Horserace Betting Levy Board recommendation for the 1993 covering season. Common code of practice for equine viral arteritis.
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Animals↗
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Clinical, virological and serological responses of donkeys to intranasal inoculation with the KY-84 strain of equine arteritis virus.
The clinical, virological and serological responses of seven female donkeys (Equus asinus) to inoculation with the KY-84 strain of equine arteritis virus (EAV), a strain that causes moderate to severe clinical signs in horses, was investigated. In the donkeys, the only clinical signs observed were fever (mainly 3-9 days after inoculation), mild depression in four animals, and a slight nasal or ocular discharge in three. All of the donkeys became infected with EAV as shown by recovery of the virus for periods of up to 14 days from the nasopharynx and buffy coat and, in three out of four donkeys sampled, from the cervix or vagina. Virus replication in the donkey appeared to mirror that previously described for the horse. The donkeys had "sero-converted" to EAV by the 10th day after inoculation. Additional studies are needed to obtain a better understanding of the pathogenesis of EAV in donkeys.
Administration, Intranasal↗
Resistance of castrated male horses to attempted establishment of the carrier state with equine arteritis virus.
Twelve geldings all became infected when inoculated intranasally with the KY-84 strain of equine arteritis virus (EAV), a strain previously shown to be capable of establishing the carrier state in the stallion. With the exception of one animal that showed no effects other than pyrexia, all of the geldings developed clinical signs characteristic of equine viral arteritis (EVA). The geldings were febrile for varying periods within the range of 2-10 days after inoculation. Viraemia occurred from day 2 onwards, for periods varying from 9 to at least 19 days. Nasal shedding of virus began 2-4 days after inoculation and persisted for periods ranging from 7-14 days. All geldings "seroconverted" to EAV by day 11, with serum neutralization titres ranging from 8 to 64. The titres ranged from 8 to 32 after 4 weeks. Low concentrations of EAV were detected in the kidney and blood of one gelding killed 30 days after inoculation and in the blood of another killed after 57 days. Virus was not isolated from any tissue or fluid sample collected from the remaining 10 geldings, all of which were killed between days 30 and 148. The findings confirm that persistent EAV infection is unlikely to occur in geldings and support the results of previous studies, which demonstrated that testosterone plays an essential role in the establishment and maintenance of the carrier state.
Animals↗
Relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt.
The relationship between stage of reproductive tract maturity and susceptibility to the experimental establishment of persistent infection with equine arteritis virus (EAV) was investigated in 21 prepubertal and 15 peripubertal colts. Five of six prepubertal colts inoculated intranasally remained infected in the reproductive tract from post-challenge day 28 to 93 and two of six from post-challenge day 120 to 180. No virus was detected in five of these animals killed on post-challenge day 210. Each of two peripubertal colts remained infected in the reproductive tract at post-challenge day 60 and one of nine was found to be persistently infected with EAV 15 months after challenge. These findings confirm that the virus can replicate in the reproductive tract of a significant proportion of colts for a variable period of time after clinical recovery in the absence of circulating concentrations of testosterone equivalent to those found in sexually mature stallions. Long-term persistent infection with EAV does not appear to occur in colts exposed to the virus before the onset of peripubertal development. We suggest that colts should be vaccinated at approximately 6 months of age, before peripubertal development but after the disappearance of maternally acquired antibodies.
Age Factors↗
Pathological changes associated with equine arteritis virus infection of the reproductive tract in prepubertal and peripubertal colts.
The nature and extent of changes associated with equine arteritis virus (EAV) infection of the reproductive tract was documented in 21 prepubertal and 15 peripubertal colts. This study was part of an investigation into the relationship between stage of reproductive tract maturity and susceptibility to the experimental establishment of persistent infection with EAV. After intranasal challenge with a field isolate of EAV, all colts developed clinical signs of equine viral arteritis (EVA) from which they recovered rapidly. Clinical signs during the acute phase consisted of fever, serous to mucopurulent ocular and nasal discharge, oedema of the limbs, scrotum or prepuce, scleral injection, conjunctivitis, icterus, cough, diarrhoea, stiff gait, lethargy, inappetence and depression. At necropsy, the most significant macroscopic lesions included excessive accumulation of fluid within the thoracic and abdominal cavities, lymph node enlargement and oedema of the reproductive tract. Colts killed 7 to 14 days after challenge had acute necrotizing vasculitis involving the testes, epididymides, vasa deferentia, ampullae, prostatic lobes, vesicular glands and bulbourethral glands. Vasculitis was characterized by striking fibrinoid necrosis of small muscular arteries with extravasation of erythrocytes and proteinaceous material into the media, adventitia and perivascular tissues. Colts examined on days 28-180 had lymphocytic and plasmacytic inflammatory cell infiltrates in the lamina propria and muscularis of the epididymides and accessory sex glands. The vascular lesions found during the acute phase of EAV infection contrasted with the multifocal lympho-plasmacytic infiltrates found within the parenchyma of the reproductive tract during the chronic phase. One peripubertal colt was found to be persistently infected with EAV 15 months after challenge. This colt had marked lympho-plasmacytic infiltrates in the ampullae at necropsy.
