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

Since winter 2019, SARS-CoV-2 has emerged, spread, and evolved all around the globe. We explore 4 y of evolutionary epidemiology of this virus, ranging from the applied public health challenges to the more conceptual evolutionary biology perspectives. Through this review, we first present the spread and lethality of the infections it causes, starting from its emergence in Wuhan (China) from the initial epidemics all around the world, compare the virus to other betacoronaviruses, focus on its airborne transmission, compare containment strategies ("zero-COVID" vs. "herd immunity"), explain its phylogeographical tracking, underline the importance of natural selection on the epidemics, mention its within-host population dynamics. Finally, we discuss how the pandemic has transformed (or should transform) the surveillance and prevention of viral respiratory infections and identify perspectives for the research on epidemiology of COVID-19.

The nucleocapsid protein (NP) of SARS-CoV-2, an RNA-binding protein, is capable of undergoing liquid-liquid phase separation (LLPS) during viral infection, which plays a crucial role in virus assembly, replication, and immune regulation. In this study, we developed a homogeneous time-resolved fluorescence (HTRF) method for identifying inhibitors of the SARS-CoV-2 NP-RNA interaction. Using this HTRF-based approach, we identified two natural products, sennoside A and sennoside B, as effective blockers of this interaction. Bio-layer interferometry assays confirmed that both sennosides directly bind to the NP, with binding sites located within the C-terminal domain. Additionally, fluorescence recovery after photobleaching (FRAP) experiments revealed that sennoside B significantly inhibited RNA-induced LLPS of the NP, while sennoside A displayed comparatively weaker activity. Thus, the developed HTRF-based assay is a valuable tool for identifying novel compounds that disrupt the RNA-binding activity and LLPS of the SARS-CoV-2 NP. Our findings may facilitate the development of antiviral drugs targeting SARS-CoV-2 NP.

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

The COVID-19 pandemic underscores the urgent need to develop broad-spectrum antivirals against coronaviruses (CoVs) to prepare for future outbreaks. In this study, we presented a systematic approach to developing broad-spectrum Mpro inhibitors, with a focus on high-risk CoVs. We optimised S-217622 as a lead compound, with the goal of enhancing conserved interactions within the S1, S2, and S3/S4 pockets of Mpro, leading to significantly improved inhibitory potency against representative CoVs. Compound 25 exhibited submicromolar activity across all ten CoVs, with IC50 values below 0.1 μM for six of them. The X-ray co-crystal structure of SARS-CoV-2 Mpro in complex with compound 25 revealed the structural basis of conserved interactions contributing to its broad-spectrum activity. This study demonstrates the feasibility of reinforcing conserved interactions to develop Mpro inhibitors with broad-spectrum activity and provides valuable strategies for combating future pandemics caused by unknown CoVs.

The identification of a novel virus related to the family Tombusviridae, provisionally named human tombus-like virus (hTLV), is significant in the context of ongoing surveillance for respiratory pathogens. Meta-transcriptomic sequencing was utilized to detect respiratory pathogens in patients with lower respiratory tract infections (LRIs) in Jinan, China, from 2022 to 2023. The additional hTLV infections were identified through retrospective analysis of meta-transcriptome data collected in Beijing, China, from 2016 to 2019, prior to the COVID-19 outbreak. Phylogenetic analyzes indicated that hTLVs were clustered with a Jingmen tombus-like virus 2 but in a distinct clade. The hTLVs genomes consist of a single-stranded positive-sense RNA genomes of 4.7-4.8 kb in size, and contained four putative open reading frames (ORF1-4). The RNA-dependent RNA polymerase protein of hTLV shared significant sequence similarity containing three conserved motifs with 15, 24, and 15 amino acids, respectively. The hTLV genome included the canonical Gly376-Asp377-Asp378 (GDD) catalytic residues, which were a unifying feature of viruses in the family Tombusviridae. The main clinical manifestations of the 23 patients were fever, cough, expectoration and dyspnea, with varying degrees of lung infection or abnormalities in other laboratory indicators. Serological studies showed that fourfold rise in IgG titers in sera of a patient between acute and convalescent phase by ELISA. Identification of the pathogens for acute respiratory tract infections is essential for timely public health interventions and clinical management. The discovery of a novel virus, hTLV, in patients with LRIs highlights the continuous emergence of new respiratory pathogens in humans.

