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The Equid alphaherpesvirus type 1 (EHV-1) infection can have devastating economic consequences in the horse industry due to large-scale outbreaks of abortions, perinatal foal mortality, and myeloencephalopathy. The present study analyzed the genome of two isolates obtained from aborted fetuses in Argentina, E/745/99 and E/1297/07. The E745/99 genome shares 98.2% sequence identity with Ab4, a reference EHV-1 strain. The E/1297/07 genome shares 99.8% identity with NY03, a recombinant strain containing part of ORF64 and part of the intergenic region from Equid alphaherpesvirus-4 (EHV-4). The E/1297/07 genome has the same breakpoints as other United States and Japanese recombinants, including NY03. The recombinant regions have varying numbers of tandem repeat sequences and different minor parental sequences (EHV-4), suggesting distinct origins of the recombinant events. These are the first complete genomes of EHV-1 from Argentina and South America available in the Databases., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: 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., Results: 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., Conclusion: 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., (© 2024. The Author(s).)

Equid Gamma herpesvirus (eGHV) infections have been reported worldwide and may be correlated with clinical signs, e.g., affecting the respiratory tract in young horses. eGHV are shed by healthy horses as well as horses with respiratory tract disease. The prevalence in healthy Swiss horses is unknown to date but this data would provide valuable information for causal diagnosis in clinical cases and formulation of biosecurity recommendations. Nasal swabs from 68 healthy horses from 12 Swiss stables and 2 stables near the Swiss border region in Germany were analyzed by panherpes nested PCR. Positive samples were sequenced. A multivariable model was used to determine if sex, age, breed, canton, or stable had a significant effect on the shedding status of each detected eGHV. Overall, the eGHV prevalence was 59% ( n = 68); the prevalence for equid herpesvirus-2 (EHV-2), equid herpesvirus-5 (EHV-5) and asinine herpesvirus-5 (AHV-5) was 38%, 12% and 9%, respectively. Co-infections with multiple eGHVs were observed in 25% of the positive samples. The odds of shedding EHV-2 decreased with age ( p = 0.01) whereas the odds of shedding AHV-5 increased with age ( p = 0.04). Breed, sex, canton, or stable had no significant association with eGHV shedding. As EHV-2 shedding was common in healthy horses a positive PCR result must be interpreted with caution regarding the formulation of biosecurity recommendations and causal diagnosis. As EHV-5 and AHV-5 shedding was less common than EHV-2, a positive test result is more likely to be of clinical relevance. Shedding of multiple eGHV complicates the interpretation of positive test results in a horse.

The objective of this study was to determine if the genotype of equine herpesvirus-1 (EHV-1) impacted clinical disease and outcome of horses with laboratory confirmed equine herpesvirus myeloencephalopathy (EHM). Medical records from 65 horses diagnosed with EHM from 2011 to 2019 were reviewed for signalment, presence and severity of clinical signs (lethargy, fever, ataxia, urinary incontinence) and outcome. Horses were further grouped based on the EHV-1 genotype into neuropathic (D 752 ) or non-neuropathic (N 752 ) EHV-1 infection. Between the two EHV-1 genotype groups, age and sex distributions were similar, although breed distribution was different (Quarter Horses and Saddlebreds were overrepresented and Warmbloods were underrepresented in the EHV-1 D 752 group compared to the EHV-1 N 752 group; P = 0.009). Lethargy, fever, ataxia and outcome were not significantly different between the two EHV-1 genotype groups (P > 0.05). However, urinary incontinence was significantly more frequently reported in horses infected with the D 752 genotype of EHV-1 (P=0.04). Contrary to previous studies, the present study showed no difference in frequency of genotype (D 752 or N 752 ) among 65 horses with EHM and, with the exception of urinary incontinence, no difference in clinical disease or outcome related to the EHV-1 genotype., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Equine herpesvirus 1 (EHV-1) is an Alphaherpesvirus infecting not only horses but also other equid and non-equid mammals. It can cause respiratory distress, stillbirth and neonatal death, abortion, and neurological disease. The different forms of disease induced by EHV-1 infection can have dramatic consequences on the equine industry, and thus the virus represents a great challenge for the equine and scientific community. This report describes the progress of a major EHV-1 outbreak that took place in Normandy in 2009, during which the three forms of disease were observed. A collection of EHV-1 strains isolated in France and Belgium from 2012 to 2018 were subsequently genetically analysed in order to characterise EHV-1 strain circulation. The open reading frame 30 (ORF30) non-neuropathogenic associated mutation A 2254 was the most represented among 148 samples analysed in this study. ORF30 was also sequenced for 14 strains and compared to previously published sequences. Finally, a more global phylogenetic approach was performed based on a recently described Multilocus Sequence Typing (MLST) method. French and Belgian strains were clustered with known strains isolated in United Kingdom and Ireland, with no correlation between the phylogeny and the time of collection or location. This new MLST approach could be a tool to help understand epidemics in stud farms., Competing Interests: The authors declare no conflict of interest.

Equid alphaherpesvirus 1 (EHV-1) is an important pathogen of horses. It is spread worldwide and causes significant economic losses. The ORF33 gene has a conserved region that is often used as target in diagnostic PCR protocols. Single nucleotide point (SNP) mutations in ORF30 are usually used to distinguish between neuropathogenic and non-neuropathogenic genotypes. An ORF68 SNP-based scheme has been used for grouping different isolates. Recently, the highest number of variable sites in EHV-1 from the UK has been found in ORF34. In this study, EHV-1 positive samples from Italian horses with a history of abortion were investigated by amplifying and sequencing the ORF30 , ORF33 , ORF34 and ORF68 genes. Most animals were infected by the neuropathogenic type A2254G. A 118 bp deletion was found at nucleotide positions 701-818 of the ORF68 gene, making impossible to assign the samples to a known group. Sequencing of the ORF34 gene with a newly designed nested PCR showed new SNPs. Analysis of these sequences and of those obtained from genetic databases allowed the identification of at least 12 groups. These data add depth to the knowledge of EHV-1 genotypes circulating in Italy., Competing Interests: The authors declare no conflict of interest.

