Equine Arteritis Virus (EAV) Outbreak in a Show Stallion Population.

(1) Background: Equine arteritis virus (EAV) infection causes reproductive losses and systemic vasculitis in susceptible equidae. The intact male becomes the virus' reservoir upon EAV infection, as it causes a chronic-persistent infection of the accessory sex glands. Infected semen is the main source of virus transmission. (2) Here, we describe acute EAV infection and spread in a stallion population after introduction of new members to the group. (3) Conclusions: acute clinical signs, acute phase detection of antigen via (PCR) nasal swabs or (EDTA) blood, and seroconversion support the idea of transmission via seminal fluids into the respiratory tract(s) of others. This outbreak highlights EAV's horizontal transmission via the respiratory tract. This route should be considered in a chronic-persistently infected herd, when seronegative animals are added to the group.

Reproductive effects of arteriviruses: equine arteritis virus and porcine reproductive and respiratory syndrome virus infections.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are the most economically important members of the family Arteriviridae. EAV and PRRSV cause reproductive and respiratory disease in equids and swine, respectively and constitute a significant economic burden to equine and swine industries around the world. Furthermore, they both cause abortion in pregnant animals and establish persistent infection in their natural hosts, which fosters viral shedding in semen leading to sexual transmission. The primary focus of this article is to provide an update on the effects of these two viruses on the reproductive tract of their natural hosts and provide a comparative analysis of clinical signs, virus-host interactions, mechanisms of viral pathogenesis and viral persistence.

Zoonotic Potential of Simian Arteriviruses.

Wild nonhuman primates are immediate sources and long-term reservoirs of human pathogens. However, ethical and technical challenges have hampered the identification of novel blood-borne pathogens in these animals. We recently examined RNA viruses in plasma from wild African monkeys and discovered several novel, highly divergent viruses belonging to the family Arteriviridae. Close relatives of these viruses, including simian hemorrhagic fever virus, have caused sporadic outbreaks of viral hemorrhagic fever in captive macaque monkeys since the 1960s. However, arterivirus infection in wild nonhuman primates had not been described prior to 2011. The arteriviruses recently identified in wild monkeys have high sequence and host species diversity, maintain high viremia, and are prevalent in affected populations. Taken together, these features suggest that the simian arteriviruses may be "preemergent" zoonotic pathogens. If not, this would imply that biological characteristics of RNA viruses thought to facilitate zoonotic transmission may not, by themselves, be sufficient for such transmission to occur.

Evidence that in vitro susceptibility of CD3+ T lymphocytes to equine arteritis virus infection reflects genetic predisposition of naturally infected stallions to become carriers of the virus.

We investigated the correlation between in vitro susceptibility of CD3(+) T lymphocytes to equine arteritis virus (EAV) infection and establishment of persistent infection among 14 stallions following natural infections. The data showed that carrier stallions with a CD3(+) T lymphocyte susceptibility phenotype to in vitro EAV infection may be at higher risk of becoming carriers than those that lack this phenotype (P = 0.0002).

Infection of embryos following insemination of donor mares with equine arteritis virus infective semen.

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.

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).

Effective removal of equine arteritis virus from stallion semen.

REASONS FOR PERFORMING STUDY: A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. OBJECTIVES: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. METHODS: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing technique. The sperm preparations were tested by PCR for the presence of EAV. The procedure was repeated using an ejaculate from a known shedding stallion, testing processed and unprocessed aliquots by PCR and virus isolation. RESULTS: Virus-free sperm preparations were obtained using the double sperm processing technique. The 'swim-up' step is apparently required to ensure complete virus removal. CONCLUSIONS: The double semen processing technique is potentially a useful and simple tool for the removal of EAV from the semen of shedding stallions. POTENTIAL RELEVANCE: The inclusion of density gradient centrifugation and 'swim-up' in protocols for the processing of semen for artificial insemination could help prevent the transmission of viral diseases carried in semen, such as EAV.

Antibodies against equine herpesviruses and equine arteritis virus in Burchell's zebras (Equus burchelli ) from the Serengeti ecosystem.

A total of 51 sera from a migratory population of Burchell's zebras (Equus burchelli) were collected in the Serengeti National Park (Tanzania) between 1999 and 2001 to assess levels of exposure to equine herpesvirus types 1, 2, 4, 9 (EHV-1, -2, -4, -9), EHV-1 zebra isolate T965, and equine arteritis virus (EAV). Using virus-specific neutralizing antibody tests, seroprevalence was high for EHV-9 (60% of 45), moderate for EAV (24% of 51), and lower for the EHV-1-related zebra isolate (17% of 41), EHV-1 (14% of 49), and EHV-4 (2% of 50). No evidence for exposure to EHV-2 was found (0% of 51). The high level of exposure to EHV-9 is interesting because evidence of infection with this virus has not been previously described in any wild equine population. Although the epidemiology of EHV-9 in Burchell's zebras is presently unknown, our results suggest that in East Africa, this species may be a natural host of EHV-9, a neuropathogenic virus that was only recently isolated from captive Thomson's gazelles (Gazella thomsoni) in Japan. There is currently no evidence that EHV-9 induced mortality in Burchell's zebras in the Serengeti, but because of the reported virulence of this virus for more susceptible species such as Thomson's gazelles, viral transmission from infected zebras to ungulates may result in mortality.

