Continual evolution of SARS-CoV-2 spike drives the emergence of Omicron variants that show increased spreading and immune evasion. Understanding how the variants orientate themselves towards host immune defence is crucial for controlling future pandemics. Herein, we demonstrate that human cathelicidin LL-37, a crucial component of innate immunity, predominantly binds to the S2 subunit of SARS-CoV-2 spike protein, occupying sites where TMPRSS2 typically binds. This binding impedes TMPRSS2-mediated priming at site S2' and subsequent membrane fusion processes. The mutation N764K within S2 subunit of Omicron variants reduces affinity for LL-37 significantly, thereby diminishing binding capacity and inhibitory effects on membrane fusion. Moreover, the early humoral immune response enhanced by LL-37 is observed in mice against SARS-CoV-2 spike but not Omicron BA.4/5 spike. These findings reveal the mechanism underlying interactions amongst LL-37, TMPRSS2 and SARS-CoV-2 and VOCs, and highlight the distinct mutation for Omicron variants to evade the fusion activity inhibition by host innate immunity.

Transmembrane Protease, Serine-2 (TMPRSS2) and TMPRSS11D are human proteases that enable SARS-CoV-2 and Influenza A/B virus infections, but their biochemical mechanisms for facilitating viral cell entry remain unclear. We show these proteases spontaneously and efficiently cleave their own zymogen activation motifs, activating their broader protease activity on cellular substrates. We determine TMPRSS11D co-crystal structures with a native and an engineered activation motif, revealing insights into its autocleavage activation and distinct substrate binding cleft features. Leveraging this structural data, we develop nanomolar potency peptidomimetic inhibitors of TMPRSS11D and TMPRSS2. We show that a broad serine protease inhibitor that underwent clinical trials for TMPRSS2-targeted COVID-19 therapy, nafamostat mesylate, was rapidly cleaved by TMPRSS11D and converted to low activity derivatives. In this work, we develop mechanistic insights into human protease viral tropism and highlight both the strengths and limitations of existing human serine protease inhibitors, informing future drug discovery efforts targeting these proteases.

The COVID-19 pandemic, caused by SARS-CoV-2, has unveiled a range of symptoms beyond the respiratory system, including significant gastrointestinal manifestations.

This study explores the prevalence and intensity of gastroesophageal symptoms in post-COVID-19 patients and the integrity of the esophageal epithelial barrier.

We conducted a prospective longitudinal cohort study with 55 patients hospitalized due to COVID-19 at a University Hospital. Patients were evaluated during hospitalization and between 3 and 6 months post-discharge, using validated questionnaires for gastrointestinal and gastroesophageal reflux symptoms. Additionally, 25 of these patients underwent upper digestive endoscopy, with esophageal mucosal biopsies analyzed for transepithelial electrical resistance (TER), permeability, and expression of inflammatory cytokines and cell junction proteins. Data expressed as mean EPM, inference by two-way ANOVA.

Results were considered statistically significant at p < 0.05. There were significant increases in heartburn and acid reflux symptoms in post-COVID-19 patients, as measured by the GSRS questionnaire. Biopsies from post-COVID patients revealed increased esophageal permeability when compared to non-COVID patients in acidic media (pH 2: non-COVID-19: 717.8 ± 168.2 vs. post-COVID-19: 1377.6 ± 316.4), suggesting compromised mucosal barrier. Furthermore, IL-8 levels and expression of Claudin-2 were elevated in these patients.

The data suggested that COVID-19 infection may cause lasting damage to the esophageal epithelial barrier, increasing its permeability and provoking an exacerbated inflammatory response. These changes may explain the prevalence of post-infection gastroesophageal symptoms. Our findings underscored the importance of continuous monitoring and the development of therapeutic strategies to mitigate gastroesophageal effects in patients recovering from COVID-19.

Long-term immunosuppressive therapy typically increases the risk of viral infection, yet during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, inflammatory bowel disease (IBD) patients showed reduced severe coronavirus disease 2019 (COVID-19) susceptibility. This suggests potential overlapping molecular mechanisms between IBD and COVID-19 that warrant investigation.

From April 2020 to April 2022, we enrolled 363 IBD patients and 146 healthy donors. Serum samples were analyzed by enzyme-linked immunoadsorption assay to determine the presence of anti-SARS-CoV-2 antibodies and to measure concentrations of the host-soluble factors sACE2 and mannose-binding lectin (MBL), which have SARS-CoV-2 neutralizing activity. Furthermore, colonic mucosa biopsies were analyzed by real-time PCR to confirm the upregulation of MBL2 as well as to assess the expression of genes encoding SARS-CoV-2 entry molecules (ie, ACE2, TMPRSS2, TMPRSS4, ADAM17, AGTR1).

