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1.
ABSTRACT: Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) glycoprotein H (gH) has been hypothesized to play a role in direct fusion of the virus envelope with cellular membranes. To investigate gH's role in infection, an EHV-1 mutant lacking gH was created and the gH genes were exchanged between EHV-1 and EHV-4 to determine if gH affects cellular entry and/or host range. In addition, a serine-aspartic acid-isoleucine (SDI) integrin-binding motif present in EHV-1 gH was mutated as it was presumed important in cell entry mediated by binding to alpha4beta1 or alpha4beta7 integrins. We here document that gH is essential for EHV-1 replication, plays a role in cell-to-cell spread and significantly affects plaque size and growth kinetics. Moreover, we could show that alpha4beta1 and alpha4beta7 integrins are not essential for viral entry of EHV-1 and EHV-4, and that viral entry is not affected in equine cells when the integrins are inaccessible.  相似文献   

2.
Some aspects of the biology of equine herpesvirus type 2 (EHV-2) were investigated by examination of the persistent cell-associated viremia stage of the infection. The EHV-2 infection of leukocytes was latent, because free virus was not retrieved without first cultivating harvested leukocytes in vitro. A virus infective center (IC) assay was developed to enumerate latently infected cells in the leukocyte population. This assay proved to be simple and reproducible and revealed a linear relationship between IC plaques formed and the number of cells inoculated, except where large numbers of cells (greater than 4 X 10(6)) were inoculated per 10 cm2 dish. This reduction at high cell densities of IC/10(6) cells inoculated was dependent on cells obtained from an EHV-2-infected horse. There was considerable variation in the numbers of IC/10(6) leukocytes harvested from different horses, but little variation in the harvests from the same horse at different times. There seemed to be a direct relationship between serum-neutralization titers and IC numbers. Transfer of viable infected leukocytes to 2 fetuses failed to establish EHV-2 infection. Infection of equine fetal kidney cells with EHV-2 virus failed to produce detectable Fc receptors on the cell surface.  相似文献   

3.
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   

4.
A silent cycle of equine herpesvirus 1 infection was described following epidemiological studies of unvaccinated mares and foals on a Hunter Valley stud farm. Following the introduction of routine vaccination with an inactivated whole virus equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4) vaccine in 1997, a subsequent study identified excretion of EHV-1 and EHV-4 in nasal swab samples tested by PCR from vaccinated mares and their unweaned, unvaccinated foals. The current sero-epidemiological investigation of vaccinated mares and their young foals found serological evidence of EHV-1 and EHV-4 infection in mares and foals in the first 5 weeks of life. The results further support that EHV-1 and EHV-4 circulate in vaccinated populations of mares and their unweaned foals and confirms the continuation of the cycle of EHV-1 and EHV-4 infection.  相似文献   

5.
The mucosal surfaces are important sites of entry for a majority of pathogens, and viruses in particular. The migration of antigen presenting cells (APCs) from the apical side of the mucosal epithelium to the lymph node is a key event in the development of mucosal immunity during viral infections. However, the mechanism by which viruses utilize the transmigration of these cells to invade the mucosa is largely unexplored. Here, we establish an ex vivo explant model of monocytic cell transmigration across the nasal mucosal epithelium and lamina propria. Equine nasal mucosal CD172a+ cells (nmCD172a+ cells), blood-derived monocytes and monocyte-derived DCs (moDCs) were labeled with a fluorescent dye and transferred to the apical part of a polarized mucosal explant. Confocal imaging was used to monitor the migration patterns of monocytic cells and the effect of equine herpesvirus type 1 (EHV-1) on their transmigration. We observed that 16–26% of mock-inoculated nmCD172a+ cells and moDCs moved into the nasal epithelia, and 1–7% moved further in the lamina propria. The migration of EHV-1 inoculated monocytic cells was not increased in these tissues compared to the mock-inoculated monocytic cells. Immediate early protein positive (IEP+) cells were observed beneath the basement membrane (BM) 48 hours post addition (hpa) of moDCs and nmCD172a+ cells, but not blood-derived monocytes. Together, our finding demonstrate that monocytic cells may become infected with EHV-1 in the respiratory mucosa and transport the virus from the apical side of the epithelium to the lamina propria en route to the lymph and blood circulation.  相似文献   

6.
The prevalence of equine respiratory virus infections among a suspected population of race horses was examined using polymerase chain reaction (PCR). One or more of five equine respiratory viruses were detected in the nasal swabs of 45 of 89 horses (50.6%), and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 5 (EHV-5), equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) were 5.6%, 7.9%, 39.0%, 2.2%, and 6.7%, respectively. Among the 45 infected horses, 7 were co-infected with EHV and/or equine rhinitisvirus (ERV). Equine influenzavirus and equine arteritisvirus were not detected in any samples. Specific antibodies to EHV-1 and/or EHV-4 were detected in 59 of 73 tested sera (80.8%), using a virus neutralization test. This investigation suggests that equine respiratory viruses are endemic at Seoul Race Park and that the impact of viral infections on race horses’ health in Republic of Korea should be evaluated.  相似文献   

