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Background. Iron deficiency (ID) is a significant, high-prevalence comorbidity in chronic heart failure (HF) that represents an independent predictor of a worse prognosis. However, a clear-cut diagnosis of ID in HF patients is not assured. The soluble transferrin receptor (sTfR) is a marker that reflects tissue-level iron demand and may be an early marker of ID. However, the impact of sTfR levels on clinical outcomes in non-anemic HF patients with a normal systemic iron status has never been evaluated. Methods. This is a post hoc analysis of an observational, prospective cohort study of 1236 patients with chronic HF of which only those with normal hemoglobin levels and a normal systemic iron status were studied. The final cohort consisted of 215 patients. Tissue ID was defined as levels of sTfR > 75th percentile (1.65 mg/L). Our aim was to describe the association between sTfR and clinical outcomes (all-cause death and HF hospitalization) and to explore its association with a wide array of serum biomarkers. Results. The sTfR level (HR 1.48, 95% CI 1.13–1.96, p = 0.005) and tissue ID (HR 2.14, 95% CI 1.22–3.75, p = 0.008) was associated with all-cause death. However, we found no association between sTfR levels and the risk of HF hospitalization. Furthermore, high sTfR levels were associated with a worse biomarker profile indicating myocardial damage (troponin and NT-proBNP), systemic inflammation (CRP and albumin), and impaired erythropoiesis (erythropoietin). Conclusions. In this cohort, the presence of tissue ID defined by sTfR levels is an independent factor for all-cause death in patients with normal systemic iron parameters.

The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer.

Plitidepsin, a marine-derived cyclic-peptide, inhibits SARS-CoV-2 replication at nanomolar concentrations by targeting the host protein eukaryotic translation elongation factor 1A. Here, we show that plitidepsin distributes preferentially to lung over plasma, with similar potency against across several SARS-CoV-2 variants in preclinical studies. Simultaneously, in this randomized, parallel, open-label, proof-of-concept study (NCT04382066) conducted in 10 Spanish hospitals between May and November 2020, 46 adult hospitalized patients with confirmed SARS-CoV-2 infection received either 1.5 mg (n = 15), 2.0 mg (n = 16), or 2.5 mg (n = 15) plitidepsin once daily for 3 d. The primary objective was safety; viral load kinetics, mortality, need for increased respiratory support, and dose selection were secondary end points. One patient withdrew consent before starting procedures; 45 initiated treatment; one withdrew because of hypersensitivity. Two Grade 3 treatment-related adverse events were observed (hypersensitivity and diarrhea). Treatment-related adverse events affecting more than 5% of patients were nausea (42.2%), vomiting (15.6%), and diarrhea (6.7%). Mean viral load reductions from baseline were 1.35, 2.35, 3.25, and 3.85 log10 at days 4, 7, 15, and 31. Nonmechanical invasive ventilation was required in 8 of 44 evaluable patients (16.0%); six patients required intensive care support (13.6%), and three patients (6.7%) died (COVID-19-related). Plitidepsin has a favorable safety profile in patients with COVID-19.

[This corrects the article DOI: 10.1371/journal.pone.0279815.].

Abstract Aims There is little information about the influence of gender on quality of life (QoL) in heart failure. The purpose of this study was to evaluate whether the health‐related QoL gap between men and women can be explained by the interaction between psychosocial factors and clinical determinants in a real‐word cohort of patients with chronic heart failure. Methods and results We conducted a single‐centre, observational, prospective cohort study of 1236 consecutive patients diagnosed with chronic heart failure recruited between 2004 and 2014. To assess QoL, we used the Minnesota Living with Heart Failure Questionnaire (MLHFQ). Female gender was associated with worse global QoL compared to male gender (MLHFQ overall summary score: 49 ± 23 vs. 43 ± 24; P value