Intestinal mucosa expression of ACE2, TMPRSS2, and TMPRSS4 genes was significantly lower in IBD than in healthy individuals, regardless of the type of medication, while ADAM17 and AGT1R were similar across groups. Serum sACE2 levels changed minimally, whereas circulating MBL levels were significantly higher in CD and somewhat elevated in UC patients versus controls. Parallel trends in MBL2 gene expression were observed in IBD patients' intestinal mucosa.

Overall, our study indicates that the presence of higher basal circulating levels of MBL in CD patients and the decreased intestinal mucosa expression of the SARS-CoV-2 receptor ACE2 and the host cell priming proteases TMPRSS2 and TMPRSS4 in both CD and UC patients may reduce COVID-19 risk, underscoring the potential protective role of these biomarkers in IBD populations against SARS-CoV-2 infection.

Sensing of cytosolic, double-stranded (ds) DNA or dsRNA molecules derived from microbial or endogenous sources triggers cell-intrinsic innate immunity, but sensors recognizing both cytosolic dsDNA and dsRNA are sparsely reported. Here we find that full-length human SAMD9 protein directly binds to synthetic or viral dsDNA or dsRNA. Overexpression of SAMD9 from various vertebrate species leads to robust production of interferons and pro-inflammatory cytokines. By contrast, loss of endogenous SAMD9 impairs the interferon responses to cytosolic dsDNA and dsRNA stimulation in multiple cell types and enhances the infectivity of pathogenic dsDNA and dsRNA viruses. Mice lacking Samd9l, the human SAMD9 homolog, show increased viral load and severe clinical manifestations of rotavirus and reovirus infections. Rotavirus-encoded non-structural protein 1 targets SAMD9 for proteasomal degradation. Collectively, our data demonstrate that SAMD9 may serve as a pattern-recognition receptor for cytosolic dsDNA and dsRNA across different domains of life and represents a potential target of viral innate immune evasion.

COVID-19 has caused millions of deaths globally; however, the characterization of molecular biomarkers of severe disease remains of great scientific importance. The aim of this study was to capture the transcriptional differences of the whole blood gene expression between COVID-19 patients with mild and severe disease, using Next Generation Sequencing technologies, on admission and after 7 days. The genes which were differentially expressed in severe compared to mild patients were used for Gene Ontology (GO) enrichment analysis. Gene expression data were used to estimate the cell abundance of 22 immune cell types via digital cytometry. GO terms related to the response to molecules of bacterial origin, such as intestine-derived lipopolysaccharide (LPS), were enriched, among other dysregulated pathways, which are well described as paramount mechanisms of severe manifestations of COVID-19. The neutrophil population increased in patients with severe disease, whereas the monocyte, CD8+ T cell, and activated Natural Killer (NK) cell populations were depleted. These cell population dynamics are also indicative of severe COVID-19 and intestinal bacterial translocation. This study elucidates the molecular basis of severe COVID-19 and highlights intestinal bacterial translocation as a potential driver of severe disease.

SARS-CoV-2 viral infection is regulated by the host cell receptors ACE2 and TMPRSS2, and therefore the effect of various natural and synthetic compounds on these receptors has recently been the subject of investigations. Cyclodextrins, naturally occurring polysaccharides derived from starch, are soluble in water and have a hydrophobic cavity at their center enabling them to accommodate small molecules and utilize them as carriers in the food, supplements, and pharmaceutical industries to improve the solubility, stability, and bioavailability of target compounds. In the current study, computational molecular simulations were used to investigate the ability of α-, β- and γ-Cyclodextrins on human cell surface receptors. Cell-based experimental approaches, including expression analyses at mRNA and protein levels and virus replication, were used to assess the effect on receptor expression and virus infection, respectively. We found that none of the three CDs could dock effectively to human cell surface receptor ACE2 and viral protease Mpro (essential for virus replication). On the other hand, α- and β-CD showed strong and stable interactions with TMPRSS2, and the expression of both ACE2 and TMPRSS2 was downregulated at the mRNA and protein levels in cyclodextrin (CD)-treated cells. A cell-based virus replication assay showed ∼20% inhibition by β- and γ-CD. Taken together, the study suggested that (i) downregulation of expression of host cell receptors may not be sufficient to inhibit virus infection (ii) activity of the receptors and virus protein Mpro may play a critical and clinically relevant role, and hence (iii) newly emerging anti-Covid-19 compounds warrant multimodal functional analyses.

The ectodomain of the Omicron SARS-CoV-2 spike has an increased positive surface charge, favoring binding to the host cell surface, but may affect the stability of the ectodomain. Thermal stability studies identified two transitions associated with the flexibility of the receptor binding domain and the unfolding of the whole ectodomain, respectively. Despite destabilizing effects of some mutations, compensatory mutations maintain ECD stability and functional advantages thus supporting viral fitness.