Aging↗
Venereal infection of mares by equine arteritis virus and use of killed vaccine against the infection.
Venereal infection with equine arteritis virus (EAV) was established in each of seven mares by inoculation via the cervix with 20 ml of viral suspension (> or = 8 x 10(6) plaque-forming units; PFU), following treatment with prostaglandin and oestradiol. A dose of < or = 8 x 10(5) PFU produced infection in only five of eight mares. Serum neutralizing antibody developed in mares manifesting clinical signs of equine viral arteritis (EVA), and a weak antibody was detectable in one apparently healthy mare inoculated with 8 x 10(5) PFU. Virus isolation was demonstrated not only in the buffy coat but also in nasal swabs of infected mares. EAV was isolated frequently from the body tissues of the mares (killed 10 to 34 days post-inoculation) up to day 12, but rarely from the reproductive tissues later than day 12. The virus persisted longest in the splenic and deep inguinal lymph nodes, followed by the spleen and internal iliac lymph nodes. Four mares immunized with a killed vaccine for EVA showed no clinical disease after venereal challenge with EAV; the virus was recovered from the buffy coat of three mares and from the nasal swab of one of them, but not from the remaining animal.
Animals↗
Virulent and avirulent strains of equine arteritis virus induce different quantities of TNF-alpha and other proinflammatory cytokines in alveolar and blood-derived equine macrophages.
Equine arteritis virus (EAV) infects endothelial cells (ECs) and macrophages in horses, and many of the clinical manifestations of equine viral arteritis (EVA) reflect vascular injury. To further evaluate the potential role of EAV-induced, macrophage-derived cytokines in the pathogenesis of EVA, we infected cultured equine alveolar macrophages (AMphi), blood monocyte-derived macrophages (BMphi), and pulmonary artery ECs with either a virulent (KY84) or an avirulent (CA95) strain of EAV. EAV infection of equine AMphi, BMphi, and ECs resulted in their activation with increased transcription of genes encoding proinflammatory mediators, including interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha. Furthermore, the virulent KY84 strain of EAV induced significantly higher levels of mRNA encoding proinflammatory cytokines in infected AMphi and BMphi than did the avirulent CA95 strain. Treatment of equine ECs with the culture supernatants of EAV-infected AMphi and BMphi also resulted in EC activation with cell surface expression of E-selectin, whereas infection of ECs with purified EAV alone caused only minimal expression of E-selectin. The presence of TNF-alpha in the culture supernatants of EAV-infected equine AMphi, BMphi, and ECs was confirmed by bioassay, and the virulent KY84 strain of EAV induced significantly more TNF-alpha in all cell types than did the avirulent CA95 strain. Thus, the data indicate that EAV-induced, macrophage-derived cytokines may contribute to the pathogenesis of EVA in horses, and that the magnitude of the cytokine response of equine AMphi, BMphi, and ECs to EAV infection reflects the virulence of the infecting virus strain.
Animals↗
Lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand RNA viruses.
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Amino Acid Sequence↗
The serologic response of horses to equine arteritis virus as determined by competitive enzyme-linked immunosorbent assays (c-ELISAs) to structural and non-structural viral proteins.
In an effort to further characterize the humoral immune response of horses to equine arteritis virus (EAV), direct and competitive enzyme-linked immunosorbent assays (c-ELISAs) were developed using monoclonal and polyclonal anti-sera to structural (G(L), N and M) and non-structural (nsp1) viral proteins. A nsp1-specific monoclonal antibody was produced to facilitate development of a c-ELISA to this protein. Data obtained using the various c-ELISAs confirm that the M protein is a major target of the antibody response of horses to EAV. However, none of the c-ELISAs that were developed were as sensitive in detecting EAV-specific antibodies in horse sera as the existing serum neutralization test.
