Equine Herpesvirus Type 1 Strain Research Articles

First identification and isolation of equine herpesvirus type 1 in aborted fetal lung tissues of donkeys.

Equine herpesvirus type 1 (EHV-1) is commonly associated with horse abortion. Currently, there are no reported cases of abortion resulting from EHV-1 infection in donkeys. This was the first survey-based study of Chinese donkeys. The presence of EHV-1 was identified by PCR. This survey was conducted in Chabuchar County, North Xinjiang, China, in 2020. A donkey EHV-1 strain (Chabuchar/2020) was successfully isolated in MDBK cells. Seventy-two of 100 donkey sera were able to neutralize the isolated EHV-1. Moreover, the ORF33 sequence of the donkey-origin EHV-1 Chabuchar/2020 strain showed high levels of similarity in both its nucleotide (99.7‒100%) and amino acid (99.5‒100%) sequences, with those of horse EHV-1 strains. EHV-1 Chabuchar/2020 showed significant consistency and was classified within cluster 1 of horse EHV-1 strains. Further, analysis of the expected ORF30 nucleotide sequence revealed that donkey EHV-1 strains contained guanine at position 2254, resulting in a change to aspartic acid at position 752 of the viral DNA polymerase. Therefore, these strains were classified as horse neuropathogenic strains. Lastly, a phylogenetic tree was constructed using the partial ORF68 nucleotide sequences, showing that the identified donkey EHV-1 strain and the EHV-1 strain found in aborted Yili horses in China comprised a novel independent VIII group. This study showed the first isolation and identification of EHV-1 as an etiological agent of abortions in donkeys. Further analysis of the ORF33, ORF30, and ORF68 sequences indicated that the donkey EHV-1 contained the neuropathogenic genotype of strains in the VIII group. It is thus important to be aware of EHV-1 infection in the donkey population, even though the virus has only been identified in donkey abortions in China.

Molecular characterization of equine herpes viruses type 1 and 4 among Arabian horse populations in Egypt during the period between 2021 and 2022.

Equine herpesvirus type 1 (EHV-1) is a major cause of abortion and respiratory disease. Equine herpesvirus type 4 (EHV-4), on the other hand, is exclusively associated with respiratory disease in horse populations worldwide, particularly in Egypt and Arabian countries. This study aims to investigate the circulation of EHV-1 and EHV-4 in the Arabian horse population through molecular detection and genetic characterization of EHV-1 and/or EHV-4 that may threaten the stability of horse industry. A total of 80 samples including 50 nasal swabs, 10 vaginal swabs and 20 whole blood samples were collected from vaccinated and registered pure-bred Arabian adult horses from different studs in the governorates of northern Egypt (Cairo, Dakahlyia and Qalyubia) from 2021 to 2022. The collected samples were screened using consensus PCR for detection of EHVs using specific primers targeting DNA polymerase gene. The positive samples were subjected to conventional PCR for detection of EHV-1 and/or EHV-4using specific primers targeting glycoprotein (gB) gene. EHV-1 and EHV-4 amplicons were partially sequenced and phylogenetically analyzed using Sanger method. Consensus PCR revealed that 48 out of 80 samples were positive for EHVs with percentage of 60%. Typing of the selected positive samples using conventional PCR showed that 29 out of 80 were positive for EHV-1 with percentage 36.25%, while 24 out of 80 samples were positive for EHV-4 with percentage 30%. Mixed infections with both viruses were detected in five samples. The amplified products were sequenced using Sanger method and submitted to GenBank under accession number OM362231MG-1 for EHV-1 strain and OM362232 MG-4 for EHV-4 strain. Sequence analysis and alignments of the amplified fragments of the EHV-1 and EHV-4 glycoprotein B (gB) gene to that of GenBank-derived reference strains revealed a high degree of similarity. According to the phylogenetic tree, the obtained sequences of EHV-1 and 4 in the current study showed homogeneity with local Egyptian and foreign EHV-1 and 4 strains and heterogeneity with EHV-2 and 5. The current investigation showed that molecular methods are appropriate assays for an efficient and accurate diagnosis of EHVs. Furthermore, it supports earlier research findings about the prevalence of EHV-1 and 4 in Arabian horse populations in Egypt.

