Allelic Variation in CXCL16 Determines CD3+ T Lymphocyte Susceptibility to Equine Arteritis Virus Infection and Establishment of Long-Term Carrier State in the Stallion

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. Following natural infection, 10–70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3+ T lymphocytes that are susceptible to in vitro EAV infection and that this phenotypic trait is associated with long-term carrier status following exposure to the virus. In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions (XM_00154756; c.715 A → T, c.801 G → C, c.804 T → A/G, c.810 G → A) within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles (EqCXCL16Sa, EqCXCL16Sb) encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P<0.000001) and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R) was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence) in the male reproductive tract. EqCXCL16Sa and EqCXCL16Sb exert a dominant mode of inheritance. Most importantly, the protein isoform EqCXCL16S but not EqCXCL16R can function as an EAV cellular receptor. Although both molecules have equal chemoattractant potential, EqCXCL16S has significantly higher scavenger receptor and adhesion properties compared to EqCXCL16R.

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

“…Furthermore, genetic association studies have demonstrated the correlation between CXCL16S and EAV carrier status in stallions. Moreover, we have determined that CXCL16S has receptor activity for EAV whereas CXCL16R does not (42,92). Therefore, CXCL16S clearly plays a central role in the establishment and maintenance of persistent infection in the stallion reproductive tract.…”

“…Although the mechanisms that determine why some stallions clear the virus from the reproductive tract while others become carriers is far from being fully understood, we have recently demonstrated that the ability of EAV to persist is strongly influenced by host genetics (42). In support of this conclusion it was initially shown that long-term persistence of EAV in the stallion reproductive tract is strongly correlated with possession of a subpopulation of CD3 ϩ T lymphocytes that is susceptible to EAV infection in vitro (39).…”

“…These alleles differ by four nonsynonymous nucleotide substitutions within exon 1. The resultant proteins associated with CD3 ϩ T lymphocyte EAV susceptibility or resistance are designated CXCL16S and CXCL16R, respectively (41,42). Furthermore, genetic association studies have demonstrated the correlation between CXCL16S and EAV carrier status in stallions.…”

“…On the other hand, stallions that lack this subpopulation are at significantly lower risk of becoming persistently infected (39). Furthermore, a genome-wide association study (GWAS) con-ducted by this laboratory revealed that the ability of EAV to infect CD3 ϩ T lymphocytes and establish long-term carrier status in stallions is strongly associated with four nucleotide changes in the CXCL16 gene located on equine chromosome 11 (41,42). To our knowledge this is the first demonstration of a correlation between a viral carrier state and a host genetic component.…”

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

“…The carrier state is testosterone dependent and can last from several weeks or months (viral shedding for Ͻ1 year postinfection; short-term carriers) to several years or even lifelong (viral shedding for Ͼ1 year postinfection; long-term carriers), despite the development of a strong neutralizing antibody response in serum (1, 5, 11-15, 20, 21). Recently, it has been demonstrated that host genetics are implicated in the outcome of infection (i.e., the short-or long-term carrier state) in the stallion (22,23). Carrier stallions constitute the natural reservoir for EAV and, thus, play a key epidemiological role, as they ensure the maintenance and perpetuation of the virus in equine populations between breeding seasons (1,11,12).…”