Development and Evaluation of a One-Step Multiplex Real-Time TaqMan\u3csup\u3e®\u3c/sup\u3e RT-qPCR Assay for the Detection and Genotyping of Equine G3 and G14 Rotaviruses in Fecal Samples

Background: Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and are significantly time-consuming, costly and labor intensive. Here, we developed the first one-step multiplex TaqMan® real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes for the rapid detection and G-typing directly from fecal specimens. Methods: A one-step multiplex TaqMan® RT-qPCR assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes was designed. The analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA containing the target sequences while the analytical specificity was determined using RNA and DNA derived from a panel of group A rotaviruses along with other equine viruses and bacteria. The clinical performance of this multiplex assay was evaluated using a panel of 177 fecal samples and compared to a VP7-specific standard RT-PCR assay and Sanger sequencing. Limits of detection (LOD), sensitivity, specificity, and agreement were determined. Results: The multiplex G3 and G14 VP7 assays demonstrated high specificity and efficiency, with perfect linearity. A 100-fold difference in their analytical sensitivity was observed when compared to the singleplex assays; however, this difference did not have an impact on the clinical performance. Clinical performance of the multiplex RT-qPCR assay demonstrated that this assay had a high sensitivity/specificity for every target (100% for NSP3, \u3e 90% for G3 VP7 and \u3e 99% for G14 VP7, respectively) and high overall agreement (\u3e 98%) compared to conventional RT-PCR and sequencing. Conclusions: This new multiplex RT-qPCR assay constitutes a useful, very reliable tool that could significantly aid in the rapid detection and G-typing of ERVA strains circulating in the field

Equine Arteritis Virus Long-Term Persistence Is Orchestrated by CD8\u3csup\u3e+\u3c/sup\u3e T Lymphocyte Transcription Factors, Inhibitory Receptors, and the CXCL16/CXCR6 Axis

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a “hub” gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract

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

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8(+) T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a "hub" gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8(+) T lymphocyte response and unique lymphocyte homing in the reproductive tract

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

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 CXCL16 gene (CXCL16S) 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 Elicits a Mucosal Antibody Response in the Reproductive Tract of Persistently Infected Stallions

Equine arteritis virus (EAV) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. We have recently demonstrated that EAV persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. In the present study, we demonstrated that EAV elicits a mucosal antibody response in the reproductive tract during persistent infection with homing of plasma cells into accessory sex glands. The EAV-specific immunoglobulin isotypes in seminal plasma included IgA, IgG1, IgG3/5, and IgG4/7. Interestingly, seminal plasma IgG1 and IgG4/7 possessed virus-neutralizing activity, while seminal plasma IgA and IgG3/5 did not. However, virus-neutralizing IgG1 and IgG4/7 in seminal plasma were not effective in preventing viral infectivity. In addition, the serological response was primarily mediated by virus-specific IgM and IgG1, while virus-specific serum IgA, IgG3/5, IgG4/7, and IgG6 isotype responses were not detected. This is the first report characterizing the immunoglobulin isotypes in equine serum and seminal plasma in response to EAV infection. The findings presented herein suggest that while a broader immunoglobulin isotype diversity is elicited in seminal plasma, EAV has the ability to persist in the reproductive tract, in spite of local mucosal antibody and inflammatory responses. This study provides further evidence that EAV employs complex immune evasion mechanisms during persistence in the reproductive tract that warrant further investigation

Evaluation of a Field-Deployable Reverse Transcription-Insulated Isothermal PCR for Rapid and Sensitive On-Site Detection of Zika Virus

Background: The recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources. Methods: We developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively. Results: These assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/μl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83). Conclusions: The ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8\u3csup\u3e+\u3c/sup\u3e T and CD21\u3csup\u3e+\u3c/sup\u3e B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract

Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages, and dendritic cells (Iba-1+ and CD83+), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation

Seaweed polysaccharide-based hydrogels used for the regeneration of articular cartilage

This manuscript provides an overview of the in vitro and in vivo studies reported in the literature focusing on seaweed polysaccharides based hydrogels that have been proposed for applications in regenerative medicine, particularly, in the field of cartilage tissue engineering. For a better understanding of the main requisites for these specific applications, the main aspects of the native cartilage structure, as well as recognized diseases that affect this tissue are briefly described. Current available treatments are also presented to emphasize the need for alternative techniques. The following part of this review is centered on the description of the general characteristics of algae polysaccharides, as well as relevant properties required for designing hydrogels for cartilage tissue engineering purposes. An in-depth overview of the most well known seaweed polysaccharide, namely agarose, alginate, carrageenan and ulvan biopolymeric gels, that have been proposed for engineering cartilage is also provided. Finally, this review describes and summarizes the translational aspect for the clinical application of alternative systems emphasizing the importance of cryopreservation and the commercial products currently available for cartilage treatment.Authors report no declarations of interest. Authors thank the Portuguese Foundation for Science and Technology (FCT) for the PhD fellowship of Elena G. Popa (SFRH/BD/64070/2009) and research project (MIT/ECE/0047/2009). The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS