A Screening Study Identified Decitabine as an Inhibitor of Equid Herpesvirus 4 That Enhances the Innate Antiviral Response.

Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.

Resurgence Following Higher or Lower Quality Alternative Reinforcement.

Resurgence is a temporary increase in a previously suppressed target behavior following a worsening in reinforcement conditions. Previous studies have examined how higher rates or magnitudes of alternative reinforcement affect suppression of the target behavior and subsequent resurgence. However, there has been no investigation of the effects of higher versus lower qualities of alternative reinforcement on resurgence. Using a three-phase resurgence preparation with rats, the present experiments examined the effects of an alternative reinforcer that was of higher (Experiment 1) or lower (Experiment 2) quality than the reinforcer that had previously maintained the target behavior. The results of both experiments showed greater reductions in target behavior with a higher quality alternative reinforcer and larger increases in target responding when a higher quality alternative reinforcer was removed. Along with prior findings with higher rates and magnitudes of alternative reinforcement, these findings suggest that variations in reinforcer dimensions that increase the efficacy of alternative reinforcement also tend to increase resurgence when alternative reinforcement is removed. The results are discussed in terms of the resurgence as choice in context model and in terms of potential clinical implications.

Emergency vascular repairs in trauma: Predictors of poor prognosis and a novel scoring system.

BACKGROUND: Vascular injuries comprise 1% to 4% of all trauma patients, and there are no widely used risk-stratification tools. We sought to establish predictors of revascularization failures and compare outcomes of trauma and vascular surgeons. METHODS: We performed a single-institution, case-control study of consecutive patients with traumatic arterial injuries who underwent open repair between 2016 and 2021. Multivariable logistic regression was used to investigate covariates impacting the primary composite outcome of repair failure/revision, amputation, or in-hospital mortality. RESULTS: Among 165 patients, the median age was 34 years, 149 (90%) were male, and 99 (60%) suffered penetrating injury. Popliteal (46%) and superficial femoral (44%) arterial injuries were most common. Interposition graft/bypass was the most frequent repair (n = 107 [65%]). Revascularization failure was observed in 24 patients (15%). Compared with trauma surgeons, vascular surgeons more frequently repaired blunt injuries (66% vs. 20%, p < 0.001), anterior tibial (18% vs. 5%, p = 0.012), or tibioperoneal injuries (28% vs. 4%, p < 0.001), with a below-knee bypass (38% vs. 20%, p = 0.019). Revascularization failure occurred in 10% (9 of 93) of repairs by trauma surgeons and 21% (13 of 61) of repairs by vascular surgeons. Mangled Extremity Severity Score >8 (odds ratio, 15.6; 95% confidence interval, 4.4-55.9; p < 0.001) and concomitant laparotomy or orthopedic procedure (odds ratio, 6.7; 95% confidence interval, 1.6-28.6; p = 0.010) were independently associated with revascularization failure. A novel composite scoring system (UT Houston Score) was developed by combining Mangled Extremity Severity Score, concomitant procedure, mechanism of injury, and injury location. This score demonstrated a sensitivity of 100% with a score of 0 and a specificity of 95% with a score of >3. CONCLUSION: After traumatic arterial injury, trauma surgeons repaired less-complex injuries but with fewer revascularization failures than vascular surgeons. The UT Houston Score may be used to risk stratify patients to determine who may benefit from vascular surgery consultation. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level III.

Studying longitudinal neutralising antibody levels against Equid herpesvirus 1 in experimentally infected horses using a novel pseudotype based assay.

