Air pollution is a major global environment and health concern. Recent studies have suggested an association between air pollution and COVID-19 mortality and morbidity. In this context, a close association between increased levels of air pollutants such as particulate matter ≤2.5 to 10 µM, ozone and nitrogen dioxide and SARS-CoV-2 infection, hospital admissions and mortality due to COVID 19 has been reported. Air pollutants can make individuals more susceptible to SARS-CoV-2 infection by inducing the expression of proteins such as angiotensin converting enzyme (ACE)2 and transmembrane protease, serine 2 (TMPRSS2) that are required for viral entry into the host cell, while causing impairment in the host defence system by damaging the epithelial barrier, muco-ciliary clearance, inhibiting the antiviral response and causing immune dysregulation. The aim of this review is to report the epidemiological evidence on impact of air pollutants on COVID 19 in an up-to-date manner, as well as to provide insights on in vivo and in vitro mechanisms.

Understanding SARS-CoV-2 epidemiology in companion animals is critical for evaluating their role in viral transmission and their potential as sentinels for human infections. This large-scale serosurvey analyzed serum samples from 706 cats and 2,396 dogs collected across the USA in 2023 using a surrogate virus neutralization test (sVNT) to detect SARS-CoV-2 antibodies. Overall, 5.7% of cats and 4.7% of dogs tested positive for antibodies, with younger animals (under 12 months) showing significantly lower seropositivity rates (p = 0.0048). Additionally, we analyzed 153 positive samples for variant-specific antibody responses using six sVNT kits targeting the Delta variant and five Omicron sublineages. Among cats, 67.5% showed antibodies to Delta, with positivity rates for Omicron sublineages as follows: BA.1 (62.5%), BA.2 (42.5%), BA.4/BA.5 (77.5%), XBB (52.5%), and XBB.1.5 (45.0%). In dogs, 55.8% were positive for Delta, and Omicron sublineage rates were BA.1 (46.0%), BA.4/BA.5 (37.2%), XBB (58.4%), BA.2 (13.3%), and XBB.1.5 (9.7%). Given the close contact between companion animals and humans, and the persistence of antibodies against various SARS-CoV-2 variants and sublineages, our findings suggest that seroprevalence in cats and dogs may serve as valuable tool for tracking COVID-19 epidemiology.

N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification in eukaryotic RNA and is also present in various viral RNAs, where it plays a crucial role in regulating the viral life cycle. However, the molecular mechanisms through which viruses regulate host RNA m6A methylation are not fully understood. In this study, we reveal that SARS-CoV-2 and HCoV-OC43 infection enhance host m6A modification by activating the mTORC1 signalling pathway. Specifically, the viral non-structural protein nsp14 upregulates the expression of S-adenosylmethionine synthase MAT2A in an mTORC1-dependent manner. This mTORC1-MAT2A axis subsequently stimulates the synthesis of S-adenosylmethionine (SAM). The increase of SAM then enhances the m6A methylation of host RNA and facilitates viral replication. Our findings uncover a molecular mechanism by which viruses regulate host m6A methylation and provide insights into how SARS-CoV-2 hijacks host cellular epitranscriptomic modifications to promote its replication.

Packaging signals (PSs) of coronaviruses (CoVs) are specific RNA elements recognized by nucleocapsid (N) proteins that direct the selective packaging of genomic RNAs (gRNAs). These signals have been identified in the coding regions of the nonstructural protein 15 (Nsp 15) in CoVs classified under Embecovirus, a subgenus of betacoronaviruses (beta-CoVs). The PSs in other alpha- and beta-CoVs have been proposed to reside in the 5'-proximal regions of gRNAs, supported by comprehensive phylogenetic evidence. However, experimental data remain limited. In this study, we investigated the interactions between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) 5'-proximal gRNA transcripts and N proteins using electrophoretic mobility shift assays (EMSAs). Our findings revealed that the in vitro synthesized 5'-proximal gRNA transcripts of CoVs can shift from a major conformation to alternative conformations. We also observed that the conformer comprising multiple stem-loops (SLs) is preferentially bound by N proteins. Deletions of the 5'-proximal structural elements of CoV gRNA transcripts, SL1 and SL5a/b/c in particular, were found to promote the formation of alternative conformations. Furthermore, we identified RNA-binding peptides from a pool derived from SARS-CoV N protein. These RNA-interacting peptides were shown to preferentially bind to wild-type SL5a RNA. In addition, our observations of N protein condensate formation in vitro demonstrated that liquid-liquid phase separation (LLPS) of N proteins with CoV-5'-UTR transcripts was influenced by the presence of SL5a/b/c. In conclusion, these results collectively reveal previously uncharacterized binding features between the 5'-proximal transcripts of CoV gRNAs and N proteins.

