Equine viral arteritis control scheme: a brief review with emphasis on laboratory aspects of the scheme in New Zealand

AIM: To review laboratory aspects of the equine viral arteritis (EVA) control scheme in New Zealand between 1989 and 2002. METHODS: The optimisation and performance of the virus neutralisation test (VNT) for equine arteritis virus (EAV) antibody, and the cell culture test to detect EAV in semen were analysed. Laboratory data and control scheme results were reviewed. RESULTS: Using optimised tests, it has been shown that antibody prevalence in Standardbred horses has steadily declined from 54% to <20%. Prevalences in Thoroughbred horses have remained at a low level of around 3%. The number of horses shedding EAV (all Standardbreds) has steadily declined from a maximum at any one time of 20 to the current figure of three. CONCLUSION: Eradication of EVA from the horse population in New Zealand is achievable in the near future.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Epizotiology and phylogeny of equine arteritis virus in hucul horses

The aim of the study was to determine the situation of equine arteritis virus (EAV) infections in hucul horses. A total of 176 horses (154 mares and 22 stallions) from the biggest hucul horse stud in Poland were tested. Antibodies against EAV were detected in 97 (55.1%) horses. The EAV seroprevalence among mares was 53.2% while in stallions - 68.2%. The percentage of positive mares increased with their age, thus amongst the mares of less than 2 years of age the percentage was 32.5%, while in the group of 3-5 years old increased to 59.4% and in the mares in the age of 6-10 years and older than 10 years 89.5% and 95% were seropositive, respectively. Among 11 seropositive stallions five were supposed to be shedders of EAV with their semen. It is likely that those persistently infected stallions were the reservoirs of the virus in the stud. Genetic studies using of ORF5 gene showed high homology between the viruses detected in the semen of those stallions what suggested lateral transmission between the stallions sharing the same stable. Persistent infection in an immature stallion, which has not yet been used for breeding, was established as a result of infection via respiratory route. Phylogenetic analysis confirmed that all hucul viruses shared the same ancestor and as most of EAV strains dominating in Polish horse population belonged to the European origin EAV subgroup (EU-1).     

Differentiation of strains of equine arteritis virus of differing virulence to horses by growth in equine endothelial cells

OBJECTIVE: To compare growth characteristics of strains of equine arteritis virus (EAV) of differing virulence to horses in rabbit kidney (RK)-13 cells and equine endothelial cells (EECs) cultured from the pulmonary artery of a foal. SAMPLE POPULATION: 13 strains of EAV, including 11 field isolates of differing virulence to horses; the highly virulent, horse-adapted Bucyrus strain; and the modified-live virus (MLV) vaccine derived from it. PROCEDURE: The growth characteristics of the 13 strains were compared in EECs and RK-13 cells. Viral nucleoprotein expression, cytopathogenicity, and plaque size were compared to determine whether growth characteristics of the 13 strains were predictive of their virulence to horses. RESULTS: Cytopathogenicity, viral nucleoprotein expression, and plaque size induced by all 13 viruses were similar in RK-13 cells, whereas virulent strains of EAV caused significantly larger plaques in EECs than did the avirulent strains of EAV. Paradoxically, the highly attenuated MLV vaccine and 1 field isolate of EAV caused plaques in EECs that were larger than those caused by any of the other viruses, and sequence analysis confirmed the field isolate of EAV to be indistinguishable from the MLV vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: With the notable exception of the MLV vaccine, growth of the various strains of EAV in EECs was predictive of their individual virulence to horses. Thus, EECs provide a relevant and useful model to further characterize determinants of virulence and attenuation amongst strains of EAV.     

Highly diverse type of equine arteritis virus (EAV) from the semen of a South African donkey: short communication

Equine arteritis virus (EAV) was detected by RT-nested PCR in semen samples from a naturally infected South African donkey. Sequence analysis of the amplified ORF5 fragment revealed only 60 to 70% nucleotide identity to a panel of EAV reference sequences. The unique donkey EAV sequence was also found to be stable during passage in horses. The sequence data reported in this study indicate that the South African donkey variant might represent a new genotype of EAV. The distinct genetic properties of the South African asinine strain of EAV suggest a divergent evolution of this arterivirus in various host species or, alternatively, a possible role for African donkeys in the emergence of EAV in horses.     