Infection with equid herpesvirus 1 (EHV-1), a DNA virus of the Herpesviridae family represents a significant welfare issue in horses and a great impact on the equine industry. During EHV-1 infection, entry of the virus into different cell types is complex due to the presence of twelve glycoproteins (GPs) on the viral envelope. To investigate virus entry mechanisms, specific combinations of GPs were pseudotyped onto lentiviral vectors. Pseudotyped virus (PV) particles bearing gB, gD, gH and gL were able to transduce several target cell lines (HEK293T/17, RK13, CHO-K1, FHK-Tcl3, MDCK I & II), demonstrating that these four EHV-1 glycoproteins are both essential and sufficient for cell entry. The successful generation of an EHV-1 PV permitted development of a PV neutralisation assay (PVNA). The efficacy of the PVNA was tested by measuring the level of neutralising serum antibodies from EHV-1 experimentally infected horses (n = 52) sampled in a longitudinal manner. The same sera were assessed using a conventional EHV-1 virus neutralisation (VN) assay, exhibiting a strong correlation (r = 0.82) between the two assays. Furthermore, PVs routinely require -80 °C for long term storage and a dry ice cold-chain during transport, which can impede dissemination and utilisation in other stakeholder laboratories. Consequently, lyophilisation of EHV-1 PVs was conducted to address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions for different time periods. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests. Results indicated that lyophilisation could be used to stably preserve such complex herpesvirus pseudotypes, even after weeks of storage at room temperature, and that reconstituted EHV-1 PVs could be successfully employed in antibody neutralisation tests.

Aversive control versus stimulus control by punishment.

Estes (1944) reported that adding electric shock punishment to extinction hastened response suppression but that responding increased when shock was removed. This result contributed to a view that reinforcement and punishment are asymmetrical processes because punishment has only indirect and temporary suppressive effects. Azrin and Holz (1966) suggested the result might be interpreted instead as shock serving as a discriminative stimulus for the absence of reinforcement. Here, to further examine potential stimulus control by punishment in a similar preparation, two groups of rats initially responded for food plus punishment and a third group for food alone. Reinforcement was then removed for all groups for the remaining three phases. With P and N denoting punishment and no punishment, the four phases for the three groups were: P-P-N-N, P-N-P-N, and N-P-N-N. We found some evidence for stimulus control by shock deliveries for group N-P-N-N (as suggested by Azrin and Holz), but all other changes in responding appeared due to introduction or removal of the aversive properties of shock. Although punishment may indeed have temporary effects under many circumstances, we argue that the view that this implies asymmetrical reinforcement and punishment processes was based on the flawed assumption that reinforcement has direct strengthening effects.

Oral Administration of Valganciclovir Reduces Clinical Signs, Virus Shedding and Cell-Associated Viremia in Ponies Experimentally Infected with the Equid Herpesvirus-1 C2254 Variant.

Equid alphaherpesvirus-1 (EHV-1) is one of the main pathogens in horses, responsible for respiratory diseases, ocular diseases, abortions, neonatal foal death and neurological complications such as equine herpesvirus myeloencephalopathy (EHM). Current vaccines reduce the excretion and dissemination of the virus and, therefore, the extent of an epizooty. While their efficacy against EHV-1-induced abortion in pregnant mares and the decreased occurrence of an abortion storm in the field have been reported, their potential efficacy against the neurological form of disease remains undocumented. No antiviral treatment against EHV-1 is marketed and recommended to date. This study aimed to measure the protection induced by valganciclovir (VGCV), the prodrug of ganciclovir, in Welsh mountain ponies experimentally infected with an EHV-1 ORF30-C2254 strain. Four ponies were administered VGCV immediately prior to experimental EHV-1 infection, while another four ponies received a placebo. The treatment consisted in 6.5 mg/kg body weight of valganciclovir administered orally three times the first day and twice daily for 13 days. Clinical signs of disease, virus shedding and viraemia were measured for up to 3 weeks. The severity of the cumulative clinical score was significantly reduced in the treated group when compared with the control group. Shedding of infectious EHV-1 was significantly reduced in the treated group when compared with the control group between Day + 1 (D + 1) and D + 12. Viraemia was significantly reduced in the treated group when compared with the control group. Seroconversion was measured in all the ponies included in the study, irrespective of the treatment received. Oral administration of valganciclovir induced no noticeable side effect but reduced clinical signs of disease, infectious virus shedding and viraemia in ponies experimentally infected with the EHV-1 C2254 variant.

The effects of large, small, and thinning magnitudes of alternative reinforcement on resurgence.

