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1.
Infection with Equid Herpesvirus type 1 (EHV-1) leads to respiratory disease, abortion, and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single nucleotide polymorphism (A2254/G2254) in the genome region of the open reading frame 30 (ORF30), which results in an amino acid variation (N752/D752) of the EHV-1 DNA polymerase, is significantly associated with the neuropathogenic potential of naturally occurring strains. In order to estimate the prevalence of the EHV-1 neuropathogenic genotype in our country, we analyzed the ORF30 genome region of Argentinean EHV-1 isolates. The study was carried out by real time allelic discrimination PCR in 90 equine EHV-1-positive samples, being 89 from 54 cases of abortion outbreaks (two of which were in association with neurological disease) and one from the respiratory tract of a healthy horse in training. Our results indicate that 7% (4/54) of the abortion outbreaks studied were induced by the neuropathogenic (G2254) genotype of EHV-1 and 50% (2/4) of them were associated with simultaneous neurological disease. This information emphasizes the necessity to extreme the hygienic and preventive measures to diminish EHV-1 infections and consequently reduce the risk of epizootic neurological disease as has been recently observed in other countries.  相似文献   

2.
Latent equine herpesvirus type 1 (EHV-1) infection is common in horse populations worldwide and estimated to reach a prevalence nearing 90% in some areas. The virus causes acute outbreaks of disease that are characterized by abortion and sporadic cases of myeloencephalopathy (EHM), both severe threats to equine facilities. Different strains vary in their abortigenic and neuropathogenic potential and the simultaneous occurrence of EHM and abortion is rare. In this report, we present clinical observations collected during an EHV-1 outbreak caused by a so-called “neuropathogenic” EHV-1 G2254/D752 polymerase (Pol) variant, which has become more prevalent in recent years and is less frequently associated with abortions. In this outbreak with 61 clinically affected horses, 6/7 pregnant mares aborted and 8 horses developed EHM. Three abortions occurred after development of EHM symptoms. Virus detection was performed by nested PCR targeting gB from nasal swabs (11 positive), blood serum (6 positive) and peripheral blood mononuclear cells (9 positive) of a total of 42 horses sampled. All 6 fetuses tested positive for EHV-1 by PCR and 4 by virus isolation. Paired serum neutralization test (SNT) on day 12 and 28 after the index case showed a significant (≥ 4-fold) increase in twelve horses (n = 42; 28.6%). This outbreak with abortions and EHM cases on a single equine facility provided a unique opportunity for the documentation of clinical disease progression as well as diagnostic procedures.  相似文献   

3.
A single non-synonymous nucleotide substitution of guanine (G) for adenine (A) at position 2254 in the viral DNA polymerase gene (encoded by open reading frame [ORF] 30) of equine herpesvirus type 1 (EHV-1) has been significantly associated with neuropathogenic potential in strains of this virus. To estimate the prevalence of EHV-1 strains with the neuropathogenic genotype (ORF30 G(2254)) in the Hidaka district--a major horse breeding area in Japan--we analyzed the ORF30 genomic region in cases of EHV-1 infection in this area during the years 2001-2010. Of the 113 cases analyzed, 3 (2.7%) were induced by ORF30 G(2254) strains. This prevalence is lower than those observed in the U.S.A. (10.8-19.4%), Argentina (7.4%), France (24%), and Germany (10.6%).  相似文献   

4.
Equine herpesvirus myeloencephalitis (EHM) remains one of the most devastating manifestations of equine herpesvirus type 1 (EHV-1) infection but our understanding of its pathogenesis remains rudimentary, partly because of a lack of adequate experimental models. EHV-1 infection of the ocular vasculature may offer an alternative model as EHV-1-induced chorioretinopathy appears to occur in a significant number of horses, and the pathogenesis of EHM and ocular EHV-1 may be similar. To investigate the potential of ocular EHV-1 as a model for EHM, and to determine the frequency of ocular EHV-1, our goal was to study: (1) Dissemination of virus following acute infection, (2) Development and frequency of ocular lesions following infection, and (3) Utility of a GFP-expressing virus for localization of the virus in vivo. Viral antigen could be detected following acute infection in ocular tissues and the central nervous system (experiment 1). Furthermore, EHV-1 infection resulted in multifocal choroidal lesions in 90% (experiment 2) and 50% (experiment 3) of experimentally infected horses, however ocular lesions did not appear in vivo until between 3 weeks and 3 months post-infection. Taken together, the timing of the appearance of lesions and their ophthalmoscopic features suggest that their pathogenesis may involve ischemic injury to the chorioretina following viremic delivery of virus to the eye, mirroring the vascular events that result in EHM. In summary, we show that the frequency of ocular EHV-1 is 50-90% following experimental infection making this model attractive for testing future vaccines or therapeutics in an immunologically relevant age group.  相似文献   

