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.

The constant mutation of SARS-CoV-2 has led to the continuous appearance of viral variants and their pandemics and has improved the development of vaccines with a broad spectrum of antigens to curb the spread of the virus. The work described here suggested a novel vaccine with a virus-like structure (VLS) composed of combined mRNA and protein that is capable of stimulating the immune system in a manner similar to that of viral infection. This VLS vaccine is characterized by its ability to specifically target dendritic cells and/or macrophages through S1 protein recognition of the DC-SIGN receptor in cells, which leads to direct mRNA delivery to these innate immune cells for activation of robust immunity with a broad spectrum of neutralizing antibodies and immune protective capacity against variants. Research on its composition characteristics and structural features has suggested its druggability. Compared with the current mRNA vaccine, the VLS vaccine was identified as having no cytotoxicity at its effective application dosage, while the results of safety observations in animals revealed fewer adverse reactions during immunization.

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone significant genomic mutations, contributing to resistance against existing 2019 coronavirus disease (COVID-19) treatments. In a previous study, we identified N-0385, a potent host-directed inhibitor of transmembrane serine protease 2 (TMPRSS2), which has therapeutic efficacy towards SARS-CoV-2 infection. However, in further evaluation of its preclinical druggability, N-0385 displayed unfavorable pharmacokinetic properties, including high bioavailability (99 %) following intranasal (IN) administration. This can lead to substantial systemic exposure and potential adverse effects due to off-target interactions. Here, we designed a library of peptidomimetic compounds with P3 site modifications on an optimized scaffold. We sought to maintain sub-nanomolar potency against TMPRSS2 (Kis < 2 nM), reduce pseudovirus infection, while addressing the lack of selectivity and excessive lung uptake. Notably, inhibitor 9, which contains Asp at the P3 position, achieved a two-fold increase in TMPRSS2 inhibitory potency (Ki = 0.13 ± 0.03 nM), a >700-fold selectivity over Factor Xa (FXa), and showed superior selectivity against other proteases (matriptase, transmembrane serine protease 6 (TMPRSS6), thrombin, furin, and tPA). Despite concerns about the role of FXa in the coagulation cascade, compound 9 had no impact on coagulation or thrombolysis 2 h after in vitro treatment. In the air-liquid interface (ALI) model of the lung epithelium, compound 9 displayed a 1.5-fold decrease in permeability compared to N-0385 and demonstrated sustained stability in lungs (11 h) and plasma (13 h). Taken together, our data demonstrate that continued optimization of this type of inhibitors will lead to improved therapeutics for the treatment of SARS-CoV-2 infection by IN administration.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiated a global pandemic resulting in an estimated 775 million infections with over 7 million deaths, it has become evident that COVID-19 is not solely a pulmonary disease. Emerging evidence has shown that, in a subset of patients, certain symptoms - including chest pain, stroke, anosmia, dysgeusia, diarrhea and abdominal pain - all indicate a role of vascular, neurological and gastrointestinal (GI) pathology in the disease process. Many of these disease processes persist long after the acute disease has been resolved, resulting in 'long COVID' or post-acute sequelae of COVID-19 (PASC). The molecular mechanisms underlying the acute and systemic conditions associated with COVID-19 remain incompletely defined. Appropriate animal models provide a method of understanding underlying disease mechanisms at the system level through the study of disease progression, tissue pathology, immune system response to the pathogen and behavioral responses. However, very few studies have addressed PASC and whether existing models hold promise for studying this challenging problem. Here, we review the current literature on cardiovascular, neurological and GI pathobiology caused by COVID-19 in patients, along with established animal models of the acute disease manifestations and their prospects for use in PASC studies. Our aim is to provide guidance for the selection of appropriate models in order to recapitulate certain aspects of the disease to enhance the translatability of mechanistic studies.

Glucose-regulated protein 94 (Grp94/gp96) is endoplasmic reticulum (ER) resident form of the 90 kDa heat shock protein 90 (Hsp90) that is responsible for folding, maturation and stabilization of more than 400 client proteins. Grp94 has been implicated for various diseases including metastatic cancer, primary open-angle glaucoma, and infectious diseases. In fact, Grp94 plays critical roles in different stages of viral infection cycle. It chaperones receptor proteins and viral glycoproteins that are necessary for viral entry and replication. Beyond its role in protein homeostasis, Grp94 modulates host cellular processes such as apoptosis and immune responses, which are often exploited by viruses to sustain infection. This work provides an overview of the roles of Grp94 in viral pathogenesis across various viruses and its involvement in immune modulation with the development of Grp94-selective inhibitors and their potential as anti-viral therapeutics.