Porcine deltacoronavirus (PDCoV) is increasingly prevalent in newborn piglets with diarrhea. With the development of research on the virus and the feasibility of PDCoV cross-species transmission, the biosafety and the development of pig industry have been greatly affected. In this study, a PDCoV strain CH/LNFX/2022 was isolated from diarrheal newborn piglets at a farm in China. A genome-wide based phylogenetic analysis suggests that 97.5% to 99.2% homology existed in the whole genomes of other strains. Five amino acid mutations are seen for the first time in the S protein. By constructing 3D models, it was found that the S1-NTD/CTD and S2-HR-C regions produced structural alterations. Protein functional analysis showed that the structural changes of the three regions changed the epitope of S protein, the O-GalNAc glycosylation site and the 3C-like protease cleavage site. In addition, oral administration of 107 TCID50 CH/LNFX/2022 to newborn piglets successfully reproduced obvious clinical signs of piglets, such as diarrhea and dehydration. Meanwhile, PDCoV antigen was detected by immunofluorescence in the small intestine, and microscopic lesions and intestinal mucosal barrier destruction were detected by histological observation and scanning electron microscopy. Our study confirmed that porcine coronavirus strains increased pathogenicity through evolution, damaged the intestinal barrier of newborn piglets, and caused diarrhea in pigs. This study provided the candidate strains and theoretical basis for establishing the prevention and control system of vaccine and diagnostic methods for piglet diarrhea.

Complement hyperactivation and thrombotic microangiopathy are closely associated with severe COVID-19. Endothelial dysfunction is a key mechanism underlying thrombotic microangiopathy. To address the relationship between endothelial injury, complement activation and thrombotic microangiopathy of severe COVID-19, we wonder whether, and if so, what and how SARS-CoV-2 factors make endothelial cells (ECs) sensitive to complement-mediated cytotoxicity. We revealed that multiple SARS-CoV-2 proteins enhanced complement-mediated cytotoxicity to ECs by inhibiting membrane complement regulatory proteins (CRPs) and enhancing the deposition of complement-recognizing component FCN1. By screening with CRISPR/Cas9-gRNA libraries, we identified that ADAMTS9, SYAP1, and HIGD1A as intrinsic regulators of CD59 on ECs, which were inhibited by the SARS-CoV-2 M, NSP16, and ORF9b proteins. IFN-γ, GM-CSF, and IFN-α upregulated CD55 and CD59, while IFN-γ antagonized the inhibition of CD59 by the three SARS-CoV-2 proteins. So, the deficiency of IFN-γ weakened the protection of ECs by CRPs against complement-mediated injury which may be enhanced during infection. Our findings illustrated the regulation of protection against complement-mediated attack on self-cells by SARS-CoV-2 infection and immune responses, providing insights into endothelial injury, thrombotic microangiopathy, and potential targets for treating severe COVID-19.

Peptide-like 3CLpro covalent binding inhibitors are the most effective antiviral drugs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Their covalent warheads were designed based on the addition reaction activity of the aldehyde (ketone) carbonyl or its derivative structures. These addition reactions between the warheads and the thiol of the 3CLpro are reversible, and the resulting hemimonothioacetals are chemically unstable. Herein, after DFT calculation, we designed thiol ester warheads using the principle of ester exchange reaction. Then, the warhead fluorescence probe binding experiment suggested these adducts of thiol ester warheads and 3CLpro protein are more stable than the hemimonothioacetals mentioned earlier. Therefore, new 3CLpro inhibitors were subsequently designed through a structure-based drug design method employing those thiol ester warheads. Those 3CLpro inhibitors demonstrated potent 3CLpro inhibitory activities and anti-coronavirus HCoV-OC43 activities. Among them, B16 stands out as the most promising, demonstrating not only the strongest anti-coronavirus HCoV-OC43 activity but also being a moderate inhibitor of CYP3A4, suggesting that B16 does not require co-administration with ritonavir in the treatment of SARS-CoV-2 infection. This work demonstrates the significant potential of thiol esters as novel chemical warheads in designing covalent binding inhibitors for 3CLpro and beyond.

