Detection of equine herpesvirus antibodies in large-scale donkey farms in Liaocheng area.

BACKGROUND: Equine herpesvirus (EHV) can cause respiratory, reproductive and neurological diseases in equine animals, including donkeys. The main pathogens responsible for these diseases are EHV type 1 (EHV-1) and EHV-4. In this study, we collected serum samples from 230 donkeys on 27 large-scale donkey farms to detect EHV-1 and EHV-4 antibodies. We analyzed the presence of EHV antibodies based on region, age and season. RESULTS: Out of the 27 farms, 62.96% (17/27) tested positive for EHV. Of the 230 donkeys tested, 2.61% (6/230) were positive only for EHV-1, 5.22% (12/230) were positive only for EHV-4, and 4.78% (11/230) were positive for both EHV-1 and EHV-4. The highest percentage of positive donkeys (21.28%) was found in Dong'e County. The seropositivity rate among donkeys aged 1-4 years was significantly higher compared to the group of donkeys aged 0-1 year (p < 0.05). Additionally, the positive rate was significantly higher in fall and winter compared to spring and summer (p < 0.05). CONCLUSIONS: Altogether, our findings indicate that large-scale donkey farms in the Liaocheng area have a high prevalence of EHV antibodies. Since Liaocheng is an important donkey trading market in Shandong Province, it is crucial to consider the risk of disease transmission based on our test results. This will help in early detection and prevention of EHV outbreaks.

Impact of equine herpesvirus-1 ORF15 (EUL45) on viral replication and neurovirulence.

Equine herpesvirus 1 (EHV-1) causes respiratory illness, fetal loss, perinatal mortality, and myeloencephalopathy. This study investigated ORF15's impact on virus infectivity and neurovirulence. The Ab4p neurovirulent strain of EHV1 was used as a backbone to create Ab4p attB, Ab4p∆ORF15, and Ab4p∆ORF15R chimeras via BAC DNA transfection into RK-13 cells. Viral growth kinetics, plaque size, transcription, and growth were assessed in MDBK cells, mouse neurons, and fetal equine brain cells. Neurovirulence was evaluated post-intranasal inoculation into male CBA/N1 SPF mice, measuring signs, virus titers, and histopathological changes. Deletion of EUL45 (Ab4p-∆EUL45) reduced viral replication efficiency, resulting in decreased release and smaller plaques. EUL45 deletion also upregulated neighbouring genes (EUL46 and EUL44). Ab4p-∆EUL45 exhibited reduced virulence and poor growth in neural cells compared to wild-type viruses. This study sheds light on EUL45's role in EHV-1, viral replication, and regulation of EUL46 and EUL44 expression, suggesting potential as a vaccine candidate.

Aspergillus Fumigatus Spore Proteases Alter the Respiratory Mucosa Architecture and Facilitate Equine Herpesvirus 1 Infection.

Numerous Aspergillus fumigatus (Af) airborne spores are inhaled daily by humans and animals due to their ubiquitous presence. The interaction between the spores and the respiratory epithelium, as well as its impact on the epithelial barrier function, remains largely unknown. The epithelial barrier protects the respiratory epithelium against viral infections. However, it can be compromised by environmental contaminants such as pollen, thereby increasing susceptibility to respiratory viral infections, including alphaherpesvirus equine herpesvirus type 1 (EHV-1). To determine whether Af spores disrupt the epithelial integrity and enhance susceptibility to viral infections, equine respiratory mucosal ex vivo explants were pretreated with Af spore diffusate, followed by EHV-1 inoculation. Spore proteases were characterized by zymography and identified using mass spectrometry-based proteomics. Proteases of the serine protease, metalloprotease, and aspartic protease groups were identified. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed that Af spores induced the desquamation of epithelial cells and a significant increase in intercellular space at high and low concentrations, respectively. The increase in intercellular space in the epithelium caused by Af spore proteases correlated with an increase in EHV-1 infection. Together, our findings demonstrate that Af spore proteases disrupt epithelial integrity, potentially leading to increased viral infection of the respiratory epithelium.

Investigation of the Use of Environmental Samples for the Detection of EHV-1 in the Stalls of Subclinical Shedders.