The global pandemic of SARS-CoV-2 has highlighted the necessity for innovative therapeutic solutions. This research presents a new formulation utilising the metal-organic framework MIL-101(Al)-NH2, which is loaded with hypericin, aimed at addressing viral and bacterial challenges. Hypericin, recognised for its antiviral and antibacterial efficacy, was encapsulated to mitigate its hydrophobicity, improve bioavailability, and utilise its photodynamic characteristics. The MIL-101(Al)-NH2 Hyp complex was synthesised, characterised, and evaluated for its biological applications for the first time. The main objective of this study was to demonstrate the multimodal potential of such a construct, in particular the effect on SARS-CoV-2 protein levels and its interaction with cells. Both in vitro and in vivo experiments demonstrated the effective transport of hypericin to cells that express ACE2 receptors, thereby mimicking mechanisms of viral entry. In addition, hypericin found in the mitochondria showed selective phototoxicity when activated by light, leading to a decrease in the metabolic activity of glioblastoma cells. Importantly, the complex also showed antibacterial efficacy by selectively targeting Gram-positive Staphylococcus epidermidis compared to Gram-negative Escherichia coli under photodynamic therapy (PDT) conditions. To our knowledge, this study was the first to demonstrate the interaction between hypericin, MIL-101(Al)-NH2 and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which inhibits cellular uptake and colocalises with ACE2-expressing cells. Therefore, the dual functionality of the complex - targeting the viral RBD and the antibacterial effect via PDT - emphasises its potential to mitigate complications of viral infections, such as secondary bacterial infections. In summary, these results suggest that MIL-101(Al)-NH2 Hyp is a promising multifunctional therapeutic agent for antiviral and antibacterial applications, potentially contributing to the improvement of COVID-19 treatment protocols and the treatment of co-infections.

The rapid global spread of SARS-CoV-2 has demanded innovative approaches to treatment and prevention. This article reviews the current landscape of COVID-19 therapeutics and vaccines, emphasizing the role of biotechnological products, particularly lectins and protease inhibitors. SARS-CoV-2, a single-stranded RNA virus, infects host cells via its spike (S) protein, which binds to the angiotensin-converting enzyme 2 (ACE2) receptor. This interaction is facilitated by host proteases like TMPRSS2, which are critical for viral entry. Treatments for COVID-19 primarily focus on antiviral drugs, anti-inflammatory agents, and monoclonal antibodies. Protease inhibitors that target viral enzymes like Mpro and PLpro have demonstrated potential. Additionally, vaccines, including mRNA-based, DNA-based, and those using viral vectors or inactivated viruses, are essential for preventing new infections. Lectins, proteins that bind specifically to carbohydrates, have emerged as potential antiviral agents. They can impede viral entry by binding to glycoproteins on the virus's surface or modulate immune responses. Studies indicate that lectins like cyanovirin-N and griffithsin exhibit significant antiviral activity against SARS-CoV-2. While most of the research on these biotechnological products is still in preclinical or early stages, their potential for treating and preventing COVID-19 is substantial. Further investigation and clinical trials are crucial to validate their efficacy and safety. This article underscores the need for continued exploration of novel therapeutic strategies to combat the evolving COVID-19 pandemic. However, the review is limited by the scarcity of clinical data on these products, highlighting the need for translational research.

Angiotensin-converting enzyme 2 (ACE2) was discovered 25 years ago as a negative regulator of the renin-angiotensin system, opposing the effects of angiotensin II. Beyond its well-demonstrated roles in cardiovascular regulation and COVID-19 pathology, ACE2 is involved in a plethora of physiopathological processes. In this review, we summarize the latest discoveries on the role of ACE2 in glucose homeostasis and regulation of metabolism. In the endocrine pancreas, ACE2 is expressed at low levels in β-cells, but loss of its expression inhibits glucose-stimulated insulin secretion and impairs glucose tolerance. Conversely, overexpression of ACE2 improved glycemia, suggesting that recombinant ACE2 might be a future therapy for diabetes. In the skeletal muscle of ACE2-deficient mice a progressive triglyceride accumulation was observed, whereas in diabetic kidney the initial increase in ACE2 is followed by a chronic reduction of expression in kidney tubules and impairment of glucose metabolism. At the intestinal level dysregulation of the enzyme alters the amino acid absorption and intestinal microbiome, whereas at the hepatic level ACE2 protects against diabetic fatty liver disease. Not least, ACE2 is upregulated in adipocytes in response to nutritional stimuli, and administration of recombinant ACE2 decreased body weight and increased thermogenesis. In addition to tissue-specific regulation of ACE2 function, the enzyme undergoes complex cellular posttranslational modifications that are changed during diabetes evolution, with at least proteolytic cleavage and ubiquitination leading to modifications in ACE2 activity. Detailed characterization of ACE2 in a cellular and tissue-specific manner holds promise for improving therapeutic outcomes in diabetes and metabolic disorders.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a devastating global pandemic for 4 years and is now an endemic disease. With the emergence of new viral variants, COVID-19 is a continuing threat to public health despite the wide availability of vaccines. The virus-encoded trimeric spike protein (S protein) mediates SARS-CoV-2 entry into host cells and also induces strong immune responses, making it an important target for development of therapeutics and vaccines. In this Review, we summarize our latest understanding of the structure and function of the SARS-CoV-2 S protein, the molecular mechanism of viral entry and the emergence of new variants, and we discuss their implications for development of S protein-related intervention strategies.

