Experimental infection with equine herpesvirus type 1 (EHV-1) induces chorioretinal lesions

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.     

Equine herpesvirus-1 infection induces IFN-gamma production by equine T lymphocyte subsets

A commercial bovine IFN-gamma-specific monoclonal antibody was used to measure antigen-specific IFN-gamma production by equine lymphocytes. Paired PBMC samples were collected from six ponies prior to and 10 days after challenge infection with equine herpesvirus-1 (EHV-1). Each sample was stimulated in vitro with EHV-1, virus-free medium, or PMA and ionomycin, and labelled with monoclonal antibodies specific for various equine lymphocyte subset markers. Following fixation, intracellular IFN-gamma was detected using a FITC-conjugated bovine IFN-gamma-specific monoclonal antibody. In vitro restimulation of PBMC with EHV-1 induced IFN-gamma production by a significantly higher percentage of total (CD5(+)) T lymphocytes, and CD4(+) and CD8(+) T lymphocyte subsets among post-EHV-1 infection PBMC samples compared to pre-infection samples. This response was associated with an increase in virus-specific CTL activity, a critical immune effector for the control of EHV-1 infection and disease. No significant increase in IFN-gamma production by B lymphocytes was observed. These data demonstrate that EHV-1 challenge infection of ponies results in increased production of IFN-gamma by virus-specific T lymphocytes, and that this response can be quantitated using flow cytometry.     

Comparison of methods for the diagnosis of equine herpesvirus type 1 infection

The objective of the investigations was to study the occurrence of the equine herpesvirus type 1 (EHV-1) infection in aborted equine fetuses and in newborn foals and to compare the sensitivity of virus isolation, immunohistochemistry and histology in 101 cases and of fetal serology in 68 cases in the diagnosis of the infection. Out of the 93 aborted equine fetuses and 8 weak foals, 15 (14.9%) (14 fetuses and 1 foal) proved to be EHV-1 infected by immunohistochemical and 13 (12.9%) by virological investigation. Characteristic microscopic changes were seen in several organs in all cases, while intranuclear inclusion bodies could be found only in 25 (35.2%) of the 71 virus-positive tissue samples. Four (5.9%) cases proved to be positive by fetal serological investigation, but none of these cases showed any EHV-1 specific lesions and in none of these cases could the virus be detected by virus isolation or by immunohistochemistry. According to the results, fetal serology does not seem to be a useful test in virus-positive cases, while the immunohistochemical method seems to be a reliable and a slightly more sensitive method than virus isolation in the diagnosis of EHV-1 infection.     

Equine Multinodular Pulmonary Fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: a case report

A standardbred gelding with a history of 10 days pyrexia and lethargy was referred to the Equine Hospital at the Swedish University of Agricultural Sciences in Uppsala, Sweden.The horse had tachypnea with increased respiratory effort and was in thin body condition. Laboratory findings included leukocytosis, hyperfibrinogenemia and hypoxemia. Thoracic radiographs showed signs of pneumonia with a multifocal nodular pattern, which in combination with lung biopsy findings indicated Equine Multinodular Pulmonary Fibrosis (EMPF). EMPF is a recently described disease in adult horses with clinical signs of fever, weight loss and respiratory problems. The pathological findings include loss of functional pulmonary parenchyma due to extensive nodular interstitial fibrosis which has been related to infection with the equine herpesvirus type 5 (EHV-5). In this case, lung biopsy and tracheal wash samples tested positive for both asinine herpesvirus type 5 (AHV-5) and EHV-5 using PCR assays. The horse failed to respond to treatment and was euthanized for humane reasons. Postmortem examination confirmed the diagnosis of EMPF. This case suggests that not only EHV-5 alone should be considered in association with the development of this disease.     

Meningoencephalomyelitis in horses associated with equine herpesvirus 1 infection.

During an outbreak of abortion caused by equine herpesvirus 1, a neurologic disease characterized clinically by dullness and ataxia occurred in several mares. Equine herpesvirus 1 was isolated from brain and lung of two severely affected mares. Histologically, both mares had disseminated meningoencephalomyelitis characterized by necrotizing arteritis, focal malacia in grey and white matter of brain and spinal cord, and accumulation of lymphocytes and neutrophils in paravertebral ganglia. Eosinophilic intranuclear inclusion bodies occurred in foci of necrosis in thyroid adenomas of both mares.     