Respiratory viral infections are a major global public health concern, and current antiviral therapies still have limitations. In recent years, research has revealed significant similarities between the immune systems of the gut and lungs, which interact through the complex physiological network known as the "gut-lung axis." As one of the largest immune organs, the gut, along with the lungs, forms an inter-organ immune network, with strong parallels in innate immune mechanisms, such as the activation of pattern recognition receptors (PRRs). Furthermore, the gut microbiota influences antiviral immune responses in the lungs through mechanisms such as systemic transport of gut microbiota-derived metabolites, immune cell migration, and cytokine regulation. Studies have shown that gut dysbiosis can exacerbate the severity of respiratory infections and may impact the efficacy of antiviral therapies. This review discusses the synergistic role of the gut-lung axis in antiviral immunity against respiratory viruses and explores potential strategies for modulating the gut microbiota to mitigate respiratory viral infections. Future research should focus on the immune mechanisms of the gut-lung axis to drive the development of novel clinical treatment strategies.

Diarrhea is the predominant symptom of acute gastroenteritis resulting from enteric infections and a leading cause of death in infants and young children. However, the role of the host response in diarrhea pathogenesis is unclear. Using rotavirus and neonatal mice as a model, we found that oral inoculation of UV-inactivated replication-defective rotavirus consistently induced watery diarrhea by robust activation of cytosolic double-stranded RNA sensing pathways and type III interferon (IFN-λ) secretion. Diarrhea was significantly diminished in mice lacking the IFN-λ receptor. Mechanistically, IFN-λ signaling downregulates the expression of Dra, a chloride and bicarbonate exchanger, which contributes to reduced water absorption. We confirmed these findings in mice inoculated with reovirus, as well as in donor-derived human intestinal organoids and human biopsy samples. Our data highlight a mechanism of rapid diarrhea induction by host innate immune sensing in the gastrointestinal tract and suggest that diarrhea induction is an active host defense strategy to eliminate the pathogen.

Organoids have revolutionized the whole field of biology with their ability to model complex three-dimensional human organs in vitro. Intestinal organoids were especially consequential as the first successful long-term culture of intestinal stem cells, which raised hopes for translational medical applications. Despite significant contributions to basic research, challenges remain to develop intestinal organoids into clinical tools for diagnosis, prognosis, and therapy. In this review, we outline the current state of translational research involving adult stem cell and pluripotent stem cell derived intestinal organoids, highlighting the advances and limitations in disease modeling, drug-screening, personalized medicine, and stem cell therapy. Preclinical studies have demonstrated a remarkable functional recapitulation of infectious and genetic diseases, and there is mounting evidence for the reliability of intestinal organoids as a patient-specific avatar. Breakthroughs now allow the generation of structurally and cellularly complex intestinal models to better capture a wider range of intestinal pathophysiology. As the field develops and evolves, there is a need for standardized frameworks for generation, culture, storage, and analysis of intestinal organoids to ensure reproducibility, comparability, and interpretability of these preclinical and clinical studies to ultimately enable clinical translation.

Despite the importance of vaccination- and infection-elicited antibodies (Abs) to SARS-CoV-2 immunity, current mouse models do not fully capture the dynamics of Ab-mediated immunity in vivo, including potential contributions of the neonatal Fc receptor, encoded by FCGRT.

We generated triple knock-in (TKI) mice expressing human ACE2, TMPRSS2, and FCGRT; and evaluated the protective efficacy of anti-SARS-CoV-2 monoclonal Abs (mAbs) and plasma from individuals with immunity elicited by vaccination alone plus SARS-CoV-2 infection-induced (hybrid) immunity.

A human anti-SARS-CoV-2 mAb harbouring a half-life-extending mutation, but not the wild-type mAb, exhibited prolonged half-life in TKI mice and protected against lung infection with Omicron BA.2, validating the utility of these mice for evaluating therapeutic Abs. Pooled plasma from individuals with hybrid immunity to Delta, but not from vaccinated-only individuals, cleared infectious Delta from the lungs of TKI mice (P < 0.01), even though the two plasma pools had similar Delta-binding and -neutralising Ab titres in vitro. Similarly, plasma from individuals with hybrid Omicron BA.1/2 immunity, but not hybrid Delta immunity, decreased lung infection (P < 0.05) with BA.5 in TKI mice, despite the plasma pools having comparable BA.5-binding and -neutralising titres in vitro. Depletion of receptor-binding domain-targeting Abs from hybrid immune plasma abrogated their protection against infection.

These results demonstrate the utility of TKI mice as a tool for the development of anti-SARS-CoV-2 mAb therapeutics, show that in vitro neutralisation assays do not accurately predict in vivo protection, and highlight the importance of hybrid immunity for eliciting protective anti-receptor-binding domain Abs.

