Interobserver agreement during clinical magnetic resonance imaging of the equine foot.

BACKGROUND: Agreement between experienced observers for assessment of pathology and assessment confidence are poorly documented for magnetic resonance imaging (MRI) of the equine foot. OBJECTIVES: To report interobserver agreement for pathology assessment and observer confidence for key anatomical structures of the equine foot during MRI. STUDY DESIGN: Exploratory clinical study. METHODS: Ten experienced observers (diploma or associate level) assessed 15 equine foot MRI studies acquired from clinical databases of 3 MRI systems. Observers graded pathology in seven key anatomical structures (Grade 1: no pathology, Grade 2: mild pathology, Grade 3: moderate pathology, Grade 4: severe pathology) and provided a grade for their confidence for each pathology assessment (Grade 1: high confidence, Grade 2: moderate confidence, Grade 3: limited confidence, Grade 4: no confidence). Interobserver agreement for the presence/absence of pathology and agreement for individual grades of pathology were assessed with Fleiss' kappa (k). Overall interobserver agreement for pathology was determined using Fleiss' kappa and Kendall's coefficient of concordance (KCC). The distribution of grading was also visualised with bubble charts. RESULTS: Interobserver agreement for the presence/absence of pathology of individual anatomical structures was poor-to-fair, except for the navicular bone which had moderate agreement (k = 0.52). Relative agreement for pathology grading (accounting for the ranking of grades) ranged from KCC = 0.19 for the distal interphalangeal joint to KCC = 0.70 for the navicular bone. Agreement was generally greatest at the extremes of pathology. Observer confidence in pathology assessment was generally moderate to high. MAIN LIMITATIONS: Distribution of pathology varied between anatomical structures due to random selection of clinical MRI studies. Observers had most experience with low-field MRI. CONCLUSIONS: Even with experienced observers, there can be notable variation in the perceived severity of foot pathology on MRI for individual cases, which could be important in a clinical context.

Integrin-αvß6 targeted peptide-toxin therapy in a novel αvß6-expressing immunocompetent model of pancreatic cancer.

Previously we reported that a novel αvß6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvß6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvß6. We report that orthotopic implantation of the αvß6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

TGFß-mediated MMP13 secretion drives myoepithelial cell dependent breast cancer progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Virtually all women with DCIS are treated, despite evidence suggesting up to half would remain with stable, non-threatening, disease. Overtreatment thus presents a pressing issue in DCIS management. To understand the role of the normally tumour suppressive myoepithelial cell in disease progression we present a 3D in vitro model incorporating both luminal and myoepithelial cells in physiomimetic conditions. We demonstrate that DCIS-associated myoepithelial cells promote striking myoepithelial-led invasion of luminal cells, mediated by the collagenase MMP13 through a non-canonical TGFß - EP300 pathway. In vivo, MMP13 expression is associated with stromal invasion in a murine model of DCIS progression and is elevated in myoepithelial cells of clinical high-grade DCIS cases. Our data identify a key role for myoepithelial-derived MMP13 in facilitating DCIS progression and point the way towards a robust marker for risk stratification in DCIS patients.

Ligand-bound integrin αvß6 internalisation and trafficking.

The integrin αvß6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvß6. As αvß6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvß6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvß6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvß6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvß6-dependent migration (αvß6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvß6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvß6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvß6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvß6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvß6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvß6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvß6-specific therapeutics and potentially the types of therapeutic load.

Mechanostimulation of breast myoepithelial cells induces functional changes associated with DCIS progression to invasion.

Women with ductal carcinoma in situ (DCIS) have an increased risk of progression to invasive breast cancer. Although not all women with DCIS will progress to invasion, all are treated as such, emphasising the need to identify prognostic biomarkers. We have previously shown that altered myoepithelial cells in DCIS predict disease progression and recurrence. By analysing DCIS duct size in sections of human breast tumour samples, we identified an associated upregulation of integrin ß6 and an increase in periductal fibronectin deposition with increased DCIS duct size that associated with the progression of DCIS to invasion. Our modelling of the mechanical stretching myoepithelial cells undergo during DCIS progression confirmed the upregulation of integrin ß6 and fibronectin expression in isolated primary and cell line models of normal myoepithelial cells. Our studies reveal that this mechanostimulated DCIS myoepithelial cell phenotype enhances invasion in a TGFß-mediated upregulation of MMP13. Immunohistochemical analysis identified that MMP13 was specifically upregulated in DCIS, and it was associated with progression to invasion. These findings implicate tissue mechanics in altering the myoepithelial cell phenotype in DCIS, and that these alterations may be used to stratify DCIS patients into low and high risk for invasive progression.

