Genetic variation in open reading frame 2 of field isolates and laboratory strains of equine arteritis virus.

The open reading frame 2 (ORF2) of three laboratory strains, the live attenuated vaccine virus, and 18 field isolates of equine arteritis virus (EAV) from Europe and North America was sequenced. The ORF2 of EAV encodes the Gs protein that is abundantly expressed in infected cells but constitutes less than 2% of the virion protein mass. Variation of ORF2 among the isolates facilitated phylogenetic analysis that largely confirmed results of an earlier study based on sequence divergence of ORF5 of the same isolates of EAV, despite exposure of the proteins encoded by ORF2 (Gs) and ORF5 (GL) to potentially different selective pressures in vivo. The data indicate that the Gs protein is highly conserved between isolates, considerably more so than the GL protein, consistent with an important role of the Gs protein in virus replication.

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.

Genomic variability among globally distributed isolates of equine arteritis virus.

Equine arteritis virus (EAV), a non-arthropod borne togavirus, has been shown to have a global distribution. To date, no major antigenic variation has been demonstrated between EAV isolates from different geographic origins. In this study, the genomic RNA of EAV isolates obtained from horses of different breeds in various countries around the world was oligonucleotide fingerprinted. Comparisons of these fingerprints were used to determine the extent of genomic variation among such isolates. Comparisons among isolates from North American horses revealed, for the most part, oligonucleotide homologies of less than 60%. Only 29 of the 98 comparisons revealed greater than 60% oligonucleotide homology. Nonetheless, several comparisons indicated a close epidemiologic relationship between isolates from horses of different breeds located in different states. Though all European isolates were of Standardbred origin and were from horses located in northern European countries, the majority had oligonucleotide homologies of less than 60%. Where oligonucleotide homology was apparent, it was, with one exception, greater than 70%. The two isolates from New Zealand had 93.2% oligonucleotide homology. This is indicative of an extremely close epidemiologic relationship. Comparisons between EAV isolates from around the world revealed oligonucleotide homologies between viruses from North America, Europe and New Zealand. In several instances, this homology was greater than 70% and in one case greater than 80%. No oligonucleotide homology was evident in comparisons involving the virus from South Africa. The high level of genomic conservation between certain EAV isolates of disparate geographic origins may reflect dissemination of the virus associated with the international movement of horses. The extent of genomic variation demonstrated between most of the EAV isolates used in this study confirms the need for further investigation of genomic heterogeneity among strains of this virus before techniques that rely upon nucleic acid hybridization can be effectively applied as diagnostic procedures.

Serologic response of horses to the structural proteins of equine arteritis virus.

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, an apparently emerging disease of equids. In this study, the antibody response of horses to the structural proteins of EAV was evaluated using gradient-purified EAV virions and baculovirus-expressed recombinant EAV structural proteins (G(L), G(S), M, N) as antigens in a Western immunoblotting assay. Thirty-three sera from horses that previously had been naturally or experimentally infected with EAV were evaluated, including samples from mares, geldings, and both persistently and nonpersistently infected stallions. Sera also were evaluated from 4 horses that had been vaccinated with the commercial modified live EAV vaccine. The data suggest that the serologic response of individual horses to EAV may vary with the infecting virus strain and duration of infection. The M protein was most consistently recognized by the various serum samples, whereas the response to the N and G(L) proteins was variable and the G(S) protein was bound by only 1 serum sample. The immunoblotting assay definitively established the protein specificity of the humoral response of horses to EAV; however, it clearly is less sensitive than the standard serum neutralization (SN) test--2 of the 37 sera that were seropositive by the SN test failed to react in the immunoblot assay with any EAV structural protein. Furthermore, the G(L) protein expresses the known neutralization determinants of EAV, yet only 22 of the 37 sera that had SN antibodies bound the G(L) protein in the immunoblotting assay. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection of horses.

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.

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.

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.

Demonstration of the carrier state in naturally acquired equine arteritis virus infection in the stallion.

