurrent single intravenous dose of dexamethasone (approximately .05 mg/kg, group 3), vaccination and 3 intravenous doses of dexamethasone at 24 hours intervals (group 4). Serum SAA levels were measured on day 1 and day 3. Antibody levels against EIV (hemagglutination inhibition assay, Kentucky 2014 antigen) and EHV-1 (multiplex ELISA targeting total IgG and IgG 4/7) were measured on day 1 and day 30. RESULTS: Significantly increased mean antibody titers after vaccination were only noted against EIV and only after the vaccination alone (n = 14, prevaccine mean [prvm] 166.9, SD 259.6, 95% CI 16.95-316.8; postvaccine mean [povm] 249.1, SD 257.2, 95% confidence interval [CI] 100.6-397.6, P = .02) and the single dose dexamethasone (n = 14, prvm 93.14, SD 72.2, CI 51.45-134.8; povm 185.1, SD 118, CI 116.7-253.6, P = .01), but not after multiple doses of dexamethasone (n = 14, prvm 194.3, SD 258.3, CI 45.16-343.4; povm 240.0, SD 235.7, CI 103.9-376.1, P > .05). CONCLUSION: The effect of dexamethasone on the postvaccine antibody response varies depending on the dosing frequency and the antigen-specific antibody type.

Comparison of antibody and antigen response to intranasal and intramuscular EHV-1 modified-live vaccination in healthy adult horses.

During neurological EHV-1 outbreaks, modified-live vaccines (MLV) are often administrated intranasally in an off-label fashion to healthy cohort horses in order to achieve rapid mucosal immunity. Thus, the goal of the present study was to determine if a commercially available EHV-1 MLV given intranasally to healthy horses would trigger a measurable systemic and/or mucosal antibody response. Eight healthy adult horses were given the EHV-1 MLV vaccine intranasally, while 8 healthy adult horses received the vaccine intramuscularly. An additional 8 healthy horses served as unvaccinated controls. EHV-1 specific antibodies (total IgG, IgG4/7, IgG1 and IgA) were measured in blood and nasal secretions prior to vaccine administration and 14- and 30-days post-vaccine administration. Further, nasal secretions and whole blood were tested for the presence of EHV-1 DNA by qPCR prior to and 5 days after vaccine administration. EHV-1 was detected by qPCR for the first 48 hours post-intranasal vaccine administration in nasal secretions in a total of three horses. Total EHV-1 IgG and IgG4/7 antibody values in serum increased only in horses receiving the intramuscular MLV. Antibody values at 14- and 30-days post vaccine administration were not different from values prior to vaccine administration in horses receiving the intranasal vaccine. The results support the intramuscular use of the EHV-1 MLV as recommended by the manufacturer. Intranasal vaccination with the study-specific EHV-1 MLV did not induce an increase in systemic or nasal antibodies, therefore, this vaccine route seems suboptimal and should not be used to vaccinate adult horses that have received multiple EHV-1 vaccinations and have pre-existing antibodies against EHV-1.

Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay.

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 °C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests.

Phylogenomic assessment of 23 equid alphaherpesvirus 1 isolates obtained from USA-based equids.

BACKGROUND: Equid alphaherpesvirus 1 (EHV-1) is a global viral pathogen of domestic equids which causes reproductive, respiratory and neurological disease. Few isolates acquired from naturally infected USA-based hosts have been fully sequenced and analyzed to date. An ORF 30 (DNA polymerase) variant (A2254G) has previously been associated with neurological disease in host animals. The purpose of this study was to perform phylogenomic analysis of EHV-1 isolates acquired from USA-based hosts and compare these isolates to previously sequenced global isolates. METHODS: EHV-1 was isolated from 23 naturally infected USA-based equids (6 different states, 15 disease outbreaks) with reproductive (22/23) or neurological disease (1/23). Following virus isolation, EHV-1 DNA was extracted for sequencing using Illumina MiSeq. Following reference-based assembly, whole viral genomes were annotated and assessed. Previously sequenced EHV-1 isolates (n = 114) obtained from global host equids were included in phylogenomic analyses. RESULTS: The overall average genomic distance was 0.0828% (SE 0.004%) for the 23 newly sequenced USA isolates and 0.0705% (SE 0.003%) when all 137 isolates were included. Clade structure was predominantly based on geographic origin. Numerous nucleotide substitutions (mean [range], 179 [114-297] synonymous and 81 [38-120] non-synonymous substitutions per isolate) were identified throughout the genome of the newly sequenced USA isolates. The previously described ORF 30 A2254G substitution (associated with neurological disease) was found in only one isolate obtained from a host with non-neurological clinical signs (reproductive disease), six additional, unique, non-synonymous ORF 30 substitutions were detected in 22/23 USA isolates. Evidence of recombination was present in most (22/23) of the newly sequenced USA isolates. CONCLUSIONS: Overall, the genomes of the 23 newly sequenced EHV-1 isolates obtained from USA-based hosts were broadly similar to global isolates. The previously described ORF 30 A2254G neurological substitution was infrequently detected in the newly sequenced USA isolates, most of which were obtained from host animals with reproductive disease. Recombination was likely to be partially responsible for genomic diversity in the newly sequenced USA isolates.