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.

Exogenous porcine viruses.

Porcine organs, cells and tissues provide a viable source of transplants in humans, though there is some concern of public health risk from adaptation of swine infectious agents in humans. Limited information is available on the public health risk of many exogenous swine viruses, and reliable and rapid diagnostic tests are available for only a few of these. The ability of several porcine viruses to cause transplacental fetal infection (parvoviruses, circoviruses, and arteriviruses), emergence or recognition of several new porcine viruses during the last two decades (porcine circovirus, arterivirus, paramyxoviruses, herpesviruses, and porcine respiratory coronavirus) and the immunosuppressed state of the transplant recipients increases the xenozoonoses risk of humans to porcine viruses through transplantation. Much of this risk can be eliminated with vigilance and sustained monitoring along with a better understanding of pathogenesis and development of better diagnostic tests. In this review we present information on selected exogenous viruses, highlighting their characteristics, pathogenesis of viral infections in swine, methods for their detection, and the potential xenozoonoses risk they present. Emphasis has been given in this review to swine influenza virus, paramyxovirus (Nipah virus, Menagle virus, LaPiedad paramyxovirus, porcine paramyxovirus), arterivirus (porcine reproductive and respiratory syndrome virus) and circovirus as either they represent new swine viruses or present the greatest risk. We have also presented information on porcine parvovirus, Japanese encephalitis virus, encephalomyocarditis virus, herpesviruses (pseudorabies virus, porcine lymphotropic herpesvirus, porcine cytomegalovirus), coronaviruses (TGEV, PRCV, HEV, PEDV) and adenovirus. The potential of swine viruses to infect humans needs to be assessed in vitro and in vivo and rapid and more reliable diagnostic methods need to be developed to assure safe supply of porcine tissues and cells for xenotransplantation.

Transplacental lactate dehydrogenase-elevating virus (LDV) transmission: immune inhibition of umbilical cord infection, and correlation of fetal virus susceptibility with development of F4/80 antigen expression.

Maternal-to-fetal transmission of the murine lactate dehydrogenase-elevating virus (LDV) has been previously shown to be regulated by maternal immunity as well as gestational age. For the present study, the role of maternal immunity in placental and umbilical cord virus protection was studied, and virus targeting of umbilical cord and fetal macrophages was correlated with expression of the F4/80 macrophage phenotypic marker. The results showed that LDV-infected macrophages appeared in umbilical cord by 24 h post-infection of pregnant mice, and some LDV-infected macrophages displayed the F4/80 phenotype. This potential reservoir of virus for the fetus was inhibited by passive immunization of pregnant mice with IgG anti-LDV antibodies, which rapidly concentrated in the placenta and umbilical cord. Probing of umbilical cord cells with antibodies directed at MHC genetic markers demonstrated the presence of both maternal and fetal cells in umbilical cords. A strong developmental correlation was observed between fetal F4/80 expression and LDV susceptibility, at about 13.6 days of gestation. These results demonstrate immune suppression of free and cell-associated virus in umbilical cord, thus defining a potentially important mechanism for immune protection of the fetus from transplacental virus infection. The results also clarify the developmental basis for fetal susceptibility to LDV infection.

Equine viral arteritis: further characterization of the carrier state in stallions.

Further characterization of the carrier state in stallions infected with equine arteritis virus revealed that there is considerable variation in the frequency of its occurrence among breeds. The frequency ranged from 12.5% (Holsteiner stallions) to 72.7% (Dutch Warmblood stallions), with a mean occurrence of 40.8% in the seropositive stallions (n=561) examined. More than 70% of the virus shedders were Standardbred stallions. The carrier state was not confirmed in any of the stallions that had been vaccinated against equine viral arteritis nor was there any evidence of intermittent virus shedding by carrier stallions. Most (98.2%) of the semen isolates of equine arteritis virus were obtained on first passage in RK-13 cell culture and most of the samples had very high virus infectivity titres. Intermediate term (3.5-7.0 months) and long-term (> or =1 year) carrier states were confirmed in various horse breeds. Long-term persistence of equine arteritis virus in individual stallions was common, and some animals continued to shed the virus in semen for 4-12 years. Spontaneous clearance of the carrier state was observed in 27 stallions after periods ranging from several months to many years. There was a considerable difference in the rate of clearance of the carrier state between Standardbred (4.3%) and Thoroughbred (42.3%) stallions. Reduction and eventual elimination of the carrier stallion reservoir of equine arteritis virus is the key to the success of any control programme for this disease.

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 stability of equine arteritis virus during horizontal and vertical transmission in an outbreak of equine viral arteritis.