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes six accessory proteins (3a, 6, 7a, 7b, 8, and 9b) for which limited information is available on their role in pathogenesis. We showed that the deletion of open reading frames (ORFs) 6, 7a, or 7b individually did not significantly impact viral pathogenicity in humanized K18-hACE2 transgenic mice. In contrast, the deletion of ORF8 partially attenuated SARS-CoV-2, resulting in reduced lung pathology and 40% less mortality, indicating that ORF8 is a critical determinant of SARS-CoV-2 pathogenesis. Attenuation of SARS-CoV-2-∆8 was not associated with a significant decrease in replication either in the lungs of mice or in organoid-derived human airway cells. An increase in the interferon signaling at early times post-infection (1 dpi) in the lungs of mice and a decrease in the pro-inflammatory and interferon response at late times post-infection, both in the lungs of mice (6 dpi) and in organoid-derived human airway cells [72 hours post-infection (hpi)], were observed. The early, but not prolonged, interferon response along with the lower inflammatory response could explain the partial attenuation of SARS-CoV-∆8. The presence of ORF8 in SARS-CoV-2 was associated with an increase in the number of macrophages in the lungs of mice. In addition, the supernatant of SARS-CoV-2-WT (wild-type)-infected organoid-derived cells enhanced the activation of macrophages as compared to SARS-CoV-2-∆8-infected cells. These results show that ORF8 is a virulence factor involved in inflammation that could be targeted in COVID-19 therapies. IMPORTANCE The relevance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF8 in the pathogenesis of COVID-19 is unclear. Virus natural isolates with deletions in ORF8 were associated with wild milder disease, suggesting that ORF8 might contribute to SARS-CoV-2 virulence. This manuscript shows that ORF8 is involved in inflammation and in the activation of macrophages in two experimental systems: humanized K18-hACE2 transgenic mice and organoid-derived human airway cells. These results identify ORF8 protein as a potential target for COVID-19 therapies.

Engineering of reverse genetics systems for newly emerged viruses allows viral genome manipulation, being an essential tool for the study of virus life cycle, virus-host interactions and pathogenesis, as well as for the development of effective antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent human coronavirus that has caused the coronavirus disease (COVID-19) pandemic. The engineering of a full-length infectious cDNA clone and a fluorescent replicon of SARS-CoV-2 Wuhan-Hu-1, using a bacterial artificial chromosome, is reported. Viral growth and genetic stability in eleven cell lines were analyzed, showing that both VeroE6 cells overexpressing transmembrane serin protease 2 (TMPRSS2) and human lung derived cells resulted in the optimization of a cell system to preserve SARS-CoV-2 genetic stability. The recombinant SARS-CoV-2 virus and a point mutant expressing the D614G spike protein variant were virulent in a mouse model. The RNA replicon was propagation-defective, allowing its use in BSL-2 conditions to analyze viral RNA synthesis. The SARS-CoV-2 reverse genetics systems developed constitute a useful tool for studying the molecular biology of the virus, the development of genetically defined vaccines and to establish systems for antiviral compounds screening.

In this work, authors provide a proof-of-concept study showing that deubiquitinating enzyme inactivation in MERS-CoV leads to attenuation in mice, and protection against a lethal challenge.

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. Case presentation In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a two-tube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, next-generation sequencing (NGS) covered > 90% SARS-CoV-2 genome sequence, allowing to classify it as variant B.1.177. Remarkably, the sequence revealed the Ile402Val substitution in the spike protein (S), of potential concern because it mapped in the receptor binding domain (RBD) that mediates virus interaction with the cell. NGS reads mapping to bacterial genomes showed that the dog fecal microbiome fitted best the characteristic microbiome of dog’s acute hemorrhagic diarrhea. Conclusion Our findings exemplify dog infection stemming from the human SARS-CoV-2 pandemic, providing nearly complete-genome sequencing of the virus, which is recognized as belonging to the B.1.177 variant, adding knowledge on variant circulation in a geographic region and period for which there was little viral variant characterization. A single amino acid substitution found in the S protein that could have been of concern is excluded to belong to this category given its rarity and intrinsic nature. The dog’s pathology suggests that SARS-CoV-2 could affect the gastrointestinal tract of the dog.

The emergent human coronavirus SARS-CoV-2 and its resistance to current drugs makes the need for new potent treatments for COVID-19 patients strongly necessary. Dextran sulfate (DS) polysaccharides have long demonstrated antiviral activity against different enveloped viruses in vitro. However, their poor bioavailability has led to their abandonment as antiviral candidates. Here, we report for the first time the broad-spectrum antiviral activity of a DS-based extrapolymeric substance produced by the lactic acid bacterium Leuconostoc mesenteroides B512F. Time of addition assays with SARS-CoV-2 pseudoviruses in in vitro models confirm the inhibitory activity of DSs in the early stages of viral infection (viral entry). In addition, this exopolysaccharide substance also reports broad-spectrum antiviral activity against several enveloped viruses such as SARS-CoV-2, HCoV229E, HSV-1, in in vitro models and in human lung tissue. The toxicity and antiviral capacity of DS from L. mesenteroides was tested in vivo in mouse models which are susceptible to SARS-CoV-2 infection. The described DS, administered by inhalation, a new route of administration for these types of polymers, shows strong inhibition of SARS-CoV-2 infection in vivo, significantly reducing animal mortality and morbidity at non-toxic doses. Therefore, we suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.

