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The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed time-points. Four of these miRNAs (eca-miR-1247-3p, eca-miR-134-5p, eca-miR-382-5p, and eca-miR-433-3p) were found to be enriched in the serum of pregnant mares at Day 25 relative to non-pregnant mares. To further assess the accuracy of these miRNAs in differentiating pregnant (25 d) from non-pregnant mares, receiver operating characteristic (ROC) analysis was performed for each of these miRNAs, revealing that eca-miR-1247-3p and eca-miR-134-5p had the highest accuracy (AUCROC = 0.92 and 0.91, respectively; p < 0.05). Moreover, eca-miR-1247-3p, eca-miR-134-5p, eca-miR-409-3p, and eca-miR-379-5p were enriched in the serum of Day 45 pregnant mares. Among those miRNAs, eca-miR-1247-3p and eca-miR-409-3p retained the highest accuracy as shown by ROC analysis. GO analysis revealed that these miRNAs are mainly implicated in nervous system development as well as organ development. Using in situ hybridization, we localized eca-miR-409-3p in the developing embryo (25 d) and extra-embryonic membranes (25 and 45 d). In conclusion, the present study is the first to elucidate the circulating maternal profile of C24MC-associated miRNAs throughout pregnancy and to suggest that serum eca-miR-1247-3p, eca-miR-134-5p, and eca-miR-409-3p could be used as pregnancy-specific markers during early gestation (25 and 45 d). Overall, the high abundance of these embryo-derived miRNAs in the maternal circulation suggests an embryo-maternal communication during the equine early pregnancy.

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.

Increasing evidence suggests that overlapping genes are much more common in eukaryotic genomes than previously thought. These different-strand overlapping genes are potential sense-antisense (SAS) pairs, which might have regulatory effects on each other. In the present study, we identified the SAS loci in the equine genome using previously generated stranded, paired-end RNA sequencing data from the equine chorioallantois. We identified a total of 1261 overlapping loci. The ratio of the number of overlapping regions to chromosomal length was numerically higher on chromosome 11 followed by chromosomes 13 and 12. These results show that overlapping transcription is distributed throughout the equine genome, but that distributions differ for each chromosome. Next, we evaluated the expression patterns of SAS pairs during the course of gestation. The sense and antisense genes showed an overall positive correlation between the sense and antisense pairs. We further provide a list of SAS pairs with both positive and negative correlation in their expression patterns throughout gestation. This study characterizes the landscape of sense and antisense gene expression in the placenta for the first time and provides a resource that will enable researchers to elucidate the mechanisms of sense/antisense regulation during pregnancy.

BackgroundThe human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation.ResultsWe demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in gestation. Evaluation of one member of C24MC (miR-409-3p) by in situ hybridization demonstrated that its cellular localization predominantly involved the chorion and allantoic epithelium and vascular endothelium. Additionally, expression of predicted target transcripts for C24MC-associated miRNAs was evaluated by RNA sequencing. Expression analysis of a subset of predicted mRNA targets showed a negative correlation with C24MC-associated miRNAs expression levels during gestation, suggesting the reciprocal control of these target transcripts by this miRNA cluster. Predicted functional analysis of these target mRNAs indicated enrichment of biological pathways related to embryonic development, endothelial cell migration and angiogenesis. Expression patterns of selected target mRNAs involved in angiogenesis were confirmed by RT-qPCR.ConclusionThis is the first report evaluating C24MC kinetics during pregnancy. The findings presented herein suggest that the C24MC may modulate angiogenic transcriptional profiles during placental development in the horse.

An 18-y-old American Saddlebred mare was admitted with fever and acute onset of neurologic signs including grade 3 of 5 ataxia, difficulty in prehension, and dull mentation. Because of financial restraints, desired testing could not be performed; the horse's condition declined despite supportive treatment, and euthanasia was elected. Postmortem examination revealed petechiae and ecchymoses in the meninges and neuroparenchyma of the encephalon. Blast-like neoplastic round cells were identified within the vasculature and areas of hemorrhage in the neuroparenchyma, the intestinal submucosa, and other organs, including the liver, kidney, lung, and mesenteric lymph node. Necrotizing enterocolitis and acute fibrinonecrotizing bacterial pneumonia were also noted. Of the atypical round cells in the encephalon, >70% expressed ionized calcium–binding adapter molecule 1 (Iba1), 10–20% expressed myeloperoxidase (MPO), and <10% expressed PAX5, CD3, CD20, CD79a, or MUM1. The bone marrow was diffusely effaced by neoplastic round cells expressing Iba1, and ~70% of these cells expressed MPO with no expression of CD3 or CD20. CD172a also immunolabeled a portion of the neoplastic cells. These findings were consistent with the diagnosis of acute myeloid leukemia-M1 with an unusual neurologic presentation. [ABSTRACT FROM AUTHOR]

