Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

Distinct Ventral Pallidal Neural Populations Mediate Separate Symptoms of Depression.

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.

Establishment and characterization of equine mammary organoids using a method translatable to other non-traditional model species.

Mammary organoid (MaO) models are only available for a few traditional model organisms, limiting our ability to investigate mammary gland development and cancer across mammals. This study established equine mammary organoids (EqMaOs) from cryopreserved mammary tissue, in which mammary tissue fragments were isolated and embedded into a 3D matrix to produce EqMaOs. We evaluated viability, proliferation and budding capacity of EqMaOs at different time points during culture, showing that although the number of proliferative cells decreased over time, viability was maintained and budding increased. We further characterized EqMaOs based on expression of stem cell, myoepithelial and luminal markers, and found that EqMaOs expressed these markers throughout culture and that a bilayered structure as seen in vivo was recapitulated. We used the milk-stimulating hormone prolactin to induce milk production, which was verified by the upregulation of milk proteins, most notably ß-casein. Additionally, we showed that our method is also applicable to additional non-traditional mammalian species, particularly domesticated animals such as cats, pigs and rabbits. Collectively, MaO models across species will be a useful tool for comparative developmental and cancer studies.

Interferon inhibits a model RNA virus via a limited set of inducible effector genes.

Interferons control viral infection by inducing the expression of antiviral effector proteins encoded by interferon-stimulated genes (ISGs). The field has mostly focused on identifying individual antiviral ISG effectors and defining their mechanisms of action. However, fundamental gaps in knowledge about the interferon response remain. For example, it is not known how many ISGs are required to protect cells from a particular virus, though it is theorized that numerous ISGs act in concert to achieve viral inhibition. Here, we used CRISPR-based loss-of-function screens to identify a markedly limited set of ISGs that confer interferon-mediated suppression of a model alphavirus, Venezuelan equine encephalitis virus (VEEV). We show via combinatorial gene targeting that three antiviral effectors-ZAP, IFIT3, and IFIT1-together constitute the majority of interferon-mediated restriction of VEEV, while accounting for < 0.5% of the interferon-induced transcriptome. Together, our data suggest a refined model of the antiviral interferon response in which a small subset of "dominant" ISGs may confer the bulk of the inhibition of a given virus.

An Equine Protein Atlas Highlights Synovial Fluid Proteome Dynamics during Experimentally LPS-Induced Arthritis.

In human proteomics, substantial efforts are ongoing to leverage large collections of mass spectrometry (MS) fragment ion spectra into extensive spectral libraries (SL) as a resource for data independent acquisition (DIA) analysis. Currently, such initiatives in equine research are still missing. Here we present a large-scale equine SL, comprising 6394 canonical proteins and 89,329 unique peptides, based on data dependent acquisition analysis of 75 tissue and body fluid samples from horses. The SL enabled large-scale DIA-MS based quantification of the same samples to generate a quantitative equine protein distribution atlas to infer dominant proteins in different organs and body fluids. Data mining revealed 163 proteins uniquely identified in a specific type of tissue or body fluid, serving as a starting point to determine tissue-specific or tissue-type-specific proteins. We showcase the SL by highlighting proteome dynamics in equine synovial fluid samples during experimental lipopolysaccharide-induced arthritis. A fuzzy c-means cluster analysis pinpointed SERPINB1, ATRN, NGAL, LTF, MMP1, and LBP as putative biomarkers for joint inflammation. This SL provides an extendable resource for future equine studies employing DIA-MS.

Fatal West Nile Virus Infection in Horse Returning to United Kingdom from Spain, 2022.

We report fatal West Nile virus (WNV) infection in a 7-year-old mare returning to the United Kingdom from Spain. Case timeline and clustering of virus sequence with recent WNV isolates suggest that transmission occurred in Andalusía, Spain. Our findings highlight the importance of vaccination for horses traveling to WNV-endemic regions.

Molecular Epidemiology of Western Equine Encephalitis Virus, South America, 2023-2024.

Western equine encephalitis virus (WEEV) is a mosquitoborne virus that reemerged in December 2023 in Argentina and Uruguay, causing a major outbreak. We investigated the outbreak using epidemiologic, entomological, and genomic analyses, focusing on WEEV circulation near the Argentina‒Uruguay border in Rio Grande do Sul state, Brazil. During November 2023‒April 2024, the outbreak in Argentina and Uruguay resulted in 217 human cases, 12 of which were fatal, and 2,548 equine cases. We determined cases on the basis of laboratory and clinical epidemiologic criteria. We characterized 3 fatal equine cases caused by a novel WEEV lineage identified through a nearly complete coding sequence analysis, which we propose as lineage C. Our findings highlight the importance of continued surveillance and equine vaccination to control future WEEV outbreaks in South America.