Coronaviruses (CoVs) are enveloped and positive-stranded RNA viruses with a large genome (∼ 30kb). CoVs include essential genes, such as the replicase and four genes coding for structural proteins (S, M, N and E), and genes encoding accessory proteins, which are variable in number, sequence and function among different CoVs. Accessory proteins are non-essential for virus replication, but are frequently involved in virus-host interactions associated with virulence. The scientific literature on CoV accessory proteins includes information analyzing the effect of deleting or mutating accessory genes in the context of viral infection, which requires the engineering of CoV genomes using reverse genetics systems. However, a considerable number of publications analyze gene function by overexpressing the protein in the absence of other viral proteins. This ectopic expression provides relevant information, although does not acknowledge the complex interplay of proteins during virus infection. A critical review of the literature may be helpful to interpret apparent discrepancies in the conclusions obtained by different experimental approaches. This review summarizes the current knowledge on human CoV accessory proteins, with an emphasis on their contribution to virus-host interactions and pathogenesis. This knowledge may help the search for antiviral drugs and vaccine development, still needed for some highly pathogenic human CoVs.

Cardiovascular disease is a common problem in cancer patients that is becoming more widely recognized. This may be a consequence of prior cardiovascular risk factors but could also be secondary to the anticancer treatments. With the goal of offering a multidisciplinary approach to guaranteeing optimal cancer therapy and the early detection of related cardiac diseases, and in light of the recent ESC Cardio-Oncology Guideline recommendations, we developed a Cardio-Oncology unit devoted to the prevention and management of these specific complications. This document brings together important aspects to consider for the development and organization of a Cardio-Oncology program through our own experience and the current evidence.

ABSTRACT Coronaviruses (CoVs) of genera α, β, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.

Background and aimsHeart failure (HF) programs successfully reduce 30-day readmissions. However, conflicting data exist about its sustained effects afterwards and its impact on mortality. We evaluated whether the impact of a new nurse-led coordinated transitional HF program extends to longer periods of time, including 90 and 180 days after discharge.Methods and resultsWe designed a natural experiment to undertake a pragmatical evaluation of the implementation of the program. We compared outcomes between patients discharged with HF as primary diagnosis in Period #1 (pre-program; Jan 2017-Aug 2017) and those discharged during Period #2 (HF program; Sept 2017-Jan 2019). Primary endpoint was the composite of all-cause death or all-cause hospitalization 90 and 180 days after discharge. 440 patients were enrolled: 123 in Period #1 and 317 in Period #2. Mean age was 75±9 years. There were more females in Period #2 (p = 0.025), with no other significant differences between periods. The primary endpoint was significantly reduced in the HF program group, at 90 [adjusted OR 0.31 (0.18-0.53), p ConclusionA new nurse-led coordinated transitional bundle of interventions model reduces the composite endpoint of all-cause death and all-cause hospitalization both at 90 and 180 days after a discharge for HF, also in high-risk populations. Such a decrease is driven by a reduction of CV and HF hospitalization. Reduction of all-cause mortality was also observed when the full model including a more exhaustive discharge planning process was implemented.

Coronaviruses (CoVs) have the largest genome among RNA viruses and store large amounts of information without genome integration as they replicate in the cell cytoplasm. The replication of the virus is a continuous process, whereas the transcription of the subgenomic mRNAs is a discontinuous one, involving a template switch, which resembles a high frequency recombination mechanism that may favor virus genome variability. The origin of the three deadly human CoVs SARS-CoV, MERS-CoV and SARS-CoV-2 are zoonotic events. SARS-CoV-2 has incorporated in its spike protein a furine proteolytic site that facilitates the activation of the virus in any tissue, making this CoV strain highly polytropic and pathogenic. Using MERS-CoV as a model, a propagation-deficient RNA replicon was generated by removing E protein gene (essential for viral morphogenesis and involved in virulence), and accessory genes 3, 4a, 4b and 5 (responsible for antagonism of the innate immune response) to attenuate the virus: MERS-CoV-Δ[3,4a,4b,5,E]. This RNA replicon is strongly attenuated and elicits sterilizing protection after a single immunization in transgenic mice with the receptor for MERS-CoV, making it a promising vaccine candidate for this virus and an interesting platform for vector-based vaccine development. A strategy could be developed for the design of RNA replicon vaccines for other human pathogenic coronaviruses.

