Detection of equine arteritis virus (EAV)-specific cytotoxic CD8+ T lymphocyte precursors from EAV-infected ponies.

Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a (51)Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain (51)Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8(+)/CD4(+) ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8(+) T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus-host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection.

In vitro characterisation of high and low virulence isolates of equine herpesvirus-1 and -4.

Basic in vitro characteristics of high and low virulence isolates of equine herpesviruses-1 and -4 were investigated with particular reference made to the Ab4 and V592 isolates of EHV-1 as both have distinct endotheliotropism and clinical outcomes in pony challenge studies. Additionally, some EHV-4 isolates that showed variations in clinical outcome were included in some experiments. The aim of the study was to identify an in vitro characteristic that would differentiate strains of known virulence. Such a system could then be applied to vaccine and virulence studies as an effective screening tool. Viral growth kinetics in a variety of cell culture systems, plaque size, ability to replicate in fetal endothelium in organ culture, and sensitivity to acyclovir were compared. No reliable marker system that differentiated between higher and lower virulence isolates of EHV-1 and EHV-4 was identified.

Evaluation of a prototype sub-unit vaccine against equine arteritis virus comprising the entire ectodomain of the virus large envelope glycoprotein (G(L)): induction of virus-neutralizing antibody and assessment of protection in ponies.

An Escherichia coli-expressed recombinant protein (6hisG(L)ecto) comprising the entire ectodomain (aa 18-122) of equine arteritis virus (EAV) glycoprotein G(L), the immunodominant viral antigen, induced higher neutralizing antibody titres than other G(L)-derived polypeptides when compared in an immunization study in ponies. The potential of the recombinant G(L) ectodomain to act as a sub-unit vaccine against EAV was evaluated further in three groups of four ponies vaccinated with doses of 35, 70 or 140 microg of protein. All vaccinated animals developed a virus-neutralizing antibody (VNAb) response with peak titres 1-2 weeks after the administration of a booster on week 5 (VNAb titres of 1.8-3.1), 13 (VNAb titres of 1.4-2.9) or 53 (VNAb titres of 1.2-2.3). Vaccinated and unvaccinated control ponies were infected with EAV at different times post-vaccination to obtain information about the degree of protection relative to the levels of pre-challenge VNAb. Vaccination conferred varying levels of protection, as indicated by reduced or absent pyrexia, viraemia and virus excretion from the nasopharynx. The degree of protection correlated well with the levels of pre-challenge VNAb and, in particular, with levels of virus excretion. These results provide the first evidence that a sub-unit vaccine protects horses against EAV. The use of the sub-unit vaccine in combination with a differential diagnostic test based on other EAV antigens would enable serological discrimination between naturally infected and vaccinated equines.

Virulence of the V592 isolate of equid herpesvirus-1 in ponies.

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.

Replication of equid herpesvirus-1 (EHV-1) in the testes and epididymides of ponies and venereal shedding of infectious virus.

Six Welsh Mountain pony colts were infected intranasally with the Ab4 isolate of EHV-1. Clinical and virological monitoring demonstrated mild upper respiratory tract disease, with nasal shedding of virus and establishment of a cell-associated viraemia. Detailed pathological examination of the urogenital tract was performed post mortem on days 4-9 post-infection (PI). EHV-1 was isolated from the epididymis on day 8 and the testis on day 9 PI, with viral replication in endothelial cells of these organs and an associated necrotizing vasculitis and thrombosis. Productive viral infection of germinal epithelium was not observed. In a further study, three Welsh Mountain pony stallions were infected intranasally with Ab4, which again resulted in mild upper respiratory tract disease and the establishment of a cell-associated viraemia. Semen samples were collected up to day 60 PI. Two stallions showed a decrease in the proportion of morphologically normal sperm. Significant numbers of inflammatory cells were observed in the sperm-rich fraction of ejaculates collected from one stallion between days 16 and 28 PI; infectious virus was recovered from the semen of this animal between days 17 and 25 PI, after the cessation of viraemia. The affected stallion appeared clinically normal over the period of venereal EHV-1 shedding.

Replication of equid herpesvirus-1 in the vaginal tunics of colts following local inoculation.

Equid herpesvirus-1 (EHV-1; Ab4 isolate) was inoculated unilaterally into the cavum vaginale of four pony colts under general anaesthesia. The animals were monitored daily for evidence of scrotal or testicular swelling and euthanased electively on days 3, 4, 6 and 12 after infection. Detailed pathological examination of the male genital tract was carried out. In animals examined at days 3 and 4 after infection, replication of EHV-1 was detected bilaterally in mesothelial and endothelial cells of the parietal and visceral vaginal tunics. The mesothelial infection had resolved by day 12 after infection, with no evidence of direct extension to deeper genital organs. None of the four colts showed significant scrotal or testicular swelling.

An immunohistological study of the uterus of mares following experimental infection by equid herpesvirus 1.

Twelve Welsh Mountain pony mares in late gestation were infected intranasally with EHV-1 (AB4 isolate) at dose rates from 10(3) to 10(7.3) TCID50. This resulted in 3 cases of paresis, at Days 9, 10 and 12 after inoculation, and 5 abortions, at Days 6, 9, 18, 19 and 20. Euthanasia was performed between Days 6 and 21, with collection of uterine specimens for histopathology, virus isolation and immunoperoxidase staining from the pregnant horn, non-pregnant horn and body. EHV-1 replication in endometrial vessels was detected as early as Day 6 and was maximal at Days 9-11, when widespread thromboischaemic damage was present. By Days 15-19 in mares remaining pregnant, EHV-1 antigen expression in the endometrium was sparse, despite residual lesions but little associated thrombosis. Endometrial vascular pathology varied considerably in degree and extent, and no consistent predilection sites for replication within the uterus were apparent.