Short communication: Retrospective analysis of obligatory testing results for Equine virus arteritis reveals a decrease of its seroprevalence in stallions used for artificial insemination.

Equine viral arteritis (EVA) can induce a persistent carrier state in stallions which then shed the virus via semen. About 30 years ago, obligatory EVA testing of stallions used for artificial insemination (AI) was implemented in the European Union. Information on the efficacy of these regulations on the prevalence of EVA in stallions are not yet available. Therefore, we retrospectively analyzed results of serological and virus antigen testing for EVA in sires of different age and breed referred to Vetmeduni Vienna for semen preservation or veterinary diagnostic procedures between 2001 and 2021. For analysis, stallions were grouped by age (1-5, 6-8, 9-12, >12 years) and breed. The EVA antibody titer was determined by serum neutralization test and semen was analyzed for EVA virus by PCR and/or virus isolation test. Of 308 stallions tested, 14.9% (n = 46) were EVA seropositive and in 12 stallions EVA virus was detected in semen (26% of seropositive stallions). The incidence of seropositive stallions decreased over time (P < 0.05, χ2 test). Differences in the seroprevalence of EVA antibodies existed among stallion age groups (P < 0.01, Fisher's test) with the highest percentage of seropositive stallions being > 12 years old (43.5%). The EVA antibody titer increased with age (P < 0.01, Kruskal-Wallis test), potentially reflecting repeated virus challenge. In conclusion, analysis of monitoring results revealed a decrease of EVA seroprevalence and virus shedding in a European sire population. As monitoring for EVA was the only measure implemented Europe-wide, testing might be a major contributor to this development.

Development of a chronic focal equine arteritis virus infection of a male reproductive tract cell line.

Equine arteritis virus (EAV) is an Alphaarterivirus (family Arteriviridae, order Nidovirales) that frequently causes an influenza-like illness in adult horses, but can also cause the abortions in mares and death of newborn foals. Once primary infection has been established, EAV can persist in the reproductive tract of some stallions. However, the mechanisms enabling this persistence, which depends on testosterone, remain largely unknown. We aimed to establish an in vitro model of non-cytopathic EAV infection to study viral persistence. In this work, we infected several cell lines originating from the male reproductive tract of different species. EAV infection was fully cytopathic for 92BR (donkey cells) and DDT1 MF-2 (hamster cells) cells, and less cytopathic for PC-3 cells (human cells); ST cells (porcine cells) seemed to eliminate the virus; LNCaP (human cells) and GC-1 spg (murine cells) cells were not permissive to EAV infection; finally, TM3 cells (murine cells) were permissive to EAV infection without any overt cytopathic effects. Infected TM3 cells can be maintained at least 7 days in culture without any subculture. They can also be subcultured over 39 days (subculturing them at 1:2 the first time at 5 dpi and then every 2-3 days), but in this case, the percentage of infected cells remains low. Infected TM3 cells may therefore provide a new model to study the host-pathogen interactions and to help determine the mechanisms involved in EAV persistence in stallion reproductive tract.

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.

Molecular investigation of allelic variants of EqCXCL16 gene in equine arteritis virus infected stallions of selected horse breeds in Poland.

Susceptibility to long-term persistent infection with Equine Arteritis Virus (EAV) in stallions is related with EqCXCL16 gene alleles of the host. In our study EqCXCL16 gene alleles were determined for 63 EAV shedders and 126 non-shedders of various horse breeds. In total, 60 (31.7%) out of 189 tested stallions were identified as carriers of susceptible variants of EqCXCL16 by real time PCR and Sanger sequencing. The presence of susceptible genotype was related to horse breed with the highest percentage in Wielkopolska breed, Polish coldblood and Silesian breed horses. Strong correlation between EqCXCL16 susceptible genotypes and EAV shedding in semen (p < .0001) was observed.

Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors.

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.

Detection of Lactate Dehydrogenase Elevating Virus in a Mouse Vivarium Using an Exhaust Air Dust Health Monitoring Program.

Lactate dehydrogenase elevating virus (LDV) continues to be one of the most common contaminants of cells and cell byproducts. As such, many institutions require that tumor cell lines, blood products, and products derived or passaged in rodent tissues are free of LDV as well as other pathogens that are on institutional exclusion lists prior to their use in rodents. LDV is difficult to detect by using a live-animal sentinel health monitoring program because the virus does not reliably pass to sentinel animals. After switching to an exhaust air dust health monitoring system, our animal resources center was able to detect a presumably long-standing LDV infection in a mouse colony. This health monitoring system uses IVC rack exhaust air dust collection media in conjunction with PCR analysis. Ultimately, the source of the contamination was identified as multiple LDV-positive patient-derived xenografts and multiple LDV-positive breeding animals. This case study is the first to demonstrate the use of environmental PCR testing as a method for detecting LDV infection in a mouse vivarium.