The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on N-glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and continues to pose serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication and its conservation, making it an attractive drug target. Here, we employed a structure-based drug design strategy to develop and optimize novel inhibitors targeting SARS-CoV-2 Mpro. By fully exploring occupation of the S1, S2, and S3/S4 binding pockets, we identified eight promising inhibitors with half-maximal inhibitory concentration (IC50) values below 20 nM. The cocrystal structure of Mpro with compound 10 highlighted the crucial roles of the interactions within the S3/S4 pockets in inhibitor potency enhancement. These findings demonstrated that expanding the utilization of these binding pockets was an effective strategy for developing noncovalent small molecule inhibitors that target SARS-CoV-2 Mpro. Compound 4 demonstrated outstanding in vitro antiviral activity against wild-type SARS-CoV-2 with an EC50 of 9.4 nM. Moreover, oral treatment with compounds 1 and 9 exhibited excellent antiviral potency and substantially ameliorated virus-induced tissue damage in the lungs of Omicron BA.5-infected K18-human ACE2 (K18-hACE2) transgenic mice, indicating that these novel noncovalent inhibitors could be potential oral agents for the treatment of COVID-19.

Since 2020, numerous compounds have been investigated for their potential use in treating SARS-CoV-2 infections. By identifying the molecular targets during the virus replication process, rationally designed anti-SARS-CoV-2 agents are developed. Among these targets, the main protease (Mpro) is a crucial enzyme required for virus replication, and its highly conserved characteristic make it an important drug target for the development of anti-SARS-CoV-2 drugs. Herein, we utilized warhead-based design strategy to conduct the structural optimization of M-1 developed through virtual screening, leading to a series of novel Mpro inhibitors with 4-(quinolin-2-yl)aniline scaffold. Among them, M-32 exhibited good SARS-CoV-2 Mpro inhibitory activity (IC50 = 5.2 μM) with a nearly 25-fold increase. Isothermal titration calorimetry (ITC) directly proved that M-32 binds directly to SARS-CoV-2 Mpro in an entropy-driven manner. Mass spectrometry (MS) further confirmed the covalent binding ability of M-32 to Mpro. Meanwhile, M-32 effectively inhibited the replication of SARS-CoV-2 in Vero E6 cells (EC50 = 5.29 μM).

The COVID-19 pandemic exposed the fragility of today's marketed treatments for respiratory infections. As a primary site of infection, the upper airways may represent a key access route for the control and treatment for these conditions. The present study aims to explore and identify, through a patent review, the novelty of therapies for COVID-19 that use the intranasal route for drug administration. A search was carried out in Wipo and Espacenet, using the descriptors "COVID-19 OR SARS-CoV 2" AND "treatment OR therapy" AND NOT "vaccine OR immunizing" and the classification "A61K9/0043". Of the 151 patents identified, we excluded 73 duplicates, and 36 documents that meet the criteria adopted for exclusion (not nasally administered formulations, vaccines, post COVID-19 treatments, uncertain route of administration or form). We identified 78 unique patents on patent databases, of which 42 were selected for this review. The documents revealed the use of the intranasal pathway not only for drug repositioning but also for using plant-derived and biological molecules. Overall, the new formulations explore a variety of known drugs and natural products incorporated in drug carrier systems and devices for drug delivery and administration. Thus, the intranasal route remains a promising strategy for drug delivery, offering direct access to the primary infection site and warranting further exploration.

The production of coronavirus disease 2019 vaccines can be achieved by transient expression of the spike (S) protein of severe acute respiratory syndrome coronavirus 2 in agroinfiltrated leaves of Nicotiana benthamiana. Relying on bacterial vector Agrobacterium tumefaciens, this process is favoured by co-expression of viral silencing suppressor P19. Upon expression, the S protein enters the cell secretory pathway, before being trafficked to the plasma membrane where formation of coronavirus-like particles (CoVLPs) occurs. We previously characterized the effects of influenza virus hemagglutinin forming VLPs through similar processes. However, leaf samples were only collected after 6 days of expression, and it is unknown whether influenza VLPs (HA-VLPs) and CoVLPs induce similar responses. Here, time course sampling was used to profile responses of N. benthamiana leaf cells expressing P19 only, or P19 and the S protein. The latter triggered early but transient activation of the unfolded protein response and waves of transcription factor genes involved in immunity. Accordingly, defence genes were induced with different expression kinetics, including those promoting lignification, terpene biosynthesis, and oxidative stress. Cross-talk between stress hormone pathways also occurred, including repression of jasmonic acid biosynthesis genes after agroinfiltration, and dampening of salicylic acid responses upon S protein accumulation. Overall, HA-VLP- and CoVLP-induced responses broadly overlapped, suggesting nanoparticle production to have the most effects on plant immunity, regardless of the virus surface proteins expressed. Taking advantage of RNAseq inferences, we finally show the co-expression of Kunitz trypsin inhibitors to reduce CoVLP-induced defence and leaf symptoms, with no adverse effect on plant productivity.