Resurgence refers to an increase of a previously reinforced target behavior following the worsening of conditions for a more recently reinforced alternative behavior. There is evidence to suggest that alternative reinforcers of greater magnitude are more effective at reducing target responding but may also result in more resurgence when removed. Similar effects have been observed with high rates of alternative reinforcement. However, in clinical settings, reinforcement rate thinning is used to reduce the likelihood of resurgence associated with higher rates of alternative reinforcement. Given the clinical importance of alternative reinforcer magnitude, it is necessary to evaluate how reinforcer magnitude thinning may impact resurgence as well. Following Phase 1 in which target responding was reinforced, rats earned either large (six pellets), small (one pellet), or thinned (reduced from six pellets to one across sessions) magnitude reinforcement for alternative responding during target-response extinction in Phase 2. Then, alternative responding was placed on extinction for all groups in Phase 3. Target responding was comparably elevated at the end of Phase 2 for groups Small and Thin compared to group Large. In Phase 3, resurgence was evident only in group Large but target responding remained relatively elevated in groups Small and Thin. These results provide additional evidence of the important interplay between conditions of alternative reinforcement and the persistence and resurgence of target responding.

Equine Herpesvirus 1 Variant and New Marker for Epidemiologic Surveillance, Europe, 2021.

Equine herpesvirus 1 isolates from a 2021 outbreak of neurologic disease in Europe have a mutation, A713G, in open reading frame 11 not detected in 249 other sequences from equine herpesvirus 1 isolates. This single-nucleotide polymorphism could help identify horses infected with the virus strain linked to this outbreak.

EQUID ALPHAHERPESVIRUS 9 OUTBREAK ASSOCIATED WITH MORTALITY IN A GROUP OF GREVY'S ZEBRA (EQUUS GREVYI) HOUSED IN A MIXED-SPECIES EXHIBIT.

A herd of seven captive-born Grevy's zebras (Equus grevyi) experienced an outbreak of nasal discharge and sneezing. Clinical signs, including lethargy and anorexia, were severe and acute in three animals, including a 16-mo-old male that died within 48 h. Treatment of two severely affected zebras included valacyclovir (40 mg/kg PO), meloxicam (0.6 mg/kg IM/PO), and cefquinome (2.5 mg/kg IM q48h). An adult female improved rapidly, and clinical signs resolved within 48 h of treatment. Administration of valacyclovir pellets was very complicated in a 2-mo-old female, and death occurred within 48 h. Histologic examination of the two individuals that died revealed severe fibrinonecrotic interstitial pneumonia with prominent hyaline membranes and type II pneumocyte hyperplasia. Additionally, the 16-mo-old male presented systemic endothelial activation with vascular thrombosis and necrosis and mild nonsuppurative meningoencephalitis. Herpesviral DNA was detected in the lungs of both individuals by nested polymerase chain reaction. The nucleic acid sequence of the amplicons showed 100% similarity with previously published equid alphaherpesvirus 9 sequences. Three additional animals developed mild nasal discharge only and recovered spontaneously. The zebras shared housing facilities with other species, including white rhinoceros (Ceratotherium simum), reticulated giraffe (Giraffa camelopardalis reticulata), and several antelope species. None of these animals showed clinical signs. Additionally, nasal swabs and whole blood samples were collected from cohoused white rhinoceroses (n = 3) and springboks (Antidorcas marsupialis, n = 3) as well as nasal swabs from cohoused reticulated giraffes (n = 4). Nucleic acid sequence from equid herpesviruses was not detected in any of these samples. The source of the infection in the zebras remains unclear.

Resurgence of alcohol seeking following abstinence induced by punishment in male and female rats.

