Equine pegiviruses cause persistent infection of bone marrow and are not associated with hepatitis.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.

Pathogenesis, MicroRNA-122 Gene-Regulation, and Protective Immune Responses After Acute Equine Hepacivirus Infection.

BACKGROUND AND AIMS: Equine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses. APPROACH AND RESULTS: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV-specific T cells were identified. Additionally, an interferon-stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA (miR), miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. CONCLUSIONS: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.

Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet+ B cells.

BACKGROUND: Traditional laboratory model organisms represent a small fraction of the diversity of multicellular life, and findings in any given experimental model often do not translate to other species. Immunology research in non-traditional model organisms can be advantageous or even necessary, such as when studying host-pathogen interactions. However, such research presents multiple challenges, many stemming from an incomplete understanding of potentially species-specific immune cell types, frequencies, and phenotypes. Identifying and characterizing immune cells in such organisms is frequently limited by the availability of species-reactive immunophenotyping reagents for flow cytometry, and insufficient prior knowledge of cell type-defining markers. RESULTS: Here, we demonstrate the utility of single-cell RNA sequencing (scRNA-Seq) to characterize immune cells for which traditional experimental tools are limited. Specifically, we used scRNA-Seq to comprehensively define the cellular diversity of equine peripheral blood mononuclear cells (PBMC) from healthy horses across different breeds, ages, and sexes. We identified 30 cell type clusters partitioned into five major populations: monocytes/dendritic cells, B cells, CD3+PRF1+ lymphocytes, CD3+PRF1- lymphocytes, and basophils. Comparative analyses revealed many cell populations analogous to human PBMC, including transcriptionally heterogeneous monocytes and distinct dendritic cell subsets (cDC1, cDC2, plasmacytoid DC). Remarkably, we found that a majority of the equine peripheral B cell compartment is comprised of T-bet+ B cells, an immune cell subpopulation typically associated with chronic infection and inflammation in human and mouse. CONCLUSIONS: Taken together, our results demonstrate the potential of scRNA-Seq for cellular analyses in non-traditional model organisms and form the basis for an immune cell atlas of horse peripheral blood.

Relevant Equine Renal Anatomy, Physiology, and Mechanisms of Acute Kidney Injury: A Review.

Regulation of renal blood flow is by both extrinsic and intrinsic systems. Intrinsic regulation occurs via the afferent and efferent arterioles and tubuloglomerular feedback mechanisms with activation of the juxtaglomerular apparatus. Mechanisms of acute kidney injury are frequently associated with changes in renal blood flow. Acute tubular necrosis and apoptosis are common in horses following ischemic or toxic insults and in sepsis-associated acute kidney injury. Sepsis-associated renal injury often has a complex mechanism of disease involving both functional and obstructive changes in intrarenal circulation. Acute interstitial nephritis may occur following Leptospira sp infection or can be secondary to tubular necrosis.

Acute Kidney Injury and Renal Failure in Horses.

Nephrotoxic and hemodynamically mediated disorders are the most common causes of acute renal failure (ARF) in horses and foals. Leptospira spp. is the most common infectious cause of ARF. Initial treatments for ARF include elimination of nephrotoxic drugs, correction of predisposing disorders, and fluid therapy to promote diuresis. Horses and foals with polyuric ARF often have a good prognosis, while those with oliguric or anuric ARF have a guarded to poor prognosis. When fluid therapy is unsuccessful in improving urine production, various drugs treatments have been used in an attempt to increase urine production, but none are consistently effective in converting oliguria to polyuria.

Correction: miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence.

[This corrects the article DOI: 10.1371/journal.ppat.1006694.].

Clinical Pathology in the Adult Sick Horse: The Gastrointestinal System and Liver.

The gastrointestinal tract and liver comprise key components of the equine digestive system and together have important functions in metabolism, digestion, absorption, detoxification, and synthesis. Disorders of the gastrointestinal tract and liver are common in clinical practice and can cause a variety of clinical signs. Hematologic and biochemical analysis can be helpful for identifying organ dysfunction, narrowing down the differential diagnostic list, and monitoring progress and response to treatment. This article details hematologic and biochemical tests that are important in the evaluation of intestinal and hepatic diseases and reviews bloodwork trends frequently observed in adult horses affected by enteropathy or hepatopathy.

miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence.

Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5'UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5' end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity.

What Do We Know About Hepatitis Viruses in Horses?

Theiler disease (serum hepatitis or idiopathic acute hepatic necrosis) has long been suspected to have a viral etiology. Four viruses have been described in association with hepatitis in horses. Further investigation suggests equine pegivirus and Theiler disease-associated virus (a second pegivirus) are neither hepatotropic nor pathogenic. Nonprimate hepacivirus (NPHV) causes subclinical disease in experimental models and has been associated with hepatitis in some clinical cases. Equine parvovirus-hepatitis (EqPV-H) experimentally causes subclinical-to-clinical liver disease and is found in the vast majority of Theiler disease cases. EqPV-H is likely of clinical significance, whereas the significance of NPHV is unknown.

New Parvovirus Associated with Serum Hepatitis in Horses after Inoculation of Common Biological Product.

Equine serum hepatitis (i.e., Theiler's disease) is a serious and often life-threatening disease of unknown etiology that affects horses. A horse in Nebraska, USA, with serum hepatitis died 65 days after treatment with equine-origin tetanus antitoxin. We identified an unknown parvovirus in serum and liver of the dead horse and in the administered antitoxin. The equine parvovirus-hepatitis (EqPV-H) shares <50% protein identity with its phylogenetic relatives of the genus Copiparvovirus. Next, we experimentally infected 2 horses using a tetanus antitoxin contaminated with EqPV-H. Viremia developed, the horses seroconverted, and acute hepatitis developed that was confirmed by clinical, biochemical, and histopathologic testing. We also determined that EqPV-H is an endemic infection because, in a cohort of 100 clinically normal adult horses, 13 were viremic and 15 were seropositive. We identified a new virus associated with equine serum hepatitis and confirmed its pathogenicity and transmissibility through contaminated biological products.

Characterization of nonprimate hepacivirus and construction of a functional molecular clone.

Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼ 3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3'-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3'-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3'-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver cultures or after electroporation of selectable replicons. However, intrahepatic RNA inoculation of a horse initiated infection, yielding high RNA titers in the serum and liver. Delayed seroconversion, slightly elevated circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel hepacivirus in its natural host.

Experimental transmission of equine hepacivirus in horses as a model for hepatitis C virus.

UNLABELLED: Equine hepacivirus (EHCV; nonprimate hepacivirus) is a hepatotropic member of the Flaviviridae family that infects horses. Although EHCV is the closest known relative to hepatitis C virus (HCV), its complete replication kinetics in vivo have not been described, and direct evidence that it causes hepatitis has been lacking. In this study, we detected EHCV in 2 horses that developed post-transfusion hepatitis. Plasma and serum from these horses were used to experimentally transmit EHCV to 4 young adult Arabian horses, two 1-month-old foals (1 Arabian and 1 Arabian-pony cross), and 2 foals (1 Arabian and 1 Arabian-pony cross) with severe combined immunodeficiency (SCID). Our results demonstrated that EHCV had infection kinetics similar to HCV and that infection was associated with acute and chronic liver disease as measured by elevations of liver-specific enzymes and/or by histopathology. Although most of these animals were coinfected with equine pegivirus (EPgV), also a flavivirus, EPgV viral loads were much lower and often undetectable in both liver and blood. Three additional young adult Arabian-pony crosses and 1 SCID foal were then inoculated with plasma containing only EHCV, and evidence of mild hepatocellular damage was observed. The different levels of liver-specific enzyme elevation, hepatic inflammation, and duration of viremia observed during EHCV infection suggested that the magnitude and course of liver disease was mediated by the virus inoculum and/or by host factors, including breed, age, and adaptive immune status. CONCLUSION: This work documents the complete infection kinetics and liver pathology associated with acute and chronic EHCV infection in horses and further justifies it as a large animal model for HCV.

Identification of a previously undescribed divergent virus from the Flaviviridae family in an outbreak of equine serum hepatitis.