Equid alphaherpesvirus 1 (EHV-1) infections can have a major impact on the horse industry and equine welfare by causing abortion or respiratory or neurologic disease. A single nucleotide polymorphism (A 2254 →G 2254 ) in open reading frame (ORF) 30, encoding the catalytic subunit of the DNA polymerase, has been shown to be a strong predictive marker for neuropathogenicity. Given that a previously established real-time PCR (rtPCR) protocol yielded unsatisfactory results concerning determination of the EHV-1 genotype, we developed and evaluated a new conventional PCR protocol enabling identification of the genotype by sequencing and restriction enzyme analysis (REA). Thirty samples from horses with signs typical for EHV-1 infection were tested by rtPCR and our new conventional PCR. The results showed that compared to rtPCR, the conventional PCR protocol combined with sequencing and REA was more reliable concerning unambiguous determination of the EHV-1 genotype. Results of our new assay confirmed previous findings, according to which the non-neuropathogenic genotype A 2254 is predominantly found in animals with fever, respiratory signs, and abortions or perinatal mortality, whereas the neuropathogenic genotype G 2254 is primarily detected in animals suffering from neurologic disease. In some samples, results pointed towards coinfection with both genotypes. Further studies are required in order to elucidate the significance of infections with genotype A 2254 and G 2254 in neurologic and non-neurologic cases, respectively.

Equid alpha-herpesviruses (EHV) are responsible for different diseases in equine population. EHV-1 causes respiratory diseases, abortions and nervous disorders, EHV-4 causes respiratory diseases and sporadic abortion, while EHV-3 is responsible of equine coital exanthema. In view of the lack of efficacy of vaccines against EHV-1 and EHV-4 and in the absence of vaccines against EHV-3, the use of antiviral treatment is of great interest. In this study, we documented the interest of the Real-Time Cell Analysis (RTCA) technology to monitor the cytopathic effects induced by these viruses on equine dermal cells, and established the efficacy of this method to evaluate the antiviral effect of aciclovir (ACV) and ganciclovir (GCV). In addition, the RTCA technology has also been found appropriate for the high-throughput screening of small molecules against EHV, allowing the identification of spironolactone as a novel antiviral against EHV., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Equid alphaherpesvirus 1 (EHV-1) is one of the main infectious causative agents of abortion in mares and can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). The neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase (ORF30 D 752 ). Another gene, ORF68 is frequently used for phylogenetic analysis of EHV-1., Methods: 27 EHV-1 strains isolated from aborted equine fetuses in Poland, collected between 1993 and 2017, were subjected to PCR targeting the open reading frames (ORFs) 30 and 68 of the EHV-1 genome. PCR products obtained were sequenced and SNPs were analyzed and compared to sequences available in GenBank., Results: None of the analyzed sequences belonged to the ORF30 D 752 neuropathogenic genotype: all EHV-1 belonged to the non-neuropathogenic variant N 752 . On the basis of ORF68 sequences, the majority of EHV-1 sequences (76.9%) cannot be assigned to any of the known groups; only six sequences (23.1%) clustered within groups II and IV., Conclusions: EHV-1 strains obtained from abortion cases belong to the non-neuropathogenic genotype. Many EHV-1 ORF68 sequences have similar SNPs to those already described in Poland, but a clear geographical distribution was not observed. A single particular ORF68 sequence type was observed in strains isolated from 2001 onwards.

Equine herpesvirus 1 (EHV1) is considered as a major pathogen of Equidae , causing symptoms from mild respiratory disease to late-term abortion and neurological disorders. Different EHV1 strains circulating in the field have been characterized to be of abortigenic or neurovirulent phenotype. Both variants replicate in a plaque-wise manner in the epithelium of the upper respiratory tract (URT), where the abortigenic strains induce more prominent viral plaques, compared to the neurovirulent strains. Considering the differences in replication at the URT, we hypothesized that abortigenic strains may show an increased ability to modulate the type I IFN secretion/signaling pathway, compared to strains that display the neurovirulent phenotype. Here, we analyze IFN levels induced by abortigenic and neurovirulent EHV1 using primary respiratory epithelial cells (EREC) and respiratory mucosa ex vivo explants. Similar levels of IFNα (~70 U/ml) were detected in explants inoculated with both types of EHV1 strains from 48 to 72 hpi. Second, EREC and mucosa explants were treated with recombinant equine IFNα (rEqIFNα) or Ruxolitinib (Rux), an IFN signaling inhibitor, prior to and during inoculation with abortigenic or neurovirulent EHV1. Replication of both EHV1 variants was suppressed by rEqIFNα. Further, addition of Rux increased replication in a concentration-dependent manner, indicating an IFN-susceptibility for both variants. However, in two out of three horses, at a physiological concentration of 100 U/ml of rEqIFNα, an increase in abortigenic EHV1 replication was observed compared to 10 U/ml of rEqIFNα, which was not observed for the neurovirulent strains. Moreover, in the presence of Rux, the plaque size of the abortigenic variants remained unaltered, whereas the typically smaller viral plaques induced by the neurovirulent variants became larger. Overall, our results demonstrate the importance of IFNα in the control of EHV1 replication in the URT for both abortigenic and neurovirulent variants. In addition, our findings support the speculation that abortigenic variants of EHV1 may have developed anti-IFN mechanisms that appear to be absent or less pronounced in neurovirulent EHV1 strains.