The chronic carrier state was virologically confirmed in 15 thoroughbred stallions naturally infected with equine arteritis virus based on the results of test matings and, or, isolations of the virus from semen. Carrier stallions were shown to shed equine arteritis virus in the semen for at least one to two years. Existence of a short-term or convalescent carrier state was also demonstrated in five additional stallions. The frequency of the long-term carrier state in stallions naturally infected with equine arteritis virus was 35 per cent; it varied considerably between groups of stallions on different farms. The carrier stallion would appear to play an important epidemiological role in the dissemination and perpetuation of equine arteritis virus.

Lateral transmission of equine arteritis virus among Lipizzaner stallions in South Africa.

REASONS FOR PERFORMING STUDY: A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. OBJECTIVES: To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. METHODS: EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF 5-specific primers, subjected to sequence and phylogenetic analysis. RESULTS: Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stallions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from bedding or fomites contaminated with semen. CONCLUSIONS: These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. POTENTIAL RELEVANCE: The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV- infected semen.

Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.

Pathologic features of horses given avirulent equine arteritis virus intramuscularly.

Twenty horses that were seronegative for equine arteritis virus antibodies were inoculated IM with live equine arteritis virus vaccine. The inoculation did not cause clinical signs of disease. A mild, transient febrile reaction developed in 6 horses, 3 of which were in poor condition before inoculation. Six horses, 2 of which were in poor condition before inoculation, experienced mild lymphopenia. Necropsy revealed mild lesions in the lymph nodes of 6 horses (3 of which were in poor condition before inoculation). Maximum concentrations of virus were detected in the lymph nodes and were consistently present from postvaccination day 3 through 8. Lesser concentrations of virus were detected in the spleen of 5 horses, liver and kidney of 4, abdominal fluid of 3, pleural fluid of 2, and lungs and urine of 1, between postvaccination days 3 and 7. Virus was not detected in the brain, nasal tract, or serum of any of the horses.

Responses of horses vaccinated with avirulent modified-live equine arteritis virus propagated in the E. Derm (NBL-6) cell line to nasal inoculation with virulent virus.

Nineteen horses with no prior experience with equine arteritis virus (EAV) were inoculated IM with an avirulent live-virus vaccine against equine viral arteritis; the vaccinal virus had been passaged serially 131 times in primary cell cultures of equine kidney, 111 times in primary cell cultures of rabbit kidney, and 16 times in an equine dermis cell line (EAV HK-131/RK-111/ED-16). Three or 4 of the vaccinated horses each, along with appropriate nonvaccinated controls, were inoculated nasally with virulent EAV at each of months 3, 6, 9, 12, 18, and 24 after they were vaccinated. The following was concluded: Vaccination did not induce clinical signs of disease in any horse and, thus, seemed safe for use in the field. All vaccinated horses (n = 19) developed serum-neutralizing antibodies to EAV. Fourteen of the vaccinated horses were completely protected from clinical arteritis when exposed to large doses of virulent EAV. Four were partially protected, and one had little or no protection. Six of 13 nonvaccinated horses died of acute arteritis, and the remaining 7 horses experienced severe signs of disease, but survived the infection. All horses (n = 32), whether vaccinated or not, became infected when inoculated nasally with virulent EAV. Virus was recovered from 17 of the 19 vaccinated horses, and all 19 had a secondary humoral immune response. The duration and severity of thermal reaction and persistence of virus were more transitory in vaccinated horses than in the nonvaccinated controls. Protection afforded by this vaccine can persist for at least 24 months, the maximal time after horses were vaccinated that immunity was challenged in the present study.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of viability and immunizing potency of lyophilized, modified equine arteritis live-virus vaccine.