Detection of Selected Equine Respiratory Pathogens in Stall Samples Collected at a Multi-Week Equestrian Show during the Winter Months.

The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental sponges were collected from all 53 barns by using one sponge for up to 10 stalls. Further, 2/53 barns were randomly selected for individual stall sampling in order to compare the results between individual and pooled stall samples. A total of 333/948 (35.13%, 95% CI 32.09-38.26%) pooled environmental stall sponges tested PCR-positive for at least one of the selected respiratory pathogens. Streptococcus zooepidemicus was the most commonly detected pathogen in pooled samples (28.69%, 95% CI 25.83-31.69%), followed by EHV-2 (14.45%, 95% CI 12.27-16.85%), EHV-4 (1.37%, 95% CI 0.73-2.33%), and a very small percentage of pooled stall sponges tested PCR-positive for EHV-1, ERBV, EIV, and S. equi. In individual samples, 171/464 (36.85%, 95% CI 32.45-41.42%) environmental stall sponges tested PCR-positive for at least one of the selected pathogens, following a similar frequency of pathogen detection as pooled samples. The detection frequency of true respiratory pathogens from environmental samples was higher during the winter months compared to previous studies performed during spring and summer, and this testing highlights that such pathogens circulate with greater frequency during the colder months of the year. The strategy of monitoring environmental stall samples for respiratory pathogens circumvents the often labor-intensive collection of respiratory secretions from healthy horses and allows for a more efficient assessment of pathogen buildup over time. However, environmental stall testing for respiratory pathogens should not replace proper biosecurity protocols, but it should instead be considered as an additional tool to monitor the silent circulation of respiratory pathogens in at-risk horses.

Association of equine gammaherpesvirus-5 with facial lymphohistiocytic interface dermatitis in seven adult horses from the United States.

Equine herpesvirus-5 (EHV-5) is commonly found in healthy asymptomatic horses worldwide. Although a cause-and-effect relationship has not been thoroughly determined, this virus has been associated with several disease conditions including equine multinodular pulmonary fibrosis (EMPF) and 1 case of interface dermatitis. The authors searched the New York State Animal Health Diagnostic Center database for cases of equine interface dermatitis between 2007 and 2022. Ten cases were identified and scrutinized for viral inclusion bodies which were present in 5 of 10 cases. Two similar cases with interface dermatitis and viral inclusion bodies, which were not part of a retrospective search, were from the Oregon Veterinary Diagnostic Laboratory. The authors describe a total of 7 horses with dermatitis characterized by crusted, alopecic, non-pruritic, non-painful, irregular to annular areas over the face, most commonly the muzzle, for up to several years duration. Histologically, there was a CD3+ T lymphocyte-dominated lymphohistiocytic interface dermatitis with hydropic degeneration, apoptotic keratinocytes, and pigmentary incontinence. Keratinocytes within the upper stratum spinosum and stratum granulosum had glassy pale basophilic intranuclear inclusion bodies consistent with herpesvirus. The presence of EHV-5 was confirmed by quantitative polymerase chain reaction (qPCR) and in situ hybridization in 7 horses and by electron microscopy in 1 horse. One horse later developed EMPF and was euthanized. EHV-5 was not detected with qPCR from 5 control horses and 5 horses with interface dermatitis without histologic evidence of viral inclusion bodies. These are the first cases of facial interface dermatitis associated with EHV-5 reported in the United States.

Development of multiplex gold nanoparticles biosensors for ultrasensitive detection and genotyping of equine herpes viruses.

Gold nanoparticles (GNPs) biosensors can detect low viral loads and differentiate between viruses types, enabling early diagnosis and effective disease management. In the present study, we developed GNPs biosensors with two different capping agent, citrate-GNPs biosensors and polyvinylpyrrolidone (PVP)-GNPs biosensors for detection of EHV-1 and EHV-4 in multiplex real time PCR (rPCR). Citrate-GNPs and PVP-GNPs biosensors can detect dilution 1010 of EHV-1 with mean Cycle threshold (Ct) 11.7 and 9.6, respectively and one copy as limit of detection, while citrate-GNPs and PVP-GNPs biosensors can detect dilution 1010 of EHV-4 with mean Ct 10.5 and 9.2, respectively and one copy as limit of detection. These findings were confirmed by testing 87 different clinical samples, 4 more samples were positive with multiplex GNPs biosensors rPCR than multiplex rPCR. Multiplex citrate-GNPs and PVP-GNPs biosensors for EHV-1 and EHV-4 are a significant breakthrough in the diagnosis of these virus types. These biosensors offer high sensitivity and specificity, allowing for the accurate detection of the target viruses at very low concentrations and improve the early detection of EHV-1 and EHV-4, leading to faster control of infected animals to prevent the spread of these viruses.