Equine herpesvirus-1 (EHV-1) infection is an important and highly prevalent disease in equine populations worldwide. Previously we have demonstrated that a neuropathogenic strain of EHV-1, T953, suppresses the host cell's antiviral type-I interferon (IFN) response in vitro. Whether or not this is unique to EHV-1 strains possessing the neuropathogenic genotype has been undetermined. Here, we examined whether there is any direct relationship between neuropathogenic genotype and the induced IFN-β response in equine endothelial cells (EECs) infected with 10 different strains of EHV-1. The extent of virus cell-to-cell spread following infection in EECs was also compared between the neuropathogenic and the non-neuropathogenic genotype of EHV-1. We then compared IFN-β and the total type-I IFN protein suppression between T953, an EHV-1 strain that is neuropathogenic and T445, an EHV-4 strain mainly associated only with respiratory disease. Data from our study revealed no relationship between the neuropathogenic genotype of EHV-1 and the induced IFN-β mRNA by the host cell. Results also indicate no statistically significant difference in plaque sizes of both genotypes of EHV-1 produced in EECs. However, while the T953 strain of EHV-1 was able to suppress IFN-β mRNA and type-I IFN biological activity at 12 h post-infection (hpi), EHV-4 weakly induces both IFN-β mRNA and type-I IFN biological activity. This finding correlated with a statistically significant difference in the mean plaque sizes produced by the two EHV subtypes in EECs. Our data help illuminate how EHV-1, irrespective of its genotype, evades the host cell's innate immune response thereby enabling viral spread to susceptible cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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.

Equine herpesvirus 1 (EHV1) is a common pathogen of horses that causes upper respiratory tract disease, abortion, neonatal death and neurological disease. The neurological form of disease is called equine herpesvirus myeloencephalopathy (EHM). During the past decade, the incidence of EHM has been on the rise in Europe, North America, Australia and Asia. Some EHV1 isolates causing EHM exhibit a single-nucleotide polymorphism (SNP) in the DNA polymerase gene (ORF30) at position 2254 (A 2254 to G 2254 ). Further, based on polymorphism in the ORF68, EHV1 isolates have been classified into different groups. The aim of the present study was to estimate the genetic diversity of EHV1 and to determine the prevalence of the neuropathogenic genotype of EHV1 in India. Out of 133 clinical specimens from abortion cases in northern India, 56 were positive for EHV1 infection. Analysis of the A/G SNP by real-time PCR and sequence analysis revealed that 54 of 56 samples (96.43 %) were of the non-neuropathogenic genotype (A 2254 ), while two (3.57 %) had the neuropathogenic marker (G 2254 ). Sequence analysis of the polymorphic region of ORF68 of EHV1 isolates (n = 9) from India indicated that the Delhi/1998, Tohana-2/2013, Hisar-2/2014 and Hisar-15/1990 isolates belonged to group 4, while the Jind/1996, Rajasthan/1998, Delhi-3/2007 and Tohana-5/1996 isolates clustered within group 5. One isolate (Hisar-7/1990) exhibited SNPs at positions C 710 and C 713 , forming a separate group. Here, we report for the first time the detection of neuropathogenic genotypes of EHV1 in India and show that Indian EHV1 isolates cluster within groups 4 and 5.

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.

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.

A particularly severe equine herpesvirus type 1 (EHV-1) abortion outbreak occurred at a breeding farm in northern Germany. Sixteen of 25 pregnant mares that had received regular vaccination using an inactivated vaccine aborted and two gave birth to weak non-viable foals in a span of three months, with 89% of cases occurring within 40 days after the initial abortion case. Virological examinations revealed the presence of EHV-1 in all cases of abortion and serological follow-up in mares confirmed recent infection. Molecular studies identified a neuropathogenic variant (Pol/ORF30 A2254 to G2254) that belonged to geographical group 4 of EHV-1 isolates. The abortion outbreak was preceded by a case of mild ataxia of unknown cause in a mare that aborted four months after the ataxic episode. Although vaccination of pregnant mares did not prevent abortion, good EHV-1 immune status of the population at the time of outbreak may have had an impact in the failure of manifestation of the neurological form of the disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Equid herpesvirus (EHV) type 1 is a common pathogen of horses with worldwide distribution. Although severe tracheobronchitis has been described in some field outbreaks of EHV-1 respiratory disease, many EHV-1 infections occur asymptomatically or are accompanied only by signs of mild respiratory disease. However, EHV-1 infection can also result in outcomes other than respiratory disease such as abortion, neonatal death or neurological disease. This review provides an overview of the diagnosis, treatment and prognosis for EHV-1-associated diseases, with an emphasis on neurological presentations of EHV-1 infection.

Equid herpesvirus (EHV) type 1 is a common pathogen of horses with worldwide distribution. Infection with EHV-1 can be subclinical, or can result in sociologically and economically important outcomes such as abortion, neonatal death or neurological disease. The perceived recent increase in the reported cases of EHV-1 neurological disease in the United States of America and Europe over the past decade has caused concerns amongst veterinarians and horse owners worldwide. This review provides an update on the recent developments in our understanding of the pathogenesis and epidemiology of EHV-1 and associated diseases, with an emphasis on epidemiological data from Australasia. Many aspects of the pathogenesis and epidemiology of equine herpesvirus myeloencephalopathy still remain to be elucidated. This is an active area of current research worldwide.