Congenital heart defects (CHD) are the most common major birth defects and one of the leading causes of death from congenital defects after birth. CHD can arise in pregnancy from the combination of genetic and non-genetic factors. The maternal immune activation (MIA) hypothesis is widely implicated in embryonic neurodevelopmental abnormalities. MIA has been found to be associated with the development of asthma, diabetes mellitus, and other diseases in the offspring. Given the important role of cardiac immune cells and cytokines in embryonic heart development, it is hypothesized that MIA may play a significant role in embryonic heart development. This review aims to stimulate further investigation into the relationship between MIA and CHD and to highlight the gaps in the knowledge. It evaluates the impact of MIA on CHD in the context of pregnancy complications, immune-related diseases, infections, and environmental and lifestyle factors. The review outlines the mechanisms by which immune cells and their secretome indirectly regulate the immuno-microenvironment of the embryonic heart by influencing placental development. Furthermore, the inflammatory cytokines cross the placenta to induce related reactions including oxidative stress in the embryonic heart directly. This review delineates the role of MIA in CHD and underscores the impact of maternal factors, especially immune factors, as well as the embryonic cardiac immuno-microenvironment, on embryonic heart development. This review extends our understanding of the importance of MIA in the pathogenesis of CHD and provides important insights into prenatal prevention and treatment strategies for this congenital condition.

The global impact of SARS-CoV-2 has highlighted the urgent need for novel antiviral therapies. This study integrates combinatorial chemistry, molecular docking, and deep learning to design, evaluate and synthesize new pyrazole derivatives as potential inhibitors of the SARS-CoV-2 main protease (Mpro). A library of over 60,000 pyrazole-based structures was generated through scaffold decoration to enhance chemical diversity. Virtual screening employed molecular docking (ChemPLP scoring) and deep learning (DeepPurpose), with consensus ranking to identify top candidates. Binding free energy calculations refined the selection, revealing critical structural features such as tryptamine and N-phenyl fragments for Mpro binding. High-temperature solvent-free amidation allowed the synthesis of a selected derivative. Final compounds demonstrated favorable drug-likeness properties based on Lipinski's and Veber's rules. This work highlights the integration of computational and synthetic strategies to accelerate the discovery of Mpro inhibitors and provides a framework for future antiviral development.

Coronavirus disease 2019 (COVID-19) is a deadly human viral disease with a high rate of infection, morbidity, and mortality. Although vaccines and antiviral treatments are available, hospitalizations remain steady, and concerns about long-term consequences persist. Therefore, there is a great urgency to develop novel therapies. Here, we analyzed the role of miR-155, one of the most powerful drivers of host antiviral responses including immune and inflammatory responses, in the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Endogenous microRNAs (miRNAs, miRs) are key molecules in preventing viral entry and replication while building an antiviral cellular defense. Our study reveals that miR-155 expression is elevated in patients with COVID-19. Using a mouse model transgenic for human angiotensin-converting enzyme receptor 2, we evaluated the potential of anti-miR-155 therapy. Treating SARS-CoV-2-infected mice with anti-miR-155 significantly reduced miR-155 expression, improved survival, and slightly increased body weight. Notably, these mice showed altered expression of cytokines in the lungs. These findings suggest anti-miR-155 could be a promising therapy to mitigate the cytokine storm and long-lasting symptoms induced by SARS-CoV-2 infection, improving public health outcomes and enhancing global pandemic preparedness.

In this report, a structurally unique phenothiazine (PTZ) core is linked with glutamic acid-based dendrons through a solid-phase peptide synthesis approach to access a variety of PTZ-linked dendrons conveniently. Inferior cytotoxicity of anionic surface-linked second-generation glutamic acid-based dendrons would be more desirable for various applications than respective lysine-based dendrons. Solid-phase synthesis of PTZ-linked glutamic acid-based dendrons would be a novel approach to access this class of molecules. These newly synthesized dendrons were screened as an inhibitor against the main protease (Mpro) enzyme, proposed to be the best target against SARS-CoV-2. The preliminary assay studies designated a moderate response for the Mpro inhibition, specifically by tryptophan (Trp)-enriched dendron, among other analogues, which play a vital role in combating COVID-19. Further, the experimental studies realize the essential contribution of the PTZ core in interacting with the Mpro enzyme. Molecular dynamics (MD) simulations revealed that the active dendrons formed stable complexes with Mpro, and the binding affinity of the Trp-based PTZ-linked dendrons was higher than that of the decoy dendron analogue.

