Outbreak of equine herpesvirus 4 (EHV-4) in Denmark: tracing patient zero and viral characterization.

BACKGROUND: Equine herpesvirus 4 (EHV-4) causes respiratory disease in horses, and the virus is considered endemic in the global equine population. However, outbreaks can occur when several horses are gathered in relation to shows, competitions, breeding units and at hospitals. In the spring year 2022, an EHV-4 outbreak occurred at the Large Animal Teaching Hospital, University of Copenhagen, Denmark. Nine horses were tested EHV-4 positive during the outbreak, which lasted approx. seven weeks. In addition, a tenth horse "Eq10" tested EHV-4 positive almost three weeks after the last of the outbreak horses tested positive. Detailed clinical registrations were obtained from all ten horses as well as their location and movement during hospitalization. Nasal swabs were obtained throughout the outbreak and tested by qPCR for EHV-4. Additionally, pre- and post-infection sera were tested for the presence of EHV-4 antibodies. Selected samples were characterized by partial and full genome sequencing. RESULTS: The most common clinical signs of the EHV-4 infected horses during this outbreak were pyrexia, nasal discharge, mandibular lymphadenopathy and increased lung sounds upon auscultation. Based on the locations of the horses, EHV-4 detection and antibody responses the most likely "patient zero" was identified as being "Eq1". Partial genome sequencing revealed that Eq10 was infected by another wild type EHV-4 strain, suggesting that the hospital was able to eliminate the outbreak by testing and reinforcing biosecurity measures. The complete genome sequence of the outbreak strain was obtained and revealed a closer relation to Australian and Japanese EHV-4 strains rather than to other European EHV-4 strains, however, very limited sequence data are available from Europe. CONCLUSION: The study illustrated the transmission of EHV-4 within an equine facility/hospital and provided new insights into the viral shedding, antibody responses and clinical signs related to EHV-4 infections. Finally, sequencing proved a useful tool in understanding the transmission within the hospital, and in characterizing of the outbreak strain.

Outbreak of neuropathogenic equid herpesvirus 1 causing abortions in Yili horses of Zhaosu, North Xinjiang, China.

BACKGROUND: EHV-1 is one of the most serious viral pathogens that frequently cause abortion in horses around the world. However, so far, relatively little information is available on EHV-1 infections as they occur in China. In January 2021, during an abortion storm which occurred in Yili horses at the Chinese State Studs of Zhaosu (North Xinjiang, China), 43 out of 800 pregnant mares aborted. RESULTS: PCR detection revealed the presence of EHV-1 in all samples as the possible cause of all abortions, although EHV-4, EHV-2 and EHV-5 were also found to circulate in the aborted fetuses. Furthermore, the partial ORF33 sequences of the 43 EHV-1 shared 99.3-100% and 99.0-100% similarity in nucleotide and amino acid sequences respectively. These sequences not only indicated a highly conserved region but also allowed the strains to group into six clusters. In addition, based on the predicted ORF30 nucleotide sequence, it was found that all the strains carried a guanine at the 2254 nucleotide position (aspartic acid at position 752 of the viral DNA polymerase) and were, therefore, identified as neuropathogenic strains. CONCLUSION: This study is the first one that establishes EHV-1 as the cause of abortions in Yili horses, of China. Further characterization of the ORF30 sequences revealed that all the EHV-1 strains from the study carried the neuropathogenic genotype. Totally, neuropathogenic EHV-1 infection in China's horse population should be concerned although the virus only detected in Yili horse abortions.

Epizootiological investigation of equine herpesvirus type 1 infection among Japanese racehorses before and after the replacement of an inactivated vaccine with a modified live vaccine.

