Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Socha W

PROVIDER: S-EPMC7031528 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Socha Wojciech W Sztromwasser Pawel P Dunowska Magdalena M Jaklinska Barbara B Rola Jerzy J

Scientific reports 20200219 1

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 ...[more]

PMID: 32076048

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Project description:UnlabelledPrevious studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from <3% to almost 100%. The increase in permissiveness was blocked either by transfection of HEK-EqCXCL16 cells with small interfering RNAs (siRNAs) directed against EqCXCL16 or by pretreatment with guinea pig polyclonal antibody against EqCXCL16 protein (Gp anti-EqCXCL16 pAb). Furthermore, using a virus overlay protein-binding assay (VOPBA) in combination with far-Western blotting, gradient-purified EAV particles were shown to bind directly to the EqCXCL16 protein in vitro. The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14(+) monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp anti-EqCXCL16 pAb. The collective data from this study provide confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host cell entry processes, possibly acting as a primary receptor molecule for this virus.ImportanceOutbreaks of EVA can be a source of significant economic loss for the equine industry from high rates of abortion in pregnant mares, death in young foals, establishment of the carrier state in stallions, and trade restrictions imposed by various countries. Similar to other arteriviruses, EAV primarily targets cells of the monocyte/macrophage lineage, which, when infected, are believed to play a critical role in EVA pathogenesis. To this point, however, the host-specified molecules involved in EAV binding and entry into monocytes/macrophages have not been identified. Identification of the cellular receptors for EAV may provide insights to design antivirals and better prophylactic reagents. In this study, we have demonstrated that EqCXCL16 acts as an EAV entry receptor in EAV-susceptible cells, equine monocytes. These findings represent a significant advance in our understanding of the fundamental mechanisms associated with the entry of EAV into susceptible cells.

Project description: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 (CXCL16 S and CXCL16 r ) were identified in the equine population and correlated with the susceptibility or resistance of a CD3+ T cell subpopulation in peripheral blood to in vitro EAV infection, respectively. Interestingly, CXCL16 S has been linked to the establishment of LTPI in stallions, and thus, genotyping stallions based on CXCL16 S/r would allow identification of those at the highest risk of establishing LTPI. Thus, we developed a TaqMan® allelic discrimination qPCR assay for the genotyping of the equine CXCL16 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+ T cell susceptibility phenotype to EAV infection by flow cytometry and subsequently sequenced to determine CXCL16 allelic composition. Genotyping by Sanger sequencing determined that all horses with the resistant CD3+ T cell phenotype were homozygous for CXCL16 r while horses with the susceptible CD3+ T cell phenotype carried at least one CXCL16 S allele or homozygous for CXCL16 S . In addition, genotypification with the TaqMan® allelic discrimination qPCR assay showed perfect agreement with Sanger sequencing and flow cytometric analysis. In conclusion, the new TaqMan® allelic discrimination genotyping qPCR assay can be used to screen prepubertal colts for the presence of the CXCL16 genotype. It is highly recommended that colts that carry the susceptible genotype (CXCL16 S/S or CXCL16 S/r ) are vaccinated against EAV after 6 months of age to prevent the establishment of LTPI carriers following possible natural infection with EAV.

Dataset Information

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

Publications

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

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