Major Equine Herpesvirus ‐ 1 epizootic in Europe: Identification of a marker for epidemiological surveillance

Background: Equine herpesvirus type-1 (EHV-1) is an important threat to the equine industry, as illustrated by the ongoing outbreak of neurological disease that was initially reported during a large equestrian event in Valencia, Spain in 2021. Horses returning from this event to their training yards have contributed to the spread of the virus to nine other European countries and to Qatar. Objectives: To design a “tracking” marker in order to specifically follow the dissemination of the Valencia strain, in EHV-1 infected horses with no known epidemiological link with the Valencia outbreak. Study design: Strain isolation and genome sequences’ comparison. Methods: 67 nasopharyngeal swab samples from horses stationed in Valencia were analysed by EHV-1 qPCR and a rapid A/G/C2254 (ORF30) typing test. Positive samples were used for strain isolation in vitro, and Multilocus Sequence Typing (MLST). Results: 19/67 (28%) samples were EHV-1 positive (all 19 typed as A2254). Two strains (FR/Valencia1/2021 and FR/Valencia2/2021) were successfully isolated and characterised by MLST as belonging to clade 10. Analysis of the MLST ORF sequences revealed a mutation at position 713 of the ORF11 (A713G) in FR/Valencia1/2021 and FR/Valencia2/2021, when compared with reference strains Ab4 and V592. This A713G mutation was not present in 104 ORF11 sequences obtained from Genbank (strains from the UK, USA, China, Australia, Belgium, New-Zealand, Japan or India), or 131 and 14 ORF11 sequences from strains isolated in Ireland or France, respectively. This marker allowed subsequent confirmation of suspicious epidemiological links in EHV-1 cases. Main limitation: The limited number of ORF11 sequences available in some countries. Conclusions: Although the existence of this mutation in other field strains cannot be excluded, its absence in the 249 ORF11 sequences analysed, suggests that this SNP constitutes an interesting epidemiological marker to identify horses infected with the EHV-1 strain which was associated with the outbreak in Valencia.

An Equine Herpesvirus Type 1 (EHV-1) Ab4 Open Reading Frame 2 Deletion Mutant Provides Immunity and Protection from EHV-1 Infection and Disease.

Equine herpesvirus type 1 (EHV-1) outbreaks continue to occur despite widely used vaccination. Therefore, development of EHV-1 vaccines providing improved immunity and protection is ongoing. Here, an open reading frame 2 deletion mutant of the neuropathogenic EHV-1 strain Ab4 (Ab4ΔORF2) was tested as a vaccine candidate. Three groups of horses (n = 8 each) were infected intranasally with Ab4ΔORF2 or the parent Ab4 virus or were kept as noninfected controls. Horses infected with Ab4ΔORF2 had reduced fever and nasal virus shedding compared to those infected with Ab4 but mounted similar adaptive immunity dominated by antibody responses. Nine months after the initial infection, all horses were challenged intranasally with Ab4. Previously noninfected horses (control/Ab4) displayed clinical signs, shed large amounts of virus, and developed cell-associated viremia. In contrast, 5/8 or 3/8 horses previously infected with Ab4ΔORF2 or Ab4, respectively, were fully protected from challenge infection as indicated by the absence of fever, clinical disease, nasal virus shedding, and viremia. All of these outcomes were significantly reduced in the remaining, partially protected 3/8 (Ab4ΔORF2/Ab4) and 5/8 (Ab4/Ab4) horses. Protected horses had EHV-1-specific IgG4/7 antibodies prior to challenge infection, and intranasal antibodies increased rapidly postchallenge. Intranasal inflammatory markers were not detectable in protected horses but quickly increased in control/Ab4 horses during the first week after infection. Overall, our data suggest that preexisting nasal IgG4/7 antibodies neutralize EHV-1, prevent viral entry, and thereby protect from disease, viral shedding, and cell-associated viremia. In conclusion, improved protection from challenge infection emphasizes further evaluation of Ab4ΔORF2 as a vaccine candidate.IMPORTANCE Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to preexisting local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies and lack of nasal type I interferon and chemokine induction. These host immune parameters may constitute markers of protection against EHV-1 and be utilized as indicators for improved vaccine development and informed vaccination strategies.

The deletion of the ORF1 and ORF71 genes reduces virulence of the neuropathogenic EHV-1 strain Ab4 without compromising host immunity in horses.

