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.

Outbreak of equid herpesvirus 1 abortions at the Arabian stud in Poland.

BACKGROUND: Equid herpesvirus 1 (EHV-1) infections are endemic worldwide, including Poland. Many are subclinical, but some are associated with respiratory disease, abortion, neonatal foal death, or neurological disease. We describe an outbreak of abortions in Arabian mares at a well-managed State stud farm in Poland. CASE PRESENTATION: Eight of 30 pregnant mares aborted and one gave birth to a weak foal that died within 72 h after birth. EHV-1 was isolated from all fetuses as well as from the diseased foal. All viruses belonged to the N752 variant based on the predicted open reading frame (ORF) 30 amino acid sequence. All were identical to each other and to previous EHV-1 viruses from the same stud based on the ORF68 sequence analysis. The outbreak coincided with the lapse in the routine yearly EHV-1/4 vaccinations of the mares. CONCLUSIONS: Multiple abortion due to EHV-1 infection can occur in well-managed groups of horses. Reactivation of latent EHV-1 in one of the resident mares followed by a horizontal spread was considered the most likely explanation for the outbreak. Routine vaccination is an important part of a herd-heath program.

Function of myosin during entry and egress of equid herpesvirus type 1 in primary murine neurons.

Equid herpesvirus type 1 (EHV-1) is a major pathogen of horses with a worldwide distribution, which can cause various clinical signs ranging from mild respiratory disease to neurological disorders. To initiate an effective infection, EHV-1 evolved a broad spectrum of mechanisms exploiting the host cell, including its actin filaments. An actin-myosin-driven transport has been described to precede cellular entry of different viruses. Therefore, in the present study we investigated the role of actin motor protein - myosin, during replication of two EHV-1 strains: Jan-E (wild-type EHV-1 strain isolated from aborted equine fetus) and Rac-H (attenuated strain highly adapted in cell cultures in vitro) in primary murine neurons. In order to investigate this, we used two inhibitors: blebbistatin (BLB; non-muscle myosin II inhibitor) and 2,3-butanedione monoxime (BDM; inhibitor of myosin ATPase). Our results demonstrated that limitation of Jan-E EHV-1 replication occurred in cells treated with myosin inhibitor, which confirmed the important role of actin motor proteins during the entry and egress of EHV-1 virions. Application of blebbistatin did not affect Rac-H EHV-1 replication, while BDM caused reduction of replication in murine neurons. Based on these results it can be assumed that EHV-1 virion movement was myosin-dependent.

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.

Effects of different anesthetics in the murine model of EHV-1 infection.

Mice are commonly used as an experimental model to investigate the Equid herpesvirus 1 (EHV-1) infection. This model easily reproduces the disease, and the clinical signs are more or less similar to those observed in the horse, the natural host. During natural infection, the acute course of respiratory infection is mandatory for the development of adaptive immune response. Since interactions between EHV-1 and anesthetics are possible, the study investigated whether the early events of murine pulmonary immune response could be affected by different anesthetics. Therefore, mice were experimentally infected with a unique EHV-1 strain under the effects of ether, ketamine/xylazine, or isoflurane. Clinical signs and histopathological lesions in the lungs were described, and the cell death and proliferation rates of sham-inoculated or infected animals were quantified using immunohistochemistry. Clinical signs were more severe in animals anesthetized with ether. Qualitative differences in the recruited inflammatory cells were observed following application of anesthesia. The level of infection between the infected groups was not statistically significant. However, lungs from ketamine/xylazine-anesthetized animals showed the highest cell death rates, whereas those from isoflurane-anesthetized animals showed the highest proliferation rates. It has been emphasized that anesthetics alone or their interactions with EHV-1 modify the response against the infection. An appropriate selection of the anesthetic during experimental studies is relevant to minimize wrong conclusions.

Genome-wide association study for host genetic factors associated with equine herpesvirus type-1 induced myeloencephalopathy.