An imported carrier stallion (A) from Europe was implicated in causing an extensive outbreak of equine viral arteritis (EVA) on a Warmblood breeding farm in Pennsylvania, USA. Strains of equine arteritis virus (EAV) present in the semen of two carrier stallions (A and G) on the farm were compared to those in tissues of foals born during the outbreak, as well as viruses present in the semen of two other stallions that became persistently infected carriers of EAV following infection during the outbreak. The 2822 bp segment encompassing ORFs 2-7 (nt 9807-12628; which encode the G(S), GP3, GP4, G(L), M and N proteins, respectively) was directly amplified by RT-PCR from semen samples and foal tissues. Nucleotide and phylogenetic analyses confirmed that virus present in the semen of stallion A initiated the outbreak. The genomes of viruses present in most foal tissues (10/11) and serum from an acutely infected mare collected during the outbreak were identical to that of virus present in the lung of the first foal that died of EVA. Virus in the placenta of one foal differed by one nucleotide (99.9% identity) from the predominant outbreak virus. The relative genetic stability of viruses that circulated during the outbreak contrasts markedly with the heterogeneous virus populations variously present in the semen of persistently infected stallions on the farm. These findings are consistent with the hypothesis that the carrier stallion can be a source of genetic diversity of EAV, and that outbreaks of EVA can be initiated by the horizontal aerosol transmission of specific viral variants that occur in the semen of particular carrier stallions.

Genetic diversity of equine arteritis virus.

Equine arteritis viruses (EAV) from Europe and America were compared by phylogenetic analysis of 43 isolates obtained over four decades. An additional 22 virus sequences were retrieved from GenBank. Fragments of the glycoprotein G(L) and the replicase genes were amplified by RT-PCR, prior to sequencing and construction of phylogenetic trees. The trees revealed many distinctive lineages, consistent with prolonged diversification within geographically separated host populations. Two large groups and five subgroups were distinguished. Group I consisted mainly of viruses from North America, whilst group II consisted mainly of European isolates. In most instances, where the geographic origin of the viruses appeared to be at variance with the phylogenetically predicted relationships, the horses from which the viruses were recovered had been transported between Europe and America or vice versa. Analysis of the replicase gene revealed similar phylogenetic relationships although not all of the groups were as clearly defined. Virus strains CH1 (Switzerland, 1964) and S1 (Sweden, 1989) represented separate 'outgroups' based on analysis of both genomic regions. The results of this study confirm the value of the G(L) gene of EAV for estimating virus genetic diversity and as a useful tool for tracing routes by which EAV is spread. In addition, computer-assisted predictions of antigenic sites on the G(L) protein revealed considerable variability among the isolates, especially with respect to regions associated with neutralization domains.

Passive transfer, rate of decay, and protein specificity of antibodies against equine arteritis virus in horses from a Standardbred herd with high seroprevalence.

OBJECTIVE: To determine rate of decay of passively acquired antibodies in Standardbred foals on a farm with a high seroprevalence to equine arteritis virus (EAV) and to determine whether vertical or horizontal transmission of the virus was responsible for infection on the farm. DESIGN: Repeated-measures study. ANIMALS: 46 Standardbred horses (15 brood mares and their foals, 5 stallions, and 11 young horses). PROCEDURE: Serum samples obtained from horses on the farm were evaluated by serum neutralization and western immunoblot analysis to detect EAV-specific antibodies. The half-life of passively acquired antibodies in foals was estimated by use of regression analysis. RESULTS: Most (14/15) of the mares evaluated were seropositive to EAV. After suckling, their foals were also seropositive. Mean biological half-life for passively acquired antibodies in serum samples obtained from foals was 32 days (r2 = 0.61). The foal born to a seronegative dam and all 11 young horses from the farm were seronegative to EAV. At least 2 of 5 stallions on the farm were persistently infected carriers that were shedding virus in their semen. Immunoblot analysis of seropositive serum samples most consistently recognized the M protein of EAV. CLINICAL IMPLICATIONS: Analysis of these data indicated that a modified-live EAV vaccine can be administered to foals after they are 8 months old without risk of interference from maternal antibodies, regardless of serologic status of the foal's dam. Horizontal transmission of EAV via the respiratory tract apparently was uncommon on the farm, indicating that mares primarily were infected by venereal transmission of virus from carrier stallions.

[Equine arteritis virus: clinical symptoms and prevention]. / Equine arteritis virus: klinische verschijnselen en preventie.

Sero-epidemiological surveys have revealed that equine arteritis virus (EAV) is prevalent in most European countries. The virus causes sporadic cases of respiratory disease and abortion in horses, the incidence of which has increased in recent years. Mares and geldings eliminate virus after acute infection, but 30% to 60% of stallions become persistently infected. In these animals, EAV is maintained within the reproductive tract and is shed continuously in the semen. Persistent infection with EAV in stallions has no negative consequences for fertility but mares inseminated with virus-contaminated semen can have an acute infection. These mares shed large amounts of virus in respiratory secretions and urine, leading to lateral spread of the virus to other susceptible horses. Acute infection at later stages of gestation can lead to abortion. Effective control of the spread of EAV infection depends on the identification of virus-shedding stallions. Persistently infected stallions should not be used for breeding or should be bred only to seropositive mares. Mares bred to shedding stallions should be isolated from other animals for a period of 3 weeks following insemination to prevent the lateral spread of EAV.

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.