The equine herpesvirus type 1 (EHV-1) ORF1 and ORF71 genes have immune modulatory effects in vitro. Experimental infection of horses using virus mutants with multiple deletions including ORF1 and ORF71 showed promise as vaccine candidates against EHV-1. Here, the combined effects of ORF1 and ORF71 deletions from the neuropathogenic EHV-1 strain Ab4 on clinical disease and host immune response were further explored. Three groups of EHV-1 naïve horses were experimentally infected with the ORF1/71 gene deletion mutant (Ab4ΔORF1/71), the parent Ab4 strain, or remained uninfected. In comparison to Ab4, horses infected with Ab4ΔORF1/71 did not show the initial high fever peak characteristic of EHV-1 infection. Ab4ΔORF1/71 infection had reduced nasal shedding (1/5 vs. 5/5) and, simultaneously, decreased intranasal interferon (IFN)-α, interleukin (IL)-10 and soluble CD14 secretion. However, Ab4 and Ab4ΔORF1/71 infection resulted in comparable viremia, suggesting these genes do not regulate the infection of the mononuclear cells and subsequent viremia. Intranasal and serum anti-EHV-1 antibodies to Ab4ΔORF1/71 developed slightly slower than those to Ab4. However, beyond day 12 post infection (d12pi) serum antibodies in both virus-infected groups were similar and remained increased until the end of the study (d114pi). EHV-1 immunoglobulin (Ig) G isotype responses were dominated by short-lasting IgG1 and long-lasting IgG4/7 antibodies. The IgG4/7 response closely resembled the total EHV-1 specific antibody response. Ex vivo re-stimulation of PBMC with Ab4 resulted in IFN-γ and IL-10 secretion by cells from both infected groups within two weeks pi. Flow cytometric analysis showed that IFN-γ producing EHV-1-specific T-cells were mainly CD8+/IFN-γ+ and detectable from d32pi on. Peripheral blood IFN-γ+ T-cell percentages were similar in both infected groups, albeit at low frequency (~0.1%). In summary, the Ab4ΔORF1/71 gene deletion mutant is less virulent but induced antibody responses and cellular immunity similar to the parent Ab4 strain.

An equine herpesvirus type 1 (EHV-1) vector expressing Rift Valley fever virus (RVFV) Gn and Gc induces neutralizing antibodies in sheep

Rift Valley fever virus (RVFV) is an arthropod-borne bunyavirus that can cause serious and fatal disease in humans and animals. RVFV is a negative-sense RNA virus of the Phlebovirus genus in the Bunyaviridae family. The main envelope RVFV glycoproteins, Gn and Gc, are encoded on the M segment of RVFV and known inducers of protective immunity. In an attempt to develop a safe and efficacious RVF vaccine, we constructed and tested a vectored equine herpesvirus type 1 (EHV-1) vaccine that expresses RVFV Gn and Gc. The Gn and Gc genes were custom-synthesized after codon optimization and inserted into EHV-1 strain RacH genome. The rH_Gn-Gc recombinant virus grew in cultured cells with kinetics that were comparable to those of the parental virus and stably expressed Gn and Gc. Upon immunization of sheep, the natural host, neutralizing antibodies against RVFV were elicited by rH_Gn-Gc and protective titers reached to 1:320 at day 49 post immunization but not by parental EHV-1, indicating that EHV-1 is a promising vector alternative in the development of a safe marker RVFV vaccine.

Acyclovir and trichostatin A modulate EHV-1 replication in murine neurons in vitro

Equine herpesvirus type 1 (EHV-1) is a major viral pathogen of horses, causing respiratory disease, abortions, and equine herpes myeloencephalopathy (EHM). Like other alphaherpesviruses, EHV-1 establishes latency in neurons, but mechanisms involved in this process are still elusive. In the present study, we used antiviral drug acyclovir (ACV) to completely suppress EHV-1 replication in primary murine neuron culture. Trichostatin A (TSA), a known chemical reactivator of other herpesviruses, was used to stimulate productive EHV-1 infection. Moreover, gene expression of some cytokines was simultaneously evaluated, in order to check, whether the maintenance conditions of such a model may influence host cell response. Changes observed in IFN-α, IFN-β, and IL-10 mRNA gene expression depended on the EHV-1 strain. Although infection with either of the two EHV-1 strains investigated led to in an increase in type I IFNs gene expression, only the neuropathogenic strain caused a decrease in anti-inflammatory IL-10 gene expression. Unlike EHV-1 infection, the addition of neither ACV nor TSA caused significant changes in the expression of the above genes.. We may therefore conclude that the in vitro model presented in the study is suitable for detailed investigation of the host cell-virus relationship on the molecular level.

