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Additional file 13: Supplementary Table S11. Differentially expressed cytokine or cytokine related genes in infected avian endothelial cells.

Additional file 4: Supplementary Table S2. BUSCO analysis of the final genomes.

Additional file 5: Supplementary Table S3. CEGMA analysis of the final genomes.

Additional file 7: Supplementary Table S5. List of genes known to affect plumage colour in birds.

Additional file 2: Supplementary Table S1. Heterozygosity, QV and completeness values for swan genomes.

Additional file 6: Supplementary Table S4. GO biological process annotation of inverted genes in the Mallard duck (relative to the black swan).

Additional file 3: Online Methods.

Additional file 8: Supplementary Table S6. Immune gene families are contractive in black swans compared to mute swans. The number of genes in each immune gene sub-family identified is given in each column for the corresponding species.

This case report discusses Type I hypersensitivity in ferrets following exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inoculum, observed during a study investigating the efficacy of candidate COVID-19 vaccines. Following a comprehensive internal root-cause investigation, it was hypothesized that prior prime-boost immunization of ferrets with a commercial canine C3 vaccine to protect against the canine distemper virus had resulted in primary immune response to fetal bovine serum (FBS) in the C3 preparation. Upon intranasal exposure to SARS-CoV-2 virus cultured in medium containing FBS, an allergic airway response occurred in 6 out of 56 of the ferrets. The 6 impacted ferrets were randomly dispersed across study groups, including different COVID-19 vaccine candidates, routes of vaccine candidate administration, and controls (placebo). The root-cause investigation and subsequent analysis determined that the allergic reaction was unrelated to the COVID-19 vaccine candidates under evaluation. Histological assessment suggested that the allergic response was characterized by eosinophilic airway disease; increased serum immunoglobulin levels reactive to FBS further suggested this response was caused by immune priming to FBS present in the C3 vaccine. This was further supported by in vivo studies demonstrating ferrets administered diluted FBS also presented clinical signs consistent with a hyperallergic response, while clinical signs were absent in ferrets that received a serum-free SARS-CoV-2 inoculum. It is therefore recommended that vaccine studies in higher order animals should consider the impact of welfare vaccination and use serum-free inoculum whenever possible.

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management., Author summary While it is recognized that the host response to infection plays a critical role in determining the severity and outcome of COVID-19, the host microRNA (miRNA) response to SARS-CoV-2 infection is poorly defined. Here we have used next-generation sequencing and bioinformatics to profile circulating miRNAs in 10 COVID-19 patients that were sampled longitudinally over time. COVID-19 was associated with altered expression of 55 plasma miRNAs, with miR-776-3p and miR-1275 among the most strongly down-regulated, and miR-4742-3p, miR-31-5p and miR-3215-3p the most up-regulated. An artificial intelligence methodology was used to identify a miRNA signature, consisting of miR423-5p, miR-23a-3p, miR-195-5p, which could independently classify COVID-19 patients from healthy controls with 99.9% accuracy. When applied to the ferret model of COVID-19, the same signature classified COVID-19 cases with 99.8% accuracy and could distinguish between COVID-19 and influenza A(H1N1) infection with >95% accuracy. In summary this study profiles the host miRNA response to COVID-19 and suggests that the measurement of select host molecules may have potential to independently detect disease cases.

Many viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to interleukin-6 family cytokines, and that this is mediated by the interferon-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer., Author summary Ebola virus (EBOV) continues to pose a significant risk to human health globally, causing ongoing disease outbreaks with case-fatality rates between 40 and 65%. Suppression of immune responses is a critical component of EBOV haemorrhagic fever, but understanding of EBOV impact on signalling by cytokines other than interferon is limited. We find that infectious EBOV inhibits interleukin-6 cytokine signalling via antagonism of STAT3. The antagonistic strategy uniquely combines two distinct mechanisms, which appear to reflect differing nuclear trafficking mechanisms of critical STAT3 complexes. This provides fundamental insights into the mechanisms of pathogenesis of a lethal virus, and biology of STAT3, a critical player in immunity, development, growth and cancer.