To address the human ACE2 dependence for SARS-CoV-2 infection, this study presents a novel strategy for generating ZIKV-hACE2 single-round infectious particles (SRIPs) by incorporating the hACE2 gene into a Zika virus (ZIKV) mini-replicon. SARS-CoV-2 SRIP infection was significantly enhanced in HEK293T cells pre-infected with ZIKV-hACE2, as evidenced by increased cytopathic effects and elevated mRNA and protein levels of the SARS-CoV-2 nucleocapsid (N) protein. A mouse model was also developed with this approach to investigate SARS-CoV-2 infection. Immunohistochemical and real-time RT-PCR analyses confirmed the presence of the SARS-CoV-2 N protein in the lungs of mice injected with ZIKV-hACE2 SRIPs, indicating successful infection. The mouse model displayed COVID-19-like pathological changes, including increased macrophages in BALF, severe lung damage, and elevated pro-inflammatory cytokines (IL-6 and IL-1β). These features mimic severe COVID-19 cases in humans. Additionally, treatment with nirmatrelvir resulted in a 6.2-fold reduction in viral load and a marked decrease in N protein levels. Overall, this ZIKV mini-replicon-mediated hACE2 expression model, both in vitro and in vivo, is a valuable tool for studying SARS-CoV-2 infection and evaluating therapeutic interventions. The mouse model's pathological features further underscore its relevance for in vivo research on SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) global health crisis, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented unprecedented challenges to global healthcare systems, leading to rapid advances in treatment development. This review comprehensively examines the current therapeutic approaches for managing COVID-19, including direct-acting antivirals, immunomodulators, anticoagulants, and adjuvant therapies, as well as emerging and experimental approaches. Direct-acting antivirals target various stages of the viral life cycle, offering specific intervention points, while immunomodulators aim to modulate the host's immune response, reducing disease severity. Anticoagulant therapies address the coagulopathy frequently observed in severe cases, and adjuvant treatments provide supportive care to improve overall outcomes. We also explore the challenges and limitations of implementing these treatments, such as drug resistance, variable patient responses, and access to therapies, especially in resource-limited settings. The review also discusses future perspectives, including the potential of next-generation vaccines, personalized medicine, and global collaboration in shaping future COVID-19 treatment paradigms. Continuous innovation, combined with an integrated and adaptable approach, will be crucial to effectively managing COVID-19 and mitigating the impact of future pandemics.

Angiotensin-converting enzyme 2 (ACE2) receptor plays a pivotal role in the infection of several coronaviruses, including SARS-CoV and SARS-CoV-2. We combined computational and experimental protein engineering approaches to develop ACE2-YHA, a soluble, high-affinity ACE2 decoy with pan-coronavirus preventive and therapeutic potential. Leveraging native human ACE2-SARS-CoV/SARS-CoV-2 receptor binding domain (RBD) complex homology models, we employed in silico site-saturation mutagenesis to predict key ACE2-RBD interacting residues. Subsequent generation of ACE2 mutants and high-throughput screening identified specific ACE2 residue substitutions that enhanced binding to both SARS-CoV and SARS-CoV-2 RBDs. The triple mutant ACE2-YHA demonstrated significantly enhanced binding affinity to SARS-CoV, SARS-CoV-2, and bat SARSr-CoVs' RBDs. It effectively neutralized SARS-CoV and numerous SARS-CoV-2 variants with picomolar IC50s in pseudotyped virus assays. Notably, ACE2-YHA displayed potent neutralization against major variants of concern, including Delta and Omicron, in human airway epithelia, positioning it as a promising universal decoy for current and future ACE2-binding coronavirus outbreaks.

The COVID-19 pandemic is caused by the enveloped virus SARS-CoV-2. Despite extensive investigation, the molecular mechanisms for its assembly and secretion remain largely elusive. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. SARS-CoV-2 virions are enriched in the fragmented Golgi. Blocking endoplasmic reticulum (ER) to Golgi trafficking dramatically inhibits SARS-CoV-2 assembly and secretion without reducing viral genome replication. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 protein levels. Surprisingly, GRASP55 expression reduces both viral secretion and spike number on each virion without affecting viral entry, while GRASP55 depletion displays opposite effects. In contrast, TGN46 depletion only inhibits viral secretion without affecting spike incorporation into virions. Taken together, we show that SARS-CoV-2 alters Golgi structure and function to modulate viral assembly and secretion, highlighting the Golgi as a potential therapeutic target for blocking SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein has the ability to induce multinucleated syncytia via cell-cell fusion, which is thought to be related to the pathogenesis of the coronavirus disease 2019 (COVID-19). However, the mechanism by which spike protein regulates cell fusion remains unclear. Given the close correlation between cell-cell fusion and membrane protrusions, we investigated the role of membrane-proximal actin regulators in spike-induced cell fusion. We found that while Rac-Arp2/3 dependent branched actin polymerization is required for spike-mediated cell fusion, RhoA dependent actomyosin contractility has an inhibitory effect on fusion. In addition, plasma membrane tension regulated by membrane-cortex attachment plays a negative role in spike-dependent cell fusion. Furthermore, we identified several BAR proteins, which couple membrane curvature with actin dynamics, involved in spike-induced syncytia formation. Our study suggests that these actin regulators could be promising targets for inhibiting SARS-CoV-2 spike protein-induced syncytia formation.

