Equine Herpesvirus Research Articles

Protective Role of Cepharanthine Against Equid Herpesvirus Type 8 Through AMPK and Nrf2/HO-1 Pathway Activation.

Equid herpesvirus type 8 (EqHV-8) is known to cause respiratory disease and miscarriage in horses and donkeys, which is a major problem for the equine farming industry. However, there are currently limited vaccines or drugs available to effectively treat EqHV-8 infection. Therefore, it is crucial to develop new antiviral approaches to prevent potential pandemics caused by EqHV-8. This study evaluates the antiviral and antioxidant effects of cepharanthine against EqHV-8 by employing both in vitro assays and in vivo mouse models to assess its therapeutic efficacy. To assess the effectiveness of cepharanthine against EqHV-8, we conducted experiments using NBL-6 and RK-13 cells. Additionally, we developed a mouse model to validate cepharanthine's effectiveness against EqHV-8. In our in vitro experiments, we assessed the cepharanthine's ability to inhibit infection caused by EqHV-8 in NBL-6 and RK-13 cells. Our results demonstrated that cepharanthine has a dose-dependent inhibitory effect, indicating that it possesses anti-EqHV-8 properties at the cellular level. Moreover, we investigated the mechanism through which cepharanthine exerts its protective effects. It was observed that cepharanthine effectively reduces the oxidative stress induced by EqHV-8 by activating the AMPK and Nrf2/HO-1 signaling pathways. Furthermore, when administered to EqHV-8 infected mice, cepharanthine significantly improved lung tissue pathology and reduced oxidative stress. The findings presented herein collectively highlight cepharanthine as a promising candidate for combating EqHV-8 infections.

Equine coital exanthema: New approaches to minimise the negative impact on the equine industry

SummaryEquine coital exanthema (ECE), caused by Equid alphaherpesvirus 3 (EHV‐3), is a highly contagious, venereally transmitted mucocutaneous disease, characterised by the formation of papules, vesicles, pustules and ulcers on the external genital organs of mares and stallions. The infection is endemic worldwide, and the virus is transmitted mainly through direct contact during sexual intercourse and by contaminated instruments during reproductive manoeuvres in breeding facilities. The only known biological reservoir of EHV‐3 is the latently infected horse; it is well‐known that, as for other herpesviral infections, periods of reactivation from latency, with production and shedding of infectious virus, are the source of infection for other comingling susceptible animals. Episodes of reactivation and shedding, either with or without clinical manifestation, and together with variable levels of serum antibodies, have been observed. Although the disease is relatively benign and does not result in systemic illness, infertility or abortion, it does have a negative impact on the equine industry, as it forces to temporarily withdraw affected animals, consequently disrupting mating activities in breeding enterprises. Thus, outbreaks of ECE continue to be a major problem for the equine industry, mostly in those facilities with heavily scheduled breeding calendars during reproductive seasons. For affected stallions, such disruptions may translate into significant end‐of‐season decreases in the mare‐book size. Similarly, affected mares may miss breeding opportunities. In artificial insemination and embryo transfer centres, affected mares may be reluctant to be inspected, inseminated or transferred, with the consequent loss of opportunities to become pregnant. In both scenarios (natural mating or artificial insemination), delayed foaling dates or reduced pregnancy rates may occur in mares that miss breeding opportunities because of the disease. In the interest of reducing the negative impact of EHV‐3 infections on breeding centres, advances have been made during the last years as to diagnostic methodologies, treatment and prevention.

Equine Herpesvirus Type 1 ORF76 Encoding US9 as a Neurovirulence Factor in the Mouse Infection Model.

Equine herpesvirus type 1 (EHV-1) causes rhinopneumonitis, abortion, and neurological outbreaks (equine herpesvirus myeloencephalopathy, EHM) in horses. EHV-1 also causes lethal encephalitis in small laboratory animals such as mice and hamsters experimentally. EHV-1 ORF76 is a homolog of HSV-1 US9, which is a herpesvirus kinase. Starting with an EHV-1 bacterial artificial chromosome clone of neuropathogenic strain Ab4p (pAb4p BAC), we constructed an ORF76 deletion mutant (Ab4p∆ORF76) by replacing ORF76 with the rpsLneo gene. Deletion of ORF76 had no influence on replication, cell-to-cell spread in cultured cells, or replication in primary neuronal cells. In Western blots of EHV-1-infected cell lysates, an EHV-1 US9-specific polyclonal antibody detected multiple bands ranging from 35 to 42 kDa. In a CBA/N1 mouse infection model following intranasal inoculation, the parent and Ab4p∆ORF76 revertant caused the same histopathology in the brain and olfactory bulbs. The parent, Ab4p∆ORF76, and revertant mutant replicated similarly in the olfactory mucosa, although Ab4p∆ORF76 was not transported to the olfactory bulbs and was unable to infect the CNS. These results indicated that ORF76 (US9) plays an essential role in the anterograde spread of EHV-1.