Here we describe a case of fatal amebic gastritis associated with Naegleria australiensis infection in an 11-mo-old Linnaeus's two-toed sloth (Choloepus didactylus). The sloth had a history of weight loss and intermittent diarrhea for 18 d, and subsequently died despite empirical treatment. Postmortem findings included emaciation, gastric dilation with fluid content, and fibrinonecrotic gastritis with intralesional amebic trophozoites and cysts in the glandular region of the fundus. Transmission electron microscopy ruled out Amoebozoa of the family Entamoebidae based on the presence of mitochondria in the amoeboid organisms. PCR for pan–free-living amebae followed by next-generation sequencing of the PCR product revealed 99% identity with Naegleria australiensis. Gastric amebiasis has been reported sporadically in macropods and in leaf-eating monkeys with a sacculated stomach. To our knowledge, gastric amebiasis has not been reported previously in a sloth, which also has a sacculated and multi-chambered stomach. [ABSTRACT FROM AUTHOR]

It has been proposed that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that spread through human populations as a pandemic originated in Asian bats. There is concern that infected humans could transmit the virus to native North American bats; therefore, the susceptibility of several North American bat species to the pandemic virus has been experimentally assessed. Big brown bats (Eptesicus fuscus) were shown to be resistant to infection by SARS-CoV-2, whereas Mexican free-tailed bats (Tadarida brasiliensis) became infected and orally excreted moderate amounts of virus for up to 18 d postinoculation. Little brown bats (Myotis lucifugus) frequently contact humans, and their populations are threatened over much of their range due to white-nose syndrome, a fungal disease that is continuing to spread across North America. We experimentally challenged little brown bats with SARS-CoV-2 to determine their susceptibility and host potential and whether the virus presents an additional risk to this species. We found that this species was resistant to infection by SARS-CoV-2. These findings provide reassurance to wildlife rehabilitators, biologists, conservation scientists, and the public at large who are concerned with possible transmission of this virus to threatened bat populations. [ABSTRACT FROM AUTHOR]

Western equine encephalitis virus (WEEV) is a mosquito-borne arbovirus (genus Alphavirus, family Togaviridae) that has re-emerged in South America in late 2023, causing severe disease in both horses and humans after a nearly 40-year intermission period. We here describe the virological, serological, pathological, and molecular features of WEEV infection in horses during the 2023–2024 outbreak in Argentina. WEEV-infected horses developed neurological signs with mild to severe encephalitis associated with minimal to abundant WEEV-infected cells, as demonstrated by WEEV-specific in situ hybridization. The distribution of viral RNA was multifocal, with predominance within neuronal bodies, neuronal processes, and glial cells in the medulla oblongata and thalamic regions. Phylogenetic analysis of partial nsP4 sequences from three viral isolates obtained from three different provinces of Argentina support grouping with other temporally current WEEV strains from Uruguay and Brazil under a recently proposed novel lineage. [ABSTRACT FROM AUTHOR]