Animals↗
Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA.
The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the sequence of reference strain Bucyrus, the analysed samples were 78-100% identical and showed a 84-97% nucleotide identity with Bucyrus isolate. The results demonstrate high levels of genomic heterogeneity among the extracted RNAs, but inside the fragment amplified a well-preserved region of 24 bp was found with only three mismatches in some samples, suggesting that this could be ideal as a probe for RT-PCR-ELISA. The RT-PCR-ELISA assay using the EAV 7 and 8 primer set, has proved to be sensitive, specific and above all directly applicable to semen. Additionally, the short time needed for the overall procedure makes this method suitable for diagnostic purposes.
Animals↗
Development and evaluation of ELISA procedures to detect antibodies against the major envelope protein (G(L)) of equine arteritis virus.
Enzyme-linked immunosorbant assays (ELISAs) were developed for the detection of antibodies against the major envelope glycoprotein (G(L)) of equine arteritis virus (EAV). A 6-Histidine tagged recombinant protein expressing the complete G(L) ectodomain (G(L)-6His), a glutathione-S-transferase recombinant protein expressing amino acids 55-98 of G(L) (G(L)-GST) and an ovalbumin-conjugated synthetic peptide representing amino acids 81-106 of G(L) (G(L)-OVA) were used as diagnostic antigens. An ELISA procedure was developed and optimised for each antigen. The G(L)-OVA and G(L)-6His assays showed the greatest specificity while the G(L)-GST assay was slightly more sensitive that the G(L)-OVA and G(L)-6His assays; results based on the analysis of 50 virus neutralisation positive and 50 virus neutralisation negative sera. The G(L)-OVA ELISA was selected for further evaluation since it was simpler to use than ELISAs based on recombinant antigens and did not suffer from background reactivity. The final sensitivity and specificity of the G(L)-OVA ELISA were 96.75 and 95.6%, respectively, results based on the analysis of 400 virus neutralisation positive and 400 virus neutralisation negative sera. It also detected EAV antibody (100% efficiency) in seropositive shedding stallions and, in ponies infected experimentally with the UK93 isolate of EAV, the appearance of virus neutralising antibodies and G(L)-OVA ELISA-specific immunoglobulins coincided.
Animals↗
Detection of equine arteritis virus by real-time TaqMan reverse transcription-PCR assay.
A one-tube real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of equine arteritis virus (EAV). The test was validated using the seminal plasma and nasal secretions of infected horses that were proven to contain EAV by traditional virus isolation in rabbit kidney thirteen (RK-13) cells, as well as a variety of cell culture-propagated European and North American strains of EAV. The primers and a fluorogenic TaqMan probe were designed to amplify and detect a highly conserved region of open reading frame 7 (ORF7) of EAV. The real-time TaqMan PCR assay detected EAV RNA in all samples that were confirmed to contain infectious EAV by virus isolation. The assay had an analytical sensitivity of 10 molecules of EAV RNA allowing the detection of EAV in clinical samples or tissue culture fluid (TCF) containing at least 200 viral RNA copies per ml. Thus, the one-tube real-time TaqMan RT-PCR assay provides a rapid, accurate, quantitative, convenient and high sample throughput system for diagnosis of EAV infection, in a closed-tube format that minimizes the risk of cross-contamination.
Animals↗
Screening of horse polyclonal antibodies with a random peptide library displayed on phage: identification of ligands used as antigens in an ELISA test to detect the presence of antibodies to equine arteritis virus.
A random hexapeptide fusion-phage library was screened to isolate phages that bind to antibodies present in horse sera positive for equine arteritis virus (EAV). Analysis of the peptide sequences displayed by isolated phages identified seven groups. 25% of the isolated phages used as antigens in an ELISA test were specifically recognised by a pool of sera which was positive for EAV in virus neutralisation test (VN). Five of these, when used as antigen in ELISA, detected greater than 50% of sera (n = 30) containing antibodies to EAV as detected by VN. When these five phages were pooled together and used as antigen in ELISA, the detection was improved. The sensitivity and specificity of the ELISA were 99 and 71%, respectively, compared with the EAV neutralisation test (n = 200). This study has shown the potential that phage display libraries have for identifying peptide sequences which could be used as antigen in diagnostic ELISAs.
Amino Acid Sequence↗
Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing G(L), M and N proteins expressed from recombinant baculoviruses.