Characterization of Nasal Mucosal T Cells in Horses and Their Response to Equine Herpesvirus Type 1

Equine herpesvirus type 1 (EHV-1) enters through the upper respiratory tract (URT). Mucosal immunity at the URT is crucial in limiting viral infection and morbidity. Here, intranasal immune cells were collected from horses (n = 15) during an experimental EHV-1 infection. CD4+ and CD8+ T cells were the major intranasal cell populations before infection and increased significantly by day six and fourteen post-infection, respectively. Nasal mucosal T cells were further characterized in healthy horses. Compared to peripheral blood mononuclear cells (PBMC), mucosal CD8+ T-cell percentages were elevated, while CD4+ T-cell percentages were similar. A small population of CD4+CD8+ T cells was also recovered from mucosal samples. Within the URT tissue, CD4+ cells predominantly accumulated in the epithelial layer, while most CD8+ cells resided deeper in the mucosa or the submucosa below the basement membrane. In vitro stimulation of mucosal cells from healthy horses with (n = 5) or without (n = 5) peripheral T-cell immunity against EHV-1 induced IFN-γ production in nasal T cells upon polyclonal stimulation. However, after EHV-1 re-stimulation, mucosal T cells failed to respond with IFN-γ. This work provided the first characterization of mucosal T-cell phenotypes and functions in the URT of healthy horses and during EHV-1 infection.

Breaking Latent Infection: How ORF37/38-Deletion Mutants Offer New Hope against EHV-1 Neuropathogenicity.

Equid alphaherpesvirus 1 (EHV-1) has been linked to the emergence of neurological disorders, with the horse racing industry experiencing significant impacts from outbreaks of equine herpesvirus myeloencephalopathy (EHM). Building robust immune memory before pathogen exposure enables rapid recognition and elimination, preventing infection. This is crucial for effectively managing EHV-1. Removing neuropathogenic factors and immune evasion genes to develop live attenuated vaccines appears to be a successful strategy for EHV-1 vaccines. We created mutant viruses without ORF38 and ORF37/38 and validated their neuropathogenicity and immunogenicity in hamsters. The ∆ORF38 strain caused brain tissue damage at high doses, whereas the ∆ORF37/38 strain did not. Dexamethasone was used to confirm latent herpesvirus infection and reactivation. Dexamethasone injection increased viral DNA load in the brains of hamsters infected with the parental and ∆ORF38 strains, but not in those infected with the ∆ORF37/38 strain. Immunizing hamsters intranasally with the ∆ORF37/38 strain as a live vaccine produced a stronger immune response compared to the ∆ORF38 strain at the same dose. The hamsters demonstrated effective protection against a lethal challenge with the parental strain. This suggests that the deletion of ORF37/38 may effectively inhibit latent viral infection, reduce the neuropathogenicity of EHV-1, and induce a protective immune response.

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

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. 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). 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.

Serum amyloid A increases following routine vaccination of healthy adult horses.