SARS-COV2 is related to the COVID-19 pandemic that emerged in 2019. It caused severe criticism of world health care and the economic system. Its high mutation rate increases contagiousness and gives rise to new variants, causing recurrent epidemic waves. One of the effective strategies for prevention and controlling infections is evaluating antibody existence against SARS-CoV2 to know how much individuals are protected against new infection. Therefore, it can be helpful to conduct health care strategies for vaccination and healing infected patients. In this study, we identified two immunodominant linear B cell epitopes (S179-344 and S550-670) within the S1 subunit of the spike protein using bioinformatics tools (ABCpred and BepiPred). These regions were subsequently cloned into the pET28a(+) vector, expressed in E. coli BL21 (DE3), and then purified. The recombinant antigens were then assessed for their seroreactivity using a variety of diagnostic techniques, including Western blot, dot blot, and ELISA. The study population for this assessment included 68 positive sera from patients infected with SARS-CoV2 and 63 negative control samples, both of which were screened by standard PCR. The results obtained from the ELISA assay indicated that both antigens demonstrated strong seroreactivity with high sensitivity (91-98 %) and specificity (82 %), suggesting their potential as diagnostic tools. The combination of both antigens exhibited diagnostic accuracy, further supporting their utility in clinical settings. The results of this study underscore the remarkable antigenicity of these epitopes and underscore the suitability of the assay for evaluating community immunity levels against SARS-CoV2 and vaccine efficacy.

Nirmatrelvir is a substrate-related inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro) that is clinically used in combination with ritonavir to treat COVID-19. Derivatives of nirmatrelvir, modified at the substrate P2-equivalent position, have been developed to fine-tune inhibitor properties and are now in clinical use. We report the synthesis of nirmatrelvir derivatives with a (R)-4,4-dimethyl-4-silaproline (silaproline) group at the P2-equivalent position. Mass spectrometry (MS)-based assays demonstrate that silaproline-bearing nirmatrelvir derivatives efficiently inhibit isolated recombinant Mpro, albeit with reduced potency compared to nirmatrelvir. Investigations with SARS-CoV-2 infected VeroE6 cells reveal that the silaproline-bearing inhibitors with a CF3 group at the P4-equivalent position inhibit viral progression, implying that incorporating silicon atoms into Mpro inhibitors can yield in vivo active inhibitors with appropriate optimization. MS and crystallographic studies show that the nucleophilic active site cysteine residue of Mpro (Cys145) reacts with the nitrile group of the silaproline-bearing inhibitors. Substituting the electrophilic nitrile group for a non-activated terminal alkyne shifts the inhibition mode from reversible covalent inhibition to irreversible covalent inhibition. One of the two prochiral silaproline methyl groups occupies space in the S2 pocket that is unoccupied in Mpro:nirmatrelvir complex structures, highlighting the value of sila-derivatives in structure-activity-relationship (SAR) studies. The combined results highlight the potential of silicon-containing molecules for inhibition of Mpro and, by implication, other nucleophilic cysteine enzymes.

Coinfections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus have represented a major health concern since the beginning of the COVID-19 pandemic. The continued spread and constant emergence of new SARS-CoV-2 variants mean that cocirculation and coinfection with seasonal respiratory viruses will continue. Despite the considerable contribution of influenza B virus (IBV) infections to global disease burdens, its interactions with SARS-CoV-2 remain largely unstudied. In this study, we sequentially coinfected lung epithelial cells with representative SARS-CoV-2 variants and IBV strains. We found that prior infection with IBV impaired SARS-CoV-2 D614G, Delta and Omicron BA.1 replication, but did not affect replication of the more recent Omicron EG.5.1 variant. We additionally show that pre-infection with SARS-CoV-2 reduces live attenuated influenza vaccine (LAIV) replication, suggesting vaccine effectiveness in children carrying SARS-CoV-2 pre-infections can be negatively impacted in coinfection. Both SARS-CoV-2 and IBV induced strong type III interferon (IFN) responses, whereas SARS-CoV-2 drove type I IFN production not seen in IBV infection, suggesting viral interference through specific IFN responses. Treatment with innate immune response inhibitors BX795 and Ruxolitinib abrogated viral interference between IBV and SARS-CoV-2 in coinfection, demonstrating that IFN-stimulated gene (ISG) responses play a vital role in viral interference. More specifically, we show that the magnitude and timing of ISG expression, triggered by the primary infecting virus in sequential coinfection, facilitates viral interference between IBV and SARS-CoV-2.