Impact of the host immune response on the development of equine herpesvirus myeloencephalopathy in horses.

Herpesviruses establish a well-adapted balance with their host's immune system. Despite this co-evolutionary balance, infections can lead to severe disease including neurological disorders in their natural host. In horses, equine herpesvirus 1 (EHV-1) causes respiratory disease, abortions, neonatal foal death and myeloencephalopathy (EHM) in ~10 % of acute infections worldwide. Many aspects of EHM pathogenesis and protection from EHM are still poorly understood. However, it has been shown that the incidence of EHM increases to >70 % in female horses >20 years of age. In this study we used old mares as an experimental equine EHV-1 model of EHM to identify host-specific factors contributing to EHM. Following experimental infection with the neuropathogenic strain EHV-1 Ab4, old mares and yearling horses were studied for 21 days post-infection. Nasal viral shedding and cell-associated viremia were assessed by quantitative PCR. Cytokine/chemokine responses were evaluated in nasal secretions and cerebrospinal fluid (CSF) by Luminex assay and in whole blood by quantitative real-time PCR. EHV-1-specific IgG sub-isotype responses were measured by ELISA. All young horses developed respiratory disease and a bi-phasic fever post-infection, but only 1/9 horses exhibited ataxia. In contrast, respiratory disease was absent in old mares, but all old mares developed EHM that resulted in euthanasia in 6/9 old mares. Old mares also presented significantly decreased nasal viral shedding but higher viremia coinciding with a single fever peak at the onset of viremia. According to clinical disease manifestation, horses were sorted into an EHM group (nine old horses and one young horse) and a non-EHM group (eight young horses) for assessment of host immune responses. Non-EHM horses showed an early upregulation of IFN-α (nasal secretions), IRF7/IRF9, IL-1ß, CXCL10 and TBET (blood) in addition to an IFN-γ upregulation during viremia (blood). In contrast, IFN-α levels in nasal secretions of EHM horses were low and peak levels of IRF7, IRF9, CXCL10 and TGF-ß (blood) coincided with viremia. Moreover, EHM horses showed significantly higher IL-10 levels in nasal secretions, peripheral blood mononuclear cells and CSF and higher serum IgG3/5 antibody titres compared to non-EHM horses. These results suggest that protection from EHM depends on timely induction of type 1 IFN and upregulation cytokines and chemokines that are representative of cellular immunity. In contrast, induction of regulatory or TH-2 type immunity appeared to correlate with an increased risk for EHM. It is likely that future vaccine development for protection from EHM must target shifting this 'at-risk' immunophenotype.

Genetic and antigenic characterization of two diarrhoeicdominant rotavirus A genotypes G3P[12] and G14P[12] circulating in the global equine population.

Equine rotavirus species A (ERVA) G3P[12] and G14P[12] are two dominant genotypes that cause foal diarrhoea with a significant economic impact on the global equine industry. ERVA can also serve as a source of novel (equine-like) rotavirus species A (RVA) reassortants with zoonotic potential as those identified previously in 2013-2019 when equine G3-like RVA was responsible for worldwide outbreaks of severe gastroenteritis and hospitalizations in children. One hurdle to ERVA research is that the standard cell culture system optimized for human rotavirus replication is not efficient for isolating ERVA. Here, using an engineered cell line defective in antiviral innate immunity, we showed that both equine G3P[12] and G14P[12] strains can be rapidly isolated from diarrhoeic foals. The genome sequence analysis revealed that both G3P[12] and G14P[12] strains share the identical genotypic constellation except for VP7 and VP6 segments in which G3P[12] possessed VP7 of genotype G3 and VP6 of genotype I6 and G14P[12] had the combination of VP7 of genotype G14 and VP6 of genotype I2. Further characterization demonstrated that two ERVA genotypes have a limited cross-neutralization. The lack of an in vitro broad cross-protection between both genotypes supported the increased recent diarrhoea outbreaks due to equine G14P[12] in foals born to dams immunized with the inactivated monovalent equine G3P[12] vaccine. Finally, using the structural modelling approach, we provided the genetic basis of the antigenic divergence between ERVA G3P[12] and G14P[12] strains. The results of this study will provide a framework for further investigation of infection biology, pathogenesis and cross-protection of equine rotaviruses.

Advancing equine genomics: the development of a high density Axiom_Ashwa SNP chip for Indian horses and ponies.