To measure the effect of routine vaccination on serum amyloid A (SAA) concentration in apparently healthy horses. We hypothesized that routine vaccination would increase SAA in healthy horses. 21 apparently healthy client-owned horses and 15 Kansas State University College of Veterinary Medicine-owned horses. In experiment 1 (n = 8 horses), a blinded, randomized, prospective, crossover study was performed. Horses were either vaccinated (rabies, tetanus, West Nile, Eastern and Western equine encephalomyelitis, equine herpesvirus-1/-4, influenza) or administered saline, and SAA was measured at 6, 12, and 24 hours and daily until day 10 with a commercial lateral-flow immunoassay. In experiment 2 (n = 28 horses), a prospective, observational study measured SAA after vaccination at 12 and 24 hours and daily until day 10. A linear mixed-effect model with repeated measures over time blocked by horse tested the effect of treatment on SAA. A repeated-measures correlation tested the correlation between SAA and temperature. Over time, vaccinated horses had increased model-adjusted SAA compared to unvaccinated horses without clinical evidence of adverse reaction (P < .01). In experiment 1, the model-adjusted SAA after vaccination peaked on day 2 (median, 1,872 µg/mL; IQR, 1,220.8 to 2,402.5 µg/mL) and returned to normal (< 20 µg/mL) by day 9 (median, 6 µg/mL; IQR, 0.8 to 23.5 µg/mL) after vaccination. In experiment 2, vaccinated horses had increased SAA over time; temperature and SAA were not correlated (P = .78). Results of this study indicated that routine vaccination results in increased SAA concentration and provided evidence for a period of convalescence following vaccination. Measuring SAA for 10 days following vaccination cannot be used as an indicator of illness.

Booster effect of inactivated Parapoxvirus ovis on EHV‐1 neutralising antibodies when co‐injected with equine herpesvirus 1/4 vaccine

Booster effect of inactivated <i>Parapoxvirus ovis</i> on <scp>EHV</scp>‐1 neutralising antibodies when co‐injected with equine herpesvirus 1/4 vaccine

Mucosal antibodies against equine herpesvirus type‐1 and their role in preventing infection

Mucosal antibodies against equine herpesvirus type‐1 and their role in preventing infection

Stream of revelation: detection of equine herpesvirus‐1 (EHV‐1) in urine during myeloencephalopathy outbreaks

Stream of revelation: detection of equine herpesvirus‐1 (EHV‐1) in urine during myeloencephalopathy outbreaks

Evaluating non‐invasive sampling techniques for the molecular surveillance of Equid herpesvirus (EHV) in naturally shedding yearling horses

Evaluating non‐invasive sampling techniques for the molecular surveillance of Equid herpesvirus (EHV) in naturally shedding yearling horses

Equine herpesvirus type 1 specific T cells in the upper respiratory tract

Equine herpesvirus type 1 specific T cells in the upper respiratory tract

Use of an equine neurological model of EHV‐1 to identify host immune factors that contribute to the development of equine herpesvirus myeloencephalopathy

Use of an equine neurological model of EHV‐1 to identify host immune factors that contribute to the development of equine herpesvirus myeloencephalopathy

Evaluation of a triplex assay for detecting equine influenza virus and equine herpesvirus types 1 and 4 by using a microfluidic‐chip‐based mobile real‐time PCR device for point‐of‐care testing

Evaluation of a triplex assay for detecting equine influenza virus and equine herpesvirus types 1 and 4 by using a microfluidic‐chip‐based mobile real‐time PCR device for point‐of‐care testing

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.

Immune horses rapidly increase antileukoproteinase and lack type I interferon secretion during mucosal innate immune responses against equine herpesvirus type 1.

Equine herpesvirus type 1 (EHV-1) remains a considerable concern in the equine industry, with yearly outbreaks resulting in morbidity, mortality, and economic losses. In addition to its importance in equine health, EHV-1 is a respiratory pathogen and an alphaherpesvirus, and it may serve as a model for other viruses with similar pathogenicity or phylogeny. Large animal models allow the collection of high-volume samples longitudinally, permitting in-depth investigation of immunological processes. This study was performed on bio-banked nasopharyngeal samples from an EHV-1 infection experiment, where clinical outcomes had previously been determined. Matched nucleic acid and protein samples throughout infection permitted longitudinal quantification of the protein or related proteins of selected differentially expressed genes detected during the transcriptomic screen. The results of this manuscript identified novel innate immune pathways of the upper respiratory tract during the first 24 hours of EHV-1 infection, offering a first look at the components of early mucosal immunity that are indicative of protection.

Peri-anaesthetic complications in 1798 equids undergoing high-field elective orthopaedic MRI at a tertiary referral hospital.

