The spike protein (SP) is an outward-projecting transmembrane glycoprotein on viral surfaces. SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), responsible for COVID-19 (Coronavirus Disease 2019), uses SP to infect cells that express angiotensin converting enzyme 2 (ACE2) on their membrane. Remarkably, SP has the ability to cross the blood-brain barrier (BBB) into the brain and cause cerebral damage through various pathomechanisms. To combat the COVID-19 pandemic, novel gene-based products have been used worldwide to induce human body cells to produce SP to stimulate the immune system. This artificial SP also has a harmful effect on the human nervous system.

Narrative review.

This narrative review presents the crucial role of SP in neurological complaints after SARS-CoV-2 infection, but also of SP derived from novel gene-based anti-SARS-CoV-2 products (ASP).

Literature searches using broad terms such as "SARS-CoV-2", "spike protein", "COVID-19", "COVID-19 pandemic", "vaccines", "COVID-19 vaccines", "post-vaccination syndrome", "post-COVID-19 vaccination syndrome" and "proteinopathy" were performed using PubMed. Google Scholar was used to search for topic-specific full-text keywords.

The toxic properties of SP presented in this review provide a good explanation for many of the neurological symptoms following SARS-CoV-2 infection and after injection of SP-producing ASP. Both SP entities (from infection and injection) interfere, among others, with ACE2 and act on different cells, tissues and organs. Both SPs are able to cross the BBB and can trigger acute and chronic neurological complaints. Such SP-associated pathologies (spikeopathies) are further neurological proteinopathies with thrombogenic, neurotoxic, neuroinflammatory and neurodegenerative potential for the human nervous system, particularly the central nervous system. The potential neurotoxicity of SP from ASP needs to be critically examined, as ASPs have been administered to millions of people worldwide.

Cellular innate immune pathways are formidable barriers against viral invasion, creating an environment unfavorable for virus replication. Interferons (IFNs) play a crucial role in driving and regulating these cell-intrinsic innate antiviral mechanisms through the action of interferon-stimulated genes (ISGs). The host IFN response obstructs viral replication at every stage, prompting viruses to evolve various strategies to counteract or evade this response. Understanding the interplay between viral proteins and cell-intrinsic IFN-mediated immune mechanisms is essential for developing antiviral and anti-inflammatory strategies. Human coronaviruses (HCoVs), including SARS-CoV-2, MERS-CoV, SARS-CoV, and seasonal coronaviruses, encode a range of proteins that, through shared and distinct mechanisms, inhibit IFN-mediated innate immune responses. Compounding the issue, a dysregulated early IFN response can lead to a hyper-inflammatory immune reaction later in the infection, resulting in severe disease. This review provides a brief overview of HCoV replication and a detailed account of its interaction with host cellular innate immune pathways regulated by IFN.

Porcine deltacoronavirus (PDCoV) is an emerging porcine enteric coronavirus causing significant economic losses to the pig farming industry globally. In this study, we expressed the S protein of a highly virulent PDCoV strain in the CHO eukaryotic expression system. After immunizing alpaca with the PDCoV S protein and employing the phage display library technique, a high-affinity and specific nanobody Nb3 against PDCoV S protein was successfully established by three rounds of biopanning and a