Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus

Abstract

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the gene () and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion. Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the gene () and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.

Equine arteritis virus long-term persistence is orchestrated by CD8+ T lymphocyte transcription factors, inhibitory receptors, and the CXCL16/CXCR6 axis

“…Previously, we have demonstrated that the expression of CXCL16 is enhanced in the reproductive tract of long-term persistently infected stallions [30]. In agreement with this observation, whole transcriptome analysis of the ampullae identified the differential upregulation of CXCL16 in this group compared to short-term carrier and naïve stallions (FDR ≤ 0.00005, Table 2).…”

“…The mechanisms by which EAV persists in the male reproductive tract are not fully understood and have been recently the subject of extensive investigation in our laboratory [23–25, 28–30]. It has been demonstrated that long-term persistence is associated with the presence of a specific allele encoding the chemokine CXCL16 (namely, CXCL16S ), with a dominant pattern of inheritance and with EAV receptor activity [26, 28, 29].…”

“…Additionally, EAV has the ability to persist despite the induction of a strong systemic immune response and local inflammatory and mucosal antibody responses at the site of persistence [23, 24]. Recent studies showed that long-term persistence is associated with an upregulation of CXCL16 at the site of persistence along with a downregulation of the microRNA eca-mir-128 in seminal exosomes, a putative modulator of CXCL16 [30]. These provide a strong premise that EAV employs a complex strategy to evade host immunity and that host factors play a critical role in long-term viral persistence.…”

“…Moreover, EAV persists in the male genital tract despite the presence of strong inflammatory (mediated mainly by CD8 + T lymphocytes) and EAV-specific humoral and mucosal antibody responses [23, 24]. Also, it has been recently demonstrated that EAV long-term persistent infection is associated with the specific downregulation of microRNA (miRNA) eca-mir-128 in seminal exosomes along with an enhanced expression of CXCL16 in the ampullae, a putative target of eca-mir-128, at the site of persistent infection [30]. Understanding the mechanisms of EAV persistence in the stallion reproductive tract is critical to the success of efforts to develop novel therapeutics for elimination of the carrier state.…”

Detection of SARS-CoV-2 by RNAscope® in situ hybridization and immunohistochemistry techniques

“…Four micron sections of formalin-fixed paraffin-embedded mock-infected and SARS-CoV-2-infected Vero cells were mounted on positively charged Superfrost ® Plus Slides (VWR, Radnor, PA). The RNAscope ® ISH assay was performed using an RNAscope 2.5 HD Red Detection Kit (ACD) as described previously [10,14,15]. Briefly, deparaffinized sections were subjected to target retrieval for 15 min at 98-102 °C in 1X Target Retrieval Solution, dehydration in 100% ethanol for 10 min, and Protease Plus treatment for 20 min at 40 °C in a HybEZ™ oven (ACD).…”