CXCL16-associated genetic susceptibility to equine viral arteritis in export-bound horses from Spain.

Previous 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. Outbreaks 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.

Equine viral arteritis (EVA), a disease caused by infection with the equine arteritis virus (EAV), is present in many European countries. In Spain, the last confirmed outbreak was reported in 1992 and there is a paucity of seroprevalence studies. The disease has a major impact on the equine breeding industry, which is mainly represented by Spanish Purebred (SP) horses in Spain. To estimate the seroprevalence of EAV in the breeding SP horse population in central Spain and identify potential horse and studfarm level factors associated with seropositivity to EAV. Cross-sectional study. Individual serum samples from 555 SP horses, collected between September 2011 and November 2013 at 35 studfarms, were tested using a commercially available EAV antibody ELISA and seroneutralisation as the World Organisation for Animal Health reference confirmation test for samples with positive and equivocal results. Data on factors putatively associated with seropositivity to EAV were collected via a questionnaire and examined using random effects logistic regression for analysis of clustered data. Equine arteritis virus seroprevalence in the SP breeding population in central Spain standardised for the sex distribution of the reference horse population, was estimated to be 16.8% (95% confidence interval 5.2-28.5%). Increasing numbers of breeding mares on the studfarm and increasing percentage of mares with reproductive problems during the last 12 months were identified as being positively associated with EAV seropositivity. Mares vaccinated against Equine herpesvirus-1 (EHV-1) and/or -4 (EHV-4) were also positively associated with EAV seropositivity. These findings are of importance to ensure appropriate biosecurity measures for studfarms are carried out and may help facilitate the development of an EVA surveillance programme in the SP breeding horse population.

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<0.000001) and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R) was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state (viral persistence) in the male reproductive tract. EqCXCL16Sa and EqCXCL16Sb exert a dominant mode of inheritance. Most importantly, the protein isoform EqCXCL16S but not EqCXCL16R can function as an EAV cellular receptor. Although both molecules have equal chemoattractant potential, EqCXCL16S has significantly higher scavenger receptor and adhesion properties compared to EqCXCL16R.

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.

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.

Host Factors that Contribute to Equine Arteritis Virus Persistence in the Stallion: an Update

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a notifiable respiratory and reproductive disease of equids that causes significant losses to the equine industry. This study presents a comprehensive analysis of two EAV outbreaks in the UK in 2019, combining virus isolation, sequencing and phylogenetic analysis to provide a holistic understanding of EAV dynamics in these outbreaks. Genetic characterization revealed that all outbreak strains were similar to viruses detected in the UK and Europe from 2004 to 2011, belonging to phylogroup D and clustering in two groups as expected based on epidemiological profiling. Bayesian phylogenetic analysis indicated the direction of transmission. The 2019 EAV strains showed maximum variability in glycoprotein (GP) 3, followed by GP2, non-structural protein 2, GP4 and GP5, with one strain displaying a unique truncation in GP4 at position 149, a feature not previously identified in arteriviruses. Polymorphisms in the CXCL16 gene have been implicated in differential susceptibility to the establishment of long-term carrier states of EAV in stallions. Genotypic analysis of the CXCL16 gene revealed that one horse possessed the homozygous genotype associated with resistance to persistent infection. In contrast, the remaining four horses exhibited the heterozygous genotype, which has been linked to an increased risk of developing a long-term carrier state and contributing to ongoing viral transmission. All infected horses exhibited the presence of neutralizing antibodies in their serum. This study underscores the importance of early detection of silent infections to reduce the spread and prevent clinical outbreaks.

Genomic variability among globally distributed isolates of equine arteritis virus

Prolonged Persistence and Functional Activity of Anti-BCMA CAR-T Durcabtagene Autoleucel (PHE885) Manufactured Using the T-Charge™ Platform

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.IMPORTANCE 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 CXCL16 gene (CXCL16S) 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.

Overview of the role and action mechanism of microRNA-128 in viral infections

In a study of a herpes simplex virus (HSV) vaccine, the combination of sex and prior exposure to the virus resulted in HSV naive women mounting a prominent type I interferon response associated with reduced neutralizing titers to HSV.

Accurate prediction of soluble solid content of apples from multiple geographical regions by combining deep learning with spectral fingerprint features

Neuregulin 1 (NRG1) is a multifunctional neurotrophin that mediates neurodevelopment and schizophrenia risk. The NRG1 gene undergoes extensive alternative splicing, and association of brain NRG1 type IV isoform expression with the schizophrenia-risk polymorphism rs6994992 is a potential mechanism of risk. Novel splice variants of NRG1-IV (NRG1-IVNV), with predicted unique signaling capabilities, have been cloned in fetal brain tissue. The authors investigated the temporal dynamics of transcription of NRG1-IVNV, compared with the major NRG1 isoforms, across human prenatal and postnatal prefrontal cortical development, and they examined the association of rs6994992 with NRG1-IVNV expression. NRG1 type I-IV and NRG1-IVNV isoforms were evaluated with quantitative real-time polymerase chain reaction in human postmortem prefrontal cortex tissue samples at 14 to 39 weeks gestation and postnatal ages 0-83 years. The association of rs6994992 genotype with NRG1-IVNV expression and the subcellular distribution and proteolytic processing of NRG1-IVNV isoforms were also determined. Expression of NRG1 types I, II, and III was temporally regulated during prenatal and postnatal neocortical development. NRG1-IVNV was expressed from 16 weeks gestation until age 3. Homozygosity for the schizophrenia risk allele (T) of rs6994992 conferred lower cortical NRG1-IVNV levels. Assays showed that NRG1-IVNV is a novel nuclear-enriched, truncated NRG1 protein resistant to proteolytic processing. To the authors' knowledge, this study provides the first quantitative map of NRG1 isoform expression during human neocortical development and aging. It identifies a potential mechanism of early developmental risk for schizophrenia at the NRG1 locus, involving a novel class of NRG1 proteins.

BackgroundStudies which examine quality of life (QOL) provide important insights that are needed to understand the impacts of HIV/AIDS anti-retroviral treatment (ART), comorbid conditions and other factors on the daily activities of people living with HIV/AIDS (PLH). This study aimed to determine the inter-relationships between clinical factors, behavioural, socio-demographic variables and QOL among PLH.MethodsThe secondary analysis used data collected from 293 people living with HIV/AIDS (PLH) receiving second-line ART in Johannesburg in a clinical trial which evaluated the non-inferiority of ritonavir-boosted darunavir (DRV/r 400/100 mg) compared to ritonavir-boosted lopinavir (LPV/r) over a 48 week-period. Physical functioning, cognitive and mental QOL were measured using the Aids Clinical Trial Group questionnaire. Exploratory factor analyses were used to examine the structure, the relationships between and the construct validity of QOL items. Structural equation models which tested the a priori-hypothesised inter-relationships between QOL and other variables were estimated and goodness of fit of the models to the data was assessed.ResultsPatients on darunavir presented with lower pill burden. Older patients and women were more likely to report lower QOL scores. Pill burden mediated the effects of age, sex and treatment regimen on physical functioning QOL and adverse effects; the effects of age, sex, treatment regimen and adverse effects on cognitive QOL; and the effects of sex on mental QOL.ConclusionQOL among PLH is associated with socio-demographic and clinical factors. Therefore, QOL could be enhanced by considering PLH characteristics, clinical factors such as regimen side-effects profile, management of comorbid conditions and mitigating risks such as potential adverse drug-to-drug interactions among patients on ART.