In populations of healthy show horses, the subclinical transmission and circulation of respiratory pathogens can lead to disease outbreaks. Due to recent outbreaks of equine herpesvirus-1 myeloencephalopathy (EHM) in the USA and Europe, many show organizers have instituted various biosecurity protocols such as individual horse testing, monitoring for early clinical disease and increasing hygiene and cleanliness protocols. The aim of this study was to determine the accuracy of detecting EHV-1 in the various environmental samples collected from the stalls of subclinical shedders. Four healthy adult horses were vaccinated intranasally with a modified-live EHV-1 vaccine in order to mimic subclinical shedding. Three additional horses served as non-vaccinated controls. All the horses were stabled in the same barn in individual stalls. Each vaccinated horse had nose-to-nose contact with at least one other horse. Prior to the vaccine administration, and daily thereafter for 10 days, various samples were collected, including a 6" rayon-tipped nasal swab, an environmental sponge, a cloth strip placed above the automatic waterer and an air sample. The various samples were processed for nucleic acid purification and analyzed for the presence of EHV-1 via quantitative PCR (qPCR). EHV-1 in nasal secretions was only detected in the vaccinated horses for 1-2 days post-vaccine administration. The environmental sponges tested EHV-1 qPCR-positive for 2-5 days (median 3.5 days) in the vaccinated horses and 1 day for a single control horse. EHV-1 was detected by qPCR in stall strips from three out of four vaccinated horses and from two out of three controls for only one day. EHV-1 qPCR-positive air samples were only detected in three out of four vaccinated horses for one single day. For the vaccinated horses, a total of 25% of the nasal swabs, 35% of the environmental stall sponges, 7.5% of the strips and 7.5% of the air samples tested qPCR positive for EHV-1 during the 10 study days. When monitoring the subclinical EHV-1 shedders, the collection and testing of the environmental sponges were able to detect EHV-1 in the environment with greater frequency as compared to nasal swabs, stationary strips and air samples.

Divergent strains of EHV-1 in Swedish outbreaks during 2012 to 2021.

Equid alphaherpesvirus 1 (EHV-1) is a ubiquitous and significant viral pathogen in horses worldwide, causing a range of conditions, including fever, respiratory disease, abortion in pregnant mares and the severe neurological disease called equine herpes myeloencephalopathy (EHM). Despite that EHV-1 is a notifiable animal disease in Sweden, there is limited knowledge about the circulating strains. This study aimed to analyze the genetic diversity of EHV-1 strains in equine samples from different Swedish outbreaks by partial genome sequencing. Genotyping based on three selected open reading frames ORF11, ORF30, and ORF34 in the viral genome was conducted for 55 outbreaks of EHV-1 spanning from the years 2012 to 2021. The analysis revealed 14 different genovariants, with one prominent genovariant identified in 49% of the outbreaks. Additionally, the study identified seven mutations not previously described. Three new mutations were demonstrated in ORF11, all synonymous, and four new mutations in ORF34, two synonymous, and two non-synonymous. Notably, different EHV-1 genovariants were found in five out of six studied EHM outbreaks, but clonal spreading was shown within the outbreaks. Moreover, the study demonstrated that healthy (recovered) horses that returned from an EHM outbreak at an international meeting in Valencia, Spain (2021), were positive for the virus clone responsible for the severe disease outbreak despite several weeks of quarantine. These findings shed light on the genetic diversity and transmission dynamics of the virus and significantly contribute to better understanding of the epidemiology of EHV-1 in Sweden and globally.

Establishment of a new equine embryo brain primary cell culture with long-term expansion.

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.

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.

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

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

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

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.

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.

Retrospective study on equine viral abortions in Poland between 1999 and 2022.

Introduction: Loss of pregnancy in mares is a major cause of economic and emotional impact for horse breeders. It can have many different infectious and noninfectious causes. The aim of this study was identification of the main viral causes of abortion in mares in Poland based on tissue samples from 180 aborted foetuses submitted for testing between 1999 and 2022. Material and Methods: Tissues of aborted foetuses collected from different horse studs throughout Poland were tested for the presence of equine herpesvirus types 1 and 4 (EHV-1/-4) and if negative, for equine arteritis virus (EAV). The examination was performed using a PCR/reverse transcriptase PCR (1999-2012) and a quantitative PCR (2013-2022). Results: The cause of abortion was determined to be EHV-1 in 49.4% of cases (n = 89), whereas no EHV-4- or EAV-positive cases were found. The proportion of abortions due to EHV-1 differed between regions, with the highest percentage in the Lubelskie and Wielkopolskie provinces. Conclusion: The results of the study indicate that EHV-1 is the most important viral infectious agent causing abortions in mares in Poland.

Vaccination for the prevention of equine herpesvirus-1 disease in domesticated horses: A systematic review and meta-analysis.

BACKGROUND: Equine herpes virus type 1 (EHV-1) infection in horses is associated with respiratory and neurologic disease, abortion, and neonatal death. HYPOTHESIS: Vaccines decrease the occurrence of clinical disease in EHV-1-infected horses. METHODS: A systematic review was performed searching multiple databases to identify relevant studies. Selection criteria were original peer-reviewed research reports that investigated the in vivo use of vaccines for the prevention of disease caused by EHV-1 in domesticated horses. Main outcomes of interest included pyrexia, abortion, neurologic disease, viremia, and nasal shedding. We evaluated risk of bias, conducted exploratory meta-analyses of incidence data for the main outcomes, and performed a GRADE evaluation of the quality of evidence for each vaccine subtype. RESULTS: A total of 1018 unique studies were identified, of which 35 met the inclusion criteria. Experimental studies accounted for 31/35 studies, with the remainder being observational studies. Eight vaccine subclasses were identified including commercial (modified-live, inactivated, mixed) and experimental (modified-live, inactivated, deletion mutant, DNA, recombinant). Risk of bias was generally moderate, often because of underreporting of research methods, and sample sizes were small leading to imprecision in the estimate of the effect size. Several studies reported either no benefit or minimal vaccine efficacy for the primary outcomes of interest. Meta-analyses revealed significant heterogeneity was present, and our confidence in the quality of evidence for most outcomes was low to moderate. CONCLUSIONS AND CLINICAL IMPORTANCE: Our review indicates that commercial and experimental vaccines minimally reduce the incidence of clinical disease associated with EHV-1 infection.

Hybrid sequencing discloses unique aspects of the transcriptomic architecture in equid alphaherpesvirus 1.

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5'-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

Berbamine, a bioactive alkaloid, suppresses equine herpesvirus type 1 in vitro and in vivo.

Equine herpesvirus type 1 (EHV-1) poses a global threat to equines. The anticancer agent berbamine (BBM), a bioactive alkaloid, has been shown to inhibit viral infection. However, whether BBM can inhibit EHV-1 infection remains unclear. This study investigated the effect of BBM treatment on EHV-1 infection. Quantitative PCR (qPCR), immunoblotting, the Reed-Muench method, and pathological examination were employed to study the ability of BBM to inhibit EHV-1 infection, viral DNA replication, viral protein production, virion secretion, and cytopathogenesis in vitro and in vivo. The in vitro studies revealed that 10 µM BBM effectively suppressed EHV-1 viral entry into cells, viral DNA replication, and virion secretion, while the in vivo studies verified the ability of BBM to suppress EHV-1-induced damage of brain and lung tissues and animal mortality. These findings strongly suggest that BBM could be a serious contender in the therapeutic control of EHV-1 infection of equines.

Anti-inflammatory compounds reduce equine herpesvirus type 1 replication and cell-to-cell spread.

Equine herpesvirus type 1 (EHV-1) is a highly transmissible pathogen that leads to a variety of clinical disease outcomes in infected horses. A major sequela that can occur after an EHV-1 infection is a neurological disease termed equine herpesvirus myeloencephalopathy (EHM). Clinical manifestations of EHM include fever, ataxia, incontinence, and partial to full paralysis, which may ultimately lead to the euthanization of the infected horse. To develop an effective treatment strategy for EHM, it is critical that the specific virus-host interactions that lead to EHM be investigated so that safe and effective therapeutic interventions can be developed and delivered. In this study, we examined the ability of four non-steroidal anti-inflammatory drugs (NSAIDs), a steroidal anti-inflammatory drug (dexamethasone), a Rho-kinase (ROCK) inhibitor, and a JAK/STAT inhibitor (AG490) to reduce EHV-1 virus yields and cell-to-cell spread. We show that the NSAID, flunixin meglumine (FM), and the JAK/STAT inhibitor, AG490, significantly reduced virus yields in endothelial and epithelial cell lines, and this inhibition was similar for two neurologic and two non-neurologic EHV-1 strains. In addition to reducing virus yields, AG490 and FM also significantly reduced the ability of EHV-1 to spread laterally from cell to cell.

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.

Double and quadruple deletion mutant of EHV-1 is highly attenuated and induces optimal immune response.

Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 & pUL56) associated genes either individually or in combinations; and comprehensive evaluation of mutants through in vitro characterization followed by in vivo study in murine model to adjudge the attenuation of the virus and immune responses generated by mutants vis-à-vis wild type (wt) virus. The EHV-1 mutants with deletion of IR6 and gE genes (vToH-DMV) and four genes (i.e., gE, IR6, pUL43 and pUL56) (vToH-QMV) revealed a significant reduction in plaque size with minimal loss in replication efficiency in comparison to the wt virus. Further, in vivo studies showed virus attenuation adjudged through significant reduction in clinical signs, weight loss, gross and histopathological lesions in comparison to wt virus also revealed improved immune responses estimated through serum neutralization and flow cytometric analysis of CD4 + and CD8 + cell populations. Thus it can be concluded that EHV-1 mutants viz. vToH-DMV and vToH-QMV (novel combination) are promising vaccine candidates and qualify to be studied for adjudging the protective efficacy with wt virus challenge.

Prevention of fatal equine herpesvirus type 1 encephalitis in mice by immunization with a limited-replication cycle virus.

Equine herpesvirus type 1 (EHV-1) is a devastating pathogen of horses, their natural hosts, and causes fatal encephalitis in non-natural hosts. We previously demonstrated that acylation of the tegument protein UL11 is required for viral replication in cultured cells. We created a mutant virus (EHV-1 UL12 trunc UL11 G2AC7AC9A), in which glycyl and cysteinyl residues at positions 2, 7 and 9 of UL11 that are normally acylated were replaced with alanyl residues. This virus, designated the 2/7/9 mutant, has a limited-replication cycle (LRC), in which replication stops after just a few cycles. Here, we tested whether the 2/7/9 mutant could be used as a vaccine against fatal encephalitis in a mouse model. A virulence test showed that the 2/7/9 mutant was not pathogenic in mice and elicited an antibody response. We also attempted to use the 2/7/9 mutant to immunize mice against a zebra-borne EHV-1, 94-137. Two trials were conducted, each with five immunized mice, five non-immunized and five control mice. In both trials, clinical signs and fatalities were much lower in the immunized mice than in the non-immunized mice. In addition, none of the mice in either trial developed neutralizing antibodies, indicating that the immunity induced by the 2/7/9 mutant was not due to neutralizing activity. The results indicate that the 2/7/9 LRC mutant has promise as a vaccine against EHV-1 infection non-natural hosts.

Efficacy of vaccination against equine herpesvirus type 1 (EHV-1) infection: Systematic review and meta-analysis of randomised controlled challenge trials.

BACKGROUND: Equid herpesvirus type 1 (EHV-1) infection can cause a range of disease syndromes of variable severity that can result in a lethal outcome and restriction of horse movements, especially in the case of outbreaks involving neurological disease. Vaccination is one of the tools used to control the infection. It is widely known that vaccination is not completely effective in ensuring protection against disease caused by this virus. In fact, the real efficacy of vaccination against EHV-1 related disease has not been measured and no systematic reviews exist on this topic. OBJECTIVES: To perform a systematic review and meta-analysis on the efficacy of commercial or candidate vaccines against EHV-1 in randomised controlled trials (RCT) all of which involved experimental challenge of the test subjects. STUDY DESIGN: Systematic review and meta-analysis. METHODS: RCTs were searched using the search algorithm (([equid herpesvirus* OR equine herpesvirus* OR EHV-1]) AND vaccin*) AND (trial OR experimental OR challenge) on PubMed, Science Citation Index Expanded, Scopus, and CAB Abstracts. Where appropriate, meta-analysis was performed using RevMan 5.4. RESULTS: Eight studies were selected and were analysed for their respective characteristics and possible shortcomings. The results of RCTs revealed that there was a general improvement in the clinical and virological outcomes of EHV-1 infection following vaccination, but that the effects were very slight. The reduced beneficial effect is probably amplified by the paucity of detailed data reported in the studies that did not allow for the comparison of parameters in many of the cases analysed. MAIN LIMITATIONS: The remarkable heterogeneity and the poor quality of reporting of the selected studies. CONCLUSIONS: Meta-analysis has shown that EHV-1 vaccination generally results in a slight improvement in clinical and virological outcomes, although not to a significant extent. The cumulative results have probably been affected by the lack of information on some parameters not systematically reported in the studies. An improvement in the standard of reporting and better standardisation of the data collected would likely have improved the quality of each study and enabled more effective comparison of the studies with each other.