7.
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8.
The immune response in horses following experimental infection with equine herpesvirus type 1 (EHV-1) was assessed by measuring cytotoxicity for EHV-1-infected target cells. A technique was developed, using [125I]5-iodo-2'-deoxyuridine ([125I]IUDR)-labeled equine fetal kidney cells infected with EHV-1 as the target cells. It was shown that peripheral blood leukocytes from a recovered horse were capable of lysing target cells, as measured by the loss of radio-active label. Following the experimental infection of specific-pathogen-free ponies with EHV-1, cytotoxicity was obtained with fresh autologous serum, peripheral blood leukocytes in autologous serum, and washed peripheral blood leukocytes. Cytotoxicity of the serum and peripheral blood leukocytes was detected as early as one day after infection. It is suggested that cytotoxic antibodies or cells could play an important part in restricting virus spread after infection of the horse with EHV-1.  相似文献   

9.
REASONS FOR PERFORMING STUDY: EHV-1 and EHV-4 abortion diagnosis is based upon detailed examination of the aborted fetus. However, in some cases, only the placenta is available for examination. Furthermore, the contribution of lesions in the placenta to pathogenesis and diagnosis of EHV-1 and EHV-4 abortion has been neglected. OBJECTIVES: To assess the utility of placental examination in equine herpesvirus-1 (EHV-1) and EHV-4 abortion diagnosis. METHODS: Sections of allantochorion from 49 herpesvirus abortions were analysed by PCR, in situ hybridisation and immunostaining. RESULTS: Virus-specific nested PCR confirmed the presence of viral DNA in 46 cases; 41 cases were EHV-1-positive and 5 EHV-4-positive. Microscopic changes were nonspecific. Examination of the PCR-positive sections of allantochorion revealed EHV-1 DNA by in situ hybridisation (ISH) in 21 cases and EHV-4 in 4 cases. In 2 samples, DNA of both viruses was present on PCR and ISH. Viral antigen was found by immunohistology in 15 cases. Regarding the localisation of virus in the placentae, both viral DNA and antigen of EHV-1 and EHV-4 were found in endothelial cells of chorionic villi and, occasionally, in trophoblast epithelium. In the stromal endothelium, only EHV-1 was found. CONCLUSIONS: The data indicate that examination of placentae is a useful diagnostic aid in EHV-1 and EHV-4 abortion diagnosis. POTENTIAL RELEVANCE: Virological examination of the placenta should become standard practice in equine abortion investigations, particularly in those cases where the fetus is not available for examination.  相似文献   

10.
Fifteen unweaned thoroughbred foals, born on a stud farm to vaccinated mares, were clinically monitored during their first six months of life and repeatedly tested for equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4). Nasopharyngeal swabs and blood samples were collected and screened respectively by PCR and seroneutralisation to detect the presence of the virus, explore its role as a possible cause of respiratory disease, and to assess the efficiency of the pcr for the diagnosis of this disease. The foals were divided into three groups on the basis of their clinical signs and whether they had seroconverted to EHV-1 and/or EHV-4: first, foals with no clinical signs of disease that had not seroconverted; secondly, foals with clinical signs that had seroconverted, and thirdly, foals with clinical signs that had not seroconverted. The results indicated that the viruses circulated on the stud farm despite stringent vaccination regimens against them, and confirmed their association with respiratory disease. The absence of significantly different pcr results among the three groups of foals showed that the pcr was effective in confirming the circulation of the viruses on the premises without being particularly helpful as a diagnostic tool.  相似文献   

11.
We have shown previously that equine herpesvirus 1 (EHV-1) glycoprotein D (gD) DNA elicited protective immune responses against EHV-1 challenge in murine respiratory and abortion models of EHV-1 disease. In this study, 20 horses, all with pre-existing antibody to EHV-4 and two with pre-existing antibody to EHV-1, were inoculated intramuscularly with three doses each of 50, 200 or 500microg EHV-1 gD DNA or with 500microg vector DNA. In 8 of 15 horses, inoculation with EHV-1 gD DNA led to elevated gD-specific antibody and nine horses exhibited increased virus neutralising (VN) antibody titres compared to those present when first inoculated. A lack of increase in gC-specific antibody during the 66 weeks of the experiment showed that the increase in gD-specific antibodies was not due to a natural infection with either EHV-1 or EHV-4. The increase in EHV-1 gD-specific antibodies was predominantly an IgGa and IgGb antibody response, similar to the isotype profile reported following natural EHV-1 infection.  相似文献   

