Serological Examinations of Significant Viral Infections in Domestic Donkeys at the Special Nature Reserve "Zasavica", Serbia.

The paper presents the findings of specific antibodies in the blood sera of donkeys against the following viruses: equine infectious anemia virus (EIAV), African horse sickness virus (AHSV), equine herpesvirus type 1 (EHV-1), equine influenza virus subtype H3N8 (EIV) and equine arteritis virus (EAV). The analyses were conducted during the year 2022. From a total of 199 donkeys bred in "Zasavica", blood was sampled from 53 animals (2 male donkeys and 51 female donkeys), aged 3 to 10 years. Specific antibodies against EIAV were not detected in any of the tested animals using the agar-gel immunodiffusion (AGID) assay. No specific antibodies against AHSV, tested by enzyme-linked immunosorbent assay (ELISA), or antibodies against EAV, tested by the virus neutralization test (VNT) and ELISA were detected in any of these animals. A positive serological result for EHV-1 was determined by the VNT in all animals, with antibody titer values ranging from 1:2 to 1:128, while a very low antibody titer value for EIV (subtype H3N8) of 1:16 was determined in 18 donkeys using the hemagglutination inhibition test (HI test).

The N-terminal glycine of EHV-1 UL11 is essential for the localization of UL11 and EHV-1 replication in cultured cells.

Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid (aa) protein encoded by ORF51. UL11 is modified by acylation including myristoylation and palmitoylation. Myristoylation of EHV-1 UL11 is assumed to occur on the N-terminal glycine, while palmitoylation is assumed to occur on the seventh and ninth cysteines. ORF51, which encodes the first 24 aa, overlaps ORF50 encoding UL12. We previously demonstrated that UL11 was essential for EHV-1 replication in cultured cells and that UL11 was localized at the Golgi apparatus where herpesviruses obtain their final envelope. It is unclear whether the acylation is related to the localization of EHV-1 UL11 and viral replication. In this study, we investigated the role of UL11 acylation in the intracellular localization and viral growth and replication of EHV-1. We constructed seven UL11 acylation mutant plasmids and seven UL11 acylation mutant BAC DNAs; then, we analysed the localizations of the mutant UL11s and attempted virus rescue. We found that both the N-terminal glycine and the seventh or ninth cysteine, especially N-terminal glycine, were involved in the localization of UL11 and viral replication. Taken together, these results suggest that EHV-1 viral growth requires that UL11 is modified by myristoylation of an N-terminal glycine and by palmitoylation of at least one of the cysteines, and that UL11 is localized at the Golgi apparatus. This study shows that a single amino acid in EHV-1 can determine the fate of viral replication.

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

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

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

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

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

Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus related to pseudorabies virus (PRV) and varicella-zoster virus (VZV). This virus is one of the major pathogens affecting horses worldwide. EHV-1 is responsible for respiratory disorders, abortion, neonatal foal death and equine herpes myeloencephalopathy (EHM). Over the last decade, EHV-1 has received growing attention due to the frequent outbreaks of abortions and/or EHM causing serious economical losses to the horse industry worldwide. To date, there are no effective antiviral drugs and current vaccines do not provide full protection against EHV-1-associated diseases. Therefore, there is an urgent need to gain a better understanding of the pathogenesis of EHV-1 in order to develop effective therapies. The main objective of this review is to provide state-of-the-art information on the pathogenesis of EHV-1. We also highlight recent findings on EHV-1 immune evasive strategies at the level of the upper respiratory tract, blood circulation and endothelium of target organs allowing the virus to disseminate undetected in the host. Finally, we discuss novel approaches for drug development based on our current knowledge of the pathogenesis of EHV-1.

Equine herpesvirus type 1 affects mitochondrial network morphology and reactive oxygen species generation in equine dermal cell line.

In the present study, the influence of the infection with equine herpesvirus type 1 (non-neuro-pathogenic and neuropathogenic strains of EHV-1) on the morphology and distribution of mitochondrial network in equine dermal cell line was investigated. Our results indicate that EHV-1-infection caused changes in the mitochondrial morphology manifested mostly by fission and reactive oxygen species generation.