Background. Iron deficiency (ID) is a significant, high-prevalence comorbidity in chronic heart failure (HF) that represents an independent predictor of a worse prognosis. However, a clear-cut diagnosis of ID in HF patients is not assured. The soluble transferrin receptor (sTfR) is a marker that reflects tissue-level iron demand and may be an early marker of ID. However, the impact of sTfR levels on clinical outcomes in non-anemic HF patients with a normal systemic iron status has never been evaluated. Methods. This is a post hoc analysis of an observational, prospective cohort study of 1236 patients with chronic HF of which only those with normal hemoglobin levels and a normal systemic iron status were studied. The final cohort consisted of 215 patients. Tissue ID was defined as levels of sTfR > 75th percentile (1.65 mg/L). Our aim was to describe the association between sTfR and clinical outcomes (all-cause death and HF hospitalization) and to explore its association with a wide array of serum biomarkers. Results. The sTfR level (HR 1.48, 95% CI 1.13–1.96, p = 0.005) and tissue ID (HR 2.14, 95% CI 1.22–3.75, p = 0.008) was associated with all-cause death. However, we found no association between sTfR levels and the risk of HF hospitalization. Furthermore, high sTfR levels were associated with a worse biomarker profile indicating myocardial damage (troponin and NT-proBNP), systemic inflammation (CRP and albumin), and impaired erythropoiesis (erythropoietin). Conclusions. In this cohort, the presence of tissue ID defined by sTfR levels is an independent factor for all-cause death in patients with normal systemic iron parameters. [ABSTRACT FROM AUTHOR]

The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer. [ABSTRACT FROM AUTHOR]

The soluble transferrin receptor (sTfR) is a marker of tissue iron status, which could indicate an increased iron demand at the tissue level. The impact of sTfR levels on functional capacity and quality of life (QoL) in non-anemic heart failure (HF) patients with otherwise normal systemic iron status has not been evaluated. We conducted an observational, prospective, cohort study of 1236 patients with chronic HF. We selected patients with normal hemoglobin levels and normal systemic iron status. Tissue iron deficiency (ID) was defined as levels of sTfR > 75th percentile (1.63 mg per L). The primary endpoints were the distance walked in the 6 min walking test (6MWT) and the overall summary score (OSS) of the Minnesota Living with Heart Failure Questionnaire (MLHFQ). The final study cohort consisted of 215 patients. Overall QoL was significantly worse (51 ± 27 vs. 39 ± 20, p-value = 0.006, respectively), and the 6 MWT distance was significantly worse in patients with tissue ID when compared to patients without tissue ID (206 ± 179 m vs. 314 ± 155, p-value < 0.0001, respectively). Higher sTfR levels, indicating increased iron demand, were associated with a shorter distance in the 6 MWT (standardized β = −0.249, p < 0.001) and a higher MLHFQ OSS (standardized β = 0.183, p-value = 0.008). In this study, we show that in patients with normal systemic iron parameters, higher levels of sTfR are strongly associated with an impaired submaximal exercise capacity and with worse QoL.

Basketball is a sport of deceptions, but also of numbers, and the knowledge of its internal logic has benefited for decades from the contributions of colleagues such as Sampaio and Ibáñez-Godoy. Accepting that NBA playoffs are the most demanding competition in our sport, we set two complementary goals for this study: to provide the a priory probabilities associated with field shots based on space and time, and to show the way we followed to calculate them in the R environment. Our analyses allow us to provide the probability distributions associated with field shots based on clock-time and court position, as shown in Table1. In addition, by simple logit models we can see and messure the impact that distances and moments dring the match (period and minute) have on the probability of success. The best basketball players in the world maximize the utility of their shots without being able to escape the space-time logic of the game: the balance between attack and defense comes to light in the form of probabilistic functions with high linearity, especially in terms of space, and points to interesting forward-looking questions. In this sense, our study provides general statistical basis for understanding this logic, allowing in the future to advance in better informed, bayesian study models.