Coronavirus entry into host cells enables the virus to initiate its replication cycle efficiently while evading host immune response. Cell entry is intricately associated with pH levels in the cytoplasm or endosomes. In this study, we observed that the sodium hydrogen exchanger 3 (Na+/H+ exchanger 3 or NHE3), which is strongly activated by dexamethasone (Dex) to promote cell membrane Na+/H+ exchange, was critical for cytoplasmic and endosomal acidification. Dex activates NHE3, which increases intracellular pH and blocks the initiation of coronavirus infectious bronchitis virus (IBV) negative-stranded genomic RNA synthesis. Also, Dex antiviral effects are relieved by the glucocorticoid receptor (GR) antagonist RU486 and the NHE3 selective inhibitor tenapanor. These results show that Dex antiviral effects depend on GR and NHE3 activities. Furthermore, Dex exhibits remarkable dose-dependent inhibition of IBV replication, although its antiviral effects are constrained by specific virus and cell types. To our knowledge, this is the first report to show that Dex helps suppress the entry of coronavirus IBV into cells by promoting proton leak pathways, as well as by precisely tuning luminal pH levels mediated by NHE3. Disrupted cytoplasmic pH homeostasis, triggered by Dex and NHE3, plays a crucial role in impeding coronavirus IBV replication. Therefore, cytoplasmic pH plays an essential role during IBV cell entry, probably assisting viruses at the fusion and/or uncoating stages. The strategic modulation of NHE3 activity to regulate intracellular pH could provide a compelling mechanism when developing potent anti-coronavirus drugs.IMPORTANCESince the outbreak of coronavirus disease 2019, dexamethasone (Dex) has been proven to be the first drug that can reduce the mortality rate of coronavirus patients to a certain extent, but its antiviral effect is limited and its underlying mechanism has not been fully clarified. Here, we comprehensively evaluated the effect of Dex on coronavirus infectious bronchitis virus (IBV) replication and found that the antiviral effect of Dex is achieved by regulating sodium hydrogen exchanger 3 (NHE3) activity through the influence of glucocorticoid receptor on cytoplasmic pH or endosome pH. Dex activates NHE3, leading to an increase in intracellular pH and blocking the initiation of negative-stranded genomic RNA synthesis of coronavirus IBV. In this study, we identified the mechanism by which glucocorticoids counteract coronaviruses in cell models, laying the foundation for the development of novel antiviral drugs.

Porcine epidemic diarrhea virus (PEDV) is a major coronavirus in swine, causing substantial economic losses in the industry. To deepen our understanding of the PEDV-host cell interactions, we performed whole-genome CRISPR/Cas9 screens on porcine IPEC-J2 and IPI-2I cell lines to identify key host factors essential for PEDV infection. Our study identified the Yip family 5 (YIPF5) protein as a critical host factor, where its knockout suppressed PEDV infection by specifically affecting the virus replication stage. YIPF5 interacts with viral non-structural protein (nsp) 3, 4, and 6, facilitating the formation of double-membrane vesicles (DMVs), essential for replication organelle biogenesis. The knockout of YIPF5 interferes with the interaction between nsp3 and nsp4, consequently impacting the formation of DMVs mediated by these proteins. These findings establish YIPF5 as a key host factor involved in DMV formation during PEDV infection, highlighting its potential as a therapeutic target.

Coronaviruses pose serious health threats to both humans and animals. Identifying host genes critical for porcine epidemic diarrhea virus (PEDV) infection can uncover new therapeutic targets and enhance our understanding of coronavirus pathogenesis. In this study, we conducted genome-scale CRISPR/Cas9 screens in two porcine cell lines (IPEC-J2 and IPI-2I) and identified YIPF5 as an essential host factor for PEDV replication. Our results demonstrate that YIPF5 plays a pivotal role in the formation of PEDV-induced double-membrane vesicles (DMVs), which are crucial for viral replication. These findings shed new light on the molecular mechanisms of PEDV and suggest YIPF5 as a therapeutic target.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor to enter host cells, and primary receptor recognition of the spike protein is a major determinant of the host range of SARS-CoV-2. Since the emergence of SARS-CoV-2, a considerable number of variants have emerged. However, the determinants of host tropism of SARS-CoV-2 remain elusive. We conducted infection assays with chimeric recombinant SARS-CoV-2 carrying the spike protein from 10 viral variants, assessing their entry efficiency using mammalian ACE2 orthologs from species that have close contact with humans. We found that only murine ACE2 exhibited different susceptibilities to infection with the SARS-CoV-2 variants. Moreover, we revealed that the mutation N501Y in the viral spike protein has a crucial role in determining the infectivity of cells expressing murine ACE2 and of mice in vivo. Next, we identified six amino acid substitutions at 24, 30, 31, 82, 83, and 353 in murine ACE2 that allowed for viral entry of the variants to which murine ACE2 was previously resistant. Furthermore, we showed that ACE2 from a species closely related to mice, Mus caroli, is capable of supporting entry of the viral variants that could not use murine ACE2. These results suggest that few ACE2 orthologs have different susceptibility to infection with SARS-CoV-2 variants as observed for murine ACE2. Collectively, our study reveals critical amino acids in ACE2 and the SARS-CoV-2 spike protein that are involved in the host tropism of SARS-CoV-2, shedding light on interspecies susceptibility to infection.IMPORTANCESARS-CoV-2 can infect many species besides humans, leading to the evolution of the virus and adaptation to other animal hosts, which could trigger a new COVID-19 wave. The SARS-CoV-2 spike protein utilizes ACE2 as a receptor for entry into host cells. The interaction of ACE2 with the spike protein determines the host range of SARS-CoV-2. In this study, using chimeric viruses carrying the spike protein of SARS-CoV-2 variants to infect cells expressing different ACE2 orthologs from species humans come in close contact with, we confirmed murine ACE2 alone showed different susceptibility to the variants. We identified residues in murine ACE2 and the viral spike that restrict viral entry. Furthermore, an ACE2 ortholog from a species genetically close to mice mediated entry of SARS-CoV-2 variants incapable of infecting mice. This research highlights the uniquely limited susceptibility of mice to different SARS-CoV-2 variants and provides invaluable insights into the host tropism of SARS-CoV-2.

