Equine Herpesvirus 1 Isolates Research Articles

English

Equine herpesvirus 1 (EHV-1) causes considerable economic loss to the equine industry and is spread among susceptible animals during the cycles of latency and reactivation, causing rhinopneumonitis, abortion, and neurological disease. Nucleotide polymorphisms within ORF30 and ORF68 sequences of the viral genome are associated with strain neuropathogenicity and geographical origin. A total of 142 tissue and nasal swab samples from apparently healthy unvaccinated horses were examined to ascertain EHV-1 distribution, diversity, and clinical significance considering the results of virus isolation, sequence analysis, and anamnestic data. The ORF30 and ORF68 molecular study of these circulating strains and archival isolates from abortion storms aimed to contribute to the perception of strain pathogenicity and origin. EHV-1 was detected by PCR and virus isolation in 81 and 45.1% of the analyzed samples, respectively, and 82.1% of the representative samples were neuropathogenic strains. The ORF68-based grouping was restricted by the pronounced polymorphism of Balkan EHV-1 strains, and only two isolates were assigned to group 4. The cases of abortion were caused by neuropathogenic strains that also circulate within the horse population with no documented outbreaks of disease. It was evident that strain virulence is not solely accountable for the development of clinical symptoms in affected animals. Neural tissue is significant for virus latency and reactivation, considering the number of EHV-1 isolates from apparently healthy stressed horses. Special care must be taken when accommodating together immunologically naive and latently infected horses since asymptomatic carriers silently shed EHV-1.

Evaluation of the Variability of the ORF34, ORF68, and MLST Genes in EHV-1 from South Korea

Equine herpesvirus-1 (EHV-1) is an important pathogen in horses. It affects horses worldwide and causes substantial economic losses. In this study, for the first time, we characterized EHV-1 isolates from South Korea at the molecular level. We then aimed to determine the genetic divergences of these isolates by comparing them to sequences in databases. In total, 338 horse samples were collected, and 12 EHV-1 were isolated. We performed ORF30, ORF33, ORF68, and ORF34 genetic analysis and carried out multi-locus sequence typing (MLST) of 12 isolated EHV-1. All isolated viruses were confirmed as non-neuropathogenic type, showing N752 of ORF30 and highly conserved ORF33 (99.7–100%). Isolates were unclassified using ORF68 analysis because of a 118 bp deletion in nucleotide sequence 701–818. Seven EHV-1 isolates (16Q4, 19R166-1, 19R166-6, 19/10/15-2, 19/10/15-4, 19/10/18-2, 19/10/22-1) belonged to group 1, clade 10, based on ORF34 and MLST analysis. The remaining 5 EHV-1 isolates (15Q25-1, 15D59, 16Q5, 16Q40, 18D99) belonged to group 7, clade 6, based on ORF34 and MLST analysis.

Molecular Characterisation of Equine Herpesvirus 1 Isolates from Cases of Abortion, Respiratory and Neurological Disease in Ireland between 1990 and 2017.

Multiple locus typing based on sequencing heterologous regions in 26 open reading frames (ORFs) of equine herpesvirus 1 (EHV-1) strains Ab4 and V592 was used to characterise 272 EHV-1 isolates from 238 outbreaks of abortion, respiratory or neurological disease over a 28-year period. The analysis grouped the 272 viruses into at least 10 of the 13 unique long region (UL) clades previously recognised. Viruses from the same outbreak had identical multi-locus profiles. Sequencing of the ORF68 region of EHV-1 isolates from 222 outbreaks established a divergence into seven groups and network analysis demonstrated that Irish genotypes were not geographically restricted but clustered with viruses from all over the world. Multi-locus analysis proved a more comprehensive method of strain typing than ORF68 sequencing. It was demonstrated that when interpreted in combination with epidemiological data, this type of analysis has a potential role in tracking virus between premises and therefore in the implementation of targeted control measures. Viruses from 31 of 238 outbreaks analysed had the proposed ORF30 G2254/D752 neuropathogenic marker. There was a statistically significant association between viruses of the G2254/D752 genotype and both neurological disease and hypervirulence as defined by outbreaks involving multiple abortion or neurological cases. The association of neurological disease in those with the G2254/D752 genotype was estimated as 27 times greater than in those with the A2254/N752 genotype.

Genetic characterization of equine herpesvirus 1 isolates from abortion outbreaks in India.