Equine herpesvirus type 1 (EHV-1) was detected in an Indian rhinoceros (Rhinoceros unicornis), which was euthanized because of severe neurological disease. Encephalitis was suspected and EHV-1 DNA was detected in brain, lung, and spleen tissues. The viral IR6 protein was detected in lung tissues by Western blot analysis. Phylogenetic analyses of EHV-1 sequences amplified from various tissues was nearly identical to one recently described that resulted in both non-fatal and fatal encephalitis in polar bears. This represents transmission of EHV-1 to a species that is not naturally sympatric with the natural host of the virus and broadens the host range to Asian non-equid perissodactyls., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Here, we used a murine model to describe and compare the pathogenic potential of the Argentinean equid herpesvirus 1 (EHV-1) AR8 strain with the Japanese HH1 reference strain. In AR8-inoculated animals, clinical signs began earlier, but the viremic phase was shorter. Virus isolation and DNA detection in the lungs, liver and spleen were positive for both strains at different times postinfection (pi). Infection foci produced by both strains were immunohistochemically detected in lungs from day 1 to day 4 pi. We conclude that whichever EHV-1 strain is selected to experimentally reproduce the disease, it needs appropriate standardization in order to provide valid conclusions.

The equine herpesviruses type 1 (EHV-1) and 4 (EHV-4) are ubiquitous pathogens that affect horse populations on all continents. Despite widespread vaccination, EHV-1 and EHV-4 infections remain a permanent risk. While the two viruses share a high degree of genetic and antigenic similarity, they differ significantly in host range and pathogenicity. Compared to EHV-4, which mainly infects horses and causes respiratory disease, EHV-1 has a broader host range and can result in respiratory disease, abortions, neonatal death, and equine herpesvirusmyeloencephalopathy (EHM). Recent studies have elucidated a number of mechanisms that may, at least partly, explain the differential pathogenic potential of the two viruses. While both EHV-1 and EHV-4 can escape host immune responses and establish latent infection, there are differences with respect to virus entry and their ability to interfere with the innate immune response. Understanding the virus' repertoire of immunomodulatory mechanisms may lead the way to develop more efficient vaccines., (Copyright © 2013. Published by Elsevier B.V.)

Objective: 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., Animals: 13 seronegative horses., Procedures: 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., Results: 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., Conclusions and Clinical Relevance: 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.

A single-nucleotide polymorphism (A(2254) or G(2254)) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E(2)) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69-72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E(1)) was developed by redesigning primers and probes specific to ORF30. The E(1) and E(2) rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A(2254) or G(2254)) in all archived isolates plus 168 of the clinical samples. The E(1) assay was 10 times more sensitive than E(2), with a lower detection limit of 10 infectious virus particles. Furthermore, all A(2254) and G(2254) genotypes along with samples from three cases of dual infection (A(2254)+G(2254)) were correctly identified by E(1), whereas E(2) produced 20 false dual positive results with only one actual mixed A(2254)+G(2254) genotype confirmed. Based on these findings, E(1) offers greater sensitivity and accuracy for the detection and A/G(2254) genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.

Equine herpesvirus-1 (EHV-1) can be classified into distinct groups by single nucleotide polymorphisms (SNPs) in their genomes. Only a few of these can be associated with a special attribute of the virus. Differences in the ORF30 region can determine the neuropathogenic potential, while by substitutions in the ORF68 region several strain groups can be made. In previous studies no connection was found between the neuropathogenic potential and the SNPs in ORF68, but the occurrence of members of distinct groups in different outbreaks can facilitate epidemiological investigations because the geographical distribution of a particular group is very often specific. The present study aimed at the molecular examination and grouping of 35 EHV-1 strains isolated from aborted equine fetuses in Hungary between 1977 and 2008. Genotyping was based on the comparison of nucleotide sequences of a polymorphic segment located in the ORF68 region, which had previously been found to be a useful tool for classification. After sequencing this region, the Hungarian EHV-1 isolates could be classified into seven groups. Only 23 of the 35 isolates belonged to the formerly described groups, while the SNPs of 12 isolates diverged, and four new groups could be set up. In addition, phylogenetic analysis was performed to compare the ORF68 sequences of the Hungarian strains with the sequences of isolates from Europe, America and Australia. The number of newly formed groups suggests that the further analysis of unknown EHV-1 isolates would involve the emergence of extended numbers of new groups, which can impair the usability of this grouping method.

Equid herpesvirus 1 (EHV-1) infection has a significant economic impact on equine production, causing abortion, respiratory disease, neonatal death and neurological disorders. The identification of specific 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-five EHV-1 Argentinean isolates recovered from different clinical cases between 1979 and 2007 and two reference strains were amplified 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.