Equine herpesvirus type 1 modulates inflammatory host immune response genes in equine endothelial cells

Equine herpesvirus myeloencephalopathy (EHM), a disease caused by equine herpesvirus type 1 (EHV-1), is characterized by severe inflammation, thrombosis, and hypoxia in central nervous system (CNS) endothelial cells, which can result in a spectrum of clinical signs including urinary incontinence, ataxia, and paralysis. Strains of EHV-1 that contain a single point mutation within the viral DNA polymerase (nucleotide A2254>G2254: amino acid N752→D752) are isolated from EHM afflicted horses at higher frequencies than EHV-1 strains that do not harbor this mutation. Due to the correlation between the DNA Pol mutation and EHM disease, EHV-1 strains that contain the mutation have been designated as neurologic. In this study, we measured virus replication, cell to cell spread efficacy, and host inflammatory responses in equine endothelial cells infected with 12 different strains of EHV-1. Two strains, T953 (Ohio 2003) (neurologic) and Kentucky A (KyA) (non-neurologic), have well described disease phenotypes while the remaining strains used in this study are classified as neurologic or non-neurologic based solely on the presence or absence of the DNA pol mutation, respectively. Results show that the neurologic strains do not replicate better or spread more efficiently in endothelial cells. Also, the majority of the host inflammatory genes were modulated similarly regardless of EHV-1 genotype. Analyses of host gene expression showed that a subset of pro-inflammatory cytokines, including the CXCR3 ligands CXCL9, CXCL10, and CXCL11, as well as CCL5, IL-6 and TNF-α were consistently up-regulated in endothelial cells infected with each EHV-1 strain. The identification of specific pro-inflammatory cytokines in endothelial cells that are modulated by EHV-1 provides further insight into the factors that contribute to the immunopathology observed after infection and may also reveal new targets for disease intervention.

Protective effect of intranasal vaccination of horses with a modified live equine herpesvirus type 1 vaccine

Mucosal immunity in the nasal cavity is considered to play an essential role in protecting horses from disease caused by equine herpesvirus type 1 (EHV-1), and immunoglobulin A (IgA) antibody in the nasal secretions is a key component of this mucosal immunity. Although intranasal administration of live or killed EHV-1 vaccines has been attempted with the aim of protecting horses from viral challenge, induction of virus-specific IgA after these vaccinations was not observed, and only slight mitigation of clinical signs was observed after viral challenge. Here, we measured immunological responses and performed a challenge experiment in horses intranasally immunized with a modified live EHV-1 vaccine (Nisseiken, Tokyo, Japan) that was originally licensed for intramuscular injection. Horses (n ¼ 3) were intranasally inoculated with the vaccine (1.3 106 plaque-forming units [pfu]/10 mL/head) twice with a 4-week interval. Control horses (n ¼ 2) were inoculated in the same way with dilution medium. Four weeks after the second dose had been given, the horses were challenged by intranasal inoculation of EHV-1 (strain 10-I-224, 4.0 106 pfu/10 mL/head). Sequential nasal wash samples were concentrated by ultrafiltration and subjected to ELISA to measure EHV-1-specific IgA. Viral nasal shedding and viremia were monitored by virus isolation and real-time PCR. The results of real-time PCR were considered negative if there were � 10 gene copies in the analyzed sample. EHV-1-specific IgA levels in the nasal wash increased in all of the vaccinated horses after two intranasal doses of vaccine, whereas those in the control horses remained stable. Serum virus-neutralizing titers also increased in the vaccinated horses. After viral challenge, pyrexia over 39.0 � C was observed at 1 day post-inoculation (dpi) in both of the control horses. In contrast, none of the vaccinated horses was pyretic (over 39.0 � C) throughout the experiment, although one horse had a temperature of 38.7 � C at 7 dpi. Viral nasal shedding in the control horses was detected at 1 to 6 dpi, with a highest mean gene copy number of 103.8. In contrast, viral nasal shedding in the vaccinated horses was detected only at 1 to 2 dpi, with a highest mean gene copy number of 102.3. Viremia was detected in all horses at 5 to 11 dpi, with individual durations of 2 to 6 days. Intranasal immunization of horses with the live EHV-1 vaccine resulted in the production of EHV-1-specific IgA in the nasal mucosa and virus-neutralizing antibody in the whole body. Virus replication in the nasal mucosa after viral challenge was lower in vaccinated horses than in control horses; virus-specific IgA induced by the intranasal vaccination might have been responsible for this. The absence of pyrexia over 39.0 � C in the vaccinated horses indicated that the immunity induced by intranasal vaccination was effective in protecting horses from EHV-1-induced pyrexia.