Inflammation is a pathology implicated in a wide range of human diseases. Recent years have seen tremendous progress in developing new types of anti-inflammatory agents for the treatment of inflammation of various origins. However, each has its own strengths and weaknesses. The very fact that there needs to have multiple types of anti-inflammatory agents underlines the complexity of inflammatory diseases and conditions, their molecular origins, and their treatment. Such complexity dictates the need to search for new approaches with improved potency and efficacy as well as reduced side effects. For these reasons, we are interested in exploring the possibility of generating synergy between carbon monoxide (CO), an endogenously produced cytoprotective agent, and known anti-inflammatory agents. Herein, we report the potentiating actions of CO on the anti-inflammatory effects of cortisone and dexamethasone as demonstrated in their ability to suppress the expression of TNF-α and IL-6 induced by either LPS or the S protein of SARS-CoV-2. Such effects are reflected in the substantially increased potency as well efficacy, when the efficacy of the corticosteroid alone does not allow for complete suppression of the expression of these cytokines. Further, increased attenuation of p65 phosphorylation is at least part of the molecular mechanism for the observed potentiating effects. We hope our work will stimulate a high level of activity along the same direction, leading to anti-inflammatory strategies with improved potency and efficacy and reduced side effects.

Many virus species, including Ebola virus, Marburg virus, SARS-CoV-2, dengue virus (DENV) and Zika virus (ZIKV), exploit CD209 and CD209L as alternative or attachment receptors for viral cis- or trans-infection. Thus, CD209 and CD209L may be critical targets for the development of therapeutic monoclonal blocking antibody drugs to disrupt the infection process caused by multiple viruses. Here, we produced a human chimeric monoclonal blocking antibody that simultaneously blocks CD209 and CD209L, namely 7-H7-B1. We show that 7-H7-B1 effectively blocks multiple pseudotyped or live viral infections in vitro, including SARS-CoV, SARS-CoV-2, Ebola virus, Marburg virus, ZIKV and DENV infections. However, the 7-H7-B1 mAb does not provide favourable protection against Zaire Ebola virus or ZIKV infection in hCD209 knock-in mice in vivo. Thus, our findings indicate that although CD209 and CD209L are critical for multiple viral infections in vitro, they may play only a partial role in viral infections in vivo.

New strategies are needed to prevent and control upcoming outbreaks of SARS-CoV-2 infections, independent of vaccination. SARS-CoV-2 binds to the human ACE-2 receptor through the receptor binding domain (RBD) of the spike (S) protein, allowing the virus to enter human cells and begin replication. When peptides corresponding to four regions of RBD containing previously reported ACE-2 interaction sites were explored, the sequence 392 to 421, peptide p392wt, bound strongly to ACE-2 and inhibited wild-type RBD binding to ACE-2. Interestingly, p392 peptides corresponding to mutated sequences from different SARS-CoV-2 VOCs, including the current VOC BA.5 and KP.3, bound less strongly to ACE-2, but showed partially better inhibition of the ACE-2 interaction of all tested RBDs. When studied in a SARS-CoV-2 pseudovirus assay, the p392 peptides showed a good inhibition rate of 98.8±8.1 % at a peptide concentration of ~244 μmol/L, while none of the p392 peptides inhibited antibody binding to the RBD, suggesting that peptide treatment is sufficient in the presence of anti-RBD antibodies. Interestingly these peptides were active in the presence of diluted human serum and non-toxic to human cell lines.

A series of S-adenosyl-L-homosysteine (SAH) analogs, with modification in the base and sugar moiety, have been designed, synthesized and screened as nsp14 and PLpro inhibitors of severe acute respiratory syndrome corona virus (SARS-CoV-2). The outcomes of ADMET (Adsorption, Distribution, Metabolism, Excretion, and Toxicity) studies demonstrated that the physicochemical properties of all analogs were permissible for development of these SAH analogs as antiviral agents. All molecules were screened against different SARS-CoV-2 targets using molecular docking. The docking results revealed that the SAH analogs interacted well in the active site of nsp14 protein having H-bond interactions with the amino acid residues Arg289, Val290, Asn388, Arg400, Phe401 and π-alkyl interactions with Arg289, Val290 and Phe426 of Nsp14-MTase site. These analogs also formed stable H-bonds with Leu163, Asp165, Arg167, Ser246, Gln270, Tyr274 and Asp303 residues of PLpro proteins and found to be quite stable complexes therefore behaved as probable nsp14 and PLpro inhibitors. Interestingly, analog 3 showed significant in silico activity against the nsp14 N7 methyltransferase of SARS-CoV-2. The molecular dynamics (MD) and post-MD results of analog 3 unambiguously established the higher stability of the nsp14 (N7 MTase):3 complex and also indicated its behavior as probable nsp14 inhibitor like the reference sinefungin. The docking and MD simulations studies also suggested that sinefungin did act as SARS-CoV-2 PLpro inhibitor as well. This study's findings not only underscore the efficacy of the designed SAH analogs as potent inhibitors against crucial SARS-CoV-2 proteins but also pinpoint analog 3 as a particularly promising candidate. All the study provides valuable insights, paving the way for potential advancements in antiviral drug development against SARS-CoV-2.