Spinal cord (SC) reconstruction (process to reestablish the severed neural continuity at the injury site) may provide better recovery from blunt SC injury (SCI). A miniature swine model of blunt SC compression was used to test the hypothesis that reconstruction of the SC with sural nerve in combination with surgical decompression and stabilization improves functional, macro- and microstructural recovery compared to decompression and stabilization alone. Following blunt T9-T11 SC compression injury, five adult Yucatan gilts randomly received laminectomy and polyethylene glycol (as fusogen) with (n = 3) or without (n = 2) sural nerve graft SC reconstruction. Fusogens are a heterogeneous collection of chemicals that fuse the axon membrane and are currently used to augment epineural coaptation during peripheral nerve graft reconstruction. Outcome measures of recovery included weekly sensory and motor assessments, various measurements obtained from computed tomography (CT) myelograms up to 12 weeks after injury Measurements from postmortem magnetic resonance imaging (MRI) and results from spinal cord histology performed 12 weeks after injury were also reported. Vertebral canal (VC), SC and dural sac (DS) dimensions and areas were quantified on 2-D CT images adjacent to the injury. Effort to stand and response to physical manipulation improved 7 and 9 weeks and 9 and 10 weeks, respectively, after injury in the reconstruction group. Myelogram measures indicated greater T13-T14 VC, smaller SC, and smaller DS dimensions in the reconstruction cohort, and increased DS area increased DS/VC area ratio, and higher contrast migration over time. Spinal cord continuity was evident in 2 gilts in the reconstruction cohort with CT and MRI imaging. At the SCI, microstructural alterations included axonal loss and glial scarring. Better functional outcomes were observed in subjects treated with sural nerve SC reconstruction. Study results support the use of this adult swine model of blunt SCI. Long-term studies with different nerve grafts or fusogens are required to expand upon these findings. [ABSTRACT FROM AUTHOR]

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.

Feline respiratory disease complex (FRDC) is caused by a wide range of viral and bacterial pathogens. Both Influenza A virus (IAV) and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) also induce respiratory diseases in cats. Two one-step multiplex qPCR/RT-qPCR assays were developed and validated: FRA_1 (Feline respiratory assay 1) for the detection of four viral targets and FRA_2 for the detection of three bacteria associated with FRDC. Both multiplex assays demonstrated high specificity, efficiency (93.51%–107.8%), linearity (> 0.998), analytical sensitivity (≤ 15 genome copies/μl), repeatability (coefficient of variation [CV] < 5%), and reproducibility (CV < 6%). Among the 63 clinical specimens collected from FRDC-suspected cats, 92.1% were positive for at least one pathogen and co-infection was detected in 57.1% of samples. Mycoplasma felis (61.9%) was the most found pathogen, followed by feline herpesvirus-1 (30.2%), Chlamydia felis (28.7%) and feline calicivirus (27.0%). SARS-CoV-2 was detected in two specimens. In summary, this new panel of qPCR/RT-qPCR assays constitutes a useful and reliable tool for the rapid detection of SARS-CoV-2 and viral and bacterial pathogens associated with FRDC in cats. [ABSTRACT FROM AUTHOR]

Caryospora- like organisms (CLOs) form a clade of at least 11 genotypes of related coccidia that can cause epizootic mortality in marine turtles. The biology, transmission, host species range, and host cell tropism of these organisms are still largely unknown. The goal of this study was to characterize the host cell tropism, pathologic and ultrastructural features, and phylogeny associated with the first report of a mortality event due to CLO in the freshwater red-eared slider turtle (Trachemys scripta elegans). Sudden mortalities within a clutch of captive-raised red-eared slider hatchlings (n = 8) were recorded, and deceased animals had severe segmental to diffuse, transmural, fibrinonecrotic enterocolitis and multifocal to coalescing hepatic necrosis, among other lesions associated with numerous intracytoplasmic developing stages of intralesional coccidia. Among the different developmental stages, merozoites were ultrastructurally characterized by an apical complex. A pan-apicomplexan polymerase chain reaction (PCR) yielded a 347 bp-amplicon matching the Schellackia / Caryospora -like clade with 99.1% identity to the US3 strain from green sea turtles (Chelonia mydas) and 99.1% identity to Schellackia sp. Isolate OC116. Surviving hatchlings were treated with toltrazuril sulfone (ponazuril) but were subsequently euthanized due to the risk of spreading the parasite to other chelonids in the collection. The ponazuril-treated hatchlings (n = 4) had mild proliferative anterior enteritis, with few intraepithelial coccidia in one hatchling confirmed as CLO by PCR. This is the first report of Caryospora -like coccidiosis in non-cheloniid turtles, highlighting the relevance of this disease as an emerging highly pathogenic intestinal and extra-intestinal form of coccidiosis of turtles with potential cross-species infectivity. [ABSTRACT FROM AUTHOR]