Seroprevalence of antibodies against equine arteritis virus in horses residing in the United States and imported horses

OBJECTIVE: To compare seroprevalence of antibodies against equine arteritis virus (EAV) in horses residing in the United States with that of imported horses. DESIGN: Serologic survey. SAMPLE POPULATION: Serum samples from 364 horses on 44 equine operations in California and 226 horses imported from various countries. PROCEDURE: Serum samples were collected from each imported horse and from up to 20 horses on each operation. For resident horses, the number of sampled horses on each operation was determined on the basis of the number of horses on the operation. Samples were tested for antibodies against EAV by use of a serum neutralization test. RESULTS: 1.9% of resident horses and 18.6% of imported horses were seropositive to EAV, including 16.1% of imported stallions. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that the EAV seroprevalence of horses residing in California is considerably lower than that of imported horses, including imported stallions.     

马动脉炎病毒N蛋白基因GST融合表达载体的构建及表达

采用RT-PCR方法扩增出马动脉炎病毒核衣壳蛋白基因0RF7,并将其克隆到pMDl8-T载体,构建成重组质粒pMDl8-N,在上海生物工程公司测序。结果表明,所克隆的核衣壳蛋白基因序列与EAVNC-002532株的同源性为99％。表明0RF7是EAV基因组内的保守序列,将0RF7亚克隆到原核表达载体pGEX-6P-1中,构建成重组质粒pGEX-6P-N,用pGEX-6P-N转化表达菌株BL21（DE3）,诱导表达后SDS-PAGE和Westernblotting分析表明,克隆在谷胱苷肽硫转移酶（GST）下游的核衣壳蛋白基因与GST获得了高效融合表达,表达的融合蛋白GST-N分子质量约为40ku,为马动脉炎病毒病血清学诊断方法的建立奠定了基础。     

Serological surveillance of equine viral arteritis in the United Kingdom since the outbreak in 1993

Serological analysis of blood samples submitted to the Animal Health Trust showed that during 1995, 185 of 9203 unvaccinated horses (2.0 per cent) tested positive for antibodies to equine arteritis virus (EAV), and that during 1996, 46 of 8851 unvaccinated horses (0.52 per cent) tested positive. During both years thoroughbreds were the predominant breed tested and only a small proportion of these (<0.3 per cent), consisting predominantly of imported mares, were seropositive. In contrast, among standardbred horses, from which samples were actively solicited in 1995, 84 of 454 (18.5 per cent) were seropositive. Among standardbreds there was a difference in prevalence between types of horses, with 3.7 per cent of racing horses, 25 per cent of non-racing horses and 41 per cent of stallions testing seropositive. Investigations of seropositive stallions identified during 1994 and 1995 demonstrated that clinically inapparent equine viral arteritis (EVA) had occurred previously in the UK. Of 50 seropositive stallions, nearly half were standardbreds and nearly all had been imported from either North America or the European Union. Whether 34 seropositive stallions were shedding virus in their semen was established either by test mating, by the serology of the covered mares, or by investigation by MAFF following the introduction of the Equine Viral Arteritis Order 1995. Nine of the stallions (26.5 per cent) were identified as presumptive shedders of EAV in semen and among specific breeds, viral shedding was identified in six of 15 (40 per cent) standardbreds and three of nine (33 per cent) warmbloods. In contrast with the outbreak of EVA in the UK in 1993, no signs of disease typical of EAV infection were reported during these investigations, even in mares test mated to stallions shedding the virus.     

Genetic and amino acid analysis of the GL protein of Canadian, American and European equine arteritis virus isolates.

The genetic variation in equine arteritis virus (EAV) GL protein encoding gene was investigated. Nucleic and deduced amino acid sequences from 7 different EAV isolates, including 4 eastern Canadian field isolates, were compared with those of the Bucyrus reference strain. Nucleotide sequence identities between these isolates and the Bucyrus reference strain ranged from 87.5% (Vienna isolate) to 93.9% (11958 isolate). Amino acid identities with the Bucyrus reference strain varied from 90.2% (Vienna isolate) to 95.1% (19933 isolate). A 2nd potential N-linked glycosylation site was found at position 81 in the GL protein of all EAV isolates. Three amino acid substitutions at residue position 90 (Glu-->Val), position 101 (Ala-->Val or Thr), and position 119 (Val-->Leu, Phe or Ser) were also found in all EAV isolates. Phylogenetic analysis showed that the North American EAV isolates, including the Canadian isolates, and the European prototype Vienna isolate could be classified in 2 distinct groups. Three putative sequential antigenic sites were predicted in EAV GL protein. The 1st antigenic site (TAQRFT) was located at positions 24 to 29, and the 2nd antigenic site (RYDEHTA) at positions 47 to 53. The 3rd antigenic site was predicted to be located at positions 78 to 84 and showed the less conserved amino acid sequence.     