Viral infectious diseases have been seriously affecting human life and health. SARS-CoV-2 was the pathogen that caused Coronavirus Disease 2019 (COVID-19), and the impact of COVID-19 is still existing. Enterovirus 71 (EV71) is the primary pathogen of hand, foot, and mouth disease (HFMD), and no effective direct-acting antiviral drugs targeting EV71 has been approved yet. Innate antiviral strategies play an important role in preventing virus infections depending on the powerful immune regulatory system of body, while viruses have evolved to exploit diverse methods to overcome immune response. Viral proteases, which are known in cleaving viral polyproteins, have also been found to modulate the innate immunity of host cells, thereby promoting viral proliferation. Herein, we reviewed the current development of SARS-CoV-2 3CLpro, PLpro, and EV71 3Cpro and 2Apro, mainly including structure, function, modulation of immune response, and inhibitors of these four proteases, to further deepen the understanding of viral pathogenesis and provide a new perspective for subsequent corresponding drug development.

The global outbreak of the novel coronavirus has renewed interest in related viral pathogens, including canine coronavirus (CCoV), which causes severe gastroenteritis, diarrhea, and vomiting in dogs worldwide. While cases of CCoV have been reported in China, specific instances in the Guangxi Zhuang Autonomous Region-a major center for dog breeding and consumption-have not been documented. In this study, we collected spleen tissue samples from dogs in Yulin city and conducted meta-transcriptomic sequencing. Bioinformatics analysis confirmed CCoV presence in these samples. Furthermore, virus screening and phylogenetic analyses identified the circulation of two CCoV genotypes within the dog population, revealing an overall prevalence of 14.2 %, with CCoV-IIb being the predominant genotype. Notably, two significant recombination events were detected among the analyzed strains. These findings provide valuable insights into the presence and genetic diversity of CCoV Yulin's dog populations, enhancing the understanding of its genetic variation and evolution.

Antivirals have provided important protection against COVID-19, however, the emergence of SARS-CoV-2 variants and drug-resistant mutants calls for the development of novel anti-coronavirus drugs with alternative mechanisms of action. The nonstructural protein 13 (nsp13) of SARS-CoV-2 plays a conserved role in the replication of coronaviruses and has been identified as a promising target. In this study, we report a series of 4-((quinolin-8-ylthio)methyl)benzamide derivatives as inhibitors of SARS-CoV-2 nsp13. Through structure-activity relationship (SAR) analyses, we identified compound 6r, which demonstrated potent inhibition of nsp13 with an IC50 value of 0.28 ± 0.11 μM. Collectively, we discovered a new potent SARS-CoV-2 nsp13 inhibitor, which could be taken as a promising lead compound for further drug development targeting SARS-CoV-2 nsp13.

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.

It has been five years since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we are also approaching the five-year mark of the COVID-19 pandemic. The vaccine is a significant weapon in combating infectious diseases like SARS-CoV-2. Several vaccines were developed against SARS-CoV-2, and they demonstrated efficacy and safety during these five years. The rapid development of multiple next-generation vaccine candidates in different platforms with very little time is the success story of the vaccine development endeavor. This remarkable success of rapid vaccine development is a new paradigm for fast vaccine development that might help develop infectious diseases and fight against the pandemic. With the completion of five years since the beginning of SARS-CoV-2 origin, we are looking back on the five years and reviewing the milestones, vaccine platforms, animal models, clinical trials, successful collaborations, vaccine safety, real-world effectiveness, and challenges. Lessons learned during these five years will help us respond to public health emergencies and to fight the battle against future pandemics.