Spread of equine arteritis virus among Hucul horses with different EqCXCL16 genotypes and analysis of viral quasispecies from semen of selected stallions.

Equine arteritis virus (EAV) is maintained in the horse populations through persistently infected stallions. The aims of the study were to monitor the spread of EAV among Polish Hucul horses, to analyse the variability of circulating EAVs both between- and within-horses, and to identify allelic variants of the serving stallions EqCXCL16 gene that had been previously shown to strongly correlate with long-term EAV persistence in stallions. Serum samples (n = 221) from 62 horses including 46 mares and 16 stallions were collected on routine basis between December 2010 and May 2013 and tested for EAV antibodies. In addition, semen from 11 stallions was tested for EAV RNA. A full genomic sequence of EAV from selected breeding stallions was determined using next generation sequencing. The proportion of seropositive mares among the tested population increased from 7% to 92% during the study period, while the proportion of seropositive stallions remained similar (64 to 71%). The EAV genomes from different stallions were 94.7% to 99.6% identical to each other. A number (41 to 310) of single nucleotide variants were identified within EAV sequences from infected stallions. Four stallions possessed EqCXCL16S genotype correlated with development of long-term carrier status, three of which were persistent shedders and the shedder status of the remaining one was undetermined. None of the remaining 12 stallions with EqCXCL16R genotype was identified as a persistent shedder.

Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells.

Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorporated into the virion particles as monomers and as a Gp2/Gp3-HA/Gp4 trimer. Gp3-HA localized in ER and, to a lesser extent, in the Golgi, it also co-localized with the E protein but not with the N protein. The co-localization of Gp3-HA and the E protein with ERGIC was reduced. Moreover, EAV with Gp3-HA could become a valuable research tool for identifying host cell factors during infection and the role of Gp3 in virus attachment and entry.

Complete genome sequence and analysis of a new lethal arterivirus, Trionyx sinensis hemorrhagic syndrome virus (TSHSV), amplified from an infected Chinese softshell turtle.

Trionyx sinensis hemorrhagic syndrome virus (TSHSV) is a newly discovered lethal arterivirus that causes serious disease in Trionyx sinensis in China. In this study, the complete genome sequence of TSHSV was determined by RACE cloning, and the functions of the predicted proteins were predicted. The complete genome of TSHSV was found to be 17,875 bp in length, and a 3'-end poly(A) tail was detected. Eight TSHSV hypothetical proteins (TSHSV-HPs) were predicted by gene model identification. TSHSV-HP2, 3 and 4 were associated with replicase activity, since papain-like protease (PLPs), serine-type endopeptidase, P-loop-containing nucleoside triphosphate hydrolase, and EndoU-like endoribonuclease motifs were detected. Phylogenetic analysis showed that TSHSV clusters with an arterivirus from a Chinese broad-headed pond turtle.

Equine arteritis virus long-term persistence is orchestrated by CD8+ T lymphocyte transcription factors, inhibitory receptors, and the CXCL16/CXCR6 axis.

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a "hub" gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract.

Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis.

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.

Development of an antigen-capture ELISA for the quantitation of equine arteritis virus in culture supernatant.

Quantitation of virions is one of the important indexes in virological studies. To establish a sensitive and rapid quantitative detection method for equine arteritis virus (EAV), an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed by using two EAV nucleoprotein monoclonal antibodies (mAbs), 2B9 and 2B3, prepared in this study. After condition optimization, mAb 2B9 was used as the capture antibody, and HRP-labeled 2B3 was chosen as the detecting antibody. The AC-ELISA had a good standard curve when viral particles of the Bucyrus EAV strain were used as a reference standard. The detection limit for the Bucyrus EAV strain was 36 PFU, and the method had a good linear relationship between 72-2297 PFU. The AC-ELISA could specifically detect the Bucyrus EAV strain and had no cross-reaction with other equine viruses. The sensitivity of the AC-ELISA was much higher than that of a western blotting assay but lower than that of a real-time PCR method. However, as a quantitative antigen detection method, the sensitivity of the AC-ELISA was approximately 300 times than the western blotting assay. Furthermore, the AC-ELISA assay could be successfully used in quantification of viral content in an in vitro infection assay, such as a one-step growth curve of EAV, as well as in a transfection assay, such as virus rescue from an infectious cDNA clone of EAV. These results show that the AC-ELISA established in this study is a good alternative for antigen detection of EAV, being a simple, convenient and quantitative detection method for EAV antigens.

Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus.

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion.IMPORTANCE Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

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.

Serological evidence of equine arteritis virus infection and phylogenetic analysis of viral isolates in semen of stallions from Serbia.

BACKGROUND: Equine arteritis virus (EAV) is responsible for infections in equids. It can spread easily within the horse population and has a major impact on the horse breeding industry. No EAV outbreak has ever been reported in Serbia. To determine whether EAV is nonetheless circulating there, especially in the Vojvodina region, 340 horse serum samples were subjected to serology testing to detect EAV antibodies. In parallel, semen samples from three seropositive stallions were collected to evaluate their EAV status, using RT-qPCR and virus isolation on cell culture. RESULTS: Horse sera with EAV antibodies represented 15.88% (54/340) of the tested samples, 83.23% (283/340) being negative, and just three samples (0.89%) being uninterpretable due to cytotoxicity. Only 7.2% (10/138) of horses kept by private owners on their own property were seropositive for EAV, whereas 21.8% (44/202) of horses kept on stud farms had EAV antibodies. Phylogenetic analysis showed that the Serbian EAV isolate was most closely related to isolates from the neighbouring Hungary. CONCLUSIONS: EAV is circulating in the Serbian horse population, especially among the breeding population certainly due to the use of EAV shedder stallions since there is no surveillance programme in Serbia and only limited checks on racehorses. Moreover, phylogenetic analysis indicates that the EAV isolated from a Lipizzaner stallion in Serbia is closely related to isolates from Hungary, and together form a new cluster.

Identification of a divergent genotype of equine arteritis virus from South American donkeys.

A novel equine arteritis virus (EAV) was isolated and sequenced from feral donkeys in Chile. Phylogenetic analysis indicates that the new virus and South African asinine strains diverged at least 100 years from equine EAV strains. The results indicate that asinine strains belonged to a different EAV genotype.

Equine Arteritis Virus Elicits a Mucosal Antibody Response in the Reproductive Tract of Persistently Infected Stallions.

Equine arteritis virus (EAV) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. We have recently demonstrated that EAV persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. In the present study, we demonstrated that EAV elicits a mucosal antibody response in the reproductive tract during persistent infection with homing of plasma cells into accessory sex glands. The EAV-specific immunoglobulin isotypes in seminal plasma included IgA, IgG1, IgG3/5, and IgG4/7. Interestingly, seminal plasma IgG1 and IgG4/7 possessed virus-neutralizing activity, while seminal plasma IgA and IgG3/5 did not. However, virus-neutralizing IgG1 and IgG4/7 in seminal plasma were not effective in preventing viral infectivity. In addition, the serological response was primarily mediated by virus-specific IgM and IgG1, while virus-specific serum IgA, IgG3/5, IgG4/7, and IgG6 isotype responses were not detected. This is the first report characterizing the immunoglobulin isotypes in equine serum and seminal plasma in response to EAV infection. The findings presented herein suggest that while a broader immunoglobulin isotype diversity is elicited in seminal plasma, EAV has the ability to persist in the reproductive tract, in spite of local mucosal antibody and inflammatory responses. This study provides further evidence that EAV employs complex immune evasion mechanisms during persistence in the reproductive tract that warrant further investigation.

Development and Characterization of an Infectious cDNA Clone of Equine Arteritis Virus.

Development and characterization of several infectious cDNA clones of equine arteritis virus (EAV) have been described in the literature. Here we describe the assembly of the full-length infectious cDNA clone of the virulent Bucyrus strain (VBS; ATCC VR-796) of EAV in a plasmid vector. This system allows generation of infectious in vitro-transcribed (IVT) RNA from the linearized plasmid that can be transfected or electroporated into mammalian cells to produce infectious recombinant progeny virus. This is an efficient reverse genetics system that allows easy manipulation of EAV genomes to study molecular biology of the virus and pathogenesis of equine viral arteritis.

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8+ T and CD21+ B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract.

Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages, and dendritic cells (Iba-1+ and CD83+), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation.IMPORTANCE The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallion's reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.