Frequency of latent equine herpesvirus type-1 infection among a sample of horses in the central North Island of New Zealand.

Aim: To estimate the frequency of infection with equine herpesvirus type-1 (EHV-1) among horses from the central North Island of New Zealand, including the frequency of detection of the D752 genotype.Methods: Samples of retropharyngeal lymph nodes (RLN) and submandibular lymph nodes (SLN) were dissected from the heads of 63 horses that were humanely killed for various unrelated reasons between March and November 2015. DNA extracted from these tissues was subjected to enrichment for EHV-1 sequences by hybridisation with biotin-labelled EHV-1 specific probe, followed by recovery of EHV-1 sequences on streptavidin-coated magnetic beads. Enriched samples were tested for the presence of EHV-1 using nested quantitative real-time PCR. The EHV-1 amplicons were sequenced to determine the genotype of the virus.Results: The median age of the horses was 6 (min 2, max 30) years, and 47/63 (75%) were Thoroughbreds. EHV-1 DNA was detected in RLN samples from 6/63 (10%) horses, and three of these horses were also positive for EHV-1 DNA in SLN. The remaining horses were negative for EHV-1 DNA in both RLN and SLN samples. The N752 genotype was detected in all positive samples and the D752 genotype was not detected in any of the samples.Conclusions: EHV-1 continues to circulate among horses in New Zealand. The frequency of latent EHV-1 infection among sampled horses may have been underestimated due to the sensitivity limit of the assay or because of the limited anatomical sites sampled in the study. Lack of detection of the D752 genotype suggests that infection with this genotype is not common in horses in New Zealand.Clinical Relevance: If live animals are tested for EHV-1 using SLN biopsy it should be kept in mind that negative results do not rule out the presence of latent EHV-1 infection at other sites inaccessible for testing. The RLN appear to be the preferred sample for detection of EHV-1 DNA in horses following recent euthanasia.

Molecular Surveillance of EHV-1 Strains Circulating in France during and after the Major 2009 Outbreak in Normandy Involving Respiratory Infection, Neurological Disorder, and Abortion.

Equine herpesvirus 1 (EHV-1) is an Alphaherpesvirus infecting not only horses but also other equid and non-equid mammals. It can cause respiratory distress, stillbirth and neonatal death, abortion, and neurological disease. The different forms of disease induced by EHV-1 infection can have dramatic consequences on the equine industry, and thus the virus represents a great challenge for the equine and scientific community. This report describes the progress of a major EHV-1 outbreak that took place in Normandy in 2009, during which the three forms of disease were observed. A collection of EHV-1 strains isolated in France and Belgium from 2012 to 2018 were subsequently genetically analysed in order to characterise EHV-1 strain circulation. The open reading frame 30 (ORF30) non-neuropathogenic associated mutation A2254 was the most represented among 148 samples analysed in this study. ORF30 was also sequenced for 14 strains and compared to previously published sequences. Finally, a more global phylogenetic approach was performed based on a recently described Multilocus Sequence Typing (MLST) method. French and Belgian strains were clustered with known strains isolated in United Kingdom and Ireland, with no correlation between the phylogeny and the time of collection or location. This new MLST approach could be a tool to help understand epidemics in stud farms.

Deoxynivalenol, but not fumonisin B1, aflatoxin B1 or diesel exhaust particles disrupt integrity of the horse's respiratory epithelium and predispose it for equine herpesvirus type 1 infection.