BACKGROUND: Equine herpesvirus type 1 (EHV-1) infection is a major cause of pyrexias in winter among Japanese racehorses. In 2014-2015, the Japan Racing Association (JRA) changed the EHV-1 vaccine from an inactivated vaccine to a live vaccine (both produced by Nisseiken). To evaluate the effect of changing the vaccines, the capacities of these vaccines to induce virus-neutralizing (VN) antibodies were compared, and an epizootiological investigation of EHV-1 was performed at the JRA Ritto Training Center during epizootic periods from 2010-2011 to 2016-2017. RESULTS: Three-year-old horses that received the first dose of live vaccine showed higher geometric mean (GM) VN titers (205 and 220) than those that received inactivated vaccine (83, P < 0.05). The response rates after vaccination with the live vaccine (76 and 90%) were higher than that after vaccination with inactivated vaccine (42%, P < 0.05). Four-year-old horses from 2015 to 2017 that had received the live vaccine in the previous epizootic periods had higher GM titers (205 to 246) than those from 2011 to 2014, which had received the inactivated vaccine (139 to 164, P < 0.05). The estimated numbers of horses infected with EHV-1 or EHV-4, or both, in 2011-2012 (29 [95%CI: 21-37]) and 2013-2014 (37 [95%CI: 27-47]) were higher than those in the other periods (7 [95%CI: 2-12] to 16 [95%CI: 9-23]). Likewise, the seroconversion rates to EHV-1 in horses that stayed at the training center in 2011-2012 (66.0%) and 2013-2014 (52.0%) were higher than those in the other periods (12.0 to 28.6%). CONCLUSIONS: The live EHV-1 vaccine is highly immunogenic and provides greater VN antibody responses than the inactivated vaccine. Unlike the period when the policy was to use inactivated vaccine, there was no detectable epizootic EHV-1 infection at the training center during three consecutive periods after the introduction of the live vaccine. These results suggest that the replacement of inactivated vaccine with live vaccine, together with the achievement of high vaccination coverage, reinforced the herd effect, and contributed to better control of EHV-1 epizootics in the training center.

Phylogenetic and recombination analysis of the herpesvirus genus varicellovirus.

BACKGROUND: The varicelloviruses comprise a genus within the alphaherpesvirus subfamily, and infect both humans and other mammals. Recently, next-generation sequencing has been used to generate genomic sequences of several members of the Varicellovirus genus. Here, currently available varicellovirus genomic sequences were used for phylogenetic, recombination, and genetic distance analysis. RESULTS: A phylogenetic network including genomic sequences of individual species, was generated and suggested a potential restriction between the ungulate and non-ungulate viruses. Intraspecies genetic distances were higher in the ungulate viruses (pseudorabies virus (SuHV-1) 1.65%, bovine herpes virus type 1 (BHV-1) 0.81%, equine herpes virus type 1 (EHV-1) 0.79%, equine herpes virus type 4 (EHV-4) 0.16%) than non-ungulate viruses (feline herpes virus type 1 (FHV-1) 0.0089%, canine herpes virus type 1 (CHV-1) 0.005%, varicella-zoster virus (VZV) 0.136%). The G + C content of the ungulate viruses was also higher (SuHV-1 73.6%, BHV-1 72.6%, EHV-1 56.6%, EHV-4 50.5%) compared to the non-ungulate viruses (FHV-1 45.8%, CHV-1 31.6%, VZV 45.8%), which suggests a possible link between G + C content and intraspecies genetic diversity. Varicellovirus clade nomenclature is variable across different species, and we propose a standardization based on genomic genetic distance. A recent study reported no recombination between sequenced FHV-1 strains, however in the present study, both splitstree, bootscan, and PHI analysis indicated recombination. We also found that the recently sequenced Brazilian CHV-1 strain BTU-1 may contain a genetic signal in the UL50 gene from an unknown varicellovirus. CONCLUSION: Together, the data contribute to a greater understanding of varicellovirus genomics, and we also suggest a new clade nomenclature scheme based on genetic distances.

Mobile Platform for Multiplexed Detection and Differentiation of Disease-Specific Nucleic Acid Sequences, Using Microfluidic Loop-Mediated Isothermal Amplification and Smartphone Detection.

New tools are needed to enable rapid detection, identification, and reporting of infectious viral and microbial pathogens in a wide variety of point-of-care applications that impact human and animal health. We report the design, construction, and characterization of a platform for multiplexed analysis of disease-specific DNA sequences that utilizes a smartphone camera as the sensor in conjunction with a hand-held "cradle" that interfaces the phone with a silicon-based microfluidic chip embedded within a credit-card-sized cartridge. Utilizing specific nucleic acid sequences for four equine respiratory pathogens as representative examples, we demonstrated the ability of the system to utilize a single 15 µL droplet of test sample to perform selective positive/negative determination of target sequences, including integrated experimental controls, in approximately 30 min. Our approach utilizes loop-mediated isothermal amplification (LAMP) reagents predeposited into distinct lanes of the microfluidic chip, which when exposed to target nucleic acid sequences from the test sample, generates fluorescent products that when excited by appropriately selected light emitting diodes (LEDs), are visualized and automatically analyzed by a software application running on the smartphone microprocessor. The system achieves detection limits comparable to those obtained by laboratory-based methods and instruments. Assay information is combined with the information from the cartridge and the patient to populate a cloud-based database for epidemiological reporting of test results.

Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1.

Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.

Role of gB and pUS3 in Equine Herpesvirus 1 Transfer between Peripheral Blood Mononuclear Cells and Endothelial Cells: a Dynamic In Vitro Model.

UNLABELLED: Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. In order to evaluate viral transfer between infected PBMC and EC, cocultivation assays were performed. Only EHV-1 was transferred from PBMC to EC, and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to that of cells infected with parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taking the results together, we conclude that systemic spread and EC infection by EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer. IMPORTANCE: EHV-1 is widespread throughout the world and causes substantial economic losses through outbreaks of respiratory disease, abortion, and myeloencephalopathy. Despite many years of research, no fully protective vaccines have been developed, and several aspects of viral pathogenesis still need to be uncovered. In the current study, we investigated the molecular mechanisms that facilitate the cell-associated viremia, which is arguably the most important aspect of EHV-1 pathogenesis. The newly discovered functions of gB and pUS3 add new facets to their previously reported roles. Due to the conserved nature of cell-associated viremia among numerous herpesviruses, these results are also very relevant for viruses such as varicella-zoster virus, pseudorabies virus, human cytomegalovirus, and others. In addition, the constructed mutant and recombinant viruses exhibit potent in vitro replication but have significant defects in certain stages of the disease course. These viruses therefore show much promise as candidates for future live vaccines.

A SEROLOGIC AND POLYMERASE CHAIN REACTION SURVEY OF EQUINE HERPESVIRUS IN BURCHELL'S ZEBRAS (EQUUS QUAGGA), HARTMANN'S MOUNTAIN ZEBRAS (EQUUS ZEBRA HARTMANNAE), AND THOMSON'S GAZELLES (EUDORCAS THOMSONII) IN A MIXED SPECIES SAVANNAH EXHIBIT.

Reports of equine herpesvirus (EHV) 1 and EHV-9 causing clinical disease in a wide range of species have been well documented in the literature. It is thought that zebras are the natural hosts of EHV-9 both in the wild and in captive collections. Concerns about potential interspecies transmission of EHV-1 and EHV-9 in a mixed species savannah exhibit prompted serologic and polymerase chain reaction surveys. Eighteen Burchell's zebras ( Equus quagga ), 11 Hartmann's mountain zebras ( Equus zebra hartmannae), and 14 Thomson's gazelles ( Eudorcas thomsonii ) cohabitating the same exhibit were examined for EHV-1 virus neutralization titers, and evidence of virus via EHV 1-5 polymerase chain reactions. None of the animals had previous exposure to vaccination with EHV-1 or EHV-4. All tested zebras had positive EHV-1 titers, ranging from 4 to 384. All zebras and Thomson's gazelles had negative polymerase chain reaction results for all targeted equine herpesviruses. EHV-9-specific assays are not available but EHV-1, EHV-4, and EHV-9 cross-react serologically. Positive serology results indicate a potential latent equine herpesvirus in the zebra population, which prompted initiation of an equine herpesvirus vaccine protocol, changes in pregnant zebra mare management, and equine herpesvirus polymerase chain reaction screening prior to shipment to or from the study site.

Detection of equine herpesvirus-4 and physiological stress patterns in young Thoroughbreds consigned to a South African auction sale.