Betacoronaviruses, which have caused three human outbreaks within the last two decades, are thought to originate from bats, raising the concern that bat coronaviruses could cause a novel human outbreak in the future. To determine whether the bat merbecovirus EjCoV-3 strain, previously detected in Eptesicus japonensis in Japan, has the potential to infect humans, we analyzed its cellular entry mechanism. Cellular entry of EjCoV-3 via the spike protein requires protease treatment and is mediated by an unknown receptor, other than DPP4 or ACE2. We generated cultivable recombinant EjCoV-3 using bacterial artificial chromosome-based reverse genetics and found that it efficiently replicated in human respiratory and intestinal cell cultures as well as nasal ciliated epithelium in hamsters. These findings suggest that bat merbecovirus with ACE2- and DPP4-independent cell entry has the potential to cause human infections, highlighting the importance of extensive bat surveillance for pandemic preparedness.

Betacoronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused three significant outbreaks in the past two decades and are believed to have originated from bats. To investigate the potential for future outbreaks, we generated a Japanese bat-derived MERS-related coronavirus, designated EjCoV-3, using reverse genetics. Our results showed that EjCoV-3 does not utilize ACE2 and DPP4, cell entry receptors for SARS-CoV and MERS-CoV, as a means of infection. However, we found that EjCoV-3 is the first bat merbecovirus capable of efficiently replicating in human respiratory cells and the respiratory tract of hamsters. These findings provide new insight into the potential for MERS-related coronaviruses that do not use ACE2 and DPP4 to infect the human respiratory tract, highlighting the importance of preparedness for outbreaks caused by these viruses.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that poses a substantial threat to the global swine industry. However, our current understanding of the host factors crucial for PEDV infection remains limited. To identify these host factors, we conducted a genome-wide CRISPR/Cas9 gene knockout screen using a PEDV-permissive cell line. Our results indicate that the endogenous expression of human interferon-inducible transmembrane protein 3 (IFITM3) enhances PEDV entry and replication. Silencing or eliminating endogenous IFITM3 in Huh7 cells significantly suppressed PEDV entry, whereas reintroducing IFITM3 partially restored susceptibility to PEDV. Overexpression of human IFITM3 or IFITM2, but not IFITM1, in Huh7.5 cells substantially increased PEDV entry and replication. Importantly, our results suggest that human IFITM3 influences PEDV entry at a later stage. Furthermore, the overexpression of porcine IFITM1 significantly enhanced PEDV infection in LLC-PK1 cells, whereas the overexpression of porcine IFITM2/3 did not produce similar effects. Notably, removing the C-terminal 15 amino acids of porcine IFITM2/3 resulted in increased PEDV entry. Coimmunoprecipitation analyses showed that all IFITMs interacted with the PEDV S1 protein, indicating a direct role in the viral entry process. Additionally, porcine IFITM1 colocalized with the PEDV S protein at the cell nuclear periphery and enhanced PEDV infection in porcine small intestinal organoids. Overall, our results suggest that IFITMs are critical in facilitating PEDV entry into cells. Targeting IFITMs may provide a promising strategy for controlling PEDV transmission and developing interventions to mitigate the virus's impact on the swine industry.

Understanding the mechanisms underlying porcine epidemic diarrhea virus (PEDV) infection is vital for addressing its significant impact on the swine industry. This study reveals that interferon-inducible transmembrane (IFITM) proteins, particularly human IFITM3 and porcine IFITM1, play crucial roles in facilitating PEDV entry and replication. By elucidating these molecular interactions, the research highlights the potential of IFITMs as therapeutic targets for managing PEDV infections and paves the way for antiviral strategies. Moreover, this research extends beyond PEDV management, underscoring the critical role of host factors in controlling the spread of pathogenic coronaviruses.

SARS-CoV-2, the third major coronavirus of the 21st century, causing COVID-19 disease, profoundly impacts public health and workforces worldwide. Identifying individuals at heightened risk of SARS-CoV-2 infection is crucial for targeted interventions and preparedness. This study investigated 35 SNVs within viral infection-associated genes in SARS-CoV-2 patients and uninfected controls from the Basque Country (March 2020-July 2021). Its primary aim was to uncover genetic markers indicative of SARS-CoV-2 susceptibility and explore genetic predispositions to infection. Association analyses revealed previously unreported associations between SNVs and susceptibility. Haplotype analyses uncovered novel links between haplotypes and susceptibility, surpassing individual SNV associations. Descriptive modelling identified key susceptibility factors, with rs11246068-CC (IFITM3), rs5742933-GG (ORMDL1), rs35337543-CG (IFIH1), and GGGCT (rs2070788, rs2298659, rs17854725, rs12329760, rs3787950) variation in TMPRSS2 emerging as main infection-susceptibility indicators for a COVID-19 pandemic situation. These findings underscore the importance of integrated SNV and haplotype analyses in delineating susceptibility to SARS-CoV-2 and informing proactive prevention strategies. The genetic markers profiled in this study offer valuable insights for future pandemic preparedness.