Coronavirus disease (COVID-19) is a contagious respiratory disease that is causing significant global morbidity and mortality. Understanding the impact of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection on the host metabolism is still in its infancy but of great importance. Herein, we investigated the metabolic response during viral shedding and post-shedding in an asymptomatic SARS-CoV-2 ferret model (n = 6) challenged with two SARS-CoV-2 isolates. Virological and metabolic analyses were performed on (minimally invasive) collected oral swabs, rectal swabs, and nasal washes. Fragments of SARS-CoV-2 RNA were only found in the nasal wash samples in four of the six ferrets, and in the samples collected 3 to 9 days post-infection (referred to as viral shedding). Central carbon metabolism metabolites were analyzed during viral shedding and post-shedding periods using a dynamic Multiple Reaction Monitoring (dMRM) database and method. Subsequent untargeted metabolomics and lipidomics of the same samples were performed using a Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) methodology, building upon the identified differentiated central carbon metabolism metabolites. Multivariate analysis of the acquired data identified 29 significant metabolites and three lipids that were subjected to pathway enrichment and impact analysis. The presence of viral shedding coincided with the challenge dose administered and significant changes in the citric acid cycle, purine metabolism, and pentose phosphate pathways, amongst others, in the host nasal wash samples. An elevated immune response in the host was also observed between the two isolates studied. These results support other metabolomic-based findings in clinical observational studies and indicate the utility of metabolomics applied to ferrets for further COVID-19 research that advances early diagnosis of asymptomatic and mild clinical COVID-19 infections, in addition to assessing the effectiveness of new or repurposed drug therapies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the infectious disease COVID-19, which has rapidly become an international pandemic with significant impact on healthcare systems and the global economy. To assist antiviral therapy and vaccine development efforts, we performed a natural history/time course study of SARS-CoV-2 infection in ferrets to characterise and assess the suitability of this animal model. Ten ferrets of each sex were challenged intranasally with 4.64 × 104 TCID50 of SARS-CoV-2 isolate Australia/VIC01/2020 and monitored for clinical disease signs, viral shedding, and tissues collected post-mortem for histopathological and virological assessment at set intervals. We found that SARS-CoV-2 replicated in the upper respiratory tract of ferrets with consistent viral shedding in nasal wash samples and oral swab samples up until day 9. Infectious SARS-CoV-2 was recovered from nasal washes, oral swabs, nasal turbinates, pharynx, and olfactory bulb samples within 3–7 days post-challenge; however, only viral RNA was detected by qRT-PCR in samples collected from the trachea, lung, and parts of the gastrointestinal tract. Viral antigen was seen exclusively in nasal epithelium and associated sloughed cells and draining lymph nodes upon immunohistochemical staining. Due to the absence of clinical signs after viral challenge, our ferret model is appropriate for studying asymptomatic SARS-CoV-2 infections and most suitable for use in vaccine efficacy studies.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS-CoV-2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS-CoV-2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS-CoV-2 infection following intranasal challenge.

The ‘D614G’ mutation (Aspartate-to-Glycine change at position 614) of the SARS-CoV-2 spike protein has been speculated to adversely affect the efficacy of most vaccines and countermeasures that target this glycoprotein, necessitating frequent vaccine matching. Virus neutralisation assays were performed using sera from ferrets which received two doses of the INO-4800 COVID-19 vaccine, and Australian virus isolates (VIC01, SA01 and VIC31) which either possess or lack this mutation but are otherwise comparable. Through this approach, supported by biomolecular modelling of this mutation and the commonly-associated P314L mutation in the RNA-dependent RNA polymerase, we have shown that there is no experimental evidence to support this speculation. We additionally demonstrate that the putative elastase cleavage site introduced by the D614G mutation is unlikely to be accessible to proteases.

As a clinical setting in which local live biological therapy is already well established, non-muscle invasive bladder cancer (NMIBC) presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus A21 (CVA21) is a novel intercellular adhesion molecule-1 (ICAM-1)-targeted immunotherapeutic virus. This study investigated CVA21-induced cytotoxicity in a panel of human bladder cancer cell lines, revealing a range of sensitivities largely correlating with expression of the viral receptor ICAM-1. CVA21 in combination with low doses of mitomycin-C enhanced CVA21 viral replication and oncolysis by increasing surface expression levels of ICAM-1. This was further confirmed using 300-μm precision slices of NMIBC where levels of virus protein expression and induction of apoptosis were enhanced with prior exposure to mitomycin-C. Given the importance of the immunogenicity of dying cancer cells for triggering tumor-specific responses and long-term therapeutic success, the ability of CVA21 to induce immunogenic cell death was investigated. CVA21 induced immunogenic apoptosis in bladder cancer cell lines, as evidenced by expression of the immunogenic cell death (ICD) determinant calreticulin, and HMGB-1 release and the ability to reject MB49 tumors in syngeneic mice after vaccination with MB49 cells undergoing CVA21 induced ICD. Such CVA21 immunotherapy could offer a potentially less toxic, more effective option for the treatment of bladder cancer. Keywords: bladder cancer, coxsackievirus A21, intercellular adhesion molecule-1

// Subhransu S. Sahoo 1 , Min Yuan Quah 2 , Sarah Nielsen 3 , Joshua Atkins 4 , Gough G. Au 2 , Murray J. Cairns 4 , Pravin Nahar 5 , Janine M. Lombard 6 and Pradeep S. Tanwar 1 1 Gynaecology Oncology Group, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia 2 The Picornaviral Research Unit, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia 3 Hunter Cancer Biobank, University of Newcastle, Callaghan, New South Wales, Australia 4 Discipline of Pharmacy and Experimental Pharmacology, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia 5 Department of Maternity and Gynaecology, John Hunter Hospital, New Lambton Heights, New South Wales, Australia 6 Department of Medical Oncology, Calvary Mater Newcastle, Waratah, New South Wales, Australia Correspondence to: Pradeep S. Tanwar, email: // Keywords : microenvironment, ECM, endometrial cancer, TGF-β signalling, metastasis Received : May 05, 2017 Accepted : May 07, 2017 Published : May 22, 2017 Abstract Although aggressive invasion and distant metastases are an important cause of morbidity and mortality in patients with endometrial cancer (EC), the requisite events determining this propensity are currently unknown. Using organotypic three-dimensional culture of endometrial cancer cell lines, we demonstrated anti-correlated TGF-β signalling gene expression patterns that arise among extracellular matrix (ECM)-attached cells. TGF-β pathway seemed to be active in EC cells forming non-glandular colonies in 3D-matrix but weaker in glandular colonies. Functionally we found that out of several ECM proteins, fibronectin relatively promotes Smad phosphorylation suggesting a potential role in regulating TGF-β signalling in non-glandular colonies. Importantly, alteration of TGF-β pathway induced EMT and MET in both type of colonies through slug protein. The results exemplify a crucial role of TGF-β pathway during EC metastasis in human patients and inhibition of the pathway in a murine model impaired tumour cell invasion and metastasis depicting an attractive target for therapeutic intervention of malignant tumour progression. These findings provide key insights into the role of ECM-derived TGF-β signalling to promote endometrial cancer metastasis and offer an avenue for therapeutic targeting of microenvironment derived signals along with tumour cells.

Purpose: The CANON [CAVATAK in NON–muscle-invasive bladder cancer (NMIBC)] study evaluated a novel ICAM-1–targeted immunotherapeutic-coxsackievirus A21 as a novel oncolytic agent against bladder cancer. Patients and Methods: Fifteen patients enrolled in this “window of opportunity” phase I study, exposing primary bladder cancers to CAVATAK prior to surgery. The first 9 patients received intravesical administration of monotherapy CAVATAK; in the second stage, 6 patients received CAVATAK with a subtherapeutic dose of mitomycin C, known to enhance expression of ICAM-1 on bladder cancer cells. The primary endpoint was to determine patient safety and maximum tolerated dose (MTD). Secondary endpoints were evidence of viral replication, induction of inflammatory cytokines, antitumor activity, and viral-induced changes in resected tissue. Results: Clinical activity of CAVATAK was demonstrated by induction of tumor inflammation and hemorrhage following either single or multiple administrations of CAVATAK in multiple patients, and a complete resolution of tumor in 1 patient. Whether used alone or in combination with mitomycin C, CAVATAK caused marked inflammatory changes within NMIBC tissue biopsies by upregulating IFN-inducible genes, including both immune checkpoint inhibitory genes (PD-L1 and LAG3) and Th1-associated chemokines, as well as the induction of the innate activator RIG-I, compared with bladder cancer tissue from untreated patients. No significant toxicities were reported in any patient, from either virus or combination therapy. Conclusions: The acceptable safety profile of CAVATAK, proof of viral targeting, replication, and tumor cell death together with the virus-mediated increases in “immunological heat” within the tumor microenvironment all indicate that CAVATAK may be potentially considered as a novel therapeutic for NMIBC.

Summary Oncolytic viruses are attractive biological agents for the control of human malignancy. This study assessed the capacity of Coxsackievirus A21 (CVA21) to target and destroy multiple myeloma (MM) and precursor aberrant plasma cells in vitro using established MM cell lines and 15 patient bone marrow (BM) biopsies [n = 10 MM and five monoclonal gammopathy of undetermined significance (MGUS)]. Cell surface analysis revealed that all tumour cells lines expressed high levels of intercellular adhesion molecule-1 (ICAM-1) and decay-accelerating factor (DAF), the receptor molecules to which CVA21 can bind, leading to subsequent cell-entry and infection. MM cell lines were remarkably susceptible to CVA21 lytic infection, producing 100–1000-fold increases in viral progeny within 24 h. In contrast, normal peripheral blood cells were refractile to CVA21 infection. Furthermore, challenge of patient BM biopsies with CVA21 for 48 h resulted in specific purging of up to 98·7% of CD138+ plasma cells, with no significant decrease in progenitor cell function. Data generated in this study suggests that CVA21 virotherapy may have potential applications as a systemic anti-tumour agent for MM, or in the ex vivo purging of malignant plasma cells prior to autologous stem cell transplantation.