Pneumonia is a ubiquitous disease caused by viral and bacterial infections, characterized by high levels of reactive oxygen species in inflamed areas. Therapeutic strategies targeting reactive oxygen species levels in pneumonia have limited success due to the intricate nature of lung tissues and lung inflammatory responses. Here we describe an inhalable, non-invasive therapeutic platform composed of engineered cerium-based tannic acid nanozymes bound to a self-assembling peptide. In vitro and in vivo studies show that the nanozyme is internalized mostly by activated macrophages and epithelial cells in the inflamed sites. In the oxidative environments of a mouse model of viral pneumonia, nanozyme aggregates into catalytically active structures that reduce reactive oxygen species levels and inflammatory cytokine production and promote macrophage polarization to the prohealing (M2) phenotype. Moreover, the nanozyme attenuates bacterial inflammation and reduces tissue damage in a mouse viral pneumonia model with secondary bacterial infection. Overall, this nanozyme platform is a promising strategy for treating pneumonia and its associated conditions.

Accurate diagnosis and effective antiviral strategies are critical to combat acute infection and to avoid damage to the host. Due to their restricted radiation range and energy, Auger electron emitters have shown potential as a RNA-destructing radionuclide therapy in oncology and infection. Focusing on the process of angiotensin-converting enzyme 2 (ACE2)-mediated endocytosis, Technetium-99m-labeled DX600 (99mTc-DX600) was synthesized as an Auger electron vector to specifically bind to surface-expressed ACE2 proteins on 293T-hACE2 cells (293T cells stably expressing human ACE2), and Technetium-99m-loaded microvesicles (99mTc-MVs) served as an antiviral tracer and effector in pseudovirus infection. The whole-body ACE2 expression evaluation was non-invasive, meanwhile, the enhanced green fluorescent protein expression of pseudoviruses was substantially inhibited as a result of the 99mTc-DX600 loading of microvesicles, though the mitochondrial and DNA stabilities of the host cells were not affected. Furthermore, the in vivo distribution of 99mTc-DX600 in humanized ACE2 mice was demonstrated to be both ACE2-specific and long-lasting, and an antiviral effect was fully exhibited with two cycles of intravenous injection at a dosage of 37 MBq. Taking advantage of the ACE2-mediated interaction and natural trigger mechanism of virus-induced endocytosis, 99mTc-MV represents a theranostic biosensor of Auger electrons that can expose viral RNA to lethal amounts of radiation, with the host cells receiving no detrimental radiation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced impaired antiviral immunity and excessive inflammatory responses cause lethal pneumonia. However, the in vivo roles of key pattern recognition receptors that elicit protective antiviral and fatal inflammatory responses, specifically in the lungs, are not well described. Coronaviruses possess single-stranded RNA genome that activates TLR7/8 to induce an antiviral interferon (IFN) and robust inflammatory cytokine response. Here, using wild-type and TLR7-deficient (TLR7-/-) mice infected with mouse-adapted SARS-CoV-2 (MA-CoV-2), we examined the role of TLR7 in the lung antiviral and inflammatory response and severe pneumonia. We showed that TLR7 deficiency significantly increased lung virus loads and morbidity/mortality, which correlated with reduced levels of type I IFNs (Ifna/b), type III IFNs (Ifnl), and IFN-stimulated genes (ISGs) in the lungs. A detailed evaluation of MA-CoV-2-infected lungs revealed increased neutrophil accumulation and lung pathology in TLR7-/- mice. We further showed that blocking type I IFN receptor (IFNAR) signaling enhanced SARS-CoV-2 replication in the lungs and caused severe lung pathology, leading to 100% mortality compared to infected control mice. Moreover, immunohistochemical assessment of the lungs revealed increased numbers of SARS-CoV-2 antigen-positive macrophages, pneumocytes, and bronchial epithelial cells in TLR7-/- and IFNAR-deficient mice compared to control mice. In summary, we conclusively demonstrated that despite TLR7-induced robust lung inflammation, TLR7-induced IFN/ISG responses suppress lung virus replication and pathology and provide protection against SARS-CoV-2-induced fatal pneumonia. Additionally, given the similar disease outcomes in control, TLR7-/-, and IFNAR-deficient MA-CoV-2-infected mice and coronavirus disease 2019 (COVID-19) patients, we propose that MA-CoV-2-infected mice constitute an excellent model for studying COVID-19.IMPORTANCESevere coronavirus disease 2019 (COVID-19) is caused by a delicate balance between a strong antiviral and an exuberant inflammatory response. A robust antiviral immunity and regulated inflammation are protective, while a weak antiviral response and excessive inflammation are detrimental. However, the key host immune sensors that elicit protective antiviral and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge are poorly defined. Here, we examined the role of viral RNA-mediated TLR7 activation in the lung antiviral and inflammatory responses in SARS-CoV-2-infected mice. We demonstrate that TLR7 deficiency led to a high rate of morbidity and mortality, which correlated with an impaired antiviral interferon (IFN)-I/III response, enhanced lung virus replication, and severe lung pathology. Furthermore, we show that blocking IFN-I signaling using anti-IFN receptor antibody promoted SARS-CoV-2 replication in the lungs and caused severe disease. These results provide conclusive evidence that TLR7 and IFN-I receptor deficiencies lead to severe disease in mice, replicating clinical features observed in COVID-19 patients.