This work was funded by grants from the e-Asia Joint Research Program (N10A650706 and N10A660577 to MLM, in collaboration with SS); the NIH (U19 AI142790-02S1 to EOS and SS and R44 AI157900 to KJ); the GHR Foundation (to SS and EOS); the Overton family (to SS and EOS); the Arvin Gottlieb Foundation (to SS and EOS), the Prebys Foundation (to SS); and the American Association of Immunologists Fellowship Program for Career Reentry (to FASB).

Human transmembrane serine protease 2 (TMPRSS2) has garnered substantial interest due to its clinical significance in various pathologies, notably its pivotal role in viral entry into host cells. The development of effective strategies to target TMPRSS2 is a current area of intense research and necessitates a consistent source of active TMPRSS2 with sufficient stability. Here, we comprehensively characterised human seminal-fluid extracellular vesicles (SF-EVs, also referred to as prostasomes), bearing a native source of surface-exposed, enzymatically active TMPRSS2 as demonstrated by high-sensitivity flow cytometry and a fluorometric activity assay. Additionally, we recombinantly produced human TMPRSS2 ectodomain in mammalian cells adopting a directed activation strategy. We observed comparable catalytic parameters and inhibition characteristics for both native SF-EV-associated and recombinant TMPRSS2 when exposed to serine protease inhibitor Nafamostat mesylate. Leveraging these findings, we developed a robust in vitro biochemical assay based on these SF-EVs for the screening of TMPRSS2-targeting compounds. Our results will accelerate the discovery and advancement of efficacious therapeutic approaches targeting TMPRSS2 and propel further exploration into the biological role of SF-EV-associated active TMPRSS2.

Coronavirus disease 2019 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that displays great variability in clinical phenotype. Many factors have been described to be correlated with its severity, and microbiota could play a key role in the infection, progression, and outcome of the disease. SARS-CoV-2 infection has been associated with nasopharyngeal and gut dysbiosis and higher abundance of opportunistic pathogens. To identify new prognostic markers for the disease, a multicentre prospective observational cohort study was carried out in COVID-19 patients divided into three cohorts based on symptomatology: mild (n = 24), moderate (n = 51), and severe/critical (n = 31). Faecal and nasopharyngeal samples were taken, and the microbiota was analysed. Linear discriminant analysis identified Mycoplasma salivarium, Prevotella dentalis, and Haemophilus parainfluenzae as biomarkers of severe COVID-19 in nasopharyngeal microbiota, while Prevotella bivia and Prevotella timonensis were defined in faecal microbiota. Additionally, a connection between faecal and nasopharyngeal microbiota was identified, with a significant ratio between P. timonensis (faeces) and P. dentalis and M. salivarium (nasopharyngeal) abundances found in critically ill patients. This ratio could serve as a novel prognostic tool for identifying severe COVID-19 cases.

SARS-CoV-2 is highly transmissible, having infected ~16 million children in the United States. Symptom severity is higher in infants compared to older children, possibly due to their ineligibility for vaccination. Concerns persist that mothers transmit infectious viral loads of SARS-CoV-2 through breast milk. In this review, we discuss the mechanism by which viruses transmit through breast milk, weigh the specific virulence and infectivity of SARS-COV-2, and review current guidelines for minimizing transmission in neonates.

Through available literature, we propose a stepwise pathway for vertical transmission of SARS-CoV-2. The level of risk and probability of infection is assessed based on established mechanisms, reported viral loads, and presence of transmembrane receptors.

To successfully transmit viruses through breast milk, the virus must infect the mother's breast cells, replicate in the mammary gland, be secreted into breast milk, survive contact with the infant's oral mucosa and digestive tract, infect enterocytes, replicate while evading the infant's immune system, exit the gastrointestinal tract, and enter the bloodstream for systemic infection.

We conclude that SARS-CoV-2 infection through breast milk has limited transmission risk, and benefits for infants far outweigh the risks, aligning with current AAP/WHO/CDC guidelines. Though close contact during breastfeeding and exposure to respiratory droplets pose a higher transmission risk.