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, most of these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased toward the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published functional screens and proteomics data to reveal (i) common pathways that were identified in all OMICs datasets-including regulation of Wnt signaling and gap junctions, (ii) pathways uniquely identified in this screen-including NADH oxidation, or (iii) pathways supported by this screen and proteomics data but not published functional screens-including arachionate production and MAPK signaling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 32 proteins that were determined to impact viral replication and 27 impacting late stages of infection, respectively. Additionally, a subset of proteins was tested across other coronaviruses revealing a subset of proviral factors that were conserved across pandemic SARS-CoV-2, epidemic SARS-CoV-1 and MERS-CoV, and the seasonal coronavirus OC43-CoV. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

The global COVID-19 pandemic, caused by SARS-CoV-2, has led to significant mortality, with over 6.5 million deaths worldwide. While vaccines have played a crucial role in reducing disease severity, the emergence of viral variants continues to undermine vaccine efficacy, highlighting the need for alternative antiviral strategies. This review explores the potential of RNA interference (RNAi), particularly small interfering RNAs (siRNAs), as a targeted therapeutic approach against SARS-CoV-2. siRNAs can silence specific viral genes with high precision, effectively inhibiting viral replication. We discuss the design and mechanism of siRNAs, their primary molecular targets such as the spike (S), membrane (M), and RNA-dependent RNA polymerase (RdRp) genes and summarize past and current research findings. Special emphasis is placed on delivery systems, especially lung-targeted strategies essential for respiratory infections. We also evaluate how siRNA therapeutics can overcome challenges posed by viral mutations and treatment resistance. The novelty of this work lies in its focused comparison of siRNAs with other non-coding RNAs and its integration of computational tools for siRNA design. This review presents a strategic overview of siRNA development and highlights its translational potential for the current pandemic and future coronavirus outbreaks.

Myocardial injury is a critical determinant of prognosis in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, its underlying mechanisms remain incompletely understood. In this study, we examined the effects of SARS-CoV-2-derived RNA fragments on human cardiomyocytes. We identified a 19-nucleotide sequence within the viral genome that shares complete sequence homology with the human F1F0 ATP synthase subunit alpha gene (ATP5A). This sequence was found to associate with Argonaute 2 (AGO2) and downregulate ATP5A expression via a mechanism analogous to RNA interference. Consequently, oxidative phosphorylation was suppressed in cardiomyocytes, leading to impaired myocardial maturation and the emergence of heart failure-like phenotypes. Notably, exosome-mimetic liposomal delivery of this RNA fragment to cardiomyocytes reproduced the ATP5A-suppressive effect. These findings suggest that SARS-CoV-2-derived RNA fragments may contribute to myocardial injury through the siRNA-like modulation of mitochondrial gene expression. Further validation in animal models and patient-derived materials is warranted.

SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM reveals stable prefusion and postfusion conformations of the spike, the transient fusion intermediate states during the fusion process remain poorly understood. Here, we design a near-native viral fusion system that recapitulates SARS-CoV-2 entry and use cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that depends on protease cleavage and is inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2' cleavage concurrently transition to postfusion conformations encircling the hemifusion and initial fusion pores in a distinct conical arrangement. S2' cleavage is indispensable for advancing fusion intermediates to the fully folded postfusion state, culminating in membrane integration. Subtomogram averaging reveals that the WS6 S2 antibody binds to the spike's stem-helix, crosslinks and clusters prefusion spikes, as well as inhibits refolding of fusion intermediates. These findings elucidate the entire process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.

D-xylofuranosyl nucleoside analogues bearing alkylthio and glucosylthio substituents at the C3'-position were prepared by photoinitiated radical-mediated hydrothiolation reactions from the corresponding 2',5'-di-O-silyl-3'-exomethylene uridine. Sequential desilylation and 5'-O-butyrylation of the 3'-thiosubstituted molecules produced a 24-membered nucleoside series with diverse substitution patterns, and the compounds were evaluated for their in vitro antiviral activity against three dangerous human RNA viruses, SARS-CoV-2, SINV and CHIKV. Eight compounds exhibited SARS-CoV-2 activity with low micromolar EC50 values in Vero E6 cells, and two of them also inhibited virus growth in human Calu cells. The best anti-SARS-CoV-2 activity was exhibited by 2',5'-di-O-silylated 3'-C-alkylthio nucleosides. Twelve compounds showed in vitro antiviral activity against CHIKV and fourteen against SINV with low micromolar EC50 values, with the 5'-butyryl-2'-silyl-3'-alkylthio substitution pattern being the most favorable against both viruses. In the case of the tested nucleosides, removal of the 2'-O-silyl group completely abolished the antiviral activity of the compounds against all three viruses. Overall, the most potent antiviral agent was the disilylated 3'-glucosylthio xylonucleoside, which showed excellent and specific antiviral activity against SINV with an EC50 value of 3 μM and no toxic effect at the highest tested concentration of 120 μM.