The horse's respiratory tract daily encounters a plethora of respirable hazards including air pollutants, mycotoxins and airborne pathogens. To date, the precise effect of air pollution and mycotoxins on respiratory epithelial integrity and subsequent pathogen invasion in the horse has not been studied. Here, diesel exhaust particles (DEP) and three major mycotoxins (deoxynivalenol [DON], aflatoxin B1 [AFB1] and fumonisin B1 [FB1]) were applied to the apical surfaces of both ex vivo respiratory mucosal explants and in vitro primary equine respiratory epithelial cells (EREC) cultivated at the air-liquid interface, prior to inoculation with equine herpesvirus type 1 (EHV1). DON, but not AFB1, FB1 and DEP affected epithelial integrity in both ex vivo and in vitro systems, as demonstrated by histological changes in respiratory epithelial morphology and a drop in transepithelial electrical resistance across the EREC monolayer. Further, DON-pretreated explants showed on average 6.5 ± 4.5-fold more EHV1 plaques and produced on average 1 log10 more extracellular virus particles compared to control diluent- and FB1-pretreated respiratory mucosal explants. Similarly, EHV1 infection was greatly enhanced in EREC upon pretreatment with DON. Based on our findings, we propose that inhalation of DON predisposes horses for EHV1 infection by affecting respiratory epithelial integrity.

N-terminal domain of Schmallenberg virus envelope protein Gc delivered by recombinant equine herpesvirus type 1 and modified vaccinia virus Ankara: Immunogenicity and protective efficacy in cattle.

Schmallenberg virus (SBV), which emerged in 2011 in Central Europe and subsequently spread very rapidly throughout the continent, affects predominantly ruminants. SBV is transmitted by insect vectors, and therefore vaccination is one of the major tools of disease control. Only recently, a domain connected to virus neutralization has been identified at the amino-terminal part of the viral envelope protein Gc. Here, this Gc domain delivered by recombinant EHV-1 or MVA vector viruses was tested in a vaccination-challenge trial in cattle, one of the major target species of SBV. The EHV-1-based vaccine conferred protection in two of four animals, whereas immunization using the MVA vector vaccine efficiently induced an SBV-specific antibody response and full protection against SBV challenge infection in all the vaccinated animals. Moreover, due to the absence of antibodies against SBVs N-protein, both vector vaccines enable the differentiation between vaccinated and field-infected animals making them to a promising tool to control SBV spread as well as to prevent disease in domestic ruminants.

Effect of a Histone Demethylase Inhibitor on Equine Herpesvirus-1 Activity In Vitro.

Equine herpesvirus type 1 (EHV-1) is a ubiquitous and highly contagious pathogen that causes a range of disease severities with outbreaks of notable economic impact. Given the limitations in immune protection of current vaccines and the limited effectiveness of antiviral drugs on EHV-1 infections in vivo, improved treatment measures are needed to control disease. The use of drugs that alter the epigenetic state of herpes simplex virus genome has been shown to limit viral primary infection and reactivation both in vitro and in vivo. Therefore, we tested the hypothesis that maintaining a repressive epigenetic state on the EHV-1 genome in the host equine cell would decrease viral load during lytic infection. Equine fetal kidney cells (EFKCs) or isolated peripheral blood leukocytes were treated in vitro with (a) the nucleoside analog ganciclovir; (b) the histone demethylase inhibitor OG-L002; (c) both ganciclovir and OG-L002; or (d) dimethyl sulfoxide (DMSO, vehicle control); and then infected with a clinical EHV-1 isolate. Treatment of EFKCs with ganciclovir (mean 22.3 DNA copies per cell, p = 0.0005), OG-L002 (mean 25.6, p = 0.005) or both ganciclovir and OG-L002 (mean 7.1, p = 0.0001) resulted in decreased EHV-1 viral load at 24 h post-infection (hpi) in comparison with DMSO (mean 42.0), with greater impact using the combined treatment. Further, EHV-1 gene expression at 3 hpi decreased when EFKCs were infected in the presence of ganciclovir (p = 0.04) and combined treatment of ganciclovir and OG-L002 (p = 0.0003). In contrast, under similar conditions, neither ganciclovir nor OG-L002 suppressed EHV-1 infection in leukocytes. Differences between cell types, drug penetrance, or drug turnover, may have contributed to the distinct effects observed in this study.

Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks.

Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.