5.
The objective of this study was to identify and partially characterize 3 equid herpesviruses that were isolated postmortem from zebras in Ontario, Canada in 1989, 2002, and 2007. These 3 virus isolates were characterized by plaque morphology, restriction fragment length polymorphism (RFLP) of their genomic deoxyribonucleic acid (DNA), real-time polymerase chain reaction (PCR) assay, and sequence analyses of the full length of the glycoprotein G (gG) gene (ORF70) and a portion of the DNA polymerase gene (ORF30). The isolates were also compared to 3 reference strains of equid herpesvirus 1 (EHV-1). Using rabbit kidney cells, the plaques for the isolates from the zebras were found to be much larger in size than the EHV-1 reference strains. The RFLP patterns of the zebra viruses differed among each other and from those of the EHV-1 reference strains. Real-time PCR and sequence analysis of a portion of the DNA polymerase gene determined that the herpesvirus isolates from the zebras contained a G at nucleotide 2254 and a corresponding N at amino acid position 752, which suggested that they could be neuropathogenic EHV-1 strains. However, subsequent phylogenetic analysis of the gG gene suggested that they were EHV-9 and not EHV-1.  相似文献   

6.
We compared the growth kinetics of neuropathogenic and nonneuropathogenic equine herpesvirus type 1 (EHV-1) strains in mouse cerebral cortex cells and investigated the relevance of the D/N amino acid change at position 752 of ORF30 in Japanese isolates. Neuropathogenic electropherotype P strains 01c1 and 89c25 exhibited similar growth kinetics to nonneuropathogenic P strain 90c16 in cultured neurons; however, the growth ability of type B strain 97c7 was lower than those of the other strains tested. The amino acid encoded at 752 of ORF30 in 01c1 was asparagic acid; asparagine was encoded in the other EHV-1 strains isolated from Japanese horses. The D/N(752) difference in ORF30 may not be related to replication ability in neurons.  相似文献   

7.
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8.
There has been an increase in outbreaks of neuropathogenic equine herpesvirus-1 (EHV-1) in the United States and Europe. However, the presence and frequency of neuropathogenic EHV-1 in Turkish horses are not known at present. This study aimed to investigate the frequency of EHV-1 and neuropathogenic strains of EHV-1 in the Marmara Region of Turkey. Samples were analyzed for the presence of EHV-1 and neuropathogenic EHV-1 by real-time PCR TaqMan probe assays. Overall detection rate of EHV-1 was 45.5% (51 of 112). The detection rates were 70.5% (24 of 34) in aborted fetuses, 53.3% (8 of 15) in neonatal deads, 66.6% (4 of 6) in foals, 40% (2 of 5) in dead mares, and 25% (13 of 52) in living mares. Overall detection rate of neuropathogenic EHV-1 was 7.8% (4 of 51), and the real-time PCR results were confirmed by sequencing. Neuropathogenic strains of EHV-1 were detected in the brain and lung of two mares with neurological disease but without a history of abortion, in the brain of a foal that died of respiratory disorder, and in the nasal swab from a mare with a history of abortion. On histopathology, nonpurulent meningoencephalitis, hemorrhages, and vasculitis were seen in the brain. In conclusion, results of this study indicated, for the first time, that the neuropathogenic EHV-1 is circulating in the Marmara Region of Turkey. The results of this study also show that the current risk for non-neuropathogenic strains is high, whereas risk for the neuropathogenic EHV-1-G2254 strain seems to be low. As outbreaks of EHV-1 continue in the Marmara region of Turkey, surveillance for neuropathogenic EHV-1 genotype should be maintained.  相似文献   

9.
Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for β-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.  相似文献   

10.
From restriction endonuclease characterization of the DNA of 317 isolates of equine abortion virus (equine herpesvirus-1; EHV-1) from 176 epizootically unrelated outbreaks of equine virus abortion occurring over 24 years in Kentucky, an epizootic pattern and variation of the virus have emerged. Two electropherotypes of EHV-1 (1P and 1B) accounted for greater than 90% of the nonvaccine-related abortion isolates examined. From 1960 to 1981, EHV-1 1P was the predominant isolate circulating in the central Kentucky area and the cause of greater than 80% of EHV-1-related abortions. In 1981, the occurrence of isolate 1B-related abortions began to increase and since 1982, 1B has become the most frequently recovered isolate of EHV-1 from aborted fetuses.  相似文献   