BACKGROUND: Equid herpesvirus (EHV-1) infections in horses can lead to equine herpesvirus myeloencephalopathy (EHM), characterised by neurological clinical signs. The sporadic occurrence of the disease in horse herds suggests a host genetic component. A recent study reported an association between the occurrence of EHM and genetic markers on horse chromosome 6 (ECA6). OBJECTIVES: To investigate the association of EHM with genetic host factors, especially with reference to the association reported for ECA6. STUDY DESIGN: Genome-wide association study (GWAS) was conducted based on 94 horses that had EHV-1 infections and comparing the 27 developing clinical EHM to the 67 which did not. METHODS: DNA samples were tested from 94 horses for 382,529 single nucleotide polymorphisms (SNPs) with the Affymetrix Axiom 670K SNP array to identify possible associations with EHM. The data analysis included tests for basic, additive, dominant and recessive modes of inheritance, haplotype associations and runs of homozygosity (ROH). RESULTS: Results from this study did not identify significant SNPs, haplotypes or ROH associations with the development of EHM following EHV-1 infections and excluded the involvement of a recessive genetic factor in the susceptibility to develop EHM. MAIN LIMITATIONS: Sample size and complex phenotype. CONCLUSIONS: The results exclude the involvement of a recessive genetic factor in the susceptibility to develop clinically apparent EHM but do not have the power to exclude the involvement of other, complex host genetic factors. Furthermore, there was no association between development of EHM and genes on equine chromosome 6, as previously reported.

Vaccination of foals with a modified live, equid herpesvirus-1 gM deletion mutant (RacHΔgM) confers partial protection against infection.

Equid herpesvirus-1 (EHV-1) causes respiratory and neurological disease and late gestation abortion in pregnant mares. Current vaccines contain either inactivated or live EHV-1, but fail to provide complete clinical or virological protection, namely prevention of nasopharyngeal shedding and cell-associated viraemia. Thus, the development of novel products, such as modified live virus (MLV) vaccines which stimulate virus-specific, humoral and cell mediated immune responses more effectively remains a priority. Two groups of weaned foals (n = 6 each group) were used in a longitudinal, prospective, experimental study to evaluate immune responses elicited by two vaccinations with a glycoprotein M (gM) deletion mutant of EHV-1 (RacHdeltagM). Following two concurrent intranasal and intramuscular inoculations six weeks apart, vaccinated (8.4 ±â€¯0.2 months old) and control foals (6.2 ±â€¯0.4 months) were challenge infected intranasally with EHV-1 Ab4/8 four weeks after the second vaccination and clinical signs and virological replication measured. Vaccination caused no adverse events, but did stimulate significantly higher complement fixing and virus neutralizing antibodies in serum compared with control foals at either equivalent or pre-vaccination time points. Virus-specific nasopharyngeal antibody levels and cytotoxic T lymphocyte responses were not significantly different between the groups. Following challenge infection, these immune responses were associated with a reduction in clinical signs and virological replication in the vaccinated foals, including a reduction in duration and magnitude of pyrexia, nasopharyngeal shedding and cell-associated viraemia. We conclude that the RacHΔgM MLV primed EHV-1-specific humoral immune responses in weaned foals. However, complete virological protection by vaccination against EHV-1 requires further research.

Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: Efficacy of decitabine and valganciclovir alone or in combination.

Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.

Screening of potential antiviral molecules against equid herpesvirus-1 using cellular impedance measurement: Dataset of 2,891 compounds.

Data presented in this article are associated with the research article "Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: efficacy of decitabine and valganciclovir alone and in combination" [1]. These data correspond to the in vitro screening of 2,891 potential antiviral compounds against equid herpesvirus-1 (EHV-1) based on impedance measurements using the xCELLigence® RTCA MP System. This dataset includes compounds from three different libraries: i) 1,199 compounds from the Prestwick® Chemical Library, which contains mostly US Food and Drug Administration approved drugs (Prestwick® Chemical, Illkirch, France); ii) 1,651 compounds from the Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN, Caen, France); iii) 41 compounds (called herein in-house antiviral library) selected for their effects against different human viruses. Compounds effective against EHV-1 were selected using the area under normalised curves (AUCn) and the time required for the Cell Index to decrease by 50% after virus infection (CIT50). The full dataset from the screen is made publicly available for further analyses.

Identification of a New Equid Herpesvirus 1 DNA Polymerase (ORF30) Genotype with the Isolation of a C2254/H752 Strain in French Horses Showing no Major Impact on the Strain Behaviour.

Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C2254 (H752) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as UL clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C2254 mutation. When cultured in vitro, the C2254 EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A2254 and G2254 EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C2254 strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C2254 strain and two other A/G2254 field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C2254 and A2254 strains were more sensitive to aciclovir than the G2254 strain, based on viral load measurement.