The unique horse and pony breeds of India are declining at an alarming rate. These horses have been integral to the Indian culture and customs for centuries and represent a valuable genetic resource. It is imperative to harness the potential of this equine genetic resource that urgently needs conservation. The study highlights the design and development of a high density SNP array, the Axiom_Ashwa to aid in the genetic analysis and conservation efforts for Indian horse and pony breeds. With 613,950 SNPs, this chip offers extensive genome coverage having an average inter-marker distance of 4 kb. The Axiom_Ashwa has been validated on a larger set of diverse indigenous samples as well as Thoroughbreds, demonstrating a high call rate of 99.4% and robustness for genotyping indigenous breeds. Linkage disequilibrium (LD) analysis showed higher average LD in Indian breeds compared to exotic breeds, suggesting a limited effective population size and recent bottlenecks. Phylogenetic and population stratification analyses using PCA and DAPC clearly distinguished horses, ponies and Thoroughbreds, confirming the efficacy of the Axiom_Ashwa chip. These findings underscore the urgent need for conservation efforts for Indian horse breeds, which have experienced significant drop in population size. The Axiom_Ashwa SNP chip offers advantages such as cost-effectiveness and high throughput, providing a more accurate genetic representation of Indian horses.

Alphavirus-based replicons demonstrate different interactions with host cells and can be optimized to increase protein expression.

IMPORTANCE: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.

Mammalian ANP32A and ANP32B Proteins Drive Differential Polymerase Adaptations in Avian Influenza Virus.

ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B. IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk.

Maturational competence of equine oocytes is associated with alterations in their 'cumulome'.

Assisted reproductive technologies are an emerging field in equine reproduction, with species-dependent peculiarities, such as the low success rate of conventional IVF. Here, the 'cumulome' was related to the developmental capacity of its corresponding oocyte. Cumulus-oocyte complexes collected from slaughterhouse ovaries were individually matured, fertilized by ICSI, and cultured. After maturation, the cumulus was collected for proteomics analysis using label-free mass spectrometry (MS)-based protein profiling by nano-HPLC MS/MS and metabolomics analysis by UPLC-nanoESI MS. Overall, a total of 1671 proteins and 612 metabolites were included in the quantifiable 'cumulome'. According to the development of the corresponding oocytes, three groups were compared with each other: not matured (NM; n = 18), cleaved (CV; n = 15), and blastocyst (BL; n = 19). CV and BL were also analyzed together as the matured group (M; n = 34). The dataset revealed a closer connection within the two M groups and a more distinct separation from the NM group. Overrepresentation analysis detected enrichments related to energy metabolism as well as vesicular transport in the M group. Functional enrichment analysis found only the KEGG pathway 'oxidative phosphorylation' as significantly enriched in the NM group. A compound attributed to ATP was observed with significantly higher concentrations in the BL group compared with the NM group. Finally, in the NM group, proteins related to degradation of glycosaminoglycans were lower and components of cumulus extracellular matrix were higher compared to the other groups. In summary, the study revealed novel pathways associated with the maturational and developmental competence of oocytes.

Comprehensive meta-analysis of the effects of oral medroxyprogesterone acetate plus conjugated equine oestrogens on the lipid profile in women: Insights from randomized controlled trials.

BACKGROUND: Menopause is associated with elevated cardiovascular risk due to the loss of the cardioprotective effect of oestrogens. Postmenopausal women are often prescribed hormone replacement therapy (HRT) in order to control menopause symptoms and correct hormone imbalances; however, HRT can impact serum lipids' concentrations. At present, data on the effect of the administration of medroxyprogesterone acetate plus conjugated equine oestrogens (MPACEE) on the lipid profile in females are uncertain, as the investigations conducted so far have produced conflicting results. Thus, we aimed to clarify the impact of MPACEE prescription on the serum lipids' values in women by means of a systematic review and meta-analysis of randomized controlled trials (RCTs). METHODS: We employed a random-effects model based on the DerSimonian and Laird method to determine the combined estimates of the intervention's impact on the lipid profile. The computation of the weighted mean difference (WMD) and its corresponding 95% confidence interval (CI) relied on the mean and standard deviation values from both the MPACEE and control group, respectively. RESULTS: A total of 53 RCTs were included in the meta-analysis with 68 RCT arms on total cholesterol (TC), 70 RCT arms on low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG), and 69 RCT arms on high-density lipoprotein cholesterol (HDL-C). Administration of MPACEE resulted in a significant reduction of TC (WMD = -11.93 mg/dL; 95% CI: -13.42, -10.44; p < .001) and LDL-C (WMD = -16.61 mg/dL; 95% CI: -17.97, -15.26; p < .001) levels, and a notable increase in HDL-C (WMD = 3.40 mg/dL; 95% CI: 2.93, 3.86; p < .001) and TG (WMD = 10.28 mg/dL; 95% CI: 7.92, 12.64; p < .001) concentrations. Subgroup analysis revealed that changes in the lipid profile were influenced by several factors: body mass index (for TC, HDL-C, TG), MPACEE dosages (for TC, LDL-C, HDL-C, TG), age (for TC, LDL-C, HDL-C, TG), durations of the intervention (for TC, LDL-C, HDL-C, TG), continuous/sequential administration of MPACEE (continuous for TC; sequential for LDL-C, TG) administration of MPACEE and serum lipids' concentrations before enrolment in the RCT (for TC, LDL-C, HDL-C, TG). CONCLUSIONS: MPACEE administration can influence serum lipids' concentrations in females by raising HDL-C and TG levels and reducing LDL-C and TC values. Therefore, postmenopausal women who suffer from hypercholesterolaemia might benefit from this type of HRT.