Synthesis and Systematic Study on the Effect of Different PEG Units on Stability of PEGylated, Integrin-αvß6-Specific A20FMDV2 Analogues in Rat Serum and Human Plasma.

A20FMDV2 is a 20-mer peptide that exhibits high selectivity and affinity for the tumour-related αvß6 integrin that can compete with extracellular ligands for the crucial RGD binding site, playing a role as a promising αvß6-specific inhibitor for anti-cancer therapies. Unfortunately, the clinical value of A20FMDV2 is limited by its poor half-life in blood caused by rapid renal excretion and its reported high susceptibility to serum proteases. The incorporation of poly (ethylene glycol) chains, coined PEGylation, is a well-established approach to improve the pharmacokinetic properties of drug molecules. Here, we report a systematic study on the incorporation of a varying number of ethylene glycol units (1-20) into the A20FMDV2 peptide to establish the effects of PEGylation size on the peptide stability in both rat serum and human plasma. In addition, the effect of acetyl and propionyl PEGylation handles on peptide stability is also described. Selected peptide analogues were assessed for integrin-αvß6-targeted binding, showing good specificity and activity in vitro. Stability studies in rat serum established that all of the PEGylated peptides displayed good stability, and an A20FMDV2 peptide containing twenty ethylene glycol units (PEG20) was the most stable. Surprisingly, the stability testing in human plasma identified shorter PEGs (PEG2 and PEG5) as more resistant to degradation than longer PEGs, a trend which was also observed with affinity binding to integrin αvß6.

Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion.

In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.

Long-term adaptation following influenza A virus host shifts results in increased within-host viral fitness due to higher replication rates, broader dissemination within the respiratory epithelium and reduced tissue damage.

The mechanisms and consequences of genome evolution on viral fitness following host shifts are poorly understood. In addition, viral fitness -the ability of an organism to reproduce and survive- is multifactorial and thus difficult to quantify. Influenza A viruses (IAVs) circulate broadly among wild birds and have jumped into and become endemic in multiple mammalian hosts, including humans, pigs, dogs, seals, and horses. H3N8 equine influenza virus (EIV) is an endemic virus of horses that originated in birds and has been circulating uninterruptedly in equine populations since the early 1960s. Here, we used EIV to quantify changes in infection phenotype associated to viral fitness due to genome-wide changes acquired during long-term adaptation. We performed experimental infections of two mammalian cell lines and equine tracheal explants using the earliest H3N8 EIV isolated (A/equine/Uruguay/63 [EIV/63]), and A/equine/Ohio/2003 (EIV/2003), a monophyletic descendant of EIV/63 isolated 40 years after the emergence of H3N8 EIV. We show that EIV/2003 exhibits increased resistance to interferon, enhanced viral replication, and a more efficient cell-to-cell spread in cells and tissues. Transcriptomics analyses revealed virus-specific responses to each virus, mainly affecting host immunity and inflammation. Image analyses of infected equine respiratory explants showed that despite replicating at higher levels and spreading over larger areas of the respiratory epithelium, EIV/2003 induced milder lesions compared to EIV/63, suggesting that adaptation led to reduced tissue pathogenicity. Our results reveal previously unknown links between virus genotype and the host response to infection, providing new insights on the relationship between virus evolution and fitness.

A computational framework for crack propagation in spatially heterogeneous materials.

This paper presents a mathematical formulation and numerical modelling framework for brittle crack propagation in heterogeneous elastic solids. Such materials are present in both natural and engineered scenarios. The formulation is developed in the framework of configurational mechanics and solved numerically using the finite-element method. We show the methodology previously established for homogeneous materials without the need for any further assumptions. The proposed model is based on the assumption of maximal dissipation of energy and uses the Griffith criterion; we show that this is sufficient to predict crack propagation in brittle heterogeneous materials, with spatially varying Young's modulus and fracture energy. Furthermore, we show that the crack path trajectory orientates itself such that it is always subject to Mode-I. The configurational forces and fracture energy release rate are both expressed exclusively in terms of nodal quantities, avoiding the need for post-processing and enabling a fully implicit formulation for modelling the evolving crack front and creation of new crack surfaces. The proposed formulation is verified and validated by comparing numerical results with both analytical solutions and experimental results. Both the predicted crack path and load-displacement response show very good agreement with experiments where the crack path was independent of material heterogeneity for those cases. Finally, the model is successfully used to consider the real and challenging scenario of fracture of an equine bone, with spatially varying material properties obtained from CT scanning. This article is part of a discussion meeting issue 'A cracking approach to inventing new tough materials: fracture stranger than friction'.