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)     

Assessment of correlation between in vitro CD3+ T cell susceptibility to EAV infection and clinical outcome following experimental infection

In a recent study, we demonstrated that the virulent Bucyrus strain (VBS) of EAV could infect in vitro a small population of CD3(+) T lymphocytes from some but not all horses. Furthermore, we have shown that a common haplotype is associated with this in vitro CD3(+) T cell susceptibility/resistance phenotype to EAV infection. In this study, we investigated whether the differences in the susceptibility or resistance of CD3(+) T cells in vitro correlate with the outcome and severity of clinical signs in vivo. Thus, horses were divided into two groups based on their CD3(+) T cell susceptible or resistant phenotype. Following experimental inoculation with the recombinant VBS of EAV, horses were assessed for presence and severity of clinical signs, duration and magnitude of virus shedding, as well as production of proinflammatory and immunomodulatory cytokines in peripheral blood mononuclear cells using real-time quantitative RT-PCR. The data showed that there was a significant difference between the two groups of horses in terms of cytokine mRNA expression and evidence of increased clinical signs in horses possessing the in vitro CD3(+) T cell resistant phenotype. This is the first study to provide direct evidence for a correlation between variation in host genotype and phenotypic differences in terms of the extent of viral replication, presence and severity of clinical signs and cytokine gene expression caused by infection with virulent EAV.     

Comparison of a vaccine strain and field isolates of avian reovirus by T1-oligonucleotide mapping.

Total RNA of eight avian reovirus isolates and the S1133 strain were compared by RNase T1-oligonucleotide mapping. The viruses were propagated in Vero cell cultures, and viral genomes were extracted from purified virions for comparison. Pairwise comparisons of the oligonucleotide maps showed genetic variation among reovirus isolates ranging from 78% to 99%. The T1 fingerprints of the RNA of isolates 1103, 724, 615, and 684 differed slightly from the standard S1133 strain, suggesting that the vaccine strain might have changed and became part of the circulating reoviruses. In contrast, when compared with the vaccine strain, isolates 902, 644, and 6207 showed greater differences in the fingerprint pattern. This genomic diversity may be due to the differences in immunological status of the affected avian population and/or due to simultaneous coinfection with different reovirus strains.     

Three cases of virus isolation from horse fetuses diagnosed with equine arteritis virus (EAV) abortion from stud farms with different breeds]

Three cases of abortions were diagnosed as caused by Equine Arteritis Virus (EAV) by isolation and typing of this virus from the respective fetuses. All 3 abortions were single cases, one occurring on a stud with Iceland Ponies, one with Warmbloods, one with Lipizzaner horses. On each stud horses of the respective breed were kept exclusively, therefore there existed no epidemiologic link. By means of seroneutralization tests performed on in contact horses it could be shown, that EAV had only been introduced recently into the stud with the Iceland Ponies. An extraneous mare stabled temporarily for covering by the stud's stallion could be incriminated for introducing EAV. By means of post-abortion serology it could be demonstrated that the Warmblood stud had been harbouring EAV for a longer period of time. Likewise, the Lipizzaner stud could be shown to have been persistently infected, this time on pre-abortion serums stored frozen at our Institute. On both these studs preexisting neutralizing antibodies accounted for the single case of abortion and prevented serial abortions. By investigating frozen serums taken in earlier years we could show that the Lipizzaner stallions had reacted positively to EAV for several years already. However, the gestation period of the aborting mare allowed to exclude EAV-positive semen transmitted on copulation as cause of its abortion. Both the Iceland Pony stallion as well as the Warmblood stallion could be excluded as sources of infection for the respective aborting mares as both repeatedly were seronegative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic variation in the feral horses of the Namib Desert, Namibia

Genetic variation at 7 blood-group and 10 biochemical genetic loci was examined in 30 horses from a feral herd from the Namib Desert of Namibia, Africa. The observed genetic variability was extremely low compared with that found in domestic horse breeds. The low variation was most probably a result of recent small population size and a small founding population size. Genetic comparison of the Namib horses, which were of unknown origins, to domestic horse breeds, showed that the Namib horses had the highest genetic similarity to Arabian type horses, although they did not closely resemble this type of horse in conformation.     