BACKGROUND: The prevalence of equine herpesvirus types-1 and -4 (EHV-1 and -4) in South African Thoroughbreds at auction sales is currently undefined. Commingling of young Thoroughbreds from various populations together with physiological stress related to their transport and confinement at a sales complex, may be associated with shedding and transmission of EHV-1 and -4. This prospective cohort study sampled 90 young Thoroughbreds consigned from eight farms, originating from three provinces representative of the South African Thoroughbred breeding demographic to a sales complex. Nasal swabs for quantitative real-time polymerase chain reaction (qPCR) assay to detect EHV-1 and -4 nucleic acid and blood samples for enzyme-linked immunosorbent assay for EHV-1 and -4 antibodies were collected from all horses on arrival and departure. Additional nasal swabs for qPCR were obtained serially from those displaying pyrexia and, or nasal discharge. Daily faecal samples were used for determination of faecal glucocorticoid metabolite (FGM) concentrations as a measurement of physiological stress and these values were modelled to determine the factors best explaining FGM variability. RESULTS: EHV-4 nucleic acid was detected in 14.4 % and EHV-1 from none of the animals in the study population. Most (93.3 %) and very few (1.1 %) of this population showed antibodies indicating prior exposure to EHV-4 and EHV-1 respectively. Pyrexia and nasal discharge were poor predictors for detecting EHV-4 nucleic acid. The horses' FGM concentrations increased following arrival before decreasing for most of the remaining study period including the auction process. Model averaging showed that variation in FGM concentrations was best explained by days post-arrival and transport duration. CONCLUSIONS: In this study population, sales consignment was associated with limited detection of EHV-4 nucleic acid in nasal secretions, with most showing prior exposure to EHV-4 and very few to EHV-1. The physiological stress response shown by most reflected the combination of stressors associated with transport and arrival and these are key areas for future investigation into management practices to enhance health and welfare of young Thoroughbreds during sales consignment.

Detection of equine herpesvirus in horses with idiopathic keratoconjunctivitis and comparison of three sampling techniques.

OBJECTIVES: To determine the role of equine herpesvirus (EHV) in idiopathic keratoconjunctivitis in horses and to determine whether sample collection method affects detection of EHV DNA by quantitative polymerase chain reaction (qPCR). ANIMALS STUDIED: Twelve horses with idiopathic keratoconjunctivitis and six horses without signs of ophthalmic disease. PROCEDURES: Conjunctival swabs, corneal scrapings, and conjunctival biopsies were collected from 18 horses: 12 clinical cases with idiopathic keratoconjunctivitis and six euthanized controls. In horses with both eyes involved, the samples were taken from the eye judged to be more severely affected. Samples were tested with qPCR for EHV-1, EHV-2, EHV-4, and EHV-5 DNA. Quantity of EHV DNA and viral replicative activity were compared between the two populations and among the different sampling techniques; relative sensitivities of the sampling techniques were determined. RESULTS: Prevalence of EHV DNA as assessed by qPCR did not differ significantly between control horses and those with idiopathic keratoconjunctivitis. Sampling by conjunctival swab was more likely to yield viral DNA as assessed by qPCR than was conjunctival biopsy. EHV-1 and EHV-4 DNA were not detected in either normal or IKC-affected horses; EHV-2 DNA was detected in two of 12 affected horses but not in normal horses. EHV-5 DNA was commonly found in ophthalmically normal horses and horses with idiopathic keratoconjunctivitis. CONCLUSIONS: Because EHV-5 DNA was commonly found in control horses and in horses with idiopathic keratoconjunctivitis, qPCR was not useful for the etiological diagnosis of equine keratoconjunctivitis. Conjunctival swabs were significantly better at obtaining viral DNA samples than conjunctival biopsy in horses in which EHV-5 DNA was found.

Equid herpesvirus type 4 uses a restricted set of equine major histocompatibility complex class I proteins as entry receptors.

Equid herpesvirus type 1 (EHV-1) was shown to use an unusual receptor for cellular entry - MHC-I molecules. Here, we demonstrated that the closely related EHV, EHV-4, also uses this strategy for cellular invasion, both in equine cells in culture and in the heterologous, non-permissive murine mastocytoma cell line (P815) after stable transfection with horse MHC-I genes. Using a panel of P815 cell lines transfected with individual horse MHC-I genes, we provided support for the hypothesis that EHV-1 and EHV-4 target classical polymorphic MHC-I molecules as viral entry receptors. All known equine MHC-I molecules from the two principal classical polymorphic loci specify alanine at position 173 (A173), whilst other MHC-I loci encoded different amino acids at this position and did not permit viral entry. Site-directed mutagenesis of position 173 diminished or enhanced viral entry, depending upon the initial amino acid. However, there were other, as yet undefined, constraints to this process: MHC-I genes from two non-classical loci carried A173 but did not enable viral entry in P815 transfectants. Our study suggested that the capacity to bind MHC-I molecules arose in the common ancestor of EHV-1 and EHV-4. The widespread occurrence of A173 in classical polymorphic horse MHC-I molecules indicated that horses of most MHC haplotypes should be susceptible to infection via this entry portal.