Current Perspectives on the Use of off the Shelf CAR-T/NK Cells for the Treatment of Cancer.

CAR T cells have revolutionised the treatment of haematological malignancies. Despite this, several obstacles still prohibit their widespread use and efficacy. One of these barriers is the use of autologous T cells as the carrier of the CAR. The individual production of CAR T cells results in large variation in the product, greater wait times for treatment and higher costs. To overcome this several novel approaches have emerged that utilise allogeneic cells, so called "off the shelf" CAR T cells. In this Review, we describe the different approaches that have been used to produce allogeneic CAR T to date, as well as their current pre-clinical and clinical progress.

CEACAM7 Is an Effective Target for CAR T-cell Therapy of Pancreatic Ductal Adenocarcinoma.

PURPOSE: To investigate whether CEACAM7 represents a novel therapeutic target for treating pancreatic ductal adenocarcinoma (PDAC) and to generate CEACAM7-targeting CAR T cells to test this hypothesis. EXPERIMENTAL DESIGN: We identified CEACAM7 (CGM2), a member of the CEA family of proteins with expression restricted to the colon and pancreas, as a potential CAR T-cell target for PDAC. We probed a panel of PDAC tumor sections as well as patient-derived PDAC cell cultures for CEACAM7 expression. We generated CAR-targeting CEACAM7, and assessed antitumor efficacy of CEACAM7 CAR T cells using in vitro and in vivo models. RESULTS: We show here that CEACAM7 is expressed in a large subset of PDAC tumors, with low to undetectable expression in all normal tissues tested. CEACAM7 is also expressed in primary PDAC cultures isolated from patient-derived tumors, with high expression within the cancer stem cell-enriched subset. CAR T cells targeting CEACAM7 are capable of targeting antigen-expressing tumor cells, and mediate remission in patient-derived xenograft tumors. CONCLUSIONS: We identify CEACAM7 as a potential therapeutic target in PDAC and describe the development of CEACAM7-targeted CAR T cells with efficacy against PDAC.

Clinical magnetic resonance image quality of the equine foot is significantly influenced by acquisition system.

BACKGROUND: Investigation of image quality in clinical equine magnetic resonance (MR) imaging may optimise diagnostic value. OBJECTIVES: To assess the influence of field strength and anaesthesia on image quality in MR imaging of the equine foot in a clinical context. STUDY DESIGN: Analytical clinical study. METHODS: Fifteen equine foot studies (five studies per system) were randomly selected from the clinical databases of three MR imaging systems: low-field standing (LF St), low-field anaesthetised (LF GA) and high-field anaesthetised (HF GA). Ten experienced observers graded image quality for entire studies and seven clinically important anatomical structures within the foot (briefly, grade 1: textbook quality, grade 2: high diagnostic quality, grade 3: satisfactory diagnostic quality, grade 4: non-diagnostic). Statistical analysis assessed the effect of anaesthesia and field strength using a combination of the Pearson chi-square test or Fisher's exact test and Mann-Whitney test. RESULTS: There was no difference in the proportion of entire studies of diagnostic quality between LF St (90%, 95% CI 78%-97%) and LF GA (88%, 76-95%, P = .7). No differences were evident in the proportion of diagnostic studies or median image quality gradings between LF St and LF GA when assessing individual anatomical structures (both groups all median grades = 3). There was a statistically significant difference in the proportion of entire studies of diagnostic quality between LF GA and HF GA (100%, 95% CI lower bound 94%, P = .03). There were statistically significant differences in median image quality gradings between LF GA (all median grades = 3) and HF GA (median grades = 1 (5/7 structures) or 2 (2/7 structures) for all individual anatomical structures (all P < .001). The reasons reported for reduced image quality differed between systems. MAIN LIMITATIONS: Randomised selection of cases from clinical databases. Individual observer preferences may influence image quality assessment. CONCLUSIONS: Field strength is a more important influencer of image quality than anaesthesia for magnetic resonance imaging of the equine foot in clinical patients.