11.
Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late-term abortions and equine herpesvirus myeloencephalitis (EHM). Our understanding of EHM pathogenesis is limited except for the knowledge that EHV-1 infected, circulating peripheral blood mononuclear cells (PBMC) transport virus to the central nervous system vasculature causing endothelial cell infection leading to development of EHM. Our objective was to develop a model of CNS endothelial cell infection using EHV-1 infected, autologous PBMC. PBMCs, carotid artery and brain endothelial cells (EC) from 14 horses were harvested and grown to confluency. PBMC or ConA-stimulated PBMCs (ConA-PBMCs) were infected with EHV-1, and sedimented directly onto EC monolayers ('contact'), or placed in inserts on a porous membrane above the EC monolayer ('no contact'). Cells were cultured in medium with or without EHV-1 virus neutralizing antibody. Viral infection of ECs was detected by cytopathic effect. Both brain and carotid artery ECs became infected when cultured with EHV-1 infected PBMCs or ConA-PBMCs, either in direct contact or no contact: infection was higher in carotid artery than in brain ECs, and when using ConA-PBMCs compared to PBMCs. Virus neutralizing antibody eliminated infection of ECs in the no contact model only. This was consistent with cell-to-cell spread of EHV-1 infection from leucocytes to ECs, demonstrating the importance of this mode of infection in the presence of antibody, and the utility of this model for study of cellular interactions in EHV-1 infection of ECs.  相似文献   

12.
A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.  相似文献   

13.
Equine herpesvirus type 1 (EHV-1) is responsible for respiratory disorders, abortion and myeloencephalopathy (EHM) in horses. Two pathotypes of EHV-1 strains are circulating in the field: neurovirulent (N) and non-neurovirulent (NN). For both strains, CD172a+ monocytic cells are one of the main carrier cells of EHV-1 during primary infection, allowing the virus to invade the horse’s body. Recently, we showed that EHV-1 NN strains showed a restricted and delayed replication in CD172a+ cells. Here we characterize the in vitro replication kinetics of two EHV-1 N strains in CD172a+ cells and investigate if the replication of these strains is similarly silenced as shown for EHV-1 NN strains. We found that EHV-1 N replication was restricted to 7–8% in CD172a+ cells compared to 100% in control RK-13 cells. EHV-1 N replication was not delayed in CD172a+ cells but virus production was significant lower (103.0 TCID50/105 inoculated cells) than in RK-13 cells (108.5 TCID50/105 inoculated cells). Approximately 0.04% of CD172a+ cells produced and transmitted infectious EHV-1 to neighbour cells compared to 65% of RK-13 cells. Unlike what we observed for the NN strain, pretreatment of CD172a+ cells with histone deacetylases inhibitors (HDACi) did not influence the replication of EHV-1 N strains in these cells. Overall, these results show that the EHV-1 replication of N strains in CD172a+ cells differs from that observed for NN strains, which may contribute to their different pathogeneses in vivo.  相似文献   

14.
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.  相似文献   

15.
Equine herpes myeloencephalopathy (EHM), although a relatively uncommon manifestation of equine herpesvirus-1 (EHV-1) infection, can cause devastating losses on individual farms or boarding stables. Although outbreaks of EHM have been recognized for centuries in domestic horse populations, many aspects of this disease remained poorly characterized. In recent years, an improved understanding of EHM has emerged from experimental studies and from data collected during field outbreaks at riding schools, racetracks and veterinary hospitals throughout North America and Europe. These outbreaks have highlighted the contagious nature of EHV-1 and have prompted a re-evaluation of diagnostic procedures, treatment modalities, preventative measures and biosecurity protocols for the disease. This review concentrates on these and other selected, clinically relevant aspects of EHM.  相似文献   