SARS-CoV-2 infections directly or indirectly cause unconscionable vascular events, significantly increasing the morbidity and mortality of COVID-19. Biomarkers associated with cardiac injury often elevate in individuals with COVID-19. Cytokine storm is a mechanism underlying myocardial cell injury caused by SARS-CoV-2 infections. Cell apoptosis in AC16 cells overexpressing structural and helper proteins of SARS-CoV-2 was detected by Western blot, MTT and TUNEL assay. The M protein was determined to play the most pronounced role in inducing apoptosis. Transcriptome sequencing on AC16 cells overexpressing the M protein was performed to screen differentially expressed genes (DEGs), which were further subjected to the gene set enrichment analysis. The regulatory effect of CXCL10 on cell apoptosis of AC16 cells overexpressing M protein was finally explored. Overexpression of M protein significantly increased the Bax/Bcl-2 and cleaved caspase-3/caspase-3(CC3/C3) ratios and the percentage of TUNEL-positive cells in AC16 cells, while markedly reducing cell viability. CXCL10 was the most prominent DEG in AC16 cells overexpressing M protein. Knockdown of CXCL10 partially reversed the increases in the Bax/Bcl-2 and cleaved caspase-3/caspase-3 ratios, the percentage of TUNEL-positive cells, as well as the release of pro-inflammatory cytokines in AC16 cells overexpressing M protein. The M protein of SARS-CoV-2 triggers the production of CXCL10 and apoptosis of myocardial cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is an enveloped RNA virus that caused the Coronavirus Disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike glycoprotein binds to angiotensin converting enzyme 2 (ACE2) on host cells to facilitate viral entry. However, the presence of SARS-CoV-2 in nearly all human organs - including those with little or no ACE2 expression - suggests the involvement of alternative receptors. Recent studies have identified several cellular proteins and molecules that influence SARS-CoV-2 entry through ACE2-dependent, ACE2-independent, or inhibitory mechanisms. In this review, we explore how these alternative receptors were identified, their expression patterns and roles in viral entry, and their impact on SARS-CoV-2 infection. Additionally, we discuss therapeutic strategies aimed at disrupting these virus-receptor interactions to mitigate COVID-19 pathogenesis.

Vaccination or infection by SARS-CoV-2 elicits a protective immune response against severe outcomes. It has been reported that SARS-CoV-2 infection or vaccination elicits cross-reactive antibodies against other betacoronaviruses. While plasma neutralizing capacity was studied in great detail, their Fc-effector functions remain understudied. Here, we analyzed Spike recognition, neutralization and antibody-dependent cellular cytotoxicity (ADCC) against D614G, a recent Omicron subvariant of SARS-CoV-2 (JN.1) and SARS-CoV-1. Plasma from individuals before their first dose of mRNA vaccine, and following their second, third and sixth doses were analyzed. Despite poor neutralization activity observed after the second and third vaccine doses, ADCC was readily detected. By the sixth dose, individuals could neutralize and mediate ADCC against JN.1 and SARS-CoV-1. Since previous reports have shown that Fc-effector functions were associated with survival from acute infection, these results suggest that ADCC could help in combating future SARS-CoV-2 variants as well as closely related coronaviruses.

Human body constitutes unique biological system containing specific fluid mechanics and biomechanics. Traditional cell culture techniques of 2D and 3D do not recapitulate these specific natures of the human system. In addition, they lack the spatiotemporal conditions of representing the cells. Moreover, they do not enable the study of cell-cell interactions in multiple cell culture platforms. Therefore, establishing biological system of dynamic cell culture was of great interest. Organs on chips systems were fabricated proving their concept to mimic specific organs functions. Therefore, it paves the way for validating new drugs and establishes mechanisms of emerging diseases. It has played a key role in validating suitable vaccines for Coronavirus disease (COVID-19). Herein, the concept of organs on chips, fabrication methodology and their applications are discussed.

Infection by the recent SARS-CoV-2 virus causes the COVID-19 disease with variable clinical manifestations ranging from asymptomatic or mild respiratory symptoms to severe respiratory distress and multiorgan failure. The renin-angiotensin system, responsible for maintaining homeostasis and governing several critical processes, has been considered the main system involved in the pathogenesis and progression of COVID-19. Here, we aimed to assess the possible association between variants in the RAS-related genes and COVID-19 susceptibility and severity in a sample of the Moroccan population. A total of 325 individuals were recruited in this study, with 102 outpatients, 105 hospitalized patients, and 118 healthy controls negative for SARS-CoV-2 infection, and subjected to NGS gene panel sequencing containing eleven RAS pathway genes. A total of 65 functional variants were identified, including 63 missenses, 1 splice, and 1 INDEL. Most of them were rare, with 47 (72%) found in a single individual. According to the common disease/common variant hypothesis, five common candidate variants with MAF > 10% were identified (ACE2 rs2285666, TMPRSS2 rs12329760, AGT rs699 genes, ACE rs4341, and ACE rs4343). Statistical analysis showed that the ACE rs4343 AA genotype was associated with a 2.5-fold increased risk of severe COVID-19 (p = 0.026), and the T genotype of the ACE2 rs2285666 variant showed a borderline association with susceptibility to SARS-CoV-2 in males (p = 0.097). In conclusion, our results showed that the RAS pathway genes are highly conserved among Moroccans, and most of the identified variants are rare. Among the common variants, the ACE rs4343 polymorphism would lead to a genetic predisposition for severe COVID-19.