Equine herpesvirus type 1 (EHV-1) open reading frame 59 encodes an early protein that is localized to the cytosol and required for efficient virus growth

Equine herpesvirus type 1 (EHV-1) ORF59 is predicted to encode a protein consisting of 180 amino acids. To determine whether ORF59 in fact encodes a protein, sequences encoding an HA epitope (YPYDVPDYA) was inserted at the carboxyterminus of the ORF59 protein in EHV-1 strain Ab4. Using anti-HA monoclonal antibodies, a 21-kDa band was specifically detected by western blot analysis in lysates of cells infected with a recombinant EHV-1 from strain Ab4 that carries the pORF59-HA but not in cells infected with parental Ab4. Further characterization of the protein using immunofluorescence and fractionation studies showed that pORF59 is an early protein that localizes to the cytosol in virus-infected cells. Recombinant EHV-1 lacking ORF59 (rAb4∆59) exhibited a small-plaque phenotype and could not be propagated. Our findings suggest that the ORF59 protein plays a major role in EHV-1 replication in vitro and likely in vivo.

Evaluation of metaphylactic RNA interference to prevent equine herpesvirus type 1 infection in experimental herpesvirus myeloencephalopathy in horses

To evaluate metaphylactic RNA interference to prevent equine herpesvirus type 1 (EHV-1) infection in experimental herpesvirus myeloencephalopathy in horses and to determine whether horses infected with a neuropathogenic strain of the virus that develop equine herpesvirus myeloencephalopathy (EHM) have differences in viremia. 13 seronegative horses. EHV-1 strain Ab4 was administered intranasally on day 0, and small interfering RNAs (siRNAs [EHV-1 specific siRNAs {n = 7} or an irrelevant siRNA {6}]) were administered intranasally 24 hours before and 12, 24, 36, and 48 hours after infection. Physical and neurologic examinations, nasal swab specimens, and blood samples were collected for virus isolation and quantitative PCR assay. Data from the study were combined with data from a previous study of 14 horses. No significant difference was detected in clinical variables, viremia, or detection of EHV-1 in nasal swab specimens of horses treated with the EHV-1 targeted siRNAs (sigB3-siOri2) versus controls. No significant differences in viremia were detected between horses that developed EHM and those that did not. Administration of siRNAs targeted against EHV-1 around the time of EHV-1 infection was not protective with this experimental design. Horses infected with the neuropathogenic EHV-1 strain Ab4 that developed EHM did not have a more pronounced viremia.

Strain impact on equine herpesvirus type 1 (EHV-1) abortion models: Viral loads in fetal and placental tissues and foals

Equine herpesvirus-1 (EHV-1) continues to cause both sporadic and epidemic abortions despite extensive vaccination. Lack of progress in the development of protective vaccines may be hindered by the lack of equine abortion models that employ contemporary EHV-1 strains. The objective of our experiments was to compare a contemporary EHV-1 strain with a previously described challenge strain, and to quantify EHV-1 loads in various maternal and fetal tissues. Infection experiments were performed in two groups of 7 pregnant pony mares at 270–290 days of gestation with a contemporary EHV-1 strain (University of Findlay 2003 isolate – OH03) or an EHV-1 strain isolated over 30 years ago, and previously described in abortion models (Ab4). All mares in both groups exhibited nasal viral shedding and viremia. Infection with OH03 resulted in 1/7 abortion and infection with Ab4 resulted in 5/7 abortions. In the OH03 challenge, placentas of foals delivered at term showed little detectable virus, while the aborted fetus expressed high levels of virus infection in the spleen and liver, lower levels in the lung and thymus, and lowest levels in the chorioallantois. After Ab4 challenge, high viral loads were detected in fetal and placental tissues in abortions. In the two normal deliveries, the chorioallantois contained virus levels comparable with the chorioallantois of aborted foals and both foals shed EHV-1 starting on day 4 of life, but were clinically healthy. Our results demonstrate the continued importance of strain selection for abortion models, and this study is the first report of viral load quantification using contemporary methods. Extremely high EHV-1 loads in decidua from abortions illustrate the infection risk posed to other horses.