Loop-mediated isothermal amplification-fluorescent loop primer assay for the genotyping of a single nucleotide polymorphism at position 2254 in the viral DNA polymerase gene of equid alphaherpesvirus 1.

We developed a loop-mediated isothermal amplification (LAMP)-fluorescent loop primer (FLP) assay for genotyping the A/G2254 single nucleotide polymorphism (SNP) in the viral DNA polymerase gene of species Equid alphaherpesvirus 1 (EHV-1), which is associated with the neuropathogenic potential of this virus. In addition to the use of regular LAMP primers to amplify the target region, a 5'-FAM-labeled backward loop primer (FLB) and 3'-dabcyl-labeled quencher probe (QP) were designed for annealing curve analysis of the amplification product. The QP, which contacts the FLB, is located at the SNP site and has the A2254 allele. LAMP reactions were performed at 63°C for 40 min, and the subsequent annealing curve analyses were accomplished within 20 min. The LAMP-FLP assay could clearly differentiate A2254 and G2254 genotypes according to the difference in the annealing temperature of the QP between the 2 genotypes. Good agreement between the LAMP-FLP and the real-time PCR for genotyping of this SNP was observed in the detection of EHV-1 in equine clinical samples. The newly developed assay is a simple and rapid method for detecting and differentiating EHV-1 strains with A2254 and G2254 polymorphisms and would be suitable for clinical use.

Zebra-borne neurotropic equid herpesvirus 1 meningoencephalitis in a Thomson's gazelle ( Eudorcas thomsonii).

We describe the histopathologic, immunohistochemical, and molecular features of a case of meningoencephalitis in a Thomson's gazelle ( Eudorcas thomsonii) naturally infected with zebra-borne equid herpesvirus 1 (EHV-1) and the implications for the molecular detection of zebra-borne EHV-1. A 4-y-old female Thomson's gazelle was submitted for postmortem examination; no gross abnormalities were noted except for meningeal congestion. Microscopic evaluation demonstrated multifocal nonsuppurative meningoencephalitis with intranuclear eosinophilic and amphophilic inclusion bodies and EHV-9 antigen in neurons. PCR demonstrated the presence of a herpesvirus with a nucleotide sequence 99-100% identical to the corresponding sequences of zebra-borne EHV-1 and of EHV-9 strains. To determine whether EHV-1 or EHV-9 was involved, a PCR with a specific primer set for EHV-9 ORF59/60 was used. The sequence was identical to that of 3 recognized zebra-borne EHV-1 strains and 91% similar to that of EHV-9. This isolate was designated as strain LM2014. The partial glycoprotein G ( gG) gene sequence of LM2014 was also identical to the sequence of 2 zebra-borne EHV-1 strains (T-529 isolated from an onager, 94-137 from a Thomson's gazelle). The histologic lesions of encephalitis and antigen localization in this gazelle indicate prominent viral neurotropism, and lesions were very similar to those seen in EHV-1- and EHV-9-infected non-equid species. Histologic lesions caused by EHV-9 and zebra-borne EHV-1 are therefore indistinguishable.

Microvascular lesions and changes in cell proliferation and death, and cytokine expression in the placentas of mice experimentally infected with Equid Herpesvirus 1.

This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.

Virological and serological investigation of Equid herpesvirus 1 infection in New Zealand.

Infection with equid herpesvirus 1 (EHV-1) may be asymptomatic, or may result in respiratory disease, abortion, neonatal death, or neurological disease. The aim of this study was to estimate the prevalence of EHV-1 infection, including differentiation between genotypes with aspartic acid (D) and asparagine (N) at position 752 of the DNA polymerase sequence, within a selected population of New Zealand horses. The second aim was to determine the predictive value of serology for detection of latently infected horses. Retropharyngeal lymph nodes (RLN) and trigeminal ganglia (TG) were dissected from 52 horses at slaughter and tested for the presence of EHV-1 DNA using magnetic bead, sequence-capture enrichment followed by nested PCR. Sera were tested for EHV-1 antibody using type-specific glycoprotein G ELISA. Overall, 17/52 horses tested positive for EHV-1 DNA. All but one positive PCR results were obtained from RLN samples. Fifteen of the EHV-1 positive horses harboured EHV-1 with N752 genotype, one of which was additionally infected with the D752 genotypes of the virus. Our data comprise the first detection of EHV-1 with D752 genotype in New Zealand and suggest that the "neurovirulent" variant of EHV-1 had been present in New Zealand for at least two years before the first reported outbreak of EHM. All sampled horses tested positive for EHV-4 antibody, and 11/52 tested positive for EHV-1 antibody. The strength of agreement between results of EHV-1 PCR and EHV-1 serology was "fair" (Kappa 0.259, 95% CI: -0.022-0.539), which was likely a reflection of low levels of both EHV-1 antibody in sera and EHV-1 DNA in tissues tested.