The recent increase in incidence, morbidity, and mortality of neurological disease induced by equine herpesvirus type 1 (EHV-1) has suggested a change of virulence of the virus. The exact mechanisms by which EHV-1 induces neurologic disease are not known. Environmental, viral, and host risk factors might contribute to neurological manifestation. Here, we investigated innate interferon-α (IFN-α), interleukin-10 (IL-10) and IL-4 responses after infection of equine peripheral blood mononuclear cells (PBMC) with EHV-1 using an available cytokine multiplex assay. Three viral strains representing an older isolate (RacL11), a recent abortigenic (NY03) and a neuropathogenic isolate (Ab4) were compared to identify differences in cytokine induction that might explain the increased pathogenicity of Ab4. Cytokine concentrations were also compared between foals, mares after birth, pregnant and non-pregnant mares to investigate whether immune responses to EHV-1 infection are influenced by age or pregnancy status. PBMC from all groups secreted high concentrations of anti-viral IFN-α in response to EHV-1. A reduced response was observed in foals compared to non-pregnant mares. EHV-1 infection induced moderate IL-10 and overall low IL-4 secretion. Ab4 infection resulted in a significant reduction of IL-10 responses in adult horses. IL-10 and IL-4 responses were lower in foals than in most mare groups. These data suggested that EHV-1 induces robust IFN-α secretion without major differences between viral strains. However, anti-inflammatory IL-10 production was significantly reduced after infection with neuropathogenic Ab4. The ability of this EHV-1 isolate to down-regulate IL-10 production might contribute to increased local inflammation and a higher risk for neurological manifestation of the disease after infection with Ab4., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Equine herpesvirus type 1 (EHV-1) is highly prevalent in horses and causes rhinopneumonitis, abortion, and encephalopathy. Studies on the related human herpes simplex virus and of murine models of EHV-1 suggest that chemokines play important roles in coordinating of innate and adaptive immune responses, and thus effective control of herpesvirus infection and prevention of severe clinical disease. Here, equine peripheral blood mononuclear cells (PBMC) were infected with one of three EHV-1 strains, which differ in pathogenicity (RacL11, NY03=abortogenic, Ab4=neurogenic). Changes in CCL2, CCL3, CCL5, CXCL9 and CXCL10 chemokine gene expression relative to non-infected PBMC were measured by real-time PCR. CXCL9 and CXCL10 gene expression was up-regulated 10h post infection and decreased to the level of non-infected cells after 24h. CCL2 and CCL3 were significantly down-regulated 24h post infection with NY03 and Ab4. CCL5 was up-regulated 24h after infection with RacL11. Ab4 infected PBMC had significantly lower expression of all chemokines except CCL2 24h post infection then RacL11 infected cells. While there was not a significant difference between NY03 and the other strains, there was a trend with each chemokine toward NY03 inducing less expression then RacL11 but more then Ab4. The data suggested that EHV-1 infection of PBMC induced up-regulation of inflammatory chemokines CCL5, CXCL9 and CXCL10, and down-regulation of chemotactic CCL2 and CCL3. The data also implies that different EHV-1 strains have varying effects on all five chemokines, with the nuropathogenic strain, Ab4, having the greatest suppressive potential., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Equine herpesvirus-1 (EHV-1) is a major pathogen of horses with worldwide distribution that can cause various clinical signs ranged from mild respiratory disease to neurological symptoms. Comparison of neuropathogenic and non-neuropathogenic EHV-1 strains revealed that a single non-synonymous nucleotide substitution (A/G2254) in the ORF30 region is associated with the altered functions of the viral DNA polymerase and therefore the neuropathogenicity of EHV-1 virus strains. The aim of the present study was the development of a new differentiation method of this particular single nucleotide polymorphism on the basis of the primer-probe energy transfer (PriProET) technique that has been successfully applied for the detection and classification of various DNA and RNA viruses. The results of melting temperature analysis showed an exact correlation with the sequence variations of the targeted region of ORF30, and the two genotypes (A/G2254) could be easily identified by the different peaks of melting temperatures. The new method is simple, fast, specific and robust as well as more flexible than the previous tests., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Equine herpesvirus 1 (EHV-1) is a common pathogen of the horse which may induce mild respiratory distress, abortion, neonatal death and neurological disease. A single nucleotide polymorphism in the EHV-1 DNA polymerase (ORF30 A(2254) to G(2254)) has been associated with clinical signs of Equine herpes myeloencephalopathy (EHM). The aim of this work was to analyze the ORF30 genomic region among a panel of EHV-1 DNA extract in order to estimate the prevalence of the EHV-1 neuropathogenic genotype in France. Samples coming from cases associated with EHM, horses with respiratory symptoms and aborted mares, each obtained between 2002 and 2009, were investigated. DNA was directly extracted from biological samples and allelic discrimination was performed using real-time PCR. Thirty of the 125 analysed horses (24%) presented the G(2254) genotype of ORF 30. Among them, 7/16 were provided by EHM cases, 1/24 by respiratory cases and 22/85 by abortion cases. Concerning EHM, the 7 G(2254) genotype of ORF30 were all isolated in 2009 during two outbreaks where mortality was observed. Regarding the 22 G(2254) genotype of ORF 30, 17 were identified in foetuses on which EHV-1 was detected by PCR, without any certainty of viral implication in the abortion. These findings clearly suggest that other factors need to be considered for a better understanding of the impact of DNA polymerase genotype upon EHV-1 neuropathogenic phenotype., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Equine herpesvirus-1 is a highly prevalent and frequently pathogenic infection of equids. The most serious clinical consequences of infection are abortion and equine herpesvirus myeloencephalopathy (EHM). In recent years, there has been an apparent increase in the incidence of EHM in North America, with serious consequences for horses and the horse industry. This consensus statement draws together current knowledge in the areas of pathogenesis, strain variation, epidemiology, diagnostic testing, vaccination, outbreak prevention and control, and treatment.

Equid Herpesvirus 1 (EHV-1) has long been causally implicated in the occurrence of abortion, neonatal death, respiratory disease, and neurological disorders in horses. This study analyzed for the first time the characteristics of the genomic section of Argentinian EHV-1 strains and reconstructed the phylogeny in order to establish their origin. The phylogenetic dataset included 22 Argentinian strains and four additional reference strains isolated in other countries. The intergenic region between ORF 62 and ORF 63 was amplified by PCR and sequenced. The phylogenetic analysis carried out by parsimony algorithms showed that six of the Argentinian strains had the same origin as British and Japanese strains. The mapping of symptoms caused by EHV-1 suggested that neonatal disease developed through convergent evolution, which would constitute an adaptation mechanism of the virus. This study constitutes the first analysis carried out in South-American strains that establishes the phylogenetic relationship between Argentinian strains and rebuilds the evolutionary history of symptoms. This study focuses on a very important aspect of evolution of Herpesviridae infecting perissodactyls and attempts to shed light on the evolution of symptoms, an issue of high clinical interest.