Accumulating clinical evidence suggests an intricate relationship between severe COVID-19 and preexisting metabolic complications, which share some metabolic dysregulations, including hyperglycaemia, hyperinsulinaemia and hyperlipidaemia. However, the potential role of these metabolic risk factors in SARS-CoV-2 infection and entry factor expression remains unknown. Here we report the implication of hyperglycaemia in SARS-CoV-2 infection and therapy. Hyperglycaemia, instead of hyperinsulinaemia and hyperlipidaemia, can significantly induce the expression of SARS-CoV-2 entry factors (Ace2, Tmprss2, Tmprss4, Furin and Nrp1) in liver cells, but not in lung and pancreatic cells, which is attenuated by mTOR inhibition. Correspondingly, pathological glucose levels promote SARS-CoV-2 entry into cultured hepatocytes in pseudovirus cell systems. Conversely, representative glucose-lowering drugs (metformin, dapagliflozin, sitagliptin and exenatide) are able to diminish the enhancement of entry factor expression and SARS-CoV-2 infection in cultured hepatocytes under pathological glucose conditions. Intriguingly, SARS-CoV-2 entry factors are increased in the livers of nonalcoholic fatty liver disease and diabetes patients. These results define hyperglycaemia as a key susceptibility factor for hepatic SARS-CoV-2 infection, and provide insights into the clinical application of glucose-lowering therapies in COVID-19 patients under comorbid hyperglycaemia conditions.

The persistence and infectivity of respiratory viruses in cadavers remain poorly characterized, posing significant biosafety risks for forensic and healthcare professionals. This study systematically evaluates the post-mortem stability and transmission potential of SARS-CoV-2, influenza A virus (IAV), and respiratory syncytial virus (RSV) under varying environmental conditions, providing critical insights into viral kinetics.

To assess the post-mortem stability of SARS-CoV-2, tissue samples were collected from infected cadavers at 4 ℃, room temperature (RT, 20-22 ℃), and 37 ℃ over a predetermined timeframe. Viral kinetics were analyzed using quantitative assays, while histopathology and immunohistochemistry characterized tissue-specific distribution. Additionally, comparative analyses were conducted both in vitro and in cadaveric tissues to characterize the survival dynamics of IAV and RSV under identical conditions.

SARS-CoV-2 exhibited prolonged post-mortem infectivity, persisting for up to 5 days at RT and 37 ℃ and over 7 days at 4 ℃, with the highest risk of transmission occurring within the first 72 h at RT and 24 h at 37 ℃. In contrast, RSV remained viable for 1-2 days, while IAV persisted for only a few hours post-mortem. Viral decay rates were temperature-dependent and varied across tissues, demonstrating distinct post-mortem survival kinetics.

This study presents the first comprehensive analysis of viral persistence in cadavers, revealing prolonged SARS-CoV-2 stability compared to IAV and RSV. These findings underscore the need for enhanced post-mortem biosafety protocols to mitigate occupational exposure risks in forensic and clinical settings. By elucidating viral decay dynamics across environmental conditions, this research establishes a critical foundation for infection control strategies, informing biosafety policies for emerging respiratory pathogens.

The COVID-19 pandemic remains an ongoing global health threat, yet effective treatments are still lacking. This has led to a high demand for complementary/alternative medicine, such as Chinese herbal medicines for curbing the COVID-19 pandemic. Given the dual anticancer and antiviral activities of many herbal drugs, they may hold a multifaceted potential to tackle both cancer and SARS-CoV-2. Triptolide is the major bioactive compound isolated from Tripterygium wilfordii Hook F (TwHF), a traditional Chinese medicinal herb recognized for its beneficial pharmacological properties in many diseases, including cancer and viral infection. However, its application in the clinic has been greatly limited due to its toxicity and poor water solubility. Here, from a screen of a natural compound library of Chinese Pharmacopoeia, we identified triptolide as a top candidate to inhibit cell entry of SARS-CoV-2. We demonstrated that triptolide robustly blocked viral entry at nanomolar concentrations in cellular models, with broad range activity against emerging Omicron variants of SARS-CoV-2. Mechanistically, triptolide disrupted the interaction of SARS-CoV-2 spike protein with its receptor ACE2. Furthermore, we synthesized water-soluble, triptolide-derived carbon quantum dots. Compared to triptolide, these highly biocompatible nanomaterials exhibited prominent antiviral capabilities against Omicron variants of SARS-CoV-2 with less cytotoxicity. Finally, we showed that triptolide-derived carbonized materials excelled in their anticancer properties compared to triptolide and Minnelide, a water-soluble analog of triptolide. Together, our results provide a rationale for the potential development of triptolide-carbonized derivatives as a promising antiviral candidate for the current pandemic and future outbreaks, as well as anticancer agents.