The effects of vaccination with tissue culture-derived viral vaccines on detection of antibodies to equine arteritis virus by enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to equine arteritis virus (EAV). Results from this assay produced a good correlation with results from virus neutralisation tests in horses which had not been regularly vaccinated with commercially available mammalian tissue culture-derived viral vaccines. Vaccination of some horses with tissue culture-derived vaccines induced the formation of antibodies to bovine serum. These antibodies reacted with the bovine protein contaminants in the EAV ELISA antigen, producing false-positive results. Non-viral protein contaminants were found to be closely associated with EAV in that they co-purified with the virus during gradient centrifugation.     

The occurrence of equine arteritis virus in Australia

This paper reports the first isolation of equine arteritis virus (EAV) in Australia and serological evidence of exposure to EAV in Australian horses. Twelve Standardbred stallions imported from North America were found to shed EAV in semen. One hundred and seven stallions were tested for serum antibodies to EAV and 73% of Standardbred stallions tested were seropositive as compared to 8% of Thoroughbred stallions. Serum antibody was detected in 71% of Standardbred mares, 6% of Standardbred racehorses and 1% of Thoroughbred mares and racehorses. Examination of stored serums demonstrated that EAV had been present in Australia since at least 1975.     

Virologico-serologic studies in horses with respiratory tract diseases]

Of 1081 acute and chronically respiratory diseased as well as clinically normal horses 824 sera and 257 paired serum samples collected 1986 and 1987 were tested for antibodies against several different respiratory viruses such as influenza virus A/equi 1 and 2 (Influenza 1 a. 2), equine herpesvirus type 1/4 (EHV 1/4), mammalian reovirus type 1-3 (Reovirus 1-3), equine rhinovirus type 1 (ERV 1), equine adenovirus type 1 (EAdV 1), and equine arteritis virus (EAV). The investigations resulted in an antibody prevalence of 57.2% (Influenza 1), 59.5% (Influenza 2), 81.5% (EHV 1/4), 50.3% (Reovirus 1), 43.0% (Reovirus 2), 75.9% (Reovirus 3), 97.6% (EAdV 1), 82.5% (ERV 1) and 8.7% (EAV). With exception of EAV and EAdV 1 the ratios usually were higher in diseased animals than in clinically normal horses. Antibodies to EAV and EAdV 1 were present in all groups to almost the same amount. Of 257 horses with acute respiratory illness 3 showed a significant rise of the antibody titer against Influenza 1, 30 against Influenza 2, 54 against EHV 1/4, 1 against Reovirus 1 and 3, respectively, 11 against EAdV 1 and 26 against ERV 1.     

Bioinformatics Analysis on S1 Gene of Four Porcine Epidemic Diarrhea Virus Strains

To analyse the genetic variation of porcine epidemic diarrhea virus（PEDV),one pair of primers was designed and RT-PCR was used to amplify the S1 gene epitope sequences of 4 PEDV field strains.Phylogenetic analysis showed that 4 strains were closely related to each other and belonged to the second group,and the Hunan and Henan isolates had a close relationship with JXGZ2013,GDZQ2012,GDZQ2014 strains,with the nucleotide homologies at 98.3% to 98.7%;Shanghai strain had a closed relationship with BJ-2011 strain and LZW isolates,with the higher homology at about 98.7%;Fujian isolates had a close relationship with strain of American in 2013,Japanese and Taiwan variation of China with the nucleotide homology at 99.0% to 99.5%;These results showed that a rapid variation and evolution of PEDV strains occurred in recent years,and linear B-cell epitope analysis showed that the threshold values at site 265 to 278 amino acid of 4 filed strains were higher than that of vaccine strains.The epitope differences indicated a possible of infectious that cause of patterns of epidemiology of PEDV.