Infections by enteric virus and intestinal inflammation are recognized as a leading cause of deadly gastroenteritis, and NLRP6 and NLRP9b signaling control these infection and inflammation. However, the regulatory mechanisms of the NLRP6 and NLRP9b signaling in enteric viral infection remain unexplored. In this study, we found that the E3 ligase TRIM29 suppressed type III interferon (IFN-λ) and interleukin-18 (IL-18) production by intestinal epithelial cells (IECs) when exposed to polyinosinic:polycytidylic acid (poly I:C) and enteric RNA viruses. Knockout of TRIM29 in IECs was efficient to restrict intestinal inflammation triggered by the enteric RNA viruses, rotavirus in suckling mice, and the encephalomyocarditis virus (EMCV) in adults. This attenuation in inflammation was attributed to the increased production of IFN-λ and IL-18 in the IECs and more recruitment of intraepithelial protective Ly6A+CCR9+CD4+ T cells in small intestines from TRIM29-deficient mice. Mechanistically, TRIM29 promoted K48-linked ubiquitination, leading to the degradation of NLRP6 and NLRP9b, resulting in decreased IFN-λ and IL-18 secretion by IECs. Our findings reveal that enteric viruses utilize TRIM29 to inhibit IFN-λ and inflammasome activation in IECs, thereby facilitating viral-induced intestinal inflammation. This indicates that targeting TRIM29 could offer a promising therapeutic strategy for alleviating gut diseases.

In the pharmaceutical research of viral respiratory infections, cell culture models have traditionally been used to evaluate the therapeutic effects of candidate compounds. Although cell lines are easy to handle and cost-effective, they do not fully replicate the characteristics of human respiratory organs. Recently, organoids and microphysiological systems (MPS) have been employed to overcome this limitation for in vitro testing of drugs against viral respiratory infections. Advanced disease modeling using organoids, self-organized three-dimensional (3D) cell culture models derived from stem cells, or MPS, models for culturing multiple cell types in a microfluidic device and capable of recapitulating a physiological 3D dynamic environment, can accurately replicate the complex functions of respiratory organs, thus making them valuable tools for elucidating the organ damages caused by viral respiratory infections and evaluating the efficacy of candidate drugs against them. Recently, a wide range of organoids and MPS have been developed to model the complex pathophysiology caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and assess therapeutic drugs. In this review, we evaluate the latest pharmaceutical research on coronavirus disease 2019 (COVID-19) that utilizes organoids and MPS and discuss future perspectives of their applications.

TMPRSS4, a transmembrane serine protease type II, is associated with various pathological illnesses. It has been found to activate SARS-CoV-2, enhance viral infection of human small-intestinal enterocytes and is overexpressed in different types of cancers. Therefore, this study aims to disover potential TMPRSS4 inhibitors that have better binding affinity than the approved inhibitors: 2-hydroxydiarylamide and tyroserleutide. Since no 3D-structure is known for TMPRSS4, structural models for the TMPRSS4 serine protease domain were developed. The modeled structures were validated and subjected to molecular dynamics simulations. FDA-approved, clinical/preclinical drugs and natural products were docked to the pocket of TMPRSS4. Moreover, through a systematic analysis, MD simulations and MM-GBSA binding free energy calculations revealed that the best candidates Ergotamine, S55746, NPC478048, Lifirafenib, and NPC77101 are highly stable drug candidates in complex with TMPRSS4, displaying low RMSD and RMSF values with strong binding stability. Among these compounds, Ergotamine showed the most favorable binding energy (-33.73 kcal/mol). Overall, our in silico results revealed that these compounds could act as potent TMPRSS4 inhibitors and need to be validated by future experimental studies.

The mucosal immune system, as the most extensive peripheral immune network, serves as the frontline defense against a myriad of microbial and dietary antigens. It is crucial in preventing pathogen invasion and establishing immune tolerance. A comprehensive understanding of mucosal immunity is essential for developing treatments that can effectively target diseases at their entry points, thereby minimizing the overall impact on the body. Despite its importance, our knowledge of mucosal immunity remains incomplete, necessitating further research. The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the critical role of mucosal immunity in disease prevention and treatment. This systematic review focuses on the dynamic interactions between mucosa-associated lymphoid structures and related diseases. We delve into the basic structures and functions of these lymphoid tissues during disease processes and explore the intricate regulatory networks and mechanisms involved. Additionally, we summarize novel therapies and clinical research advances in the prevention of mucosal immunity-related diseases. The review also addresses the challenges in developing mucosal vaccines, which aim to induce specific immune responses while maintaining tolerance to non-pathogenic microbes. Innovative therapies, such as nanoparticle vaccines and inhalable antibodies, show promise in enhancing mucosal immunity and offer potential for improved disease prevention and treatment.

Coronavirus disease (COVID-19), caused by severe acquired respiratory syndrome-Coronavirus-2 (SARS-CoV-2), triggered a global pandemic with severe medical and socioeconomic consequences. Although fatality rates are higher among the elderly and those with underlying comorbidities, host factors that promote susceptibility to SARS-CoV-2 infection and severe disease are poorly understood. Although individuals with certain autoimmune/inflammatory disorders show increased susceptibility to viral infections, there is incomplete knowledge of SARS-CoV-2 susceptibility in these diseases. The aim of our study was to investigate whether the autoimmunity risk gene, PTPN2, which also confers elevated risk to develop inflammatory bowel disease, affects susceptibility to SARS-CoV-2 viral uptake.