Theiler's disease is an acute hepatitis in horses that is associated with the administration of equine blood products; its etiologic agent has remained unknown for nearly a century. Here, we used massively parallel sequencing to explore samples from a recent Theiler's disease outbreak. Metatranscriptomic analysis of the short sequence reads identified a 10.5-kb sequence from a previously undescribed virus of the Flaviviridae family, which we designate "Theiler's disease-associated virus" (TDAV). Phylogenetic analysis clusters TDAV with GB viruses of the recently proposed Pegivirus genus, although it shares only 35.3% amino acid identity with its closest relative, GB virus D. An epidemiological survey of additional horses from three separate locations supports an association between TDAV infection and acute serum hepatitis. Experimental inoculation of horses with TDAV-positive plasma provides evidence that several weeks of viremia preceded liver injury and that liver disease may not be directly related to the level of viremia. Like hepatitis C virus, the best characterized Flaviviridae species known to cause hepatitis, we find TDAV is capable of efficient parenteral transmission, engendering acute and chronic infections associated with a diversity of clinical presentations ranging from subclinical infection to clinical hepatitis.

Transnasal, Endoscopically Guided Skull-Based Surgery by Pharyngotomy for Mass Removal from the Sphenopalatine Sinus in a Horse.

OBJECTIVE: To report a transnasal, endoscopically guided ventral surgical approach for accessing the cranial and caudal segments of the sphenopalatine sinus for mass removal in a horse. STUDY DESIGN: Case report. ANIMAL: Adult horse with acute onset blindness referable to a soft tissue mass within the sphenopalatine sinus. CLINICAL REPORT: A 7-year-old Warmblood gelding presented with a history of running into a fence and falling. No neurologic signs were identified at initial examination but acute blindness was noted 3 weeks later. On computed tomography (CT) the sphenopalatine sinus was filled with a large homogeneous mass with poor contrast enhancement that extended dorsally with thinning to the dorsal cortex of the sphenoid bone, just rostral to the entrance of the optic canals into the cranial cavity. Surgical access to the sphenopalatine sinus was achieved using a transnasal, endoscopically guided ventral pharyngotomy approach and the mass lesion was removed. A presumptive diagnosis of chondroma was made based on histopathology. The horse recovered well from surgery, and although it has not regained vision as of 6.5 years postoperatively, the disease has not progressed. CONCLUSION: Transnasal, endoscopically-guided ventral surgical access to the sphenopalatine sinus is possible in horses and may improve access in horses with disease extending caudally beyond the palatine portion of the sinus. Use of smaller diameter or specialized instruments, such as various endoscopic bone cutting instruments, and CT image guidance may improve sinus access by this route.

CORRELATIONS AMONG ULTRASONOGRAPHIC MEASUREMENTS OF OPTIC NERVE SHEATH DIAMETER, AGE, AND BODY WEIGHT IN CLINICALLY NORMAL HORSES.

Early diagnosis of high intracranial pressure (ICP) is critical for minimizing progressive brain injury due to reduced cerebral perfusion. In people, detecting enlargement of the optic nerve sheath diameter (ONSD) by transpalpebral ultrasonography has been found to be an accurate test for high ICP. Aims of this prospective, observational, cross-sectional study were to test hypotheses that (1) ultrasonographic measurement of ONSD would be repeatable in horses, (2) have acceptable interobserver agreement, and (3) would be correlated with age and body weight. The sample population included 48 horses without clinical signs of high ICP and with varying ages and body weights. Two observers independently performed ONSD measurements in both eyes. All measurements ranged from 2.6 to 6.5 mm. The mean difference of repeated measures within observers was ≤0.1 mm and the coefficients of variation ranged from 5.0% to 8.8%. The mean difference of measures between observers was ≤0.2 mm. After correcting for performing multiple tests, no significant rank correlation (all r < 0.4 [absolute value]) was detected between ONSD and age or body weight. However, we observed smaller ONSD in foals versus adults (all P ≤ 0.002). In the foals, all observed measures of rostrocaudal and dorsoventral ONSD were <5 mm. In the adults, all observed measures of rostrocaudal and dorsoventral ONSD were ≤6.5 mm. Findings indicated that ultrasonographic ONSD measurement is a feasible test for use in horses of varying ages and sizes. Further investigation of this ultrasonographic measure as a clinical test for horse with suspected high ICP is warranted.