Objective: To identify risk factors associated with development of clinical neurologic signs in horses exposed to equine herpesvirus-1 (EHV-1)., Animals: 36 adult horses., Procedures: Blood samples collected before and after challenge inoculation with nonneuropathogenic or neuropathogenic EHV-1 were analyzed for leukocyte-associated viremia, serum neutralizing antibody, and EHV-1-specific cytotoxic T-lymphocyte precursors (CTLPs). Associations between variables and neurologic disease and correlations between age category or breed and development of neurologic disease were examined., Results: 9 horses developed CNS signs (ataxia, hind limb paresis or paralysis, bladder atony, or recumbency). Neurologic deficits were correlated with infection by a neuropathogenic strain of EHV-1, age>20 years, high postexposure viremic load, and low preexposure concentration of CTLPs. No significant correlations were observed between preinfection titers or horse breed and postinfection development of neurologic signs., Conclusions and Clinical Relevance: Horses with high concentrations of preexisting CTLPs, regardless of age, strain of virus, or titer, were more likely to control the magnitude of postinfection leukocyte-associated viremia and subsequent development of neurologic disease; therefore, CTLPs appear to be a critical requirement for protective immunity against EHV-1-induced myeloencephalopathy. The importance of achieving immunity related to high concentrations of vaccine-induced CTLPs in horses at high risk for exposure to neuropathogenic strains of EHV-1 is indicated.

A 4-year-old donkey was evaluated for progressive neurological abnormalities consisting of depression, stupor, weakness, and recumbency. Diagnostic evaluation for viral involvement identified an asinine herpesvirus in DNA extracted from deep pharyngeal swabs. Specific primers were designed based on comparison with equine herpesviral DNA polymerase sequences and yielded an 875-base pair product from the donkey. This sequence had complete identity with short sequences of asinine herpesvirus previously identified in donkeys with interstitial pneumonia. Amino acid analysis of the entire sequence indicated high similarity with Equid herpesvirus 7 (91%), Zebra herpesvirus 1 (90%), and Equid herpesvirus 2 (89%). With supportive treatment and physical therapy, the donkey gradually recovered over 5 days of hospitalization and returned to normal function. The current case illustrates the potential of a novel asinine herpesvirus to induce neurological disease in donkeys and provides a large viral sequence allowing confident assignment of this virus to the subfamily Gammaherpesvirinae.

Viruses related to equine herpesvirus type 1 (EHV-1) were isolated from an aborted fetus of an onager (Equus hemionus) in 1984, an aborted fetus of Grevy's zebra (Equus grevyi) in 1984 and a Thomson's gazelle (Gazella thomsoni) with nonsuppurative encephalitis in 1996, all in the USA. The mother of the onager fetus and the gazelle were kept near plains zebras (Equus burchelli). In phylogenetic trees based on the nucleotide sequences of the genes for glycoproteins B (gB), I (gI), and E (gE), and teguments including ORF8 (UL51), ORF15 (UL45), and ORF68 (US2), the onager, Grevy's zebra and gazelle isolates formed a genetic group that was different from several horse EHV-1 isolates. Within this group, the onager and gazelle isolates were closely related, while the Grevy's zebra isolate was distantly related to these two isolates. The epizootiological origin of the viruses is discussed.

Equine herpesvirus 1 was isolated from an onager in 1985, a zebra in 1986 and a Thomson's gazelle in 1996 in USA. The genetic relatedness and pathogenicity of these three viruses were investigated based on the nucleotide sequences of the glycoprotein G (gG) gene, experimental infection in hamsters, and comparison with horse isolates. The gG gene sequences of EHV-1 from onager and zebra were identical. The gG gene sequences of the gazelle isolate showed 99.5% identity to those of onager and zebra isolates. The gG gene sequences of EHV-1 isolated from horses were 99.9-100% identical and 98, 98 and 97.8% similar to gG from onager, zebra and gazelle isolates, respectively. Hamsters inoculated with onager, zebra and gazelle isolates had severe weight loss, compared with hamsters inoculated with horse isolates. The histopathological findings were related to the virulence of each isolate. The results indicated that EHV-1 isolates from onager, zebra and gazelle differ from horse EHV-1 and are much more virulent in hamsters.

This communication reports the development and performance assessment of a rapid diagnostic test for identifying horses actively infected with the neurovirulent pathotype of equine herpesvirus-1 (EHV-1). The test is a real-time polymerase chain reaction (PCR)-based assay that uses EHV-1 pathotype-specific TaqMan(R) reporter probes for discrimination between neuropathogenic and non-neuropathogenic strains of EHV-1 in equine blood or nasal swabs. The diagnostic performance of the new technique was evaluated by testing specimens collected from 234 horses involved in recent outbreaks of EHV-1 myeloencephalopathy at three separate thoroughbred racetracks and one large riding/boarding stable. Side-by-side comparison of the EHV-1 pathotyping results yielded by the new single-step, PCR-based allelic discrimination technique (24-hour turn-around-time) with those generated by a multi-step, conventional nested PCR followed by nucleotide sequencing of the amplified DNA (4-day turn-around-time) revealed complete agreement between the 2 test methods. The ability to rapidly identify horses infected with neuropathogenic strains of EHV-1 using a single-step, PCR-based method has significant implications for future diagnostic evaluation of suspect animals.

To determine the genomic variation of equine herpesviruses (EHVs) isolated in Argentina between 1979 and the first half of 2004, DNA sequences from all 69 strains isolated were analysed. Sixty strains were recovered from aborted fetuses, one from leucocyte-rich plasma from a horse with respiratory signs and eight from cases of neonatal disease. The DNA was extracted from rabbit kidney epithelial (RK13) cells infected with each strain and digested with three restriction endonucleases (BamHI, Bg/II and KpnI). Two strains could be differentiated using BamHI restriction and were assigned to the EHV-1 1B prototype group. Only one of these two strains was typed EHV-1 1B with Bg/II. DNA digestion with KpnI was ineffective. The results obtained in this study demonstrate that the EHV-1 1B genome has been present in Argentina since at least 1996. The finding of two strains with this electropherotype suggests that there is genomic heterogeneity among Argentinian isolates.