Antimicrobial prophylaxis for elective orthopaedic magnetic resonance imaging (MRI) in equids is a topic of debate among practitioners and can have negative detrimental effects on patients if used unnecessarily. To describe the complications with elective orthopaedic MRI of horses, mules, and donkeys under general anaesthesia without the use of peri-anaesthetic antimicrobial prophylaxis at a single large tertiary referral centre. We hypothesised that horses, mules, and donkeys undergoing general anaesthesia for elective orthopaedic MRI, without antimicrobial prophylaxis, will not be at increased risk of complications, including increased risk of infectious respiratory disease. Retrospective cross-sectional study. This retrospective study of 1798 systemically healthy equids that underwent elective orthopaedic MRI under general anaesthesia without peri-anaesthetic antimicrobial prophylaxis between January 2009 and May 2020. A total of 1655 MRIs were included in the study, with 25 (1.5%) horses having complications. The most common complication was post-anaesthetic fever in 11 (0.7%) horses, of which 4 (0.2%) horses went on to develop pneumonia and one horse was diagnosed with equine herpesvirus (respiratory). Seven (0.4%) horses developed transient post-anaesthetic femoral neuropathy and 7 (0.4%) horses had mild post-anaesthetic colic that resolved with initial medical management. No horses were euthanised or died in this study. The retrospective nature of the study led to non-randomised case selection, and some records were incomplete. These findings suggest that peri-anaesthetic antimicrobial prophylaxis may not be necessary for performance and sport horses undergoing general anaesthesia for elective orthopaedic MRI.

Rapid Detection of Getah Virus Antibodies in Horses Using a Recombinant E2 Protein-Based Immunochromatographic Strip.

The prevalence and impact of Getah virus (GETV) are significant concerns in China. GETV can infect a wide range of animals, including horses, pigs, sheep, cattle, birds, and humans, resulting in substantial losses in the livestock and agricultural industries. GETV infection can cause the development of ulcers and inflammation in the mouth and gums of horses, which result in pain and discomfort and lead to symptoms such as reduced appetite, drooling, and difficulty chewing. As a result, there is a pressing need for efficient and rapid disease diagnosis methods. However, the currently available diagnostic methods have limitations in terms of operational time, equipment, and the experience of the individuals using them. In this study, a rapid, specific, and sensitive detection method was developed using a colloidal gold-based immunochromatographic strip (ICS) for the detection of antibodies against GETV in horses. To prepare the ICS, the antigen domain of the E2 glycoprotein of GETV was expressed using the Escherichia coli expression system after analysis with DNAstar v7.1 software. The nitrocellulose membrane was coated with rE2 protein or SPA to form the test line and control line, respectively. After optimizing the reaction conditions, the sensitivity, specificity, and repeatability of the strip were verified. The results showed that the test strip had a detection limit of up to 1:320 dilutions for GETV-positive serum, with no cross-reactivity observed with other equine-susceptible pathogens such as equine arteritis virus (EAV), equine herpesvirus-1 (EHV-I), equine infectious anemia virus (EIAV), equine influenza virus (EIV), African horse sickness virus (AHSV), and Japanese encephalitis virus (JEV). Furthermore, the ICS exhibited a concordance rate of 94.0% when testing 182 clinical serum samples compared to the virus neutralization test. Overall, this ICS diagnosis method will be an effective tool for the rapid detection of GETV in the field.

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.

Identification of the Promoter Antisense Transcript Enhancing the Transcription of the Equine Herpesvirus-1 Immediate-Early Gene.

Equine herpesvirus-1 (EHV-1) causes respiratory diseases, abortion, and encephalomyelitis in horses. The EHV-1 immediate-early (IE) protein, essential for viral replication, is transactivated by the binding of a multiprotein complex including the open reading frame 12 (ORF12) and some host factors to the IE promoter region. Promoter-associated non-coding RNAs (pancRNAs), which are transcribed from bidirectional promoters, regulate the transcription of neighboring genes in mammals and pathogens. In this study, we identified a novel pancRNA transcribed from across the areas of the 5'-untranslated region and a promoter of EHV-1 IE and named it IE pancRNA. IE pancRNA and mRNA were simultaneously expressed in EHV-1-infected RN33B-A68B2M cells. This pancRNA was also transcribed in RK13 and E. Derm cells, which are highly susceptible to EHV-1 infection. Furthermore, IE pancRNA upregulated IE gene expression in the presence of ORF12, and stable expression of IE pancRNA increased the number of EHV-1-infected RN33B-A68B2M cells. These results suggest that IE pancRNAs facilitate EHV-1 proliferation by promoting IE gene expression.