Residue 752 in DNA polymerase of equine herpesvirus type 1 is non-essential for virus growth in vitro

A single amino acid variation in the equine herpesvirus type 1 (EHV-1) DNA polymerase (Pol) (D752/N752) determines its neuropathogenic potential. Here, an EHV-1 strain RacL11 mutant with a deletion of Pol residue 752 was constructed. The deletion virus was then repaired to encode D752 or N752, respectively. The Delta752 mutant virus replicated with kinetics indistinguishable from those of D752 and N752 viruses. In addition, we could demonstrate that the deletion mutant was significantly more resistant to aphidicolin, a drug targeting Pol, compared with the N752 but not the D752 variant. In equine peripheral blood mononuclear cells, no significant difference was detected between the mutants with respect to cellular tropism or virus replication. The results demonstrated that amino acid residue 752 in EHV-1 Pol is not required for virus growth, and that only the N752 mutation confers a drug-sensitive phenotype to the virus.

A vectored equine herpesvirus type 1 (EHV-1) vaccine elicits protective immune responses against EHV-1 and H3N8 equine influenza virus

Vaccination is commonly used to control equine respiratory pathogens such as equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV). Here, we describe the generation and characterization of a recombinant EHV-1 modified live virus vaccine (MLV) based on a recent abortogenic EHV-1 strain, NY03. The immunogenicity and efficacy of the MLV was tested in horses in an EHV-1 vaccination/challenge experiment using the highly virulent neurovirulent EHV-1 strain OH03. Induction of a robust EHV-1-specific immune response was observed. Upon challenge infection, vaccinated horses were partially protected against disease as demonstrated by a significant reduction in clinical signs, nasal shedding and viremia levels. In addition, the NY03-based MLV was used to express the EIV H3 protein and immunogenicity was tested in horses. Expression of H3 was readily detected in NY03-H3-infected cells in vitro. Vaccination of horses resulted in the induction of a robust serological immune responses against two recent but genetically distinct EIV representatives, VA05 and NY-99, which were above the threshold predicted to be protective against development of clinical disease.

In vitro and in vivo characterization of equine herpesvirus type 1 (EHV-1) mutants devoid of the viral chemokine-binding glycoprotein G (gG)

Glycoprotein G (gG) of equine herpesvirus type 1 (EHV-1), a structural component of virions and secreted from virus-infected cells, was shown to bind to a variety of different chemokines and as such might be involved in immune modulation. Little is known, however, about its role in the replication cycle and infection of EHV-1 in vivo. Here we report on the function of gG in context of virus infection in vitro and in vivo. A gG deletion mutant of pathogenic EHV-1 strain RacL11 (vL11ΔgG) was constructed and analyzed. Deletion of gG had virtually no effect on the growth properties of vL11ΔgG in cell culture when compared to parental virus or a rescuant virus vL11ΔgGR, respectively, and virus titers and plaque formation were unaffected in the absence of the glycoprotein. Similarly, in the murine model of EHV-1 infection, no significant differences in virulence between the gG deletion mutant and RacL11 or vL11ΔgGR were found at high doses of infection. However, infection of mice at lower doses revealed that the gG deletion mutant was able to replicate to higher titers in lungs of infected mice. Additionally, these mice lost significantly more weight than those infected with RacL11 and a more pronounced inflammatory response in lungs was observed. Therefore we concluded that deletion of gG in EHV-1 seems to lead to an exacerbation of respiratory disease in the mouse.

Equine Herpesvirus Type 1‐Associated Myeloencephalopathy in The Netherlands: A Four‐Year Retrospective Study (1999–2003)

Equine herpesvirus type 1(EHV‐1)‐associated myeloencephalopathy (EHM) is a rare disease affecting the central nervous system and, in particular, the spinal cord of horses. Epidemiologic data are limited, and usually are restricted to the description of a single outbreak. During an observational period of 4 years, we evaluated 9 outbreaks of EHM in The Netherlands. A risk factor analysis was done on the data from 6 outbreaks. Findings were: regular appearance of EHM outbreaks in The Netherlands (2–3/y); strong association of EHM with season; and risk factors, such as breed, sex, age, and fever. Female horses, aged horses, and specific breeds were at greater risk to develop severe neurologic disease. Other breeds and horses <3 years old were never observed to suffer from EHM during these outbreaks. It was concluded that breed variation, in addition to the presence of a specific EHV‐1 strain, and environmental factors influence an outbreak of EHM on a premise.