Feline infectious peritonitis (FIP), caused by feline coronavirus (FCoV), is a fatal disease with no effective vaccine. Early detection is crucial for FIP management, and a rapid, accurate diagnostic method is urgently needed. Hence, the purpose of this study was to establish a rapid, sensitive, specific immunochromatographic strip (ICS) for clinical detection of FIP. We selected the highly conserved N protein of FIPV and expressed recombinant N protein as an immunogen to prepare monoclonal antibodies (mAbs). Five mAbs specific to FIPV were produced. The antigenic epitopes recognized by the 2B10 and 10E7 mAbs used for ICS preparation were identified, and the structure and conservation of the epitopes were analyzed. Subsequently, we paired the 2B10 and 10E7 mAbs, assembled the ICS, and implemented several optimization measures. The specificity of the ICS was confirmed by positive reactions with FIPV-positive samples and negative reactions with FHV, FPV, and FCV. Sensitivity testing detected FIPV suspensions (TCID₅₀ = 106.5/mL) diluted to 1: 512. The ICS showed 98.3 % agreement with RT-PCR results in detecting 60 suspected samples and remained stable for 6 months at room temperature. In conclusion, this study developed a simple, sensitive, and specific ICS for the detection of FIPV.

Recommendations on the use of genomics for pathogens surveillance are evidence that high-throughput genomic sequencing plays a key role to fight global health threats. Coupled with bioinformatics and other data types (e.g., epidemiological information), genomics is used to obtain knowledge on health pathogenic threats and insights on their evolution, to monitor pathogens spread, and to evaluate the effectiveness of countermeasures. From a decision-making policy perspective, it is essential to ensure the entire process's quality before relying on analysis results as evidence. Available workflows usually offer quality assessment tools that are primarily focused on the quality of raw NGS reads but often struggle to keep pace with new technologies and threats, and fail to provide a robust consensus on results, necessitating manual evaluation of multiple tool outputs.

We present PathoSeq-QC, a bioinformatics decision support workflow developed to improve the trustworthiness of genomic surveillance analyses and conclusions. Designed for SARS-CoV-2, it is suitable for any viral threat. In the specific case of SARS-CoV-2, PathoSeq-QC: (i) evaluates the quality of the raw data; (ii) assesses whether the analysed sample is composed by single or multiple lineages; (iii) produces robust variant calling results via multi-tool comparison; (iv) reports whether the produced data are in support of a recombinant virus, a novel or an already known lineage. The tool is modular, which will allow easy functionalities extension.

PathoSeq-QC is a command-line tool written in Python and R. The code is available at https://code.europa.eu/dighealth/pathoseq-qc.

Vaccines based on mRNA have been fundamental in facing the COVID-19 pandemic, however, they still raise concerns about stability and long-term efficacy. Thus, protein-based vaccines remain valid options and hence the study of effective adjuvants is crucial. Here, we developed a COVID-19 vaccine based on the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein, which is covalently conjugated to the natural polymer hyaluronan (HA) that acts as an immunological adjuvant. Vaccination of K18-hACE2 mice with HA-RBD was well tolerated, and elicited high and sustained titres of RBD-binding antibodies and SARS-CoV-2-neutralizing antibodies, without the addition of other immunostimulatory compounds. Most importantly, HA-RBD vaccination conferred long-term protection to K18-hACE2 mice after challenge with SARS-CoV-2, also in the case of two consequent infections driven by different variants. These findings demonstrate the efficacy of HA-based vaccination against COVID-19 disease, and support the promising use of HA as an efficient and well tolerated adjuvant.