The objectives of this study were to estimate the prevalence of equine herpesviruses (EHV) 1-5 in the nasal secretions (NS) of a cohort of 12 mares and their foals from birth to 6 months of age, estimate the prevalence of EHV-1-5 infection of peripheral blood mononuclear cells (PBMC) of selected foals, and investigate phylogenetic relationships amongst the various strains of EHV-2 and 5. Virus-specific PCR assays were used to detect EHV-1-5 in NS and PBMC. A homologous portion of the glycoprotein B (gB) gene of the various strains of EHV-2 and 5 was sequenced and compared. EHV-2, 4, and 5 were all detected in NS from the horses, but only EHV-4 was associated with respiratory disease (P=0.005). EHV-2 and 5 infections were both common, but foals shed EHV-2 in their NS earlier in life than EHV-5 (P=0.01). Latent EHV-2 and 5 infections were detected in the PBMC of 75 and 88%, respectively, of the foals at approximately 6 months of age. The strains of EHV-2 shed in the NS of individual horses were more genetically heterogeneous than the strains of EHV-5 (95.5-99.3% versus 98.8-99.3% nucleotide identity, respectively). One-month-old foals typically shed strains of EHV-2 that were identical to those infecting their dams whereas older foals often shed virus strains that were different from those of their dams. Although herpesvirus infections were ubiquitous in this cohort of horses, there were distinct clinical consequences and clear epidemiological differences between infections with the different viruses.

Objective: To evaluate a technique for identifying horses latently infected with neuropathogenic strains of equine herpesvirus-1 (EHV-1)., Animals: 36 adult mares, 24 of which were experimentally infected as weanlings with neuropathogenic or nonneuropathogenic EHV-1., Procedures: Mandibular lymph node (MLN) tissue was obtained from each horse via biopsy during general anesthesia. Purified DNA from MLNs was tested for EHV-1 DNA by use of a magnetic bead, sequencecapture, nested PCR assay. For MLNs that contained EHV-1 DNA, the 256-bp DNA fragments amplified via sequence-capture nested PCR were sequenced to determine the nucleotide at the polymorphic site that determines pathotype (ie, neuropathotype [G(2254)] or non-neuropathotype [A(2254)])., Results: Latent viral DNA was detected in 26 of the 36 (72%) mares tested. Neuropathogenic and nonneuropathogenic EHV-1 genotypes were detected in the latently infected horses. In each mare previously infected with known EHV-1 pathotypes, the open reading frame 30 genotype of latent EHV-1 was identical to that of the strain that had been inoculated 4 to 5 years earlier. Latent viral DNA was detected in 10 of the 12 mares that were inoculated as weanlings with neuropathogenic strains of EHV-1. The detection rate of the sequence-capture PCR method for EHV-1 latency was double that of conventional nested or realtime PCR assays performed on the same MLN DNA preparations., Conclusions and Clinical Relevance: The magnetic bead, sequence-capture, nested PCR technique enabled low-threshold detection of DNA from latent neuropathogenic strains of EHV-1 in MLN specimens from live horses. The technique may be used to screen horses for latent neuropathogenic EHV-1 infection.

Equid herpes virus 1 (EHV-1) related isolates from a captive blackbuck (strain Ro-1) and Grevy's zebra (strain T965) behaved similarly to EHV-1 and EHV-9 in respect to their host cell range. Restriction enzyme analysis and a phylogenetic tree confirmed that Ro-1 and T965 were identical and more closely related to EHV-1 than to EHV-9. Differences from EHV-1 became obvious firstly, by amino acid alignments revealing two unique substitutions in the gB protein of Ro-1 and T965. Secondly, an EHV-1 type-specific monoclonal antibody did not detect its antigen on Ro-1, T965 or EHV-9 infected cells by immunohistochemistry. The results support the view that Ro-1 and T965 isolates represent a distinct, previously unrecognized species of equid herpesviruses.

Equid herpesvirus 1 (EHV-1) can cause a wide spectrum of diseases ranging from inapparent respiratory infection to the induction of abortion and, in extreme cases, neurological disease resulting in paralysis and ultimately death. It has been suggested that distinct strains of EHV-1 that differ in pathogenic capacity circulate in the field. In order to investigate this hypothesis, it was necessary to identify genetic markers that allow subgroups of related strains to be identified. We have determined all of the genetic differences between a neuropathogenic strain (Ab4) and a nonneuropathogenic strain (V592) of EHV-1 and developed PCR/sequencing procedures enabling differentiation of EHV-1 strains circulating in the field. The results indicate the occurrence of several major genetic subgroups of EHV-1 among isolates recovered from outbreaks over the course of 30 years, consistent with the proposal that distinct strains of EHV-1 circulate in the field. Moreover, there is evidence that certain strain groups are geographically restricted, being recovered predominantly from outbreaks occurring in either North America or Europe. Significantly, variation of a single amino acid of the DNA polymerase is strongly associated with neurological versus nonneurological disease outbreaks. Strikingly, this variant amino acid occurs at a highly conserved position for herpesvirus DNA polymerases, suggesting an important functional role.

Equine herpesvirus type 1 was determined as the etiological cause of an abortion storm in Czechia in 2003 after the virus strain was isolated from aborted fetus and identified by serological means and by PCR technique. Cloning and sequencing of the glycoprotein D confirmed the identity of the isolates and showed molecular relationships to known EHV-1 strains. Comparison of glycoprotein D sequences with corresponding sequence of EHV-1 reference strains (Kentucky-A and Ab1) revealed high nucleotide homology. The Czech isolate of EHV-1 virus does not differ significantly from the Ab1 strain regarding the glycoprotein D gene and does not bear the frameshift in the 3' terminus which occurs in the Kentucky-A strain.