16.
Although prevention of feline calcivirus (FCV) infection by vaccination has been attempted, and isolation of FCV, development of the disease, and a few fatal cases in vaccinated cats have been reported. Fifteen FCV strains isolated from cats that had been vaccinated with commercially available FCV vaccines (F9, FCV-255, and FC-7) were genogrouped. Molecular analysis of viral genomes involved the construction of a phylogenetic tree of capsid genes using the NJ method. Cat anti-F9 serum and rabbit anti-FCV-255 serum were used for virus neutralization tests. Molecular phylogenetic analysis of the amino acid sequences of 15 virus isolates and those of the previously published and GenBank-deposited 9 global and 14 Japanese strains showed that 8 (53%) of the 15 virus isolates as well as the vaccine strains F9 and FCV-255 belonged to genogroup I (GAI), and 7 (47%) belonged to genogroup II (GAII). Of the 8 GAI strains, 2 were isolated from cats that had been vaccinated with an F9 strain live vaccine, 5 from cats vaccinated with an FCV-255-derived vaccine, and 1 from a cat vaccinated with an FC-7-derived vaccine. Of the 7 GAII strains, 5 were isolated from cats that had been vaccinated with the F9 strain live vaccine, 1 from a cat vaccinated with the FCV-255-derived vaccine, and 1 from a cat vaccinated with the FC-7-derived vaccine. These results indicate that more vaccine breakdown strains isolated from the cats vaccinated with the F9 strain-derived vaccine belong to GAII than to GAI, whereas more vaccine breakdown strains isolated from the cats vaccinated with the FCV-255 strain-derived vaccine belong to GAI than to GAII, and that when the FC-7 strain-derived vaccine is used, the vaccine breakdown strains belong almost equally to GAI and GAII. Thus, the genogroups of virus isolates varied with the vaccine strain used (p < 0.05). On the other hand, the neutralizing titres of feline anti-F9 serum and rabbit anti-FCV-255 serum against the 15 isolates were very low, showing no relationships between neutralizing antibody titres and genogroups. The DNA sequence identities between the virus isolates and the vaccine strains were low, at 70.6–82.9%, and no strains were found to have sequences derived from the vaccine strains. Alignment of amino acid sequences showed that the GAI or GAII virus isolates from the F9-vaccinated cats differed at position 428 of the 5’ hypervariable region (HVR) of capsid region of the F9 strain, whereas those from the FCV-255-vaccinated cats differed at positions 438, 453, and 460 of the 5’HVR of capsid region E of the F9 strain. We speculate that these differences influence genogrouping. The amino acid changes within the F9 linear epitopes common to G A I and G A II were noted at positions 450, 451, 457 of 5’HVR of the capsid region E in the isolates from F9-derived vaccine-treated cats, and 449, 450, and 451 of 5’HVR of capsid region E in the isolates from FCV-255-derived vaccine-treated cats, suggesting that these amino acid changes are involved in escapes. These results suggest that alternate vaccination with the F9 and FCV-255 strains or the use of a polyvalent vaccine containing GAII strains serves to inhibit development.  相似文献   

17.
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.  相似文献   

18.
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19.
Equine herpesvirus type 1 (EHV-1) replicates extensively in the epithelium of the upper respiratory tract, after which it can spread throughout the body via a cell-associated viremia in mononuclear leukocytes reaching the pregnant uterus and central nervous system. In a previous study, we were able to mimic the in vivo situation in an in vitro respiratory mucosal explant system. A plaquewise spread of EHV-1 was observed in the epithelial cells, whereas in the connective tissue below the basement membrane (BM), EHV-1-infected mononuclear leukocytes were noticed. Equine herpesvirus type 4 (EHV-4), a close relative of EHV-1, can also cause mild respiratory disease, but a cell-associated viremia in leukocytes is scarce and secondary symptoms are rarely observed. Based on this striking difference in pathogenicity, we aimed to evaluate how EHV-4 behaves in equine mucosal explants. Upon inoculation of equine mucosal explants with the EHV-4 strains VLS 829, EQ(1) 012 and V01-3-13, replication of EHV-4 in epithelial cells was evidenced by the presence of viral plaques in the epithelium. Interestingly, EHV-4-infected mononuclear leukocytes in the connective tissue below the BM were extremely rare and were only present for one of the three strains. The inefficient capacity of EHV-4 to infect mononuclear cells explains in part the rarity of EHV-4-induced viremia, and subsequently, the rarity of EHV-4-induced abortion or EHM.  相似文献   

20.
Equine herpesvirus type 1 (EHV-1) is associated with abortions, respiratory distress, and neurological disturbances in horses. The ORF37 of EHV-1 encodes a protein homolog to UL24 gene product of human herpesvirus that has been associated with neurovirulence. In the present work, ORF37 PCR fragments derived from two Brazilian EHV-1 isolates, a German isolate and an American reference strain were sequenced and characterized by molecular phylogenetic analysis. This genomic region is highly conserved an allowed to infer genetic distances between EHV-1 strains and other animal herpesvirus.  相似文献   

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