To better approximate the human condition, animal models of relapse to drug and alcohol seeking have increasingly employed negative consequences to generate abstinence. Here we report the first demonstration of relapse to punishment-suppressed alcohol seeking induced by loss of non-drug reward (i.e., resurgence). We also report the first examination of potential sex differences in any form of relapse to alcohol seeking following suppression by punishment. Male and female rats first pressed a lever for 20 % oral alcohol. Next, lever pressing for one group continued to produce alcohol, but also produced occasional footshock. For another group, lever pressing similarly produced alcohol and occasional footshock, and a nose-poke response produced alternative non-drug reward (i.e., food). Males showed similar suppression of alcohol seeking by punishment alone and punishment + alternative non-drug reward, whereas females showed less suppression by punishment alone. Finally, when alternative reinforcement and punishment were suspended, resurgence occurred for both sexes in the group that previously had access to non-drug reward. Exposure to and then removal of punishment alone did not produce relapse for males, but it did for females. These results suggest that loss of alternative non-drug reward can generate relapse to alcohol seeking following abstinence induced by negative consequences. Future research should further examine the role of potential sex differences in sensitivity to punishment and how such differences may contribute to relapse more broadly.

Identification of a New Equid Herpesvirus 1 DNA Polymerase (ORF30) Genotype with the Isolation of a C2254/H752 Strain in French Horses Showing no Major Impact on the Strain Behaviour.

Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C2254 (H752) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as UL clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C2254 mutation. When cultured in vitro, the C2254 EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A2254 and G2254 EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C2254 strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C2254 strain and two other A/G2254 field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C2254 and A2254 strains were more sensitive to aciclovir than the G2254 strain, based on viral load measurement.

Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: Efficacy of decitabine and valganciclovir alone or in combination.

Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.

Resurgence and downshifts in alternative reinforcement rate.

Resurgence refers to an increase in a previously suppressed target behavior with a relative worsening of conditions for a more recently reinforced alternative behavior. This experiment examined the relation between resurgence and the magnitude of a reduction in the rate of reinforcement for the alternative behavior. Groups of both male and female rats initially pressed a target lever for food on a variable-interval (VI) 30-s schedule. In a second phase, responding to the target lever was extinguished for all groups and pressing an alternative lever was reinforced on a VI 10-s schedule. Next, the rate of reinforcement for alternative behavior was reduced differentially across groups by arranging extinction, VI 80-s, VI 40-s, VI 20-s, or continued VI 10-s reinforcement. Target responding increased as an exponential function of the magnitude of the reduction in alternative reinforcement rates. With the exception that males appeared to show higher rates of target responding in baseline and higher rates of alternative responding in other phases, the overall pattern of responding across phases was not meaningfully different between sexes. The pattern of both target and alternative response rates across sessions and phases was well described quantitatively by the Resurgence as Choice in Context model.

Cyp26b1 is an essential regulator of distal airway epithelial differentiation during lung development.

Proper organ development depends on coordinated communication between multiple cell types. Retinoic acid (RA) is an autocrine and paracrine signaling molecule essential for the development of most organs, including the lung. Despite extensive work detailing effects of RA deficiency in early lung morphogenesis, little is known about how RA regulates late gestational lung maturation. Here, we investigate the role of the RA catabolizing protein Cyp26b1 in the lung. Cyp26b1 is highly enriched in lung endothelial cells (ECs) throughout development. We find that loss of Cyp26b1 leads to reduction of alveolar type 1 cells, failure of alveolar inflation and early postnatal lethality in mouse. Furthermore, we observe expansion of distal epithelial progenitors, but no appreciable changes in proximal airways, ECs or stromal populations. Exogenous administration of RA during late gestation partially mimics these defects; however, transcriptional analyses comparing Cyp26b1-/- with RA-treated lungs reveal overlapping, but distinct, responses. These data suggest that defects observed in Cyp26b1-/- lungs are caused by both RA-dependent and RA-independent mechanisms. This work reports crucial cellular crosstalk during lung development involving Cyp26b1-expressing endothelium and identifies a novel RA modulator in lung development.

Molecular Surveillance of EHV-1 Strains Circulating in France during and after the Major 2009 Outbreak in Normandy Involving Respiratory Infection, Neurological Disorder, and Abortion.