RNA metabolism in the eukaryotic cell includes the formation of ribonucleoprotein complexes (RNPs) that, depending on their protein components, have a different function. Cytoplasmic RNPs, such as stress granules (SGs) or P-bodies (PBs) are quite relevant during infections modulating viral and cellular RNA expression and as key players in the host cell antiviral response. RNA helicases are abundant components of RNPs and could have a significant effect on viral infection. This review focuses in the role that RNPs and RNA helicases have during coronavirus (CoVs) infection. CoVs are emerging highly pathogenic viruses with a large single-stranded RNA genome. During CoV infection, a complex network of RNA-protein interactions in different RNP structures is established. In general, RNA helicases and RNPs have an antiviral function, but there is limited knowledge on whether the viral protein interactions with cell components are mediators of this antiviral effect or are part of the CoV antiviral counteraction mechanism. Additional data is needed to elucidate the role of these RNA-protein interactions during CoV infection and their potential contribution to viral replication or pathogenesis.

Transgenic human monoclonal antibodies derived from humanized mice against different epitopes of the Middle East respiratory syndrome coronavirus (MERS-CoV), and chimeric llama-human bispecific heavy chain-only antibodies targeting the Rift Valley fever virus (RVFV), were produced using a CHO-based transient expression system. Two lead candidates were assessed for each model virus before selecting and progressing one lead molecule. MERS-7.7G6 was used as the model antibody to demonstrate batch-to-batch process consistency and, together with RVFV-107-104, were scaled up to 200 L. Consistent expression titers were obtained in different batches at a 5 L scale for MERS-7.7G6. Although lower expression levels were observed for MERS-7.7G6 and RVFV-107-104 during scale up to 200 L, product quality attributes were consistent at different scales and in different batches. In addition to this, peptide mapping data suggested no detectable sequence variants for any of these candidates. Functional assays demonstrated comparable neutralizing activity for MERS-7.7G6 and RVFV-107-104 generated at different production scales. Similarly, MERS-7.7G6 batches generated at different scales were shown to provide comparable protection in mouse models. Our study demonstrates that a CHO-based transient expression process is capable of generating consistent product quality at different production scales and thereby supports the potential of using transient gene expression to accelerate the manufacturing of early clinical material.

No vaccines or specific antiviral drugs are authorized against Middle East respiratory syndrome coronavirus (MERS-CoV) despite its high mortality rate and prevalence in dromedary camels. Since 2012, MERS-CoV has been causing sporadic zoonotic infections in humans, which poses a risk of genetic evolution to become a pandemic virus. MERS-CoV genome encodes five accessory proteins, 3, 4a, 4b, 5 and 8b for which limited information is available in the context of infection. This work describes 4b as a virulence factor in vivo, since the deletion mutant of a mouse-adapted MERS-CoV-Δ4b (MERS-CoV-MA-Δ4b) was completely attenuated in a humanized DPP4 knock-in mouse model, resulting in no mortality. Attenuation in the absence of 4b was associated with a significant reduction in lung pathology and chemokine expression levels at 4 and 6 days post-infection, suggesting that 4b contributed to the induction of lung inflammatory pathology. The accumulation of 4b in the nucleus in vivo was not relevant to virulence, since deletion of its nuclear localization signal led to 100% mortality. Interestingly, the presence of 4b protein was found to regulate autophagy in the lungs of mice, leading to upregulation of BECN1, ATG3 and LC3A mRNA. Further analysis in MRC-5 cell line showed that, in the context of infection, MERS-CoV-MA 4b inhibited autophagy, as confirmed by the increase of p62 and the decrease of ULK1 protein levels, either by direct or indirect mechanisms. Together, these results correlated autophagy activation in the absence of 4b with downregulation of a pathogenic inflammatory response, thus contributing to attenuation of MERS-CoV-MA-Δ4b. Author summary Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human coronavirus that is still causing sporadic zoonotic infections in humans. Knowledge of virus-host interactions is required to develop specific protection strategies. This manuscript identifies 4b MERS-CoV protein as a virulence factor in vivo, independent of its nuclear localization signal, and demonstrates that the presence of 4b induces a proinflammatory response and inhibits autophagy. These results highlight the relevance of the activation of the inflammatory response and the inhibition of autophagy in the virulence of MERS-CoV, and suggest 4b as a candidate antiviral target.

Summary The surge of SARS‐CoV‐2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID‐19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 μg ml‐1) and IgG (~0.017 μg ml‐1) and is scalable and cost‐effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma‐based treatments, high‐throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency.

