AUTHOR=Thieulent Come J. , Carossino Mariano , Balasuriya Udeni B. R. , Graves Kathryn , Bailey Ernest , Eberth John , Canisso Igor F. , Andrews Frank M. , Keowen Michael L. , Go Yun Young 

TITLE=Development of a TaqMan® Allelic Discrimination qPCR Assay for Rapid Detection of Equine CXCL16 Allelic Variants Associated With the Establishment of Long-Term Equine Arteritis Virus Carrier State in Stallions

JOURNAL=Frontiers in Genetics

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2022.871875

DOI=10.3389/fgene.2022.871875

ISSN=1664-8021

ABSTRACT=<p>Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. Following natural infection, up to 70% of the infected stallions can remain persistently infected over 1 year (long-term persistent infection [LTPI]) and shed EAV in their semen. Thus, the LTP-infected stallions play a pivotal role in maintaining and perpetuating EAV in the equine population. Previous studies identified equine C-X-C motif chemokine ligand 16 (CXCL16) as a critical host cell factor determining LTPI in the stallion’s reproductive tract. Two alleles (<italic>CXCL16</italic><sup><italic>S</italic></sup> and <italic>CXCL16</italic><sup><italic>r</italic></sup>) were identified in the equine population and correlated with the susceptibility or resistance of a CD3<sup>+</sup> T cell subpopulation in peripheral blood to <italic>in vitro</italic> EAV infection, respectively. Interestingly, <italic>CXCL16</italic><sup><italic>S</italic></sup> has been linked to the establishment of LTPI in stallions, and thus, genotyping stallions based on <italic>CXCL16</italic><sup><italic>S/r</italic></sup> would allow identification of those at the highest risk of establishing LTPI. Thus, we developed a TaqMan<sup>®</sup> allelic discrimination qPCR assay for the genotyping of the equine <italic>CXCL16</italic> gene based on the identification of a single nucleotide polymorphism in position 1,073 based on NCBI gene ID: 100061442 (or position 527 based on Ensembl: ENSECAG00000018406.2) located in exon 2. One hundred and sixty horses from four breeds were screened for the CD3<sup>+</sup> T cell susceptibility phenotype to EAV infection by flow cytometry and subsequently sequenced to determine <italic>CXCL16</italic> allelic composition. Genotyping by Sanger sequencing determined that all horses with the resistant CD3<sup>+</sup> T cell phenotype were homozygous for <italic>CXCL16</italic><sup><italic>r</italic></sup> while horses with the susceptible CD3<sup>+</sup> T cell phenotype carried at least one <italic>CXCL16</italic><sup><italic>S</italic></sup> allele or homozygous for <italic>CXCL16</italic><sup><italic>S</italic></sup>. In addition, genotypification with the TaqMan<sup>®</sup> allelic discrimination qPCR assay showed perfect agreement with Sanger sequencing and flow cytometric analysis. In conclusion, the new TaqMan<sup>®</sup> allelic discrimination genotyping qPCR assay can be used to screen prepubertal colts for the presence of the <italic>CXCL16</italic> genotype. It is highly recommended that colts that carry the susceptible genotype (<italic>CXCL16</italic><sup><italic> S/S</italic></sup> or <italic>CXCL16</italic><sup><italic>S/r</italic></sup>) are vaccinated against EAV after 6 months of age to prevent the establishment of LTPI carriers following possible natural infection with EAV.</p>