The cellular receptor usage of numerous human enteroviruses can differ significantly between low-cell-culture-passaged clinical isolates and highly laboratory-passaged prototype strains. The prototype strain of coxsackievirus A21 (CVA21) displays a dual-receptor specificity as determined with a receptor complex consisting of decay-accelerating factor (DAF) and intercellular adhesion molecule 1 (ICAM-1). In this study, the cellular receptor interactions of low-cell-passage CVA21 clinical isolates with respect to their interactions with cell surface-expressed DAF and ICAM-1 were compared to those of the CVA21 prototype (Kuykendall) strain. Dual-receptor usage of DAF and ICAM-1 by CVA21 clinical isolates was confirmed by cell transfection and radiolabeled binding assays. The cellular attachment of clinical and prototype CVA21 strains to cells that coexpressed DAF and ICAM-1 was not additive compared to the viral binding to cells expressing one or other receptor. In fact, the binding data suggest there is an inhibition of CVA21 cellular attachment in environments where high-level coexpression of both DAF and ICAM-1 occurs. Antibody cross-linking of DAF rendered cells susceptible to lytic infection by the CVA21 clinical isolates. In a novel finding, three clinical isolates could, to various degrees, infect and lyse DAF-expressing cells in the absence of DAF-antibody cross-linking and ICAM-1 expression. Sequence analysis of the P1 region of clinical and prototype virus genomes identified a number of coding changes that may contribute to the observed enhanced DAF usage phenotype of the clinical CVA21 isolates. None of the amino acid changes was located in the previously postulated ICAM-1 footprint, a receptor-binding environment that was conserved on the capsid surface of all CVA21 clinical isolates. Taken together, the data suggest that community-circulating strains of CVA21 can infect target cells expressing either ICAM-1 or DAF alone and that such interactions extend tissue tropism and impact directly on viral pathogenesis.

Purpose: The incidence of malignant melanoma continues to increase worldwide; however, treatment of metastatic melanoma remains unsatisfactory, and there is an urgent need for development of effective targeted therapeutics. A potential biological target on the surface of malignant melanoma cells is the up-regulated expression of intercellular adhesion molecule (ICAM)-1 and decay-accelerating factor (DAF), relative to surrounding benign tissue. Coxsackievirus A21 (a common cold virus) targets and destroys susceptible cells via specific viral capsid interactions with surface-expressed virus receptors comprising ICAM-1 and DAF. Experimental Design: The oncolytic capacity of a genetically unmodified wild-type common cold-producing human enterovirus (Coxsackievirus A21, CAV21) was assessed against in vitro cultures and in vivo xenografts of malignant human melanoma cells. Results: In vitro studies established that human melanoma cells endogenously express elevated levels of ICAM-1/DAF and were highly susceptible to rapid viral oncolysis by CAV21 infection, whereas ICAM-1/DAF-expressing peripheral blood lymphocytes were refractile to infection. In vivo studies revealed that the tumor burden of nonobese diabetic severe combined immunodeficient mice bearing multiple s.c. melanoma xenografts was rapidly reduced by oncolysis mediated by a single administration of CAV21. The antitumor activity of CAV21 was characterized by highly efficient systemic spread of progeny CAV21, with oncolysis of tumors also occurring at sites distant to the primary site of viral administration. Conclusions: Overall, the findings presented herein demonstrate an important proof of principle using administration of replication-competent CAV21 as a potential biological oncolytic agent in the control of human metastatic melanoma.

The cellular receptor complex of coxsackievirus A21 (CVA21), a C-cluster human enterovirus, is formed by the subtle interaction of individual cellular receptors, decay accelerating factor (DAF) and intercellular adhesion molecule-1 (ICAM-1). In this receptor complex, DAF functions in the membrane sequestration of the virus, while the role of ICAM-1 is as the functional cellular internalization receptor. However, despite the elucidation of the CVA21-cell receptor interactions, there have been few definite investigations into cellular receptor usage of other coxsackie A viruses (CVAs) belonging to the C-cluster. In the present study, radiolabelled virus-binding assays demonstrated that CVA13, -15, -18 and -20, a subset of the human enterovirus C-cluster, bind directly to surface-expressed ICAM-1, but not to surface-expressed DAF. Furthermore, lytic infection of ICAM-1-expressing rhabdomyosarcoma (RD) cells by this C-cluster subset of viruses was inhibited by specific ICAM-1 monoclonal antibody blockade, except for that of CVA20. Despite possessing ICAM-1-binding capabilities, CVA20 employed an as yet unidentified internalization receptor for cell entry and subsequent productive lytic infection of ICAM-1-negative RD cells. In a further example of C-cluster cellular receptor heterogeneity, CVA13 exhibited significant binding to the surface of CHO cells expressing neither DAF nor ICAM-1. Despite a common receptor usage of ICAM-1 by this subset of C-cluster CVAs, the amino acid residues postulated to represent the ICAM-1-receptor footprint were not conserved.