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Following natural infection, some stallions continue to shed virus in the semen for long periods more than one year to lifelong after EAV is no longer present in blood and nasal secretions [ 2 , 22 ] and as such become reservoirs for the maintenance of this virus in equid populations. Therefore, we analyzed semen from 77 stallions comprising 24 different breeds that had been infected with EAV and their status as long-term carrier animals determined shedders; Table 5.

Of these, 37 were identified as long-term carriers stallions EAV seropositive and shedding detectable levels of virus in semen more than one year after initial infection while 40 were identified as non-carriers stallions seropositive for EAV, but had apparently cleared the virus from the reproductive tract [non-shedders] based on absence of detectable virus in semen post-infection. Recently, we demonstrated that the membrane-associated form of the chemokine encoded by the EqCXCL16Sa allele can function as a host-cell receptor for EAV binding and entry [ 23 ]. The results confirmed that the variant forms of EqCXCL16 were produced in apparently similar amounts and that they were both clearly associated with the plasma membrane Fig 2A.

Images are representative of three independent experiments. This result was confirmed in experiments where EAV gene expression following infection was detected by an indirect immunofluorescence assay IFA using a monoclonal antibody against viral nonstructural protein 1 nsp Therefore, while initial polyacrylamide gel separation of the cell lysates was conducted under denaturating conditions, it is conceivable that EqCXCL16S may associate strongly with other proteins.

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Statistically different results are represented with different letters, a and b. Panel d shows the graphical representation of the images from panels a, b, and c. All the images depicted were representative of three independent experiments. Subsequently, the bound virus particles were visualized using streptavidin FITC. Therefore, it was decided to determine if scavenger receptor activity differed between EqCXCL16 isoforms.

Although the experiments described above were not quantitative, they did reveal obvious differences between the two EqCXCL16 isoforms in terms of OxLDL scavenger receptor activity. Proteins were then sequentially denatured and renatured by using different concentrations of Gn-HCl. After washing, membranes were developed using the ECL method.

These interactions occurred at the same location as that occupied by EqCXCR6; this was confirmed by stripping the membrane shown in panel b and re-probing it with anti-His antibody as shown in panel c. The fact that human CXCL16 can act as a chemoattractant and recruit lymphoid cell types to sites of inflammation within the body suggests that this property may also be present in EqCXCL16 [ 25 , 27 ]. The cells that passed through the filter were counted with a fluorescent microscope and represented in a bar diagram.

Controls consisted of RPMI with 0. Experiments were repeated independently three times. Both a and b are significantly different. Membrane-associated forms of human CXCL16 have been reported to function as cellular adhesion molecules [ 25 ]. A Representative image of a well plate stained with crystal violet following EDTA treatment showing the differences in adhesion of the different HEKT-derived cells.

Furthermore, a genome wide association study GWAS based on single nucleotide polymorphism SNP detection using the Equine SNP50 BeadChip suggested an association between these phenotypic traits and a dominant genetic marker s located on equine chromosome 11 ECA11 between nucleotide positions 49,, and 49,, [ 20 ].

Whole genome sequencing of one resistant and two susceptible horses revealed 12 non-synonymous nucleotide substitutions distributed among eight previously annotated genes in the targeted region on ECA CXCL16 was implicated as the putative cause of the phenotype based on tests of the SNPs among 10 horses 8 susceptible and 2 resistant that excluded the seven other genes. If analogous to the human orthologue of CXCL16 , exon 1 should encode the N-terminal ectodomain of the chemokine and, therefore, play a major role in its biological properties.

Although there is to our knowledge no published information concerning the functional properties of EqCXCL16, the human equivalent of this protein CXCL16 is expressed as a type-I membrane protein and is comprised of several distinct domains chemokine, extracellular [mucin stalk], hydrophobic transmembrane, and intracellular cytoplasmic [ 28 ].

The protein can also exist in an unbound soluble form resulting either from cleavage with cellular metalloproteinases such as ADAM10 [ 28 ] or alternative mRNAsplicing [ 29 ]. Moreover, expression on these cell types is upregulated by inflammatory mediators and bacterial lipopolysaccharides [ 27 , 29 , 36 , 38 — 40 ].