12.
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   

13.
In vitro studies demonstrated that most equine herpesvirus 1 (EHV-1)-infected peripheral blood mononuclear cells (PBMC) do not expose viral envelope proteins on their surface. This protects them against antibody-dependent lysis. We examined whether viral envelope proteins are also undetectable on infected PBMC during cell-associated viremia. Further, surface expression of major histocompatibility complex (MHC)-I was examined, since MHC-I assists in making infected cells recognizable for cytotoxic T-lymphocytes (CTL). Four ponies, previously exposed to EHV, and two ponies that had no contact with EHV before, were inoculated with EHV-1. PBMC were collected at different time points up to 28 days post inoculation. Ninety-eight percent of the infected PBMC did not show viral envelope proteins on their surface. Moreover, infected PBMC without surface expression only produced immediate early and, at least, one early protein, ICP22, but not late envelope proteins gB and gM. This indicates that surface expression of viral envelope proteins is absent, simply because the PBMC are in an early phase of infection. The percentage of infected PBMC showing surface expression of MHC-I was similar as observed in non-infected PBMC from the same ponies (80-100%). Therefore, inefficient recognition of EHV-1-infected PBMC by CTLs does not arise from absent surface expression of MHC-I.  相似文献   

14.
The dissemination of equine herpesvirus types 1 (EHV-1) and 4 (EHV-4) among various horse populations in Japan was investigated through the isolation and typing of virus strains from horses with respiratory diseases. Type specific monoclonal antibody pools were used for the typing of isolates. The 42 strains of EHV-1 and 64 strains of EHV-4 were isolated from 4593 nasal swabs and/or blood plasma samples collected from 3326 horses during a period from 1979 to 1990. All the strains of EHV-1 were isolated from racehorses only and during the winter season exclusively, when the epizootic of respiratory diseases occurred among racehorse populations at two Training Centers of the Japan Racing Association. In contrast, the strains of EHV-4 were isolated from horses irrespective of the season, facility, or horse population; foals and yearlings in breeding farms and our institute, rearing horses in rearing farms, and racehorses. Especially, 4 strains of EHV-4 were isolated from plasma samples containing buffy coat cells. We believe this is the first reported case of EHV-4 cell-associated viremia in the world. All 87 strains isolated from aborted fetuses were identified as EHV-1. The results suggest that EHV-1 is responsible for epizootic respiratory diseases in racehorses in the winter and abortion among mares at the late stage of gestation, and that EHV-4 causes respiratory diseases throughout the year among all horse populations.  相似文献   

15.
Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.  相似文献   

16.
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   

17.
Equine herpesviruses type 1 and 4 (EHV-1 and EHV-4) are ubiquitous in the equine population. One of their main properties is their ability to establish life-long latent infections in their hosts even in those with natural or vaccine-induced immunity. However, effect of vaccination status on prevalence and tissue tropism was not established. In this study, EHV-1 and EHV-4 were detected by polymerase chain reaction and by classical virus isolation from neural, epithelial and lymphoid tissues collected from unvaccinated (33) or vaccinated (23) horses. The percentage of EHV-1- and EHV-4-positive horses between vaccinates and unvaccinates was similar. Both viruses were detected in all tissues of both groups; in particular, lymph nodes draining the respiratory tract, nasal epithelium and nervous ganglia [i.e. trigeminal ganglia (TG)], which represent the main positive sites for EHV-1 and EHV-4. In vaccinated animals, the nervous ganglia (i.e. TG) were less frequently positive than in unvaccinated animals. Detection of positive TG was strongly correlated to the presence of EHV-1 in nasal epithelium.  相似文献   

18.
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.  相似文献   

19.
This review concentrates on the epidemiology, latency and pathogenesis of, and the approaches taken to control infection of horses by equine herpesvirus types 1 (EHV-1) and 4 (EHV-4). Although both viruses may cause febrile rhinopneumonitis, EHV-1 is the main cause of abortions, paresis and neonatal foal deaths. The lesion central to these three conditions is necrotising vasculitis and thrombosis resulting from lytic infection of endothelial cells lining blood capillaries. The initiation of infection in these lesions is likely to be by reactivated EHV-1 from latently infected leukocytes. However, host factors responsible for reactivation remain poorly understood. While vaccine development against these important viruses of equines involving classical and modern approaches has been ongoing for over five decades, progress, compared to other alpha herpesviruses of veterinary importance affecting cattle and pigs, has been slow. However recent data with a live temperature sensitive EHV-1 vaccine show promise.  相似文献   

20.
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   

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