The protective antigens of equine herpesvirus type 1.

Equine herpesvirus type 1 was cultivated in swine testis cell cultures and partially purified by differential centrifugation and centrifugation in a linear sucrose density gradient. The viral envelope was separated from the nucleocapsid by treatment with Rexol 25J followed by sucrose gradient centrifugation. The envelope and nucleocapsid preparations were then electrophoresed in polyacrylamide gel after solubilization with sodium dodecyl sulphate. Hamsters were immunized with various preparations of the partially purified virus, including live or inactivated equine herpesvirus type 1 and viral envelope and nucleocapsid, all derived from the Kentucky D strain of the virus. Challenge of the immunized hamsters, with a hamster-adapted strain of equine herpesvirus type 1 demonstrated protection only in those animals which had been vaccinated with envelope-containing materials. When vaccination was carried out with fractions of electrophoresed envelope or nucleocapsid, protection was induced only by polypeptides of high molecular weight containing a glycoprotein component of the envelope of equine herpesvirus type 1.     

Impact of equine herpesvirus type 1 (EHV-1) infection on the migration of monocytic cells through equine nasal mucosa

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.     

Ataxia and paresis with equine herpesvirus type 1 infection in a herd of riding school horses

An outbreak of neurologic disease associated with serologic evidence of equine herpesvirus type 1 (EHV-1) infection occurred in a herd of 46 riding school horses. Ataxia and paresis were observed in 14 geldings and 5 barren mares. Eight affected horses had distal limb edema, 1 horse had a head tilt, and 3 others had urinary incontinence. Other clinical signs included fever, depression, and inappetance in 30 horses. Seven horses with neurologic signs were treated with acyclovir. Serum neutralizing antibody titers against EHV-1 increased 4-fold between acute and convalescent samples or exceeded 1: 256 in 19 of 44 horses, confirming recent infection. A significantly greater proportion of horses that seroconverted were mares ( P = .014). Of the 19 horses exhibiting ataxia and paresis, 17 made a complete recovery, 1 made a partial recovery, and 1 was euthanized.     

Equine herpesvirus 1 glycoprotein D expressed in E. coli provides partial protection against equine herpesvirus infection in mice and elicits virus-neutralizing antibodies in the horse

The envelope glycoprotein D of EHV-1 (EHV-1 gD) is essential for virus infectivity and entry of virus into cells and is a potent inducer of virus-neutralizing antibody. In this study, truncated EHV-1 gD (gDt) was expressed with a C-terminal hexahistidine tag in E. coli using a pET vector. Western blot analysis using an anti-gD monoclonal antibody demonstrated the presence of gDt bands at 37.5, 36, 29.5 and 28 kDa. The immunogenicity and protective efficacy of partially purified gDt was compared with gD expressed in insect cells by a recombinant baculovirus (Bac gD) using a BALB/c mouse model of EHV-1 respiratory infection. The proteins were also compared in a prime-boost protocol following an initial inoculation with gD DNA. gDt elicited similar levels of gD-specific antibody and neutralizing antibody compared with Bac gD and also provided a similar level of protection against EHV-1 challenge in mice. Inoculation of horses with gDt elicited EHV-1 gD-specific antibodies including virus-neutralizing antibody, suggesting that despite the lack of glycosylation, E. coli may be a useful vehicle for large scale production of EHV-1 gD for vaccine studies.     

Serological evidence of equine herpesvirus type 1 (EHV-1) activity in polo horses in Nigeria.

Serological evidence of Equine Herpes virus type 1 (EHV-1) activity in Polo horses in Nigeria is reported for the first time. Eighty-two percent of horses tested with known antigen had precipitating antibodies to EHV-1 while 43% of sera tested against antigen prepared from nasal discharges were positive suggesting that the virus was being excreted in the nasal discharges and probably acting as a source of infection for incontact animals as occurs in on-going acute infections. The result of this study indicates a high prevalence of EHV-1 activity among Polo horses in Nigeria and demonstrates the ubiquitous distribution of the virus in a country that has not been previously investigated.