Mesenchymal Stromal Cell Secretome Is Affected by Tissue Source and Donor Age.

Variation in mesenchymal stromal cell (MSC) function depending on their origin is problematic, as it may confound clinical outcomes of MSC therapy. Current evidence suggests that the therapeutic benefits of MSCs are attributed to secretion of biologically active factors (secretome). However, the effect of donor characteristics on the MSC secretome remains largely unknown. Here, we examined the influence of donor age, sex, and tissue source, on the protein profile of the equine MSC secretome. We used dynamic metabolic labeling with stable isotopes combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify secreted proteins in MSC conditioned media (CM). Seventy proteins were classified as classically secreted based on the rate of label incorporation into newly synthesized proteins released into the extracellular space. Next, we analyzed CM of bone marrow- (n = 14) and adipose-derived MSCs (n = 16) with label-free LC-MS/MS. Clustering analysis of 314 proteins detected across all samples identified tissue source as the main factor driving variability in MSC CM proteomes. Linear modelling applied to the subset of 70 secreted proteins identified tissue-related difference in the abundance of 23 proteins. There was an age-related decrease in the abundance of CTHRC1 and LOX, further validated with orthogonal techniques. Due to the lack of flow cytometry characterization of MSC surface markers, the analysis could not account for the potential effect of cell population heterogeneity. This study provides evidence that tissue source and donor age contribute to differences in the protein composition of MSC secretomes which may influence the effects of MSC therapy.

The mesenchymal stromal cell secretome promotes tissue regeneration and increases macrophage infiltration in acute and methicillin-resistant Staphylococcus aureus-infected skin wounds in vivo.

BACKGROUND AIMS: The prevalence of chronic wounds continues to be a burden in human medicine. Methicillin-resistant Staphylococcus aureus (MRSA) is commonly isolated from infected wounds. MRSA infections primarily delay healing by impairing local immune cell functions. This study aimed to investigate the potential of mesenchymal stromal cell (MSC)-secreted bioactive factors, defined as the secretome, to improve innate immune responses in vivo. MSCs were isolated from the bone marrow of horses, which serve as valuable translational models for wound healing. The MSC secretome, collected as conditioned medium (CM), was evaluated in vivo using mouse models of acute and MRSA-infected skin wounds. METHODS: Punch biopsies were used to create two full-thickness skin wounds on the back of each mouse. Acute wounds were treated daily with control medium or bone marrow-derived MSC (BM-MSC) CM. The antibiotic mupirocin was administered as a positive control for the MRSA-infected wound experiments. Wounds were photographed daily, and wound images were measured to determine the rate of closure. Trichrome staining was carried out to examine wound tissue histologically, and immunofluorescence antibody binding was used to assess immune cell infiltration. Wounds in the MRSA-infected model were swabbed for quantification of bacterial load. RESULTS: Acute wounds treated with BM-MSC CM showed accelerated wound closure compared with controls, as illustrated by enhanced granulation tissue formation and resolution, increased vasculature and regeneration of hair follicles. This treatment also led to increased neutrophil and macrophage infiltration. Chronic MRSA-infected wounds treated with BM-MSC CM showed reduced bacterial load accompanied by better resolution of granulation tissue formation and increased infiltration of pro-healing M2 macrophages compared with control-treated infected wounds. CONCLUSIONS: Collectively, our findings indicate that BM-MSC CM exerts pro-healing, immunomodulatory and anti-bacterial effects on wound healing in vivo, validating further exploration of the MSC secretome as a novel treatment option to improve healing of both acute and chronic wounds, especially those infected with antibiotic-resistant bacteria.

Equine coronavirus infection and replication in equine intestinal enteroids.

In this study, equine intestinal enteroids (EIEs) were generated from the duodenum, jejunum, and ileum and inoculated with equine coronavirus (ECoV) to investigate their suitability as in vitro models with which to study ECoV infection. Immunohistochemistry revealed that the EIEs were composed of various cell types expressed in vivo in the intestinal epithelium. Quantitative reverse-transcription PCR (qRT-PCR) and virus titration showed that ECoV had infected and replicated in the EIEs. These results were corroborated by electron microscopy. This study suggests that EIEs can be novel in vitro tools for studying the interaction between equine intestinal epithelium and ECoV.

An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018.

Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.

Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection.

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.