Indirect enzyme linked immunosorbant assays (ELISAs) utilizing the three major structural proteins (M, N, and G(L)) of equine arteritis virus (EAV) expressed from recombinant baculoviruses were developed. A large panel of sera collected from uninfected horses, and from animals experimentally and naturally infected with EAV or vaccinated with the modified live virus vaccine against equine viral arteritis, were used to characterize the humoral immune response of horses to the three major EAV structural proteins. The data suggest that the M protein was the major target of the equine antibody response to EAV. The responses of individual animals varied and ELISAs that utilized individual EAV structural proteins were not reliable for detecting antibodies in all sera that contained neutralizing antibodies to EAV. An ELISA based on a cocktail of all three EAV structural proteins, however, was used successfully to detect antibodies in most equine sera that were positive in the standard serum neutralization assay following natural or experimental EAV infection (100% specificity, 92.3% sensitivity). In contrast, this ELISA did not reliably detect antibodies in the sera of vaccinated horses. EAV frequently causes a persistent infection in stallions and all sera from carrier stallions evaluated in this study had obvious reactivity with the N protein, whereas seropositive non-carrier stallions, mares and geldings did not respond consistently to the N protein.
Animals↗
Viral respiratory disease.
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Adenoviridae Infections↗
Alphavirus replicon particles expressing the two major envelope proteins of equine arteritis virus induce high level protection against challenge with virulent virus in vaccinated horses.
Replicon particles derived from a vaccine strain of Venezuelan equine encephalitis (VEE) virus were used as vectors for expression in vivo of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). The immunogenicity of the different replicons was evaluated in horses, as was their ability to protectively immunize horses against intranasal and intrauterine challenge with a virulent strain of EAV (EAV KY84). Horses immunized with replicons that express both the G(L) and M proteins in heterodimer form developed neutralizing antibodies to EAV, shed little or no virus, and developed only mild or inapparent signs of equine viral arteritis (EVA) after challenge with EAV KY84. In contrast, unvaccinated horses and those immunized with replicons expressing individual EAV envelope proteins (M or G(L)) shed virus for 6-10 days in their nasal secretions and developed severe signs of EVA after challenge. These data confirm that replicons that co-express the G(L) and M envelope proteins effectively, induce EAV neutralizing antibodies and protective immunity in horses.
Animals↗
Phylogenetic characterisation of the G(L) sequences of equine arteritis virus isolated from semen of asymptomatic stallions and fatal cases of equine viral arteritis in Denmark.
The study describes for the first time the phylogenetic relationship between equine arteritis virus (EAV) isolated from asymptomatic virus-shedding stallions and fatal cases of equine viral arteritis (EVA) in an European country. EAV was isolated from three dead foals and an aborted foetus during three different outbreaks of EVA. From these fatalities, the complete open reading frame 5, encoding the EAV G(L) protein, was amplified by reverse transcription-polymerase chain reaction and subjected to nucleotide sequence analysis. Furthermore, DNA sequences were obtained from virus isolated from semen samples of seven virus-shedding, but clinically healthy, Danish stallions. DNA sequence alignment revealed an overall divergence of 0-14 and 0-10% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis including 24 previously published sequences revealed that European as well as North American "types" of EAV were present in the semen of asymptomatic carrier stallions and in fatal cases of EVA. Our results reveal that the presence of EAV-shedding stallions in Denmark represents a potential source of severe EVA.
Amino Acid Sequence↗
Bluetongue and equine viral arteritis viruses as models of virus-induced fetal injury and abortion.
A number of viruses have the capacity to cross the placenta and infect the fetus to cause, among other potential outcomes, developmental defects (teratogenesis), fetal death and abortion. Bluetongue virus (BTV) infection of fetal ruminants provides an excellent model for the study of virus-induced teratogenesis. This model has shown that only viruses modified by passage in cell culture, such as modified live virus vaccine strains, readily cross the ruminant placenta, and that the timing of fetal infection determines the outcome. Thus, cerebral malformations only occur after fetal infection at critical stages during development and the precise timing of fetal BTV infection determines the severity of the malformation present at birth. Fetal BTV infection also can result in fetal death, followed by abortion or resorption, growth retardation, or no obvious abnormalities, depending on age of the conceptus at infection. Equine arteritis virus (EAV) infection of the equine fetus causes fetal death and abortion but not teratogenesis. These two fetal viral infections are useful not only for the study of teratogenesis and fetal disease, but also to further characterize and compare the complex process that is responsible for normal induction of parturition in ruminants and horses.
Abortion, Veterinary↗