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Binding to the CXCR6 receptor is facilitated by the mucin-stalk located within the extracellular domain of the molecule [ 44 , 45 ]. The extracellular domain of HuCXCL16 also recognizes oxidized low-density lipoprotein OxLDL along with phosphatidylserine; therefore, the protein is multifunctional, acting as a scavenger receptor in addition to possessing chemokine activity [ 34 ].

Furthermore, aberrant expression of HuCXCL16 is implicated in the pathogenesis of certain viral infections, arthritis, atherosclerosis, and the metastasis of some cancers [ 46 — 50 ]. Preliminary in silico studies suggested that EqCXCL16 possesses a domain structure very similar to its human counterpart including the presence of six cysteine residues within the chemokine domain. However, a Tyr-X-Pro-Val motif in the C-terminal intracellular domain believed to act as a potential substrate for tyrosine kinase phosphorylation in human and mouse variants of the protein is replaced by Tyr-X-Pro-Val in the horse.

Tyr40Phe, p. Asp49His, p. Phe50Ile, and p. Moreover, there were dramatic differences between HEKT cells expressing each of these proteins to adhere to plastic culture vessels in the presence of EDTA. Collectively, these results indicate that EqCXCL16S, in common with its human counterpart, has both chemotactic and scavenger receptor activity, whereas the latter property is likely to be substantially reduced in the EqCXCL16R isoform [ 51 ]. This suggests amino acid residues located at positions 40 to 52 within the chemokine domain of EqCXCL16 have a direct role in viral attachment via interactions with EAV membrane surface glycoproteins.

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These studies confirm and extend our previous findings [ 28 ] that although some viruses such as HIV and severe acute respiratory syndrome SARS virus may specifically interact with CXCL16 to influence the course of viral pathogenesis [ 32 , 33 ], EAV is at present unique in its ability to utilize this chemokine as a receptor protein. However, the choice of a scavenger receptor as a portal for viral entry does have a precedent among the arteriviruses in that porcine reproductive and respiratory syndrome virus PRRSV uses CD [ 48 ].

It has been reported that CXCL16 in humans is expressed by a subpopulation s of T lymphocytes [ 49 , 50 ]. Another caveat to this hypothesis is that EAV exhibits a broad host-cell tropism and can infect several common laboratory cell lines from different species that certainly do not express EqCXCL Previous studies from our laboratory and others have shown that EAV can utilize a number of different non-related, host-specified molecules as cellular entry receptors or accessory molecules [ 52 ]. This P-statistic has limited value in that the stallions in Table 6 came from diverse breeds Table 5 , however the association of the genotype with the phenotype without regard to breed was remarkable.

Although results presented here are consistent with existence of the long-term carrier status in the majority of stallions being dependent on the membrane-bound form of EqCXCL16S acting as a cellular receptor for EAV, the fact that the EqCXCL16 allelic association is not complete suggests additional genetic, immunological, and viral factors or even environmental factors also play a role in this determination.

Furthermore, the EAV carrier state in stallions has a number of features that distinguish it from many other persistent viral infections of the male reproductive tract. These add additional layers of complexity and must be considered in any proposed mechanism. For example, recent virus isolation and immunohistochemistry studies have confirmed that EAV persists primarily in the ampulla along with other accessory sex glands rather than in immunologically privileged sites such as the Sertoli cells within the testis Carossino et al.

This is despite the fact that carrier stallions possess active immune responses against EAV, and the virus is not detectable in any organ or tissue except the reproductive tract in carrier animals. Indeed inflammatory infiltrates in close proximity to viral antigen-expressing cells are frequently observed in the stallion reproductive tract indicating that EAV persistence is a continuous, dynamic process that occurs in the presence of active local immune responses. Clearly these responses are not completely effective in clearance of the virus for reasons that are unknown at present.

Potential explanations range from some form of localized immunosuppression based on the fact that androgens such as testosterone can down-regulate immune responses, to more specific activity such as that mediated by T regulatory lymphocytes or even to differences in functional properties between EqCXCL16S and EqCXCL16R. In addition, the virus probably contributes to its own survival via antigenic drift as evidenced by the continual emergence of novel variants during the course of persistent infections [ 16 , 54 , 55 ].