Glycoprotein H and α4ß1 integrins determine the entry pathway of alphaherpesviruses.

Herpesviruses enter cells either by direct fusion at the plasma membrane or from within endosomes, depending on the cell type and receptor(s). We investigated two closely related herpesviruses of horses, equine herpesvirus type 1 (EHV-1) and EHV-4, for which the cellular and viral determinants routing virus entry are unknown. We show that EHV-1 enters equine epithelial cells via direct fusion at the plasma membrane, while EHV-4 does so via an endocytic pathway, which is dependent on dynamin II, cholesterol, caveolin 1, and tyrosine kinase activity. Exchange of glycoprotein H (gH) between EHV-1 and EHV-4 resulted in rerouting of EHV-1 to the endocytic pathway, as did blocking of α4ß1 integrins on the cell surface. Furthermore, a point mutation in the SDI integrin-binding motif of EHV-1 gH also directed EHV-1 to the endocytic pathway. Cumulatively, we show that viral gH and cellular α4ß1 integrins are important determinants in the choice of alphaherpesvirus cellular entry pathways.

Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4.

The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T m), which are 88.0 and 84.4 °C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/µl or 0.15 pg/µl for EHV-1 and 5 copies/µl or 2.5 fg/µl for EHV-4. This assay was 50-1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer pairs and as sensitive as the TaqMan-MGB probe-based assay. The validity of the real-time PCR assays was confirmed by testing 13 clinical samples. When all results of the EG, SG, and TaqMan probe-based singleplex and duplex real-time PCRs were considered together, a total of 84.6 % (11/13) horses and donkeys were positive for at least one virus. EHV-1 and EHV-4 coexisted in 81.8 % (9/11) horses. Overall, we report that the EvaGreen duplex real-time PCR is an economical and alternative diagnostic method for the rapid differentiation of EHV-1 and EHV-4 in nasal swabs.

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.

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.

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.

Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1.

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored.

A Screening Study Identified Decitabine as an Inhibitor of Equid Herpesvirus 4 That Enhances the Innate Antiviral Response.

Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.

Glycoproteins D of equine herpesvirus type 1 (EHV-1) and EHV-4 determine cellular tropism independently of integrins.

Equine herpesvirus type 1 (EHV-1) and EHV-4 are genetically and antigenically very similar, but their pathogenic potentials are strikingly different. The differences in pathogenicity between both viruses seem to be reflected in cellular host range: EHV-1 can readily be propagated in many cell types of multiple species, while EHV-4 entry and replication appear to be restricted mainly to equine cells. The clear difference in cellular tropism may well be associated with differences in the gene products involved in virus entry and/or spread from cell to cell. Here we show that (i) most of the EHV-1 permissive cell lines became resistant to EHV-1 expressing EHV-4 glycoprotein D (gD4) and the opposite was observed for EHV-4 harboring EHV-1 gD (gD1). (ii) The absence of integrins did not inhibit entry into and replication of EHV-1 in CHO-K1 or peripheral blood mononuclear cells (PBMC). Furthermore, integrin-negative K562 cells did not acquire the ability to bind to gD1 when αVß3 integrin was overexpressed. (iii) PBMC could be infected with similar efficiencies by both EHV-1 and EHV-4 in vitro. (iv) In contrast to results for equine fibroblasts and cells of endothelial or epithelial origin, we were unable to block entry of EHV-1 or EHV-4 into PBMC with antibodies directed against major histocompatibility complex class I (MHC-I), a result that indicates that these viruses utilize a different receptor(s) to infect PBMC. Cumulatively, we provide evidence that efficient EHV-1 and EHV-4 entry is dependent mainly on gD, which can bind to multiple cell surface receptors, and that gD has a defining role with respect to cellular host range of EHV-1 and EHV-4.