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 (A2254 to G2254). 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 (A2254), while two (3.57%) had the neuropathogenic marker (G2254). 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 C710 and C713, 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.

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

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

Equine Herpesvirus-1 Myeloencephalopathy, an Emerging Threat of Working Equids in Ethiopia.

Although equine herpesvirus myeloencephalopathy (EHM) is a sporadic and relatively uncommon manifestation of equine herpesvirus-1 (EHV-1), it has the potential for causing devastating outbreaks in horses. Up till now, there were no reported EHM outbreaks in donkeys and mules. This study describes the isolation and molecular characterization of EHV-1 from clinically EHM-affected horses (n=6), mules (n=3) and donkeys (n=82) in Ethiopia during outbreaks from May 2011 to December 2013. The incidence of EHM cases was higher from April to mid-June. EHM in donkeys was more severe and death without clinical signs of paralysis, and recumbency was frequently observed. The main age of affected equines ranged from 7 to 10years (n=51; 56.0%), and females (n=58; 63.7%) were more affected than males. The incidence of neuropathogenic (D752 ) and non-neuropathogenic (N752 ) variants of EHV-1 from EHM-affected equines in Ethiopia was assessed by sequencing the DNA polymerase gene (ORF30) of the EHV-1 isolates. The results indicated that from the total of 91 clinically affected equines, 90 (98.9%) of them had an ORF30 D752 genotype. An ORF30N752 variant was only found in one donkey. Analysis of ORF68 as grouping marker for geographical differences showed that the Ethiopian EHV-1 isolates belong to geographical group 4. Due to the fatal nature of EHV-1 in donkeys, it would be interesting to examine the pathogenesis of EHM in this species. At present, there is no vaccine available in Ethiopia, and therefore, outbreaks of EHV-1 should be controlled by proper management adaptations. In addition, it is important to test the efficacy of the commercial vaccines not only in horses, but also in donkeys and mules.

Molecular characterisation of the ORF68 region of equine herpesvirus-1 strains isolated from aborted fetuses in Hungary between 1977 and 2008

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.

First detection of the equine herpesvirus 1 neuropathogenic variant in Brazil

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G2254/D752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A2254/N752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Prevalence of latent, neuropathogenic equine herpesvirus‐1 in the Thoroughbred broodmare population of central Kentucky

An emerging problem of equine herpesvirus-1 (EHV-1) infection in horses in the USA is a high-mortality myeloencephalopathy that commonly occurs where large numbers of horses are stabled. EHV-1 isolates recovered from recent neurological outbreaks represent a mutant virus strain that possesses enhanced neuropathogenicity. A central question of EHV-1 myeloencephalopathy is the latency carriage rate for these mutants of EHV-1 in USA horse populations. To estimate the prevalence of neuropathogenic strains of EHV-1 as latent infections in the Thoroughbred broodmare population of central Kentucky. Submandibular lymph nodes (SMLN) were collected during post mortem examination of 132 Thoroughbred broodmares. Total DNA purified from SMLN tissue was tested for the presence of latent EHV-1 DNA by an ultrasensitive magnetic bead-based, sequence-capture, nested PCR method. Differentiation of active from latent infections by EHV-1 was achieved by detection of transcripts of EHV-1 glycoprotein B by reverse transcription PCR. Latent EHV-1 DNA was detected in the SMLN tissues of 71 (54%) of the 132 mares submitted for necropsy. Thirteen (18%) of the 71 latently infected horses harboured the neuropathogenic biovar of EHV-1. Of the 13 horses latently infected with an ORF30 mutant strain of EHV-1, 11 also carried a latent, wild-type strain of the virus in their SMLN tissues. Neuropathogenic strains of EHV-1 have established a significant presence in the Thoroughbred broodmare population of central Kentucky as latently infected carrier horses. The data also indicate that a highly sensitive DNA detection method is required to identify many instances of EHV-1 latency. The presence of a relatively large biological reservoir of latent, neuropathogenic EHV-1 has the potential for posing emerging equine health and economic threats to the future prosperity of the USA horse industry.