ECEIM consensus statement on equine kidney disease.

The aim of this consensus statement is to summarize and appraise scientific evidence and combine this with the clinical experience of a panel of experts to optimize recommendations on how to recognize and manage kidney disease in horses.

Case-control exercise challenge study on the pathogenesis of high serum gamma-glutamyl transferase activity in racehorses.

BACKGROUND: High serum γ-glutamyl-transferase (GGT) activity syndrome in racehorses has been associated with maladaption to exercise. Investigation of affected horses before and immediately after standard exercise may provide critical insight into the syndrome's pathophysiology. OBJECTIVES: To investigate blood biomarker changes in actively competing racehorses with high GGT activity associated with an exercise challenge. STUDY DESIGN: Case-control study. METHODS: High GGT case (age: 2-3 years) and normal GGT control (age: 2-7 years) pairs (3 Thoroughbred, 4 Standardbred pairs) at least 3 months into their training/racing season were included. Horses with a recent history of high GGT activity (≥50 IU/L) without additional biochemical evidence of liver disease were identified by veterinarians. Horses were tested again in the week prior to a planned exercise challenge to confirm persistent increases in GGT activity. Controls from the same stable with similar training/racing intensity and serum GGT activity ≤36 IU/L were matched with each case. Blood samples were obtained immediately before, 15 and 120 min after exercise. Pre-exercise serum samples were analysed for baseline select serum chemistries, selenium and vitamin E concentrations. Cortisol concentration and markers of oxidative status were measured in serum or plasma for all time points. Individual serum bile acid and coenzyme Q10 concentrations, plasma lipid mediator (fatty acids, oxylipids, isoprostanes) concentrations and targeted metabolomics analyses were performed using liquid chromatography-mass spectrometry. Serum viral PCR for equine hepaci- and parvovirus was performed in each animal. RESULTS: Cases had higher baseline concentrations of total glutathione, taurocholic acid, cortisol and cholesterol concentrations and higher or lower concentrations of specific oxylipid and isoprostane mediators, but there were no case-dependent changes after exercise. MAIN LIMITATIONS: Small sample size. CONCLUSIONS: Results indicated that glutathione metabolism was altered in high GGT horses. Enhanced glutathione recycling and mild cholestasis are possible explanations for the observed differences.

Diagnosis of Borrelia-associated uveitis in two horses.

Borrelia burgdorferi, the etiologic agent of Lyme disease is a tick born spirochetal infection. Clinical signs of Lyme borreliosis are uncommon in horses, but when present they are often vague and nonspecific. In horses, Lyme borreliosis has been implicated in musculoskeletal, neurological, reproductive, and ocular disorders, including uveitis, but definitive diagnosis can be challenging as the causative agent is rarely isolated and serologic tests can be unreliable and do not confirm active disease. Here, we report two cases of equine uveitis associated with B. burgdorferi based on the identification of spirochetes within ocular fluids and confirmed with PCR testing. The two cases illustrate some of the challenges encountered in the recognition and diagnosis of equine Lyme borreliosis. Although only one of many possible causes of equine uveitis, Lyme disease should be considered a differential diagnosis, especially in endemic areas. Given the possibility for false negative results of serum tests during uveitis associated with B. burgdorferi and the failure of such tests to confirm active infection, a combination of cytologic assessment, antibody, and/or PCR testing of ocular fluids may be worthwhile if the clinical suspicion for Lyme uveitis is high.

A case of Borrelia-associated cutaneous pseudolymphoma in a horse.

This case report describes a 10-year-old horse that developed multiple dermal papules over the right masseter area following removal of a tick from the same site 3 months earlier. Histological examination of a biopsy from a papule was suggestive of either a T-cell-rich B-cell lymphoma or cutaneous lymphoid hyperplasia, a form of pseudolymphoma sometimes associated with a tick bite. Positive serological testing and PCR of the biopsy sample for Borrelia in conjunction with immunohistochemical testing of the skin biopsy, the clinical history and response to treatment with doxycycline strongly supported the diagnosis of Borrelia-associated cutaneous pseudolymphoma.