Ted Kalbfleisch, Ernest Bailey, R. Frank Cook, Peter J. Timoney, Sanjay Sarkar, Udeni B. R. Balasuriya, Kathleen M. Shuck, R. Lakshman Chelvarajan, Yun Young Go, Sergey Artiushin, and John E. Eberth
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, Author Summary A variable proportion of EAV infected stallions (10–70%) may become persistently infected and continuously shed the virus exclusively in their semen after recovery from acute infection. Previous studies in our laboratory have shown that stallions with the CD3+ T lymphocyte susceptibility phenotype to in vitro EAV infection are at higher risk of becoming persistently infected carriers compared to those that lack this phenotype. Here genetic and experimental studies were used to demonstrate that CXCL16 in the horse codes for two proteins, one associated with resistance and the other associated with susceptibility of CD3+ T lymphocytes to EAV infection. The two proteins are the result of four nucleotide substitutions in exon 1 of the equine CXCL16 gene. These alleles determine the outcome of in vitro infection of CD3+ T lymphocytes with EAV and are strongly associated with the establishment and maintenance of long-term carrier state in stallions. In vitro studies demonstrated that one form of CXCL16 protein (CXCL16S) is one of the cellular receptors for EAV and has higher scavenger activity and adhesion ability as compared to the form of the protein associated with resistance (CXCL16R).