Low occurrence of equine herpesvirus 1 (EHV-1) as cause of abortion and perinatal mortality in Brazil / Baixa ocorrência de herpes-vírus equino 1 (HVE-1) como causa de abortamento e mortalidade perinatal no Brasil

Equine herpesvirus type 1 (EHV-1) is an important pathogen that causes abortion, neonatal disease, respiratory disorders, and neurological syndrome in equine populations worldwide. To evaluate EHV-1 as a cause of abortion and perinatal mortality in Brazil, tissue samples from 105 aborted equine fetuses, stillbirths, and foals up to one month of age were examined using virus isolation, immunohistochemistry (IHC), histopathology, and nested polymerase chain reaction (PCR). Two fetuses were positive for EHV-1 by PCR, one of which showed syncytia and eosinophilic intranuclear inclusion bodies in bronchial epithelia, but it was negative by virus isolation. The other showed no characteristic histological lesions, but it was positive by viral isolation. No sample was positive by IHC. The results presented low occurrence of EHV-1 in the studied population and suggested that the use of a combination of techniques increases the likelihood of an accurate diagnosis of EHV-1.(AU)

O herpes-vírus equino tipo 1 (HVE-1) é um importante agente patogênico causador de aborto, doença neonatal, distúrbios respiratórios e síndrome neurológica em populações de equinos em todo o mundo. Para avaliar a ocorrência do HVE-1 como agente causal de abortamento e mortalidade perinatal no Brasil, foram examinadas amostras de 105 fetos equinos abortados, natimortos e potros de até 1 mês de idade, utilizando as técnicas de isolamento viral, imuno-histoquímica (IHQ), histopatologia e reação em cadeia da polimerase aninhada (nested-PCR). Dois fetos foram positivos na análise de PCR, e um deles apresentou corpúsculos de inclusão viral eosinofílicos e sincícios no epitélio brônquico, porém foi negativo na análise de isolamento viral. O outro feto não apresentou lesões histológicas características de infecção herpética, mas foi positivo na análise de isolamento viral. Nenhuma amostra apresentou resultado positivo pela análise de IHQ. Os resultados demonstraram baixa ocorrência de HVE-1 na população estudada e que o uso de diferentes técnicas diagnósticas aumenta a probabilidade de um diagnóstico preciso para o HVE-1.(AU)

A Point Mutation in a Herpesvirus Co-Determines Neuropathogenicity and Viral Shedding.

A point mutation in the DNA polymerase gene in equine herpesvirus type 1 (EHV-1) is one determinant for the development of neurological disease in horses. Three recently conducted infection experiments using domestic horses and ponies failed to detect statistically significant differences in viral shedding between the neuropathogenic and non-neuropathogenic variants. These results were interpreted as suggesting the absence of a consistent selective advantage of the neuropathogenic variant and therefore appeared to be inconsistent with a systematic increase in the prevalence of neuropathogenic strains. To overcome potential problems of low statistical power related to small group sizes in these infection experiments, we integrated raw data from all three experiments into a single statistical analysis. The results of this combined analysis showed that infection with the neuropathogenic EHV-1 variant led to a statistically significant increase in viral shedding. This finding is consistent with the idea that neuropathogenic strains could have a selective advantage and are therefore systematically increasing in prevalence in domestic horse populations. However, further studies are required to determine whether a selective advantage indeed exists for neuropathogenic strains.

Prevention of equine herpesvirus myeloencephalopathy - Is heparin a novel option? A case report. / Prävention der equinen Herpesvirus-Myeloenzephalopathie ­ Ist Heparin eine vielversprechende Option? Ein Fallbericht.

Equine herpesvirus myeloencephalopathy (EHM) is a severe manifestation of equine herpesvirus 1 (EHV-1) infection. Prevention and treatment of EHM during EHV-1 outbreaks is critical, but no reliable and tested specific medication is available. Due to the thromboischemic nature of EHM and due to the fact that EHV-1 entry in cells is blocked by heparin, it was hypothesized that this compound may be useful in reduction of EHM incidence and severity. Therefore, during an acute EHV-1 outbreak with the neuropathogenic G2254/D752 Pol variant, metaphylactic treatment with heparin to prevent EHM was initiated. Clinical signs were present in 61 horses (fever n = 55; EHM n = 8; abortion n = 6). Heparin (25000 IU subcutaneously twice daily for 3 days) was given to 31 febrile horses from day 10 of the outbreak, while the first 30 horses exhibiting fever remained untreated. Treatment outcome was analyzed retrospectively. Heparin-treated horses showed a lower EHM incidence (1/31; 3.2%) than untreated horses (7/30; 23.3%; p = 0.03). Results indicate that heparin may be useful for prevention of EHM during an EHV-1 outbreak. These promising data highlight the need for randomized and possibly blinded studies for the use of heparin in EHV-1 outbreaks.