The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.

In this study, a multiplex polymerase chain reaction (PCR) procedure was developed for differentiation of strains and field isolates of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Specific oli-gonucleotide primers were combined to amplify the thymidine kinase (TK) gene region of EHV-1 and EHV-4, which would yield fragments of different lengths for each virus in the same amplification reaction. The specificity of the largest PCR amplicon for EHV-4 was confirmed by restriction digestion with HindIII. The multiplex PCR proved to be a fast and sensitive method for typing EHV-1 and EHV-4 isolates and for detection and differentiation of both viruses in field samples in which infectious virus is no longer available. The sensitivity was improved by combining cycling optimization and visualization of PCR products in ethidium bromide and silver-stained acrylamide gels.

The V592 strain of equid herpesvirus-1 (EHV-1), which was originally isolated from a fetus during an abortion epizootic, has proved to be of low virulence in infection studies. Five Welsh Mountain pony mares and one foal were challenged intranasally or by aerosol with this isolate, and monitored clinically and virologically. All six animals shed virus in nasopharyngeal mucus, and viraemia was recorded from day 7 post-infection (PI). Pathological investigations revealed mild rhinitis and bronchiolitis in the mares, with viral antigen expression in degenerating epithelial cells of the nasal mucosa and bronchioles, and in occasional monocytes in the respiratory tract-associated lymph nodes. Viral antigen expression was not detected in vascular endothelium of the mares, although vasculitis was seen to have affected small numbers of blood vessels in the dorsocaudal lung regions of a mare examined on day 10 PI. In the foal, respiratory lesions of a more localized nature included infection of vascular endothelium and associated vasculitis. The foal also had localized encephalitis affecting the olfactory lobes of the brain, with viral antigen expression in degenerating olfactory neurons and microglia. The data suggest that the relatively low virulence of strain V592 is associated with a lower degree of endotheliotropism than that shown by the highly virulent Ab4 and Army 183 isolates, and that this property is influenced by host immunity., (Copyright 2000 Harcourt Publishers Ltd.)

The aim of the present study was to clarify whether an EHV-1 induced abortion can be prognosticated by an increase of antibody titres, virus shedding and/or viraemia and whether the current abortion diagnostic is suitable. In this context the immune response post immunization and a possible reactivation were of great interest. For this purpose blood samples of 32 mares between the ages of 5-21 years were regularly investigated during a period of two years before and after vaccination and pregnancy. Neutralization tests, indirect immunofluorescence tests as well as PCR and virus isolation were used for EHV-1 diagnostics. It could be shown that the horses reacted individually to vaccination. In 14 cases a EHV-1-reactivation was suggested. An abortion prognosis was not possible even using serological, virological and molecular biological parameters. In addition, virus shedding and antibody titres were individual. An acute infection was detectable by a significant rise of antibodies and viraemia as well as virus shedding in the secretions. For the abortion diagnostics the antigen detection in combination with virus isolation and PCR from fetal lungs gave reliable results. In addition, the virological and serological investigation of the mare is recommendable. For prophylaxis we would advise a regular vaccination and strict hygiene.

Ninety-two equine herpesvirus type 1 isolates were recovered from aborted, stillborn, or neonatal foals from Ontario, Canada, from 1986 to 1992. From this total, 32 strains were randomly chosen for further study. Four or 5 isolates from each winter were selected, each from a different premises, and characterized by restriction enzyme analysis using BamHI, KpnI, BglII, HindIII, and EcoRI. Additional isolates from 2 premises and from a zebra foal were also assessed. For the strains isolated in 1986 and 1989-1992, the DNA pattern of 18 strains was similar to that of type 1P (Kentucky D) for BamHI and KpnI. None of the 32 strains studied could be differentiated by HindIII or EcoRI. Using BglII, an inconsistent fragment pattern and distribution were observed. Of the 8 strains isolated in 1987 and 1988, 7 were assigned into the 1B prototype group. The geographic distribution of 17 type 1P and 12 1B isolates was random across southern Ontario. These findings suggest that both electropherotypes can be recovered from horses in Ontario. The patterns of the additional equine isolates from the same premises were identical. The zebra isolate was different from the prototype equine herpesvirus type 1 and type 4 patterns and from all other equine isolates.

A herpesvirus was isolated from Thomson's gazelle (Gazella thomsoni) kept at a zoological garden in Japan during an outbreak of epizootic acute encephalitis. The virus, gazelle herpesvirus 1 (GHV-1), was serologically related to equine herpesvirus 1 (EHV-1). However, DNA fingerprints of GHV-1 were different from those of EHV-1 and other equine herpesviruses. Southern hybridization with probes of cloned BamHI fragments derived from UL and US segments of EHV-1 revealed differences in the DNA restriction profiles throughout the entire genome. Nucleotide sequences were determined for a conserved region of an essential envelope glycoprotein B (gB) gene and a type-specific glycoprotein G (gG) homologue gene. The predicted amino acid sequence of GHV-1 gB showed 97, 92, 61, and 57% identity to EHV-1, EHV-4, feline herpesvirus, and pseudorabies virus, respectively, indicating that GHV-1 was closer to EHV-1 than any other herpesvirus. The GHV-1 gG gene showed 93.2, 92.3, and 53% identity to EHV-1, EHV-8, and EHV-4 gGs, respectively. GHV-1 was virulent to suckling mice of the ICR strain by intracerebral inoculation and was virulent to 4-week-old BALB/c mice by intranasal inoculation, causing neurological symptoms and death. We conclude that GHV-1 is a new type of equine herpesvirus with strong neurotropism.