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

The SARS-CoV-2 virus has undergone extensive mutations over time, resulting in considerable genetic diversity among circulating strains. This diversity directly affects important viral characteristics, such as transmissibility and disease severity. During a viral outbreak, the rapid mutation rate produces a large cloud of variants, referred to as a viral quasispecies. However, many variants are lost due to the bottleneck of transmission and survival. Advances in next-generation sequencing have enabled continuous and cost-effective monitoring of viral genomes, but constructing reliable phylogenetic trees from the vast collection of sequences in GISAID (the Global Initiative on Sharing All Influenza Data) presents significant challenges. We introduce a novel graph-based framework inspired by quasispecies theory, the Variant Evolution Graph (VEG), to model viral evolution. Unlike traditional phylogenetic trees, VEG accommodates multiple ancestors for each variant and maps all possible evolutionary pathways. The strongly connected subgraphs in the VEG reveal critical evolutionary patterns, including recombination events, mutation hotspots, and intra-host viral evolution, providing deeper insights into viral adaptation and spread. We also derive the Disease Transmission Network (DTN) from the VEG, which supports the inference of transmission pathways and super-spreaders among hosts. We have applied our method to genomic data sets from five arbitrarily selected countries - Somalia, Bhutan, Hungary, Iran, and Nepal. Our study compares three methods for computing mutational distances to build the VEG, sourmash, pyani, and edit distance, with the phylogenetic approach using Maximum Likelihood (ML). Among these, ML is the most computationally intensive, requiring multiple sequence alignment and probabilistic inference, making it the slowest. In contrast, sourmash is the fastest, followed by the edit distance approach, while pyani takes more time due to its BLAST-based computations. This comparison highlights the computational efficiency of VEG, making it a scalable alternative for analyzing large viral data sets.

Emerging high-consequence infectious disease outbreaks (EHCIDs) have significantly impacted healthcare systems worldwide. Nurses specialising in outbreak response nurses play an important role in the prevention and control of EHCIDs. However, the specific competencies required for these nurses remain undefined. This study aims to develop a competency model for nurses involved in managing EHCIDs, providing a foundation for the training and organisation of specialised nurse response teams.

We recruited a geographically stratified sample of nurses from tertiary hospitals in four representative regions of China (Eastern, Western, Northern, Southern) for this cross-sectional study, conducted from April 2021 to April 2023. Using the validated Behavioural Event Interview (BEI) methodology, we conducted qualitative research with 40 nurses from hospitals designated for COVID-19 treatment. Participants were asked to describe three successful scenarios and three challenging experiences they encountered during recent pandemic responses with semi-structured interviews. We analysed the qualitative data using an iterative constant comparison method, employing a three-phase coding process (open, axial, selective) facilitated by NVivo 12.0 software. This analysis culminated in an evidence-based competency model. To ensure the model's reliability and validity, we performed categorical consistency checks and synchronous cross-validity testing‌.

The study included 20 excellent nurses and 20 average nurses. We developed a tailored nursing competency model comprising 20 total competencies, categorised into core (6 competencies), critical (7 competencies), and foundational (7 competencies). Inter-coder reliability was robust, with Cohen's κ ranging from 0.741 to 0.830, and an overall agreement of 0.773. Significant differences in competency scores (p < 0.05) were noted between high-performing and adequately-performing nurses, particularly in core and critical competencies.

The competency model for outbreak response nursing is scientifically robust and practically applicable. It is particularly crucial for effective nursing management in the face of sudden and uncertain infectious disease outbreaks.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has undergone continuous evolution, leading to the emergence of variants with altered transmissibility and immune evasion. For the non-structural proteins (Nsps) of SARS-CoV-2, there are limited structural analyses of their naturally occurring mutations. Here, we identified four non-synonymous single-nucleotide polymorphisms (nsSNPs) in the Epsilon lineage of SARS-CoV-2 within Nsp15, an endoribonuclease critical for immune evasion. Of these Epsilon nsSNPs, E266Q is in the catalytic domain. This study investigates the effects of this on enzymatic activity, structural stability, and oligomeric assembly by serial crystallography. By solving the structure of the Nsp15 hexamer at room temperature of both Nsp15-E266Q and WT in the P21 space group to 3 Å, we observed asymmetric motions within its trimer subunits, a feature not visible in previously reported higher-symmetry space groups. These asymmetric motions resemble substrate-induced conformational changes reported in RNA-bound Nsp15 structures, suggesting functional relevance. Biochemical analyses further reveal that Nsp15-E266Q exhibited significantly higher enzymatic activity and thermal stability compared to the wild-type protein. These findings highlight how mutations in Nsp15 contribute to viral replication and immune evasion, offering insights into the molecular mechanisms underlying SARS-CoV-2 variant evolution and potential therapeutic strategies.

Early prediction of critical COVID-19 disease is crucial for an optimal clinical management. The objective of this study was to optimize predictive models for critical COVID-19 disease. Clinical data, laboratory data and genetic polymorphisms were integrated into AI models to compare the performance of different machine learning algorithms.