Using samples from PTPN2 genotyped patients with inflammatory bowel disease, PTPN2-deficient mice, and human intestinal and lung epithelial cell lines, we investigated how PTPN2 affects expression of the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2), and uptake of virus-like particles expressing the SARS-CoV2 spike protein and live SARS-CoV-2 virus.

We report that the autoimmune PTPN2 loss-of-function risk variant rs1893217 promotes expression of the SARS-CoV-2 receptor, ACE2, and increases cellular entry of SARS-CoV-2 spike protein and live virus. Elevated ACE2 expression and viral entry were mediated by increased Janus kinase-signal transducers and activators of transcription signaling and were reversed by the Janus kinase inhibitor, tofacitinib.

Collectively, our findings uncover a novel risk biomarker for increased expression of the SARS-CoV-2 receptor and viral entry, and identify a clinically approved therapeutic agent to mitigate this risk.

Middle East respiratory syndrome coronavirus (MERS-CoV) infects respiratory epithelial cells in humans and camels by binding to dipeptidyl peptidase 4 (DPP4) as its entry receptor. DPP4 is a multifunctional type II membrane protein with a long ectodomain and a short six-amino-acid (aa) cytoplasmic tail. MERS-CoV is known to bind to the ectodomain of DPP4 to gain entry into the host cell. However, the role of the cytoplasmic tail in the entry process remains unclear. Here, we show that mutating or deleting individual aa residues or the entire cytoplasmic tail of DPP4 (ΔcytDPP4) does not completely prevent DPP4 from being inserted into the membrane or from allowing the binding of the MERS-CoV spike protein and pseudovirus infection. Although two mutants, ΔcytDPP4, and a single aa deleted DPP4 (ΔK6DPP4) displayed less surface presentation than wtDPP4, the spike protein could still bind and localize on different DPP4 mutants. The reduced surface expression of ΔK6DPP4 might be due to the extended transmembrane domain, which is altered by the hydrophobic tryptophan (W) residue adjacent to the deleted K6. Furthermore, HEK293T cells transiently expressing DPP4 mutants were permeable to MERS-CoV pseudovirus infection. Not only transiently expressing cells but also cells stably expressing the ΔcytDPP4 mutant were susceptible to MERS-CoV pseudoviral infection, indicating that the DPP4 cytoplasmic tail is not required for MERS-CoV entry. Overall, these data suggest that, although MERS-CoV binds to DPP4, other host factors may need to interact with DPP4 or the spike protein to trigger internalization.

Wastewater-based epidemiology (WBE) is already being adopted for the surveillance of health conditions of communities and shows great potential for the monitoring of infectious pathogens of public health importance. There is however paucity of robust data to support extensive WBE in Nigeria. This study evaluated the prevalence of clinically relevant infectious pathogens and provided antimicrobial resistance profiles of bacteria pathogens in wastewater canals in Lagos State at a single point in time.

This is a cross-sectional survey of wastewater canals in 20 Local Government Areas (LGAs) in Lagos State for detection of bacteria pathogens of public health importance including non-tuberculous mycobacteria and SARS-Cov-2 virus using cultural analysis and conventional Polymerase Chain Reaction (PCR) techniques. Descriptive epidemiological survey of communities around the canals was done using questionnaires to assess exposure pathways. Statistical analysis was done using SPSS version 27 while P value of < 0.05 was considered as significant.

Three thousand and fifty-four (3054) questionnaires were administered to 1215 (39.8%) females and 1658 (54.3%) males in communities situated around 40 canals in 20 LGAs. Although majority (81.8%) reported using water closet toilet system and pit latrine (12.5%), a few of them admitted to open defaecation [101 (3.3%)] while 299 (9.8%) engaged in open field waste disposal. SARS-CoV-2 was not detected from wastewater in this study. Two mycobacterial species that included Mycobacterium fortitium group (13, 32.5%) and Mycobacterium kansasii (11, 27.5%) were identified in 15 out of 20 LGAs sampled. A total of 123 bacteria pathogens were isolated across the 40 canals. Prominent enteropathogens isolated included Escheriachia coli (28.5%), Salmonella spp (16.3%), Vibro cholerae (10.6%) and Shigella spp (5.7%). Extended spectrum beta-lactamase genes were prominent (87.5%) in the wastewater samples with almost a half (42.5%) of the canals containing both SHV and CTX-M.

This study highlights the presence of pathogens with potential to cause epidemic in wastewater canals in Lagos State and provides evidence to inform policy and strategies for wastewater monitoring and treatment. Further studies involving longitudinal monitoring of time-based variations is needed to identify trends in pathogen loads and AMR patterns over time.