Molecular Characterization of Equine Herpesvirus 1 (EHV‐1) Isolated from Cattle Indicating No Specific Mutations Associated with the Interspecies Transmission

Interspecies trasmission of equine herpesvirus 1 (EHV-1) from horse to cattle was shown by Crandell et al. (1988). Specific mutations related to the transmission were studied by comparison of five EHV-1 isolates in cattle (BH1247, 3M20-3, G118, G1753, and 9BSV4) using polymerase chain reaction and restriction fragment length polymorphism analysis with added sequencing. G118 and 3M20-3 were the genome type EHV-1 P, while G1753 was the genome type EHV-1 B. BH1247 and 9BSV4 might be other genome types. We could not identify specific mutations related to the interspecies transmission.

Use of polarised equine endothelial cell cultures and an in vitro thrombosis model for potential characterisation of EHV-1 strain variation

Equine herpesvirus-1 (EHV-1) is responsible for respiratory disease and abortion in pregnant mares. Some high virulence isolates of EHV-1 also cause neurological disease. The pathogenesis of both abortion and neurological disease relates in part, to thrombus formation occurring in the pregnant uterus and central nervous system. The differences in disease outcome may relate to differing abilities of high and low virulence EHV-1 isolates to cause cell-associated viraemia, infect endothelial cells and cause thrombosis at sites distant from the respiratory tract. This study attempted to identify in vitro assays, which could be used to characterise the interaction between these isolates, equine endothelial cells and clotting factors. No significant difference was found between the growth kinetics of high and low virulence isolates of EHV-1 in polarised endothelial cells. For both isolates, virus was released preferentially from the apical surface of the polarised cells. The functional effects of viral infection on endothelial cells, with reference to virally-induced thrombosis were then investigated. Endothelial cells were grown on microcarrier beads, infected with EHV-1 and assayed for procoagulant activity. No significant difference in clotting time was observed between mock and EHV-1 infected endothelial cells in microcarrier cultures. Thus the degree of thrombosis may reflect a more complex interaction between endothelial cells, circulating leucocytes and other factors in the microenvironment.

Molecular Variability in Different Indian Isolates of Equine Herpesvirus-1

Three abortigenic Indian isolates of equine herpesvirus-1 (EHV-1) (Tohana, Hisar and Bikaner), along with two exotic abortigenic isolates (AB4 and V592) and another EHV-1 isolate (Jind) obtained from a case of perinatal foal mortality, were studied for variability. For this purpose, PCR and restriction endonuclease (RE) digestion techniques were used simultaneously as a DNA fingerprinting system. Nine different regions of EHV-1 virus were amplified by PCR using primer pairs specific for the regions and the products obtained from these regions were subsequently subjected to various restriction endonucleases to further assess the variability in the number of RE sites as well as in their positions. No difference was observed in all the four abortigenic isolates in terms of the size of different PCR products amplified by all the nine primer pairs, except for primer pairs 'E' and 'C'. PCR products obtained with primer pair E revealed that Tohana and Bikaner isolates were most similar while Hisar isolate was like V592 isolate. However, the PCR product obtained from Jind isolate had a size between the PCR products of Hisar and Tohan/Bikaner isolates. The primer pair 'C' used to amplify the region between 1151 to 3679 in 'Gene 1,2,3' clearly differentiated the EHV-1 isolate obtained from a case of perinatal foal mortality from isolates obtained from abortion cases. This primer pair needs to be exploited more extensively for use as a potential marker for differentiating the EHV-1 isolates, mainly the abortion cases from perinatal foal mortality ones. Restriction endonuclease studies done with PCR product of all the isolates with various primer pairs did not reveal any changes in the position or number of RE sites present in the products amplified, indicating no variation in different RE sites within the amplified PCR products. However, this study clarified that all the Indian isolates belonged to the IP group of EHV-1.

Growth and Virulence Alterations of Equine Herpesvirus 1 by Insertion of a Green Fluorescent Protein Gene in the Intergenic Region between ORFs 62 and 63

Nucleotide sequences of the intergenic region between ORF 62 and ORF 63 of equine herpesvirus 1 (EHV-1) isolates were analyzed. The sequences of this region consisted of variable and conserved domains among EHV-1 isolates. An EHV-1 mutant, Ab4-GFP, was constructed by inserting a green fluorescent protein (GFP) expression cassette flanked by lox P at both ends into the intergenic region between ORF 62 and ORF 63. Another mutant, Ab4-loxP, which contains one lox P site, was constructed by excision of the GFP cassette from the Ab4-GFP virus genome by cre enzyme. The recombinant Ab4-GFP formed smaller plaques than the wild type in MDBK cells. Virus production also decreased for Ab4-GFP in multistep growth analyses. Virulence of Ab4-GFP in both mice and hamsters was weaker than that of the wild type. Ab4-loxP exhibited properties similar to those of the wild type. These results suggest that the intergenic region between ORF 62 and ORF 63 plays various roles in the virus growth.

The Molecular Character and Comparison with German EHV-1 Viruses of Equine Herpes Virus Isolated from izmit Region

Equine Herpes Virus 1 (EHV-1) that was isolated Turkey, Izmit region has compared with 5 isolates of EHV-1 in Germany. When all of the isolates culturated in the cells of EEL and Ed-min, they had shown typical cytopathic effects of EHV-1. The DNA of these six EHV-1 isolates had been divided by the restriction enzymes which are BamHI, BgIII and ECoRI and investigated in gel electrophoresis. In the all of the isolates, section examples that had discovered by this 3 enzymes resemble each other.

Development of PCR assays to detect genetic variation amongst equine herpesvirus-1 isolates as an aid to epidemiological investigation.

A search for variable restriction sites has been carried out for equine herpesvirus-1 (EHV-1) in an attempt to develop markers which can be used to group epidemiologically related viruses into groups, and to learn more about the dynamics of EHV-1 disease. Crude viral DNA extracts of EHV-1, prepared by Hirt extraction, were digested with AluI, HaeIII, or RsaI, and Southern blotted following electrophoresis. DNA fingerprints, produced by probing the Southern blots with the EHV-1 EcoR1-I fragment, separated 56 isolates into 16 groups. The variable sites within the EcoR1-I fragment were mapped approximately using fragments from within EcoR1-I, and the precise location of the variable sites determined from the DNA sequence of this fragment. Oligonucleotide primers flanking the variable sites were synthesized, and used in PCR assays to detect variable fragments. The AluI variable fragment was found to result from the presence or absence of a single AluI site. In contrast, the variable bands seen with HaeIII and RsaI, resulted from variation in the copy number of two tandemly repeated sequences, one of which had not previously been recognized. In addition, HaeIII digests of EHV-1 isolates probed with the glycoprotein B (gB) gene of EHV-1 also separated isolates into two groups. The variable HaeIII site was mapped towards the 5'-end of the gB gene and a PCR assay established. The distribution of the variable AluI site within the EcoR1-I fragment and the HaeIII site within the gB gene were estimated on a large number of clinical isolates using PCR on unpurified viral tissue culture medium. Both sites had a good distribution and together with additional variable sites should provide the basis for the rapid DNA fingerprinting of EHV-1 isolates.

The genomic diversity among equine herpesvirus-1 strains isolated in Japan.

The DNAs from nine Japanese field isolates of equine herpesvirus-1 (EHV-1) were analyzed by digestion with the restriction endonuclease Bam HI and Southern hybridization. Comparing restriction profiles among the EHV-1 strains, there was no considerable difference between isolates before and after vaccine application, but some minor variations in the mobility of Bam HI fragments were observed. To identify these variable fragments, all genomic DNA sequences of the Japanese prototype of EHV-1 have been cloned as Bam HI restriction fragments into the plasmid pUC-18. Physical maps of the virus DNA were constructed by a combination of Southern blot analysis and double enzyme digestion of the cloned fragments. By using these cloned fragments as probes in Southern blot analysis, the areas of heterogeneity observed among the field EHV-1 isolates were located in both terminals of UL, the center of UL, IR, US and TR regions of the genome.

Alterations in the equine herpesvirus 1 genome after in vitro and in vivo virus passage.

The effect of in vitro and in vivo serial virus passage on the genetic stability of equine herpesvirus 1 (EHV-1) was investigated by restriction endonuclease analysis of the viral DNA. DNAs of EHV-1 isolates at different passage levels in cultured cells or in Syrian hamsters were compared by electrophoresis of the DNA cleavage fragments produced by restriction endonuclease digestion. No changes were observed in the restriction profile of the DNAs of EHV-1 strains after 100 sequential passages in cultured equine cells. However, serial passage of the virus in hamsters or in cells of non-equine origin quickly gave rise to alterations in the viral DNA. These changes occurring in the restriction endonuclease profiles of EHV-1 DNA during serial virus passage in non-equine cells or animals hosts could be explained by sequence additions or deletions to preexisting restriction fragments resulting in variation in their electrophoretic mobilities.