The evolutionary adaptation of pathogens to biological materials has led to an upsurge in drug-resistant superbugs that significantly threaten public health. Treating most infections is an uphill task, especially those associated with multi-drug-resistant pathogens, biofilm formation, persister cells, and pathogens that have acquired robust colonization and immune evasion mechanisms. Innovative diagnostic solutions are crucial for identifying and understanding these pathogens, initiating efficient treatment regimens, and curtailing their spread. While next-generation sequencing has proven invaluable in diagnosis over the years, the most glaring drawbacks must be addressed quickly. Many promising pathogen-associated and host biomarkers hold promise, but their sensitivity and specificity remain questionable. The integration of CRISPR-Cas9 enrichment with nanopore sequencing shows promise in rapid bacterial diagnosis from blood samples. Moreover, machine learning and artificial intelligence are proving indispensable in diagnosing pathogens. However, despite renewed efforts from all quarters to improve diagnosis, accelerated bacterial diagnosis, especially in Africa, remains a mystery to this day. In this review, we discuss current and emerging diagnostic approaches, pinpointing the limitations and challenges associated with each technique and their potential to help address drug-resistant bacterial threats. We further critically delve into the need for accelerated diagnosis in low- and middle-income countries, which harbor more infectious disease threats. Overall, this review provides an up-to-date overview of the diagnostic approaches needed for a prompt response to imminent or possible bacterial infectious disease outbreaks.

The evolution of COVID-19 pandemic has been characterized by the rapid emergence of new SARS-CoV-2 variants, each of which poses unique challenges to public health. This study analyzes the dispersion profiles during the Pre-Omicron and Post-Omicron phases in different epidemiological contexts. The Brazilian state of Espirito Santo, despite its low population density, plays a critical role as a commercial hub due to its intense port activity, which may have contributed to COVID-19 cases and mortality rates being higher than the national average. The state recorded 34,000 confirmed cases and 377 deaths per 100,000 inhabitants. Genomic surveillance revealed that the Pre-Omicron phase was dominated by the B.1.1.33 lineage, characterized by localized intraregional circulation. In contrast, the Post-Omicron phase, dominated by the BQ.1.1 lineage, exhibited greater diversity in circulating lineages, increased international interactions, and rapid viral dissemination, highlighting distinct transmission dynamics between such periods. This study highlights the need for adaptive public health strategies that account for both viral behavior and regional socioeconomic factors, while highlighting the strategic importance of Espirito Santo in monitoring SARS-CoV-2 evolution.

DNBSEQ platforms have been widely used for variation detection, including single-nucleotide variants (SNVs) and short insertions and deletions (INDELs), which is comparable to Illumina. However, the performance and even characteristics of structural variations (SVs) detection using DNBSEQ platforms are still unclear.

In this study, we assessed the detection of SVs using 40 tools on eight DNBSEQ whole-genome sequencing (WGS) datasets and two Illumina WGS datasets of NA12878. Our findings confirmed that the performance of SVs detection using the same tool on DNBSEQ and Illumina datasets was highly consistent, with correlations greater than 0.80 on metrics of number, size, precision and sensitivity, respectively. Furthermore, we constructed a "DNBSEQ" SV set (4,785 SVs) from the DNBSEQ datasets and an "Illumina" SV set (6,797 SVs) from the Illumina datasets. We found that these two SV sets were highly consistent of SV sites and genomic characteristics, including repetitive regions, GC distribution, difficult-to-sequence regions, and gene features, indicating the robustness of our comparative analysis and highlights the value of both platforms in understanding the genomic context of SVs.

Our study systematically analyzed and characterized germline SVs detected on WGS datasets sequenced from DNBSEQ platforms, providing a benchmark resource for further studies of SVs using DNBSEQ platforms.

In recent years, the role of the intestinal microbiota in regulating host health and immune balance has attracted widespread attention. This study provides an in-depth analysis of the close relationship between the intestinal microbiota and respiratory system diseases, with a focus on three common respiratory virus infections, including respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and influenza virus. The research indicates that during RSV infection, there is a significant decrease in intestinal microbial diversity, suggesting the impact of the virus on the intestinal ecosystem. In SARS-CoV-2 infection, there are evident alterations in the intestinal microbiota, which are positively correlated with the severity of the disease. Similarly, influenza virus infection is associated with dysbiosis of the intestinal microbiota, and studies have shown that the application of specific probiotics exhibits beneficial effects against influenza virus infection. Further research indicates that the intestinal microbiota exerts a wide and profound impact on the occurrence and development of respiratory system diseases through various mechanisms, including modulation of the immune system and production of short-chain fatty acids (SCFAs). This article comprehensively analyzes these research advances, providing new perspectives and potential strategies for the prevention and treatment of future respiratory system diseases. This study not only deepens our understanding of the relationship between the intestinal microbiota and respiratory system diseases but also offers valuable insights for further exploring the role of host-microbiota interactions in the development of diseases.

The coronavirus disease 2019 (COVID-19) outbreak revealed the susceptibility of elderly patients to respiratory virus infections, showing cell senescence or subclinical persistent inflammatory profiles and favoring the development of severe pneumonia.

In our study, we evaluated the potential influence of lung aging on the efficiency of replication of influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as determining the pro-inflammatory and antiviral responses of the distal lung tissue.