A review of traditional and contemporary assays for direct and indirect detection of Equid herpesvirus 1 in clinical samples.

Equid herpesvirus 1 (EHV-1) is one of the most economically important equine viral pathogens. Its clinical manifestations in horses vary from acute upper respiratory tract disease, abortion, or neonatal death, to neurological disease termed equine herpesviral myeloencephalopathy, which may lead to paralysis and a fatal outcome. Successful identification of EHV-1 infection in horses depends on a variety of factors such as suitable case selection with emphasis on timing of sample collection, selection of appropriate sample(s) based on the clinical manifestations, application of relevant diagnostic technique(s) and/or test(s), and careful evaluation and interpretation of laboratory results. Several traditional serologic and virus isolation assays have been described; however, these assays have inherent limitations that prevent rapid and reliable detection of EHV-1. The advent of molecular biologic techniques has revolutionized the diagnosis of infectious diseases in humans and animal species. Specifically, polymerase chain reaction (PCR)-based assays have allowed detection of nucleic acid in clinical specimens precisely and rapidly as compared to the traditional methods that detect the agent or antigen, or agent-specific antibodies in serum. The new molecular methods, especially real-time PCR, can be a very useful means of EHV-1 detection and identification. Veterinarians involved in equine practice must be aware of the advantages and disadvantages of various real-time PCR assays, interpretation of viral genetic marker(s), and latency in order to provide the best standard of care for their equine patients.

Genomic study of Argentinean Equid herpesvirus 1 strains / Estudio genómico de cepas argentinas de Herpesvirus equino 1

Equid herpesvirus 1 (EHV-1) infection has a signifcant economic impact on equine production, causing abortion, respiratory disease, neonatal death and neurological disorders. The identifcation of specifc EHV-1 genes related to virulence and pathogenicity has been the aim of several research groups. The purpose of the present study was to analyze different genomic regions of Argentinean EHV-1 strains and to determine their possible relationship with virulence or clinical signs. Twenty-fve EHV-1 Argentinean isolates recovered from different clinical cases between 1979 and 2007 and two reference strains were amplifed and sequenced. The sequence alignments were carried out using Clustal X version 1.92 and the putative amino acid sequences were deduced using Bio-Edit version 7.05. Minor changes were observed. No changes that could be involved in the different virulence in the mouse model of three EHV-1 Argentinean strains were found. No genetic variants were observed. The genomic regions analyzed are unsuitable for differentiation between abortigenic strains and those isolated from neonatal deaths.

La infección por Herpesvirus equino 1 (EHV-1) tiene un signifcativo impacto económico en la producción equina mundial al causar abortos, enfermedad respiratoria, muertes perinatales y desórdenes neurológicos. La identifcación de genes específcos relacionados con la virulencia y patogenicidad de este virus ha sido el propósito de varios grupos de investigación. En este trabajo se analizaron diferentes regiones genómicas de cepas argentinas de EHV-1 para determinar la posible relación entre la estructura genómica y la virulencia o los signos clínicos producidos. Veinticinco cepas aisladas de diferentes casos clínicos observados entre los años 1979 y 2007 y dos cepas de referencia fueron amplifcadas y secuenciadas. El alineamiento de las secuencias se realizó con el programa Clustal X versión 1.92; el programa Bio-Edit versión 7.05 permitió deducir la secuencia de aminoácidos. Solo se observaron cambios menores, no se encontraron variaciones que pudieran estar relacionadas con la diferencia de virulencia observada previamente en el modelo ratón. No se hallaron variantes genómicas. Las regiones genómicas analizadas no permitieron diferenciar cepas abortigénicas de aquellas aisladas de muertes neonatales.