Abstract Background Iron deficiency (ID) is common in patients with heart failure (HF) and is associated with poor outcomes, yet its role in the pathophysiology of HF is not well-defined. We sought to determine the consequences of HF neurohormonal activation in iron homeostasis and mitochondrial function in cardiac cells. Methods HF was induced in C57BL/6 mice by using isoproterenol osmotic pumps and embryonic rat heart-derived H9c2 cells were subsequently challenged with Angiotensin II and/or Norepinephrine. The expression of several genes and proteins related to intracellular iron metabolism were assessed by Real time-PCR and immunoblotting, respectively. The intracellular iron levels were also determined. Mitochondrial function was analyzed by studying the mitochondrial membrane potential, the accumulation of radical oxygen species (ROS) and the adenosine triphosphate (ATP) production. Results Hearts from isoproterenol-stimulated mice showed a decreased in both mRNA and protein levels of iron regulatory proteins, transferrin receptor 1, ferroportin 1 and hepcidin compared to control mice. Furthermore, mitoferrin 2 and mitochondrial ferritin were also downregulated in the hearts from HF mice. Similar data regarding these key iron regulatory molecules were found in the H9c2 cells challenged with neurohormonal stimuli. Accordingly, a depletion of intracellular iron levels was found in the stimulated cells compared to non-stimulated cells, as well as in the hearts from the isoproterenol-induced HF mice. Finally, neurohormonal activation impaired mitochondrial function as indicated by the accumulation of ROS, the impaired mitochondrial membrane potential and the decrease in the ATP levels in the cardiac cells. Conclusions HF characteristic neurohormonal activation induced changes in the regulation of key molecules involved in iron homeostasis, reduced intracellular iron levels and impaired mitochondrial function. The current results suggest that iron could be involved in the pathophysiology of HF.

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) and the closely related SARS-CoV-2 are emergent highly pathogenic human respiratory viruses causing acute lethal disease associated with lung damage and dysregulated inflammatory responses. SARS-CoV envelope protein (E) is a virulence factor involved in the activation of various inflammatory pathways. Here, we study the contribution of host miRNAs to the virulence mediated by E protein. Small RNAseq analysis of infected mouse lungs identified miRNA-223 as a potential regulator of pulmonary inflammation, since it was significantly increased in SARS-CoV-WT virulent infection compared to the attenuated SARS-CoV-ΔE infection. In vivo inhibition of miRNA-223-3p increased mRNA levels of pro-inflammatory cytokines and NLRP3 inflammasome, suggesting that during lung infection, miRNA-223 might contribute to restrict an excessive inflammatory response. Interestingly, miRNA-223-3p inhibition also increased the levels of the CFTR transporter, which is involved in edema resolution and was significantly downregulated in the lungs of mice infected with the virulent SARS-CoV-WT virus. At the histopathological level, a decrease in the pulmonary edema was observed when miR-223-3p was inhibited, suggesting that miRNA-223-3p was involved in the regulation of the SARS-CoV-induced inflammatory pathology. These results indicate that miRNA-223 participates in the regulation of E protein-mediated inflammatory response during SARS-CoV infection by targeting different host mRNAs involved in the pulmonary inflammation, and identify miRNA-223 as a potential therapeutic target in SARS-CoV infection. IMPORTANCE The SARS-CoV-2 pandemic has emphasized the need to understand the mechanisms of severe lung inflammatory pathology caused by human deadly coronaviruses in order to design new antiviral therapies. Here, we identify miRNA-223-3p as a host miRNA involved in the regulation of lung inflammatory response mediated by envelope (E) protein during SARS-CoV infection. miRNAs downregulate the expression of cellular mRNAs and participate in complex networks of mRNA–miRNA interactions that regulate cellular processes. The inhibition of miRNA-223 in infected mice by intranasal administration of antisense RNAs led to changes in the expression of host factors involved in inflammation (cytokines, chemokines, and NLRP3 inflammasome) and in the resolution of lung edema ion transporter CFTR. These results confirmed the contribution of miRNA-223 to the regulation of SARS-CoV-induced pathogenic processes and support the therapeutic potential of inhibiting miRNAs during coronavirus infection using RNA interference approaches.

ABSTRACT Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. IMPORTANCE The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.