Equine herpesvirus type 1 modified live virus vaccines: quo vaditis?

Infections of horses with equine herpesvirus type 1 (EHV-1) have garnered new attention over the last few years. Devastating outbreaks occurring worldwide, primarily of the neurologic form of the disease, have resulted in a reassessment of the control strategies, and particularly the prophylactic measures, that are necessary to keep the infection and spread of disease in check. Most of the available EHV-1 vaccines are based on preparations of inactivated virus, which are applied monovalently for prevention of EHV-1-caused abortion in pregnant mares or as part of multivalent vaccines to prevent respiratory disease. Despite the importance of an induction of cytotoxic immune responses for protection against EHV-1-induced disease, only two modified live virus vaccine preparations, which are both based on the avirulent EHV-1 strain RacH and were developed more than 40 years ago, are commercially available. Current efforts focus on exploiting the available infectious bacterial artificial chromosome clones of various EHV-1 strains to engineer a new generation of modified live virus vaccines. Both more efficient and long-lasting anti-EHV-1 immunity and delivery of immunogens of other pathogens are attempted and within immediate reach. The improvement of modified live virus vaccines will likely be a major focus of research in the future, and will hopefully help to more completely protect horses against one of the most important and devastating viral diseases.

Equine Herpesvirus Type 1–Associated Myeloencephalopathy In The Netherlands: A Four-Year Retrospective Study (1999–2003)

Equine herpesvirus type 1(EHV-1)-associated myeloencephalopathy (EHM) is a rare disease affecting the central nervous system and, in particular, the spinal cord of horses. Epidemiologic data are limited, and usually are restricted to the description of a single outbreak. During an observational period of 4 years, we evaluated 9 outbreaks of EHM in The Netherlands. A risk factor analysis was done on the data from 6 outbreaks. Findings were: regular appearance of EHM outbreaks in The Netherlands (2-3/y); strong association of EHM with season; and risk factors, such as breed, sex, age, and fever. Female horses, aged horses, and specific breeds were at greater risk to develop severe neurologic disease. Other breeds and horses <3 years old were never observed to suffer from EHM during these outbreaks. It was concluded that breed variation, in addition to the presence of a specific EHV-1 strain, and environmental factors influence an outbreak of EHM on a premise.

Equine herpesvirus type 1 (EHV-1) glycoprotein K is required for efficient cell-to-cell spread and virus egress

The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HΔgK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HΔgK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early–late kinetics, and multiple forms of the protein exhibiting M rs between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK.

Stem cell-mediated tolerance induction in transplantation

Equine herpesvirus myeloencephalopathy (EHM), a disease caused by equine herpesvirus type 1 (EHV-1), is characterized by severe inflammation, thrombosis, and hypoxia in central nervous system (CNS) endothelial cells, which can result in a spectrum of clinical signs including urinary incontinence, ataxia, and paralysis. Strains of EHV-1 that contain a single point mutation within the viral DNA polymerase (nucleotide A2254 > G2254: amino acid N752 → D752) are isolated from EHM afflicted horses at higher frequencies than EHV-1 strains that do not harbor this mutation. Due to the correlation between the DNA Pol mutation and EHM disease, EHV-1 strains that contain the mutation have been designated as neurologic. In this study, we measured virus replication, cell to cell spread efficacy, and host inflammatory responses in equine endothelial cells infected with 12 different strains of EHV-1. Two strains, T953 (Ohio 2003) (neurologic) and Kentucky A (KyA) (non-neurologic), have well described disease phenotypes while the remaining strains used in this study are classified as neurologic or non-neurologic based solely on the presence or absence of the DNA pol mutation, respectively. Results show that the neurologic strains do not replicate better or spread more efficiently in endothelial cells. Also, the majority of the host inflammatory genes were modulated similarly regardless of EHV-1 genotype. Analyses of host gene expression showed that a subset of pro-inflammatory cytokines, including the CXCR3 ligands CXCL9, CXCL10, and CXCL11, as well as CCL5, IL-6 and TNF-α were consistently up-regulated in endothelial cells infected with each EHV-1 strain. The identification of specific pro-inflammatory cytokines in endothelial cells that are modulated by EHV-1 provides further insight into the factors that contribute to the immunopathology observed after infection and may also reveal new targets for disease intervention.