Data from 155 inpatients were analyzed, 23 of whom developed critical disease. A univariate analysis was performed to assess potential correlations between seven SNPs, nine clinical variables and 10 laboratory parameters at admission.

Of the 7 SNPs, only three SNPs demonstrated a significant association with critical disase, namely: rs77534576, rs10774671 and rs10490770. The ensemble models exhibited the best performance: Random Forest (AUC=0.989), XGBoost (AUC=0.954) and AdaBoost (AUC=0.927). Variable importance varied across models, with age, C-reactive protein, heart diseases and the three SNPs being the most influential features. The predictive power of models improved with the integration of the three SNPs, as compared to previous studies where genetic data were not included. Internal validation confirmed the superiority and stability of the ensemble models.

Machine learning models may help predict progression into critical COVID-19-disease. The predictive power of models improves when SNPs associated with COVID-19 severity are integrated with laboratory and clinical data. Prior to implementation in clinical practice, larger studies in different populations are needed to validate and support the generalization of these results.

The SARS-CoV-2 pandemic has caused a global health crisis due to its high pathogenicity. The SARS-Unique Domain (SUD) in the non-structural protein Nsp3 of SARS-CoV-2 is hypothesized to play a critical role in viral replication and pathogenesis by interacting with host RNA G-quadruplex (G4) structures, but the molecular mechanisms remain unclear. In this study, we used a multidisciplinary approach, including fluorescence assays, CD, single-molecule FRET, SAXS, G4-unfolding experiments and MD simulations, to investigate the interaction between SUD and RNA G4 structures. We found that SUD exhibited a strong binding affinity for RNA three-layer G4 structures with 3'-tail preference but did not unfold G4; instead, it stabilized the G4 conformation, suggesting a role in modulating viral RNA stability and translation. SAXS revealed that G4 binds to a surface groove formed by the N- and C-termini of SUD, enhancing its conformational stability. MD simulations identified key interaction sites and confirmed the induced-fit binding mechanism. These findings provide critical insights into the role of SUD in modulating viral RNA stability and translation, and offers potential targets for antiviral strategies targeting SUD/G4 interactions.

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.

The COVID-19 disease has spread rapidly across the world within just six months, affecting 169 million people and causing 3.5 million deaths globally (2021). The most affected countries include the USA, Brazil, India, and several European countries such as the UK and Russia. Healthcare professionals face new challenges in finding better ways to manage patients and save lives. In this regard, more comprehensive research is needed, including genomic and proteomic studies, personalized medicines and the design of suitable treatments. However, finding novel molecular entities (NME) using a standard or de novo strategy to drug development is a time-consuming and costly process. Another alternate strategy is discovering new therapeutic uses for old/existing/available medications, known as drug repurposing. There are a variety of computational repurposing methodologies, and some of them have been used to counter the coronavirus disease pandemic of 2019 (COVID-19). This review article compiles recently published data on the origin, transmission, pathogenesis, diagnosis, and management of the coronavirus by drug repurposing and vaccine development approach. We have attempted to screen probable drugs in clinical trials by using literature survey. This systematic review aims to create priorities for future research of drugs repurposed and vaccine development for COVID-19.

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

Pancreatic cancer is a highly aggressive gastrointestinal malignancy, often termed the "king of cancers" due to its notoriously high mortality rate. Its clinical characteristics, including late diagnosis, low surgical resectability, high recurrence rates, significant chemoresistance, and poor prognosis have collectively driven the persistent rise in incidence and mortality. Despite ongoing advancements in therapeutic strategies, the management of pancreatic cancer, particularly at advanced stages, remains challenging. Chemotherapy remains the mainstay of current treatment. However, the prevalent problem of chemotherapy resistance poses a significant obstacle to effective treatment. Metabolic reprogramming, characterized by alterations in glucose metabolism, lipid biosynthesis, and amino acid utilization, supports the high energy demands and rapid proliferation of cancer cells. Emerging evidence suggests that these metabolic changes, possibly mediated by epigenetic mechanisms, also contribute to tumorigenesis and metastasis. These findings highlight the critical role of metabolic alterations in pancreatic cancer pathogenesis. This review explores the relationship between metabolic reprogramming and chemotherapy resistance, discussing underlying mechanisms and summarizing preclinical studies and drug development targeting metabolism. The aim is to provide a comprehensive perspective on potential therapeutic strategies for pancreatic cancer.