The continued emergence of SARS-CoV-2 variants and the threat of future Sarbecovirus zoonoses have spurred the design of vaccines that can induce broad immunity against multiple coronaviruses. Here, we use computational methods to infer ancestral phylogenetic reconstructions of receptor binding domain (RBD) sequences across multiple Sarbecovirus clades and incorporate them into a multivalent adenoviral-vectored vaccine. Mice immunized with this pan-Sarbecovirus vaccine are protected in the upper and lower respiratory tracts against infection by historical and contemporary SARS-CoV-2 variants, SARS-CoV, and pre-emergent SHC014 and Pangolin/GD coronavirus strains. Using genetic and immunological approaches, we demonstrate that vaccine-induced protection unexpectedly is conferred principally by CD4+ and CD8+ T cell-mediated anamnestic responses. Importantly, prior mRNA vaccination or SARS-CoV-2 respiratory infection does not alter the efficacy of the mucosally delivered pan-Sarbecovirus vaccine. These data highlight the promise of a phylogenetic approach for antigen and vaccine design against existing and pre-emergent Sarbecoviruses with pandemic potential.

Human enteric viruses, such as norovirus, adenovirus, rotavirus, and enterovirus, are crucial targets in controlling biological contamination in water systems worldwide. Due to their small size and low concentrations in water, effective virus concentration and detection methods are essential for ensuring microbial safety. This paper reviews the typical and innovative methods for concentrating and detecting human enteric viruses, highlights viral contamination levels across different water bodies, and discusses the removal efficiencies of virus through various treatment technologies. The application and current gaps of quantitative microbial risk assessment (QMRA) for evaluating the risks of human enteric viruses is also explored. Innovative methods such as digital polymerase chain reaction and isothermal amplification show promise in sensitivity and convenience, however, distinguishing between infectious and non-infectious viruses should be a key focus of future detection techniques. The highest concentrations of human enteric viruses were detected in wastewater, ranging from 103 to 106 copies/L, while drinking water showed significantly lower concentrations, often below 102 copies/L. QMRA studies suggest that exposure to human enteric viruses, whether through contaminated drinking water, occupational contact, or accidental wastewater discharge, could result in a life expectancy of 1.96 × 10-4 to 4.53 × 10-1 days/year.

SARS-CoV-2 infection poses a major threat to public health, and understanding the mechanism of viral replication and virion release would help identify therapeutic targets and effective drugs for combating the virus. Herein, we identified E3 ubiquitin-protein ligase Itchy homolog (ITCH) as a central regulator of SARS-CoV-2 at multiple steps and processes. ITCH enhances the ubiquitination of viral envelope and membrane proteins and mutual interactions of structural proteins, thereby aiding in virion assembly. ITCH-mediated ubiquitination also enhances the interaction of viral proteins to the autophagosome receptor p62, promoting their autophagosome-dependent secretion. Additionally, ITCH disrupts the trafficking of the protease furin and the maturation of cathepsin L, thereby suppressing their activities in cleaving and destabilizing the viral spike protein. Furthermore, ITCH exhibits robust activation during the SARS-CoV-2 replication stage, and SARS-CoV-2 replication is significantly decreased by genetic or pharmacological inhibition of ITCH. These findings provide new insights into the mechanisms of the SARS-CoV-2 life cycle and identify a potential target for developing treatments for the virus-related diseases.

Enteric virus infection is a major public health issue worldwide. Enteric viruses have become epidemic infectious diseases in several countries. Enteric viruses primarily infect the gastrointestinal tract and complete their life cycle in intestinal epithelial cells. These viruses are transmitted via the fecal-oral route through contaminated food, water, or person to person and cause similar common symptoms, including vomiting, abdominal pain, and diarrhea. Diarrheal disease is the third leading cause of death in children under five years of age, accounting for approximately 1.7 billion cases and 443,832 deaths annually in this age group. Additionally, some enteric viruses can invade other tissues, leading to severe conditions and even death. The pathogenic mechanisms of enteric viruses are also unclear. In this review, we organized the research on trending enteric virus infections, including rotavirus, norovirus, adenovirus, Enterovirus-A71, Coxsackievirus A6, and Echovirus 11. Furthermore, we discuss the gastrointestinal effects and pathogenic mechanisms of SARS-CoV-2 in intestinal epithelial cells, given the gastrointestinal symptoms observed during the COVID-19 pandemic. We conducted a literature review on their pathogenic mechanisms, which serves as a guide for formulating future treatment strategies for enteric virus infections.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 2019 (COVID-19) pandemic, posing serious threats to global health. Effective broad-spectrum antiviral drugs for the treatment of COVID-19 are not sufficiently available. In the present study, we investigated the antiviral activity of the natural lignan diphyllin (PubChem CID 100492) against different SARS-CoV-2 variants and explored the underlying molecular mechanisms. We found that diphyllin dose-dependently inhibits the SARS-CoV-2 spike (S)-mediated entry into different types of cells. The potent inhibition was evident against spike proteins derived from the original SARS-CoV-2 and from variants of concern such as Alpha, Beta, Delta or Omicron. Accordingly, diphyllin also significantly inhibited the in vitro infection of a clinical SARS-CoV-2 virus isolate. Mechanistically, diphyllin simultaneously inhibited the endosomal entry of SARS-CoV-2 by neutralizing the endosomal acidification and reducing the activity of the cysteine protease cathepsin L (CTSL) as well as S-meditated cell surface entry by impairing furin activity. Collectively, our findings establish diphyllin as novel inhibitor of CTSL and furin proteases, resulting in a double-pronged attack on SARS-CoV-2 entry along endosomal as well as cell surface routes. Therefore, diphyllin has the potential to be advanced as an inhibitor of SARS-CoV-2 entry.