The Bucyrus strain of equine arteritis virus, previously modified to avirulence and vaccinal virus by 131 serial passages in primary cell cultures of horse kidney followed by 111 passages in primary cell cultures of rabbit kidney, was further passaged in cultures of the E. Derm (NBL-6) cell line, a continuous diploid cell line. Pools of the 16th and 25th passages of the virus in this last equine dermal cell line were lyophilized and stored in lots at 37 C, 23 to 28 C, 4C, and -20 C. The viability of the vaccinal virus deteriorated rapidly during storage at 37 C and at 23 to 28 C, but was relatively stable at 4 C and at -20 C for at least 1 year. The vaccine stored at 4 C for 9 months protected 2 horses, subsequently inoculated with avirulent virus, from arteritis, whereas the 2 nonvaccinated control horses inoculated simultaneously developed severe signs of the disease and died of acute arteritis.

Correlation between ultrasonographic findings and serum testosterone concentration in prepubertal and peripubertal colts.

Correlation between serum testosterone concentration and morphometric findings from ultrasonography of the accessory sex glands in peripubertal colts was investigated during pubertal development. Nineteen colts of initial age ranging from 5 to 12 months were monitored over a 13-month period. Serum testosterone concentration was determined on a biweekly basis, and accessory sex gland development was ultrasonographically monitored once a month. Notwithstanding individual variation, there was significant correlation (r = 0.913; P < 0.01) between increasing serum testosterone concentration and the onset of developmental changes involving the accessory sex glands. As colts entered their 2-year-old year with relatively immature reproductive tracts, compared with mature stallions, there was still a significant seasonal effect on serum testosterone concentration and accessory sex gland measurements (P < 0.05). Ultrasonography was confirmed as a valuable noninvasive method of monitoring and assessing peripubertal accessory sex gland development in colts.

Status of equine viral arteritis in Kentucky, 1985.

Clinical cases of equine arteritis virus infection have not been diagnosed in Kentucky since 1984, and there has been no indication that any of the horses involved in the 1984 epizootic have since been responsible for spread of the disease to horses in other states or other countries. Cases of abortion caused by naturally acquired infection with this virus have not been confirmed in 1984 or 1985. Neither field nor vaccine strains of equine arteritis virus have been shown to induce teratologic abnormalities or the carrier state in foals born to infected or vaccinated mares. The carrier stallion appears to have played a major epidemiologic role in the dissemination and perpetuation of the virus. A commercial modified live equine viral arteritis vaccine was found to be safe and efficacious for stallions and mares. The disease can be controlled by immunizing the stallion population and by restricting the breeding of equine arteritis virus-shedding stallions to vaccinated or seropositive mares, followed by an appropriate period of isolation from other nonvaccinated Equidae.

Serologic and molecular characterization of an abortigenic strain of equine arteritis virus isolated from infective frozen semen and an aborted equine fetus.

A virus isolated from an aborted equine fetus was determined to be antigenically distinct from several other strains of equine arteritis virus (EAV) by use of a neutralization assay with a large panel of neutralizing monoclonal antibodies. The virus was readily neutralized by polyclonal equine anti-EAV serum. Comparative nucleotide and amino acid sequence analyses indicated that the virus (WA97) isolated from the aborted fetus was virtually identical to a virus (S1971) isolated from imported semen used to inseminate another mare on the farm. Phylogenetic analysis indicated that the WA97/S1971 virus was more related to European than to North American strains of EAV. These sensitive molecular procedures may be useful for epidemiologic investigations of EAV infections. Screening and certification of stallions and frozen equine semen would prevent dissemination of pathogenic strains of EAV.

Equine viral arteritis.

Equine viral arteritis is reviewed with specific reference to clinical features, etiology, transmission, diagnosis, epidemiology, and current methods for the control of this disease. There is evidence of variation in pathogenicity among strains of equine arteritis virus. Virus transmission occurs primarily by the respiratory and venereal routes during the acute phase of the infection. The long-term carrier stallion appears to play a major epidemiological role in dissemination and perpetuation of the virus. Unlike the stallion, the carrier state has yet to be demonstrated in the mare or foal. A commercial modifiedlive equine arteritis virus vaccine has been shown to be safe and efficacious for stallions and mares. The disease can be controlled by identification and isolation of carrier stallions, immunization of seronegative stallions, and by restricting the breeding of equine arteritis virus-shedding stallions to equine arteritis virus vaccinated or seropositive mares.