Here, the authors report the isolation and characterization of two human monoclonal antibodies (mAbs) from immunized mice with trimeric spike ectodomains of three human betacoronaviruses HCoV-OC43, SARS-CoV and MERSCoV, and show that while exhibiting cross-reactivity, the mAbs only neutralize MERS-CoV but not SARS-CoV nor SARS-CoV-2, likely due to the subtle epitope differences in the spike S2 fusion subunit.

The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence. This study aimed at using the RNA-targeting CRISPR-Cas13d system to attack highly conserved sequences in the viral RNA genome, thereby preparing for future zoonotic outbreaks of other coronaviruses. We designed 29 crRNAs targeting highly conserved sequences along the complete SARS-CoV-2 genome. Several crRNAs demonstrated efficient silencing of a reporter with the matching viral target sequence and efficient inhibition of a SARS-CoV-2 replicon. The crRNAs that suppress SARS-CoV-2 were also able to suppress SARS-CoV, thus demonstrating the breadth of this antiviral strategy. Strikingly, we observed that only crRNAs directed against the plus-genomic RNA demonstrated antiviral activity in the replicon assay, in contrast to those that bind the minus-genomic RNA, the replication intermediate. These results point to a major difference in the vulnerability and biology of the +RNA versus −RNA strands of the SARS-CoV-2 genome and provide important insights for the design of RNA-targeting antivirals.

Heart failure (HF) is classified according to the degree of reduction in left ventricular ejection fraction (EF) in HF with reduced, mildly reduced, and preserved EF. Biomarkers could behave differently depending on EF type. Here, we analyze the soluble form of the AXL receptor tyrosine kinase (sAXL) in HF patients with reduced and preserved EF. Two groups of HF patients with reduced (HFrEF; n = 134) and preserved ejection fraction (HFpEF; n = 134) were included in this prospective observational study, with measurements of candidate biomarkers and functional, clinical, and echocardiographic variables. A Cox regression model was used to determine predictors for clinical events: cardiovascular mortality and all-cause mortality. sAXL circulating values predicted outcome in HF: for a 1.0 ng/mL increase in serum sAXL, the mortality hazard ratio (HR) was 1.019 for HFrEF (95% CI 1.000 to 1.038) and 1.032 for HFpEF (95% CI 1.013 to 1.052). In a multivariable Cox regression analysis, sAXL and NT-proBNP were independent markers for all-cause and cardiovascular mortality in HFpEF. In contrast, only NT-proBNP remained significant in the HFrEF group. When analyzing the event-free survival at a mean follow-up of 3.6 years, HFrEF and HFpEF patients in the higher quartile of sAXL had a reduced survival time. Interestingly, sAXL is a reliable predictor for all-cause and cardiovascular mortality only in the HFpEF cohort. The results suggest an important role for AXL in HFpEF, supporting sAXL evaluation in larger clinical studies and pointing to AXL as a potential target for HF therapy.

Abstract Aims The role of non‐invasive telemedicine (TM) combining telemonitoring and teleintervention by videoconference (VC) in patients recently admitted due to heart failure (HF) (‘vulnerable phase’ HF patients) is not well established. The aim of the Heart failure Events reduction with Remote Monitoring and eHealth Support (HERMeS) trial is to assess the impact on clinical outcomes of implementing a TM service based on mobile health (mHealth), which includes remote daily monitoring of biometric data and symptom reporting (telemonitoring) combined with VC structured, nurse‐based follow‐up (teleintervention). The results will be compared with those of the comprehensive HF usual care (UC) strategy based on face‐to‐face on‐site visits at the vulnerable post‐discharge phase. Methods and results We designed a 24 week nationwide, multicentre, randomized, controlled, open‐label, blinded endpoint adjudication trial to assess the effect on cardiovascular (CV) mortality and non‐fatal HF events of a TM‐based comprehensive management programme, based on mHealth, for patients with chronic HF. Approximately 508 patients with a recent hospital admission due to HF decompensation will be randomized (1:1) to either structured follow‐up based on face‐to‐face appointments (UC group) or the delivery of health care using TM. The primary outcome will be a composite of death from CV causes or non‐fatal HF events (first and recurrent) at the end of a 6 month follow‐up period. Key secondary endpoints will include components of the primary event analysis, recurrent event analysis, and patient‐reported outcomes. Conclusions The HERMeS trial will assess the efficacy of a TM‐based follow‐up strategy for real‐world ‘vulnerable phase’ HF patients combining telemonitoring and teleintervention.