In vitro virucidal activity of nebulized citrate-complexed silver nanoparticles against equine herpesvirus-1 and murine norovirus.

Viruses can be involved in respiratory disorders in horses, with limited therapeutic options. Citrate-complexed silver nanoparticles (C-AgNP) have shown bactericidal properties after in vitro nebulization. The aim of the present study was to assess the virucidal activity of C-AgNP after in vitro instillation or nebulization on equine herpesvirus-1 (EHV-1) and murine norovirus (MNV), the latter used as surrogate for small non-enveloped viruses. Both viruses were instilled or nebulized with C-AgNP of increasing concentrations, and titres were determined via TCID50 method. We demonstrated efficient inactivation of enveloped EHV-1 following instillation and nebulization of C-AgNP (infectivity losses of ≥ three orders of magnitude). While tenacious MNV was inactivated via 2000 ppm C-AgNP instillation, nebulized C-AgNP did not lead to reduction in MNV titres. Nebulization of C-AgNP may represent a novel virucidal therapeutic approach in horses. Further investigations are needed to assess its safety and effective concentrations for in vivo use.

Investigation of the Frequency of Detection of Common Respiratory Pathogens in Nasal Secretions and Environment of Healthy Sport Horses Attending a Multi-Week Show Event during the Summer Months.

Little information is presently available regarding the frequency of the silent shedders of respiratory viruses in healthy sport horses and their impact on environmental contamination. Therefore, the aim of this study was to investigate the detection frequency of selected respiratory pathogens in nasal secretions and environmental stall samples of sport horses attending a multi-week equestrian event during the summer months. Six out of fifteen tents were randomly selected for the study with approximately 20 horse/stall pairs being sampled on a weekly basis. Following weekly collection for a total of 11 weeks, all samples were tested for the presence of common respiratory pathogens (EIV, EHV-1, EHV-4, ERAV, ERBV, and Streptococcus equi ss equi (S. equi)) using qPCR. A total of 19/682 nasal swabs (2.8%) and 28/1288 environmental stall sponges (2.2%) tested qPCR-positive for common respiratory pathogens. ERBV was the most common respiratory virus (17 nasal swabs, 28 stall sponges) detected, followed by EHV-4 (1 nasal swab) and S. equi (1 nasal swab). EIV, EHV-1, EHV-4 and ERAV were not detected in any of the study horses or stalls. Only one horse and one stall tested qPCR-positive for ERBV on two consecutive weeks. All the other qPCR-positive sample results were related to individual time points. Furthermore, only one horse/stall pair tested qPCR-positive for ERBV at a single time point. The study results showed that in a selected population of sport horses attending a multi-week equestrian event in the summer, the frequency of the shedding of respiratory viruses was low and primarily restricted to ERBV with little evidence of active transmission and environmental contamination.

Equine Herpesvirus-1 Outbreak During a Show-Jumping Competition: A Clinical and Epidemiological Study.

A total of 752 horses were involved in the CES Valencia Spring Tour 2021. Due to an equine herpesvirus-1 (EHV-1) outbreak, the competition was cancelled and the site was locked down. The objective of this study was to describe epidemiological, clinical, diagnostic, and outcome data of the 160 horses remaining in Valencia. Clinical and quantitative polymerase chain reaction (qPCR) data were analysed for 60 horses in a retrospective case-control observational study. The risk of developing clinical manifestations was explored using a logistic regression approach. EHV-1 was detected by qPCR, genotyped as A2254 (ORF30) and isolated on cell culture. From the 60 horses, 50 (83.3%) showed fever, 30 horses (50%) showed no further signs and 20 (40%) showed neurological signs, with eight horses (16%) hospitalised, of which two died (3%). Stallions and geldings were six times more likely to develop EHV-1 infection compared to mares. Horses older than 9 years, or housed in the middle of the tent were more likely to develop EHV-1 myeloencephalopathy (EHM). These data show that for EHV-1 infection, the risk factor was male sex. For EHM the risk factors were age > 9-year old and location in the middle of the tent. These data highlight the crucial role of stable design, position, and ventilation in EHV-outbreaks. It also showed that PCR testing of the horses was important to manage the quarantine.