TLR7, which encodes a key receptor for single-stranded RNA (ssRNA) virus of the innate immune system, was recently associated with X-linked immunodeficiency and COVID-19 susceptibility. This study investigates the association between TLR7 variants and susceptibility to severe COVID-19 in a multicentric Spanish cohort. The TLR7 gene was sequenced in a cohort of 365 COVID-19 patients, stratified into two groups: one comprising mild and asymptomatic patients, considered as controls (n = 87), and the other consisting of moderate to severely affected patients hospitalized due to COVID-19 pneumonia, considered as cases (n = 278). A total of 152 unique TLR7 variants were identified, of note, six rare variants were identified in 11 cases (3.96%), all of whom belonged to the case group. The functional impact of rare TLR7 variants was assessed using a luciferase reporter assay and revealed that N215S is a loss-of-function (LOF) variant, while D332G exhibits an hypomorphic behavior. Conversely, H90Y, V219I, A448V, and R902K maintained normal signaling. No skewed X-inactivation was observed in female carriers of N215S or D332G. In addition, the common variants Q11L (rs179008), c.4-151A>G (rs179009) and c.*881C>G (rs3853839) were associated with severe pneumonia, while c.4-151A>G (rs179009) was specifically linked to Intensive Care Unit (ICU) admission. These findings highlight the role of TLR7 in antiviral immune response and its association with severe COVID-19 in men. The luciferase assay proves to be a reliable tool for evaluating TLR7 signaling, effectively distinguishing between neutral, LOF, and gain-of-function (GOF) variants. Further research is needed to better understand TLR7 variants and its implications in immunodeficiency and immune dysregulation.

Emerging evidence suggests a potential association between periodontitis and adverse outcomes in COVID-19. Both conditions share risk factors and exhibit similar immune dysregulation, including elevated pro-inflammatory cytokines, altered myeloid compartments, and T-cell dysfunction. SARS-CoV-2 uses angiotensin-converting enzyme type 2 and transmembrane protease serine 2 membrane proteins, highly expressed in the oral cavity, for cellular entry. Periodontitis may exacerbate COVID-19 through mechanisms such as oral microbe aspiration, increased viral receptor expression, and systemic inflammation. The shared immunopathogenesis, characterized by cytokine storms and perturbed immune profiles, suggests periodontitis can predispose patients to more severe COVID-19 outcomes. This article aims to review the associations between periodontitis and the severity of COVID-19 and the possible immune mechanisms involved.

The coronavirus disease 2019 pandemic has highlighted the importance and challenges of the sample collection component of the diagnostic cycle. Although combined nasopharyngeal and oropharyngeal swabs (NOS) have historically been the gold standard of sampling, the saline gargle (SG) sampling method has been evaluated and implemented in multiple jurisdictions for respiratory pathogen detection. It has proven to be user-acceptable to patients, simple to collect, and highly sensitive to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by molecular methods when compared to swabs. We performed a prospective cross-sectional study to evaluate the SG collection method against the NOS collection method for molecular detection and next-generation sequencing (NGS) of SARS-CoV-2 in Botswana. Paired SG and NOS samples were collected and underwent nucleic acid extraction prior to molecular detection. The SG had an overall sensitivity of 81.3% (95% CI: 68.8%%-96.0%), while the NOS had an overall sensitivity of 96.9% (95% CI: 84.3-99.4). Paired samples with a mean crossing threshold value of <35 also underwent NGS. SG specimens had a median genome coverage of 94.7% (interquartile range [IQR] 87.0%-99.2%) and NOS specimens had a median genome coverage of 99.6% (IQR 90.0%-99.6%). Bioinformatics analysis showed the 15 successfully matched pairs belong to clades BA.1 and BA.2 indicative of the Omicron variant. Further analysis at the nucleotide level showed a mean similarity of 99.998% ± 0.00465% between NOS and SG. This method has the potential to overcome the challenges that come with swab-based sampling for SARS-CoV-2 testing and may be an alternative in testing for other viral pathogens.