Epidermolysis bullosa.

Epidermolysis bullosa (EB) is an inherited, heterogeneous group of rare genetic dermatoses characterized by mucocutaneous fragility and blister formation, inducible by often minimal trauma. A broad phenotypic spectrum has been described, with potentially severe extracutaneous manifestations, morbidity and mortality. Over 30 subtypes are recognized, grouped into four major categories, based predominantly on the plane of cleavage within the skin and reflecting the underlying molecular abnormality: EB simplex, junctional EB, dystrophic EB and Kindler EB. The study of EB has led to seminal advances in our understanding of cutaneous biology. To date, pathogenetic mutations in 16 distinct genes have been implicated in EB, encoding proteins influencing cellular integrity and adhesion. Precise diagnosis is reliant on correlating clinical, electron microscopic and immunohistological features with mutational analyses. In the absence of curative treatment, multidisciplinary care is targeted towards minimizing the risk of blister formation, wound care, symptom relief and specific complications, the most feared of which - and also the leading cause of mortality - is squamous cell carcinoma. Preclinical advances in cell-based, protein replacement and gene therapies are paving the way for clinical successes with gene correction, raising hopes amongst patients and clinicians worldwide.

The ITGB6 gene: its role in experimental and clinical biology.

Integrin αvß6 is a membrane-spanning heterodimeric glycoprotein involved in wound healing and the pathogenesis of diseases including fibrosis and cancer. Therefore, it is of great clinical interest for us to understand the molecular mechanisms of its biology. As the limiting binding partner in the heterodimer, the ß6 subunit controls αvß6 expression and availability. Here we describe our understanding of the ITGB6 gene encoding the ß6 subunit, including its structure, transcriptional and post-transcriptional regulation, the biological effects observed in ITGB6 deficient mice and clinical cases of ITGB6 mutations.

Integrin αvß6-specific therapy for pancreatic cancer developed from foot-and-mouth-disease virus.

Goals of investigation: The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained at <5% for decades because no effective therapies have been identified. Integrin αvß6 is overexpressed in most PDAC and represents a promising therapeutic target. Thus, we attempted to develop an αvß6-specific peptide-drug conjugate (PDC) for therapy of PDAC. Methodology: We conjugated the DNA-binding pyrrolobenzodiazepine (PBD)-based payload SG3249 (tesirine) to an αvß6-specific 20mer peptide from the VP1 coat protein of foot-and-mouth-disease virus (FMDV) (forming conjugate SG3299) or to a non-targeting peptide (forming conjugate SG3511). PDCs were tested for specificity and toxicity on αvß6-negative versus-positive PDAC cells, patient-derived cell lines from tumor xenografts, and on two different in vivo models of PDAC. Immunohistochemical analyses were performed to establish therapeutic mechanism. Results: The αvß6-targeted PDC SG3299 was significantly more toxic (up to 78-fold) for αvß6-expressing versus αvß6-negative PDAC cell lines in vitro, and achieved significantly higher toxicity at equal dose than the non-targeted PDC SG3511 (up to 15-fold better). Moreover, SG3299 eliminated established (100mm3) Capan-1 PDAC human xenografts, extending the lifespan of mice significantly (P=0.005). Immunohistochemistry revealed SG3299 induced DNA damage and apoptosis (increased γH2AX and cleaved caspase 3, respectively) associated with significant reductions in proliferation (Ki67), ß6 expression and PDAC tumour growth. Conclusions: The FMDV-peptide drug conjugate SG3299 showed αvß6-selectivity in vitro and in vivo and can specifically eliminate αvß6-positive cancers, providing a promising new molecular- specific therapy for pancreatic cancer.

The ITGB6 gene: its role in experimental and clinical biology.

Integrin αvß6 is a membrane-spanning heterodimeric glycoprotein involved in wound healing and the pathogenesis of diseases including fibrosis and cancer. Therefore, it is of great clinical interest for us to understand the molecular mechanisms of its biology. As the limiting binding partner in the heterodimer, the ß6 subunit controls αvß6 expression and availability. Here we describe our understanding of the ITGB6 gene encoding the ß6 subunit, including its structure, transcriptional and post-transcriptional regulation, the biological effects observed in ITGB6 deficient mice and clinical cases of ITGB6 mutations.