Equine herpesvirus 1 (EHV-1) is an Alphaherpesvirus infecting not only horses but also other equid and non-equid mammals. It can cause respiratory distress, stillbirth and neonatal death, abortion, and neurological disease. The different forms of disease induced by EHV-1 infection can have dramatic consequences on the equine industry, and thus the virus represents a great challenge for the equine and scientific community. This report describes the progress of a major EHV-1 outbreak that took place in Normandy in 2009, during which the three forms of disease were observed. A collection of EHV-1 strains isolated in France and Belgium from 2012 to 2018 were subsequently genetically analysed in order to characterise EHV-1 strain circulation. The open reading frame 30 (ORF30) non-neuropathogenic associated mutation A2254 was the most represented among 148 samples analysed in this study. ORF30 was also sequenced for 14 strains and compared to previously published sequences. Finally, a more global phylogenetic approach was performed based on a recently described Multilocus Sequence Typing (MLST) method. French and Belgian strains were clustered with known strains isolated in United Kingdom and Ireland, with no correlation between the phylogeny and the time of collection or location. This new MLST approach could be a tool to help understand epidemics in stud farms.

Spatiotemporal heterogeneity and patterning of developing renal blood vessels.

The kidney vasculature facilitates the excretion of wastes, the dissemination of hormones, and the regulation of blood chemistry. To carry out these diverse functions, the vasculature is regionalized within the kidney and along the nephron. However, when and how endothelial regionalization occurs remains unknown. Here, we examine the developing kidney vasculature to assess its 3-dimensional structure and transcriptional heterogeneity. First, we observe that endothelial cells (ECs) grow coordinately with the kidney bud as early as E10.5, and begin to show signs of specification by E13.5 when the first arteries can be identified. We then focus on how ECs pattern and remodel with respect to the developing nephron and collecting duct epithelia. ECs circumscribe nephron progenitor populations at the distal tips of the ureteric bud (UB) tree and form stereotyped cruciform structures around each tip. Beginning at the renal vesicle (RV) stage, ECs form a continuous plexus around developing nephrons. The endothelial plexus envelops and elaborates with the maturing nephron, becoming preferentially enriched along the early distal tubule. Lastly, we perform transcriptional and immunofluorescent screens to characterize spatiotemporal heterogeneity in the kidney vasculature and identify novel regionally enriched genes. A better understanding of development of the kidney vasculature will help instruct engineering of properly vascularized ex vivo kidneys and evaluate diseased kidneys.

DNA methylation-independent growth restriction and altered developmental programming in a mouse model of preconception male alcohol exposure.

The preconception environment is a significant modifier of dysgenesis and the development of environmentally-induced disease. To date, fetal alcohol spectrum disorders (FASDs) have been exclusively associated with maternal exposures, yet emerging evidence suggests male-inherited alterations in the developmental program of sperm may be relevant to the growth-restriction phenotypes of this condition. Using a mouse model of voluntary consumption, we find chronic preconception male ethanol exposure associates with fetal growth restriction, decreased placental efficiency, abnormalities in cholesterol trafficking, sex-specific alterations in the genetic pathways regulating hepatic fibrosis, and disruptions in the regulation of imprinted genes. Alterations in the DNA methylation profiles of imprinted loci have been identified in clinical studies of alcoholic sperm, suggesting the legacy of paternal drinking may transmit via heritable disruptions in the regulation of imprinted genes. However, the capacity of sperm-inherited changes in DNA methylation to broadly transmit environmentally-induced phenotypes remains unconfirmed. Using bisulphite mutagenesis and second-generation deep sequencing, we find no evidence to suggest that these phenotypes or any of the associated transcriptional changes are linked to alterations in the sperm-inherited DNA methylation profile. These observations are consistent with recent studies examining the male transmission of diet-induced phenotypes and emphasize the importance of epigenetic mechanisms of paternal inheritance beyond DNA methylation. This study challenges the singular importance of maternal alcohol exposures and suggests paternal alcohol abuse is a significant, yet overlooked epidemiological factor complicit in the genesis of alcohol-induced growth defects, and may provide mechanistic insight into the failure of FASD children to thrive postnatally.