Similar regulation of two distinct UL24 promoters by regulatory proteins of equine herpesvirus type 1 (EHV-1).

To characterise the pattern of the transcriptional regulation of equine herpesvirus type 1 (EHV-1) UL24 by regulatory proteins, we identified two distinct promoter regions and two transcription initiation (Tci) sites located upstream of the UL24 open reading frame (ORF). The ORF proximal promoter exhibited higher cis-activity than that of the distal one. Contrary to the former, the latter performed its function dependent on an initiator (INR) due to its lack of a TATA box. Our results showed that the EHV-1 regulatory proteins EICP0, EICP22 and ETIF trans-activated the two promoters, whereas IEP and IR2P displayed negative regulation. In summary, the regulatory proteins exhibited similar regulatory patterns for the two distinct promoters of EHV-1 UL24.

Equine herpes virus type 1 (EHV-1) and 4 (EHV-4) infections in horses and donkeys in northeastern Turkey.

The herpesviruses infections in equides are caused by five different serotypes of viruses, belonging to family Herpesviridae. The goal of this study was to conduct a seroepidemiological investigation of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) in horses and donkeys raised in two provinces and their villages in northeastern Turkey. A total of 666 samples from 423 horses and 243 donkeys that were not immunized against these infections were tested with ELISA. While 52.48% of tested horse sera was found to carry specific antibodies to EHV-1, 83.69% of these serums were found to carry specific antibodies to EHV-4. 51. Eighty-five percent of analyzed donkey samples tested positive for EHV-1 and 64.20% of these samples tested positive for EHV-4 antibodies. When the horse and donkey samples were evaluated together, 52.25% were seropositive for EHV-1 and 76.58% were seropositive for EHV-4. This study showed that EHV-1 and EHV-4 infections are quite common in the horses and donkeys being raised in the areas where the study was carried out. In addition, since the area where the study was carried out in the borders of Armenia and Georgia, the high level of seropositive results for these infections leads to the conclusion that we should consider the risk of diseases spreading to neighboring countries. This is the first study to serologically identify EHV-1 and EHV-4 infections in donkeys raised in Turkey.

The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins.

Efficacy of a Parapoxvirus ovis-based immunomodulator against equine herpesvirus type 1 and Streptococcus equi equi infections in horses.

The efficacy of Zylexis®, an immunomodulator in horses based on inactivated Parapoxvirus ovis (iPPVO), was assessed using an equine herpesvirus type 1 (EHV-1) challenge model in the presence of a natural infection with Streptococcus equi equi (S. equi). Eleven horses were treated with iPPVO and twelve were kept as controls. Six horses were challenged with EHV-1 and commingled with the horses on study. Animals were dosed on Days -2, 0 (just before commingling) and Day 7. On Day 11 significantly less nasal discharge, enlarged lymph nodes, EHV-1 shedding and lower rectal temperatures were observed in the iPPVO-treated group. In addition, iPPVO-treated horses showed significantly fewer enlarged lymph nodes on Days 17 and 19, significantly less lower jaw swelling on Day 3 and significantly lower rectal temperatures on Days 12 and 13. Dyspnoea, depression and anorexia were only recorded for the control group. Following challenge seven out of 11 horses in the iPPVO treated group shed EHV-1 but on Days 11, 12, 13, 14, 15 and 16 quantitative virus detection in this group was significantly lower as compared to the controls. All animals shed S. equi but the percentage of animals with positive bacterial detection was lower in the iPPVO group than in the control group from Day 14 through Day 28. This difference was significant on Day 24. No injection site reactions or adverse events were observed. In conclusion, Zylexis administration is safe and reduced clinical signs and shedding related to both EHV-1 and S. equi infections.