Integrin-Mediated TGFß Activation Modulates the Tumour Microenvironment.

TGFß (transforming growth factor-beta) is a pleotropic cytokine with contrasting effects in cancer. In normal tissue and early tumours, TGFß acts as a tumour suppressor, limiting proliferation and inducing apoptosis. However, these effects are eventually abrogated by the loss or inactivation of downstream signalling within the TGFß pathway, and in established tumours, TGFß then acts as a tumour promotor through multiple mechanisms including inducing epithelial-to-mesenchymal transition (EMT), promoting formation of cancer-associated fibroblasts (CAFs) and increasing angiogenesis. TGFß is secrereted as a large latent complex and is embedded in the extracellular matrix or held on the surface of cells and must be activated before mediating its multiple functions. Thus, whilst TGFß is abundant in the tumour microenvironment (TME), its functionality is regulated by local activation. The αv-integrins are major activators of latent-TGFß. The potential benefits of manipulating the immune TME have been highlighted by the clinical success of immune-checkpoint inhibitors in a number of solid tumour types. TGFß is a potent suppressor of T-cell-mediated immune surveillance and a key cause of resistance to checkpoint inhibitors. Therefore, as certain integrins locally activate TGFß, they are likely to have a role in the immunosuppressive TME, although this remains to be confirmed. In this review, we discussed the role of TGFß in cancer, the role of integrins in activating TGFß in the TME, and the potential benefits of targeting integrins to augment immunotherapies.

The integrin αvß6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy.

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvß6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of ß6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10-8 ). In two separate cohorts, we showed that over 80% of PDACs expressed αvß6 protein and that paired metastases retained αvß6 expression. In vitro, integrin αvß6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvß6-positive human PDAC xenografts and transgenic mice bearing αvß6-positive PDAC with the αvß6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p < 0.0001) and increased survival (log-rank test, p < 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase-3) and suppression of the pro-tumourigenic microenvironment (suppression of TGFß signalling, fewer αSMA-positive myofibroblasts, decreased blood vessel density). These data show that αvß6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control.

Nanoscale organisation of receptor ligands has become an important approach to study the clustering behaviour of cell-surface receptors. Biomimetic substrates fabricated via different nanopatterning strategies have so far been applied to investigate specific integrins and cell types, but without multivalent control. Here we use DNA origami to surpass the limits of current approaches and fabricate nanoarrays to study different cell adhesion processes, with nanoscale spatial resolution and single-molecule control. Notably, DNA nanostructures enable the display of receptor ligands in a highly customisable manner, with modifiable parameters including ligand number, ligand spacing and most importantly, multivalency. To test the adaptability and robustness of the system we combined it with focused ion beam and electron-beam lithography nanopatterning to additionally control the distance between the origami structures (i.e. receptor clusters). Moreover, we demonstrate how the platform can be used to interrogate two different biological questions: (1) the cooperative effect of integrin and growth factor receptor in cancer cell spreading, and (2) the role of integrin clustering in cardiomyocyte adhesion and maturation. Thereby we find previously unknown clustering behaviour of different integrins, further outlining the importance for such customisable platforms for future investigations of specific receptor organisation at the nanoscale.

Absence of adaptive evolution is the main barrier against influenza emergence in horses in Asia despite frequent virus interspecies transmission from wild birds.

Virus ecology and evolution play a central role in disease emergence. However, their relative roles will vary depending on the viruses and ecosystems involved. We combined field studies, phylogenetics and experimental infections to document with unprecedented detail the stages that precede initial outbreaks during viral emergence in nature. Using serological surveys we showed that in the absence of large-scale outbreaks, horses in Mongolia are routinely exposed to and infected by avian influenza viruses (AIVs) circulating among wild birds. Some of those AIVs are genetically related to an avian-origin virus that caused an epizootic in horses in 1989. Experimental infections showed that most AIVs replicate in the equine respiratory tract without causing lesions, explaining the absence of outbreaks of disease. Our results show that AIVs infect horses but do not spread, or they infect and spread but do not cause disease. Thus, the failure of AIVs to evolve greater transmissibility and to cause disease in horses is in this case the main barrier preventing disease emergence.