However, it is also possible that variation exists between individual stallions in the efficacy of EAV-specific immune responses within the reproductive tract that can operate independently of the EqCXCL16 genotype.

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Based on the EqCXCL16 gene polymorphisms and its association with long-term carrier status, it would be possible to develop an allelic discrimination real-time PCR assay to distinguish horses that are prone to become long-term versus short-term shedders. It is interesting that while polymorphisms of CXCL16 have been reported in, for example, the human CXCL16 gene [ 44 , 51 , 56 — 58 ], these are located in regions other than exon 1.

Furthermore, these polymorphisms have not been shown to completely abrogate the chemokine, scavenger receptor properties [ 43 ]. The horse may therefore be highly unusual in possessing allelic variants of this gene with SNPs situated in exon 1 that in the case of EqCXCL16R appear to disrupt the ability of the resultant protein to act as a scavenger receptor for OxLDL. Although the molecular mechanisms associated with these phenotypic traits have not been fully elucidated, there is compelling evidence the plasma membrane-associated variant of EqCXCL16S can function as a cellular entry receptor for EAV, whereas this property is absent in the EqCXCL16R isoform of the protein.

It is interesting that the sequence associated with abrogation of the virus binding site costs the horse the scavenging capability of CXCL This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Briefly, blood 20 ml was collected aseptically using Vacutainer tubes containing 0. Staining was performed following the protocol as described previously [ 20 , 45 ].

Both viruses were propagated in EECs to generate high titer working stocks as previously described [ 63 , 64 ]. Serum neutralizing antibodies to EAV were demonstrated by microneutralization assay in both carrier and non-carrier stallions to confirm that they were seropositive for EAV [ 67 ].

Based on clinical histories, none of the stallions had been vaccinated against EAV, and seroconversion was the result of natural infection.


EAV long-term shedders were defined as those horses that had detectable EAV in their semen for more than one year following infection where date of exposure was known. Non-shedders of EAV were those which likely shed EAV in their semen during the acute phase of infection but that had ceased shedding virus at the time of initial testing for presence of the carrier state.

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To achieve 20—35 GB of raw data per lane, bp paired end sequencing across 7 lanes was conducted. Reads were mapped to the horse genome reference sequence Ecab 2. Genome annotation was from Ensembl version Equus caballus. In order to express recombinant polypeptides, plasmids were transformed into E. Jeff Stott, University of California, Davis. Briefly, for expression of the EqCXCL16R protein in eukaryotic cells, a codon-optimized full-length EqCXCL16R sequence obtained from whole genome sequencing in this study was commercially synthesized and cloned into the pJ plasmid, into which the puromycin resistance gene was incorporated, by DNA2.

This process was repeated every other day until only puromycin-resistant colonies remained.

Cells were then stained as described previously [ 72 ]. The membranes were washed again and antibody binding was visualized with an ECL-detection system using SuperSignal West Pico chemiluminescent substrate Thermo Scientific. The cell adhesion assay was performed in accordance with a published protocol [ 75 ]. Cells were then incubated with 0.

Cells were then washed in distilled water and stained with 0. The bound proteins were then denatured and gradually renatured on the membrane by sequential incubation with 6 M, 3 M, 1 M, and 0. Cells were resuspended in cold PBS pH 7. Cells were then analyzed using a Nikon inverted fluorescence microscope and the percentage of cells bound to EAV was calculated.

Differences among multiple treatment groups were analyzed by statistical analysis software Sigmaplot P-values less than 0. The authors would like to thank Ms. The authors would also like to thank Ms. Diane Furry at the Maxwell H. Lynn Ennis and Mr. Funding acquisition: UBRB. Project administration: UBRB. Supervision: UBRB. Abstract Equine arteritis virus EAV is the causative agent of equine viral arteritis EVA , a respiratory, systemic, and reproductive disease of horses and other equid species. Download: PPT. Table 1. Table 2. Table 3.