Using precision-cut lung slices (PCLS) from donors of different ages, we found that pandemic H1N1 and avian H5N1 IAV replicated in the lung parenchyma with high efficacy. In contrast to these IAV strains, SARS-CoV-2 Early isolate and Delta variant of concern (VOC) replicated less efficiently in PCLS. Interestingly, both viruses showed reduced replication in PCLS from older compared to younger donors, suggesting that aged lung tissue represents a suboptimal environment for viral replication. Regardless of the age-dependent viral loads, PCLS responded to H5N1 IAV infection by an induction of IL-6 and IP10/CXCL10, both at the mRNA and protein levels, and to H1N1 IAV infection by induction of IP10/CXCL10 mRNA. Finally, while SARS-CoV-2 and H1N1 IAV infection were not causing detectable cell death, H5N1 IAV infection led to more cytotoxicity and induced significant early interferon responses.

In summary, our findings suggest that aged lung tissue might not favor viral dissemination, pointing to a determinant role of dysregulated immune mechanisms in the development of severe disease.

Medical treatments using potent neutralizing SARS-CoV-2 antibodies have achieved remarkable improvements in clinical symptoms, changing the situation for the severity of COVID-19 patients. We previously reported an antibody, NT-108 with potent neutralizing activity. However, the structural and functional basis for the neutralizing activity of NT-108 has not yet been understood. Here, we demonstrated the therapeutic effects of NT-108 in a hamster model and its protective effects at low doses. Furthermore, we determined the cryo-EM structure of NT-108 in complex with SARS-CoV-2 spike. The single-chain Fv construction of NT-108 improved the cryo-EM maps because of the prevention of preferred orientations induced by Fab orientation. The footprints of NT-108 illuminated how escape mutations such as E484K evade from class 2 antibody recognition without ACE2 affinity attenuation. The functional and structural basis for the potent neutralizing activity of NT-108 provides insights into the rational design of therapeutic antibodies.

Although many studies have associated changes in the nasopharyngeal microbiota to patient's susceptibility to COVID-19, their results are highly variable and contradictory. Addressing the limitations in previous research responsible for that variability, this study uses 16S rRNA gene sequencing to analyze the nasopharyngeal microbiota of 395 subjects, 117 controls, and 278 COVID-19 patients, of different age groups that cover the entire lifespan and across varying disease severities. This revealed that bacterial alpha diversity decreases progressively throughout life but only in severely ill COVID-19 patients, in whose nasopharynx, moreover, several opportunistic pathogen bacterial genera are overrepresented. Notably, Scardovia wiggsiae appears only in severe COVID-19 patients over 60 years of age, suggesting its potential utility as a COVID-19 severity biomarker in the elderly, who are the most susceptible individuals to suffer from serious forms of the disease. Thus, our results provide valuable insights into age-associated dynamics within nasopharyngeal microbiota during severe COVID-19.

DPP4 was considered a canonical receptor for merbecoviruses until the recent discovery of African bat-borne MERS-related coronaviruses using ACE2. The extent and diversity of ACE2 utilization among merbecoviruses and their receptor species tropism remain unknown. Here, we reveal that HKU5 enters host cells utilizing Pipistrellus abramus (P.abr) and several non-bat mammalian ACE2s through a binding mode distinct from that of any other known ACE2-using coronaviruses. We defined the molecular determinants of receptor species tropism and identified a single amino acid mutation enabling HKU5 to utilize human ACE2, providing proof of principle for machine-learning-assisted outbreak preparedness. We show that MERS-CoV and HKU5 have markedly distinct antigenicity and identified several HKU5 inhibitors, including two clinical compounds. Our findings profoundly alter our understanding of coronavirus evolution, as several merbecovirus clades independently evolved ACE2 utilization, and pave the way for developing countermeasures against viruses poised for human emergence.

Merbecoviruses comprise four viral species with remarkable genetic diversity: MERS-related coronavirus, Tylonycteris bat coronavirus HKU4, Pipistrellus bat coronavirus HKU5, and Hedgehog coronavirus 1. However, the potential human spillover risk of animal merbecoviruses remains to be investigated. Here, we reported the discovery of HKU5-CoV lineage 2 (HKU5-CoV-2) in bats that efficiently utilize human angiotensin-converting enzyme 2 (ACE2) as a functional receptor and exhibits a broad host tropism. Cryo-EM analysis of HKU5-CoV-2 receptor-binding domain (RBD) and human ACE2 complex revealed an entirely distinct binding mode compared with other ACE2-utilizing merbecoviruses with RBD footprint largely shared with ACE2-using sarbecoviruses and NL63. Structural and functional analyses indicate that HKU5-CoV-2 has a better adaptation to human ACE2 than lineage 1 HKU5-CoV. Authentic HKU5-CoV-2 infected human ACE2-expressing cell lines and human respiratory and enteric organoids. This study reveals a distinct lineage of HKU5-CoVs in bats that efficiently use human ACE2 and underscores their potential zoonotic risk.