The ongoing emergence of SARS-CoV-2 variants poses significant challenges to existing therapeutics. The spike (S) glycoprotein is central to both viral entry and cell-to-cell transmission via syncytia formation, a process that confers resistance to neutralizing antibodies. The mechanisms underlying this resistance, particularly in relation to spike-mediated fusion, remain poorly understood.

We analyzed two clinical SARS-CoV-2 isolates differing by a single amino acid substitution in the S protein. Using biochemical and cell-based assays, we evaluated entry kinetics, syncytia formation, and the neutralizing efficacy of convalescent sera. These parameters were further correlated with S-mediated cell-cell fusion activity.

The single amino acid substitution significantly altered entry kinetics and enhanced syncytia formation. This modification did not diminished the neutralizing capacity of convalescent sera, but it increased the efficiency of S-induced cell-cell fusion. These findings highlight the mutation's impact on viral transmissibility and immune evasion.

Our study demonstrates that even minor changes in the S protein can profoundly influence SARS-CoV-2 transmissibility and resistance to antibody-mediated neutralization. Understanding the molecular basis of S-mediated cell-cell fusion is crucial for anticipating the impact of emerging variants and developing next-generation therapeutic strategies. These insights provide a framework for predicting variant fitness and optimizing treatment approaches against future SARS-CoV-2 variants.

Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread.

Enveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.

The continued emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates updating coronavirus disease 2019 (COVID-19) vaccines to match circulating strains. The immunogenicity and efficacy of these vaccines must be tested in pre-clinical animal models. In Syrian hamsters, we measured the humoral and cellular immune response after immunization with the nanoparticle recombinant Spike (S) protein-based COVID-19 vaccine (Novavax, Inc.). We also compared the efficacy of the updated monovalent XBB.1.5 variant vaccine with previous COVID-19 vaccines for the induction of XBB.1.5 and EG.5.1 neutralizing antibodies and protection against a challenge with the EG.5.1 variant of SARS-CoV-2. Immunization induced high levels of S-specific IgG and IgA antibody-secreting cells and antigen-specific CD4+ T cells. The XBB.1.5 and XBB.1.16 vaccines, but not the Prototype vaccine, induced high levels of neutralizing antibodies against the XBB.1.5, EG.5.1, and JN.1 variants of SARS-CoV-2. Upon challenge with the Omicron EG.5.1 variant, the XBB.1.5 and XBB.1.16 vaccines reduced the virus load in the lungs, nasal turbinates, trachea, and nasal washes. The bivalent vaccine (Prototype rS + BA.5 rS) continued to offer protection in the trachea and lungs, but protection was reduced in the upper airways. By contrast, the monovalent Prototype vaccine no longer offered good protection, and breakthrough infections were observed in all animals and tissues. Thus, based on these study results, the protein-based XBB.1.5 vaccine is immunogenic and increased the breadth of protection against the Omicron EG.5.1 variant in the Syrian hamster model.

As SARS-CoV-2 continues to evolve, there is a need to assess the immunogenicity and efficacy of updated vaccines against newly emerging variants in pre-clinical models such as mice and hamsters. Here, we compared the immunogenicity and efficacy between the updated XBB.1.5, the original Prototype Wuhan-1, and the bivalent Prototype + BA.5 vaccine against a challenge with the EG.5.1 Omicron variant of SARS-CoV-2 in hamsters. The XBB.1.5 and bivalent vaccine, but not the Prototype, induced serum-neutralizing antibodies against EG.5.1, albeit the titers were higher in the XBB.1.5 immunized hamsters. The presence of neutralizing antibodies was associated with complete protection against EG.5.1 infection in the lower airways and reduced virus titers in the upper airways. Compared with the bivalent vaccine, immunization with XBB.1.5 improved viral control in the nasal turbinates. Together, our data show that the updated vaccine is immunogenic and that it offers better protection against recent variants of SARS-CoV-2.