During the coronavirus disease 2019 (COVID-19) pandemic, a major challenge has been inadequate sampling for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pediatric patients posed additional challenges with sample collection, and they and others are also at risk of rare complications from swab collection. Saline gargle (SG) sampling method has been evaluated and introduced as an alternative to swab collection in several jurisdictions. Our study affirms the acceptable performance of the saline gargle method for the molecular detection of SARS-CoV-2 and also establishes that SG samples do not pose an obstacle for genomic sequencing of SARS-CoV-2. The SG method may be a reliable alternative for SARS-CoV-2 detection and next-generation sequencing, facilitating COVID-19 surveillance efforts in resource-constraint settings.

In the course of epidemics, the pathogen may mutate to acquire a higher fitness. At the same time, such a mutant is automatically in an unfavorable position because the resident virus has a head start in accessing the pool of susceptible individuals. We considered a class of tunable small-world networks, where a parameter, p (the rewiring probability), characterizes the prevalence of non-local connections, and we asked, whether the underlying network can influence the fate of a mutant virus. Under an SIR model, we considered two measures of mutant success: the expected height of the peak of mutant infected individuals, and the total number of recovered from mutant individuals at the end of the epidemic. Using these measures, we have found the existence of an optimal (for an advantageous mutant virus) rewiring probability that promotes a larger infected maximum and a larger total recovered population corresponding to the advantageous pathogen strain. This optimal rewiring probability decreases as mean degree and the infectivity of the wild type are increased, and it increases with the mutant advantage. The non-monotonic behavior of the advantageous mutant as a function of rewiring probability may shed light into some of the complex patterns in the size of mutant peaks experienced by different countries during the COVID-19 pandemic.

A series of 3-hydroxy-5-arylpyrazine-2-carboxamides, structural analogs of favipiravir, have been successfully synthesized using a green and sustainable method through a one-pot condensation reaction of arylglyoxals 1a-i and 2-aminopropanediamide 2 in an alkaline solution under heating conditions. The reaction is temperature-sensitive; when conducted at 80 °C, 5-aryl substituted pyrazine derivatives were predominantly obtained. In reactions with arylglyoxals 1a, 1d, 1h, and 1i, temperatures exceeding 80 °C produced a mixture of two regioisomeric pyrazine derivatives with significant efficiency. This method is highly desirable due to its short reaction time, simple purification of products, and the use of water as an eco-friendly solvent.

Coronaviruses have undergone evolutionary changes and mutations in response to the immune pressures exerted by vaccines and environmental factors, resulting in more severe consequences during breakthrough infections. Nevertheless, the specific correlation between the evolutionary mutations of coronaviruses and immune pressures remains ambiguous. Swine coronavirus-porcine epidemic diarrhea virus (PEDV)-has existed for decades. This study utilized in vivo preparation of polyclonal antibodies against the PEDV and identified critical neutralizing epitopes through serial in vitro passaging. Then, the recombinant mutated strains were successfully constructed. In vitro experiments confirmed the ability of the rA1273P strain to escape neutralization by polyclonal antibodies. Both in vitro cell studies and in vivo animal experiments revealed that the strain maintains virulence and pathogenicity while evading antibody pressure post-vaccination. The pathogenicity of the strain while evading immune pressure is comparable to wild-type strains. A comparison of the S protein gene between vaccine strains and clinical strains identified mutations in 1273 amino acid positions in clinical strains. In conclusion, this study identified a novel PEDV S protein neutralizing site under immune pressure through serial passaging, indicating that the 1,273th amino acid position is prone to mutation under prolonged antibody pressure, enhancing the virus's ability to escape hosts. This study offers new insights into the interpretation of coronavirus escape immune pressure and provides technical support for monitoring and predicting the variation and evolution of coronavirus.IMPORTANCECoronaviruses represent an ongoing public health threat because of high variability. Since 2010, the emergence of highly pathogenic porcine epidemic diarrhea virus (PEDV) strains has resulted in significant economic losses to the global pig industry. PEDV undergoes evolution and mutation under external immune pressure, rendering it an increasingly challenging target for prevention and control measures. Here, we prepared the polyclonal antibodies against PEDV and identified a novel neutralization epitope on the S protein (1,273th amino acids) through serial in vitro passaging. Furthermore, our findings indicate that the mutation of A1273P in the S protein did not alter the virulence of the PEDV but significantly enhanced its ability to escape and infect the host in vitro and in vivo. Finally, we found that the 1,273 amino acid position of the S gene has been mutated to varying degrees in clinical PEDV strains. This work provides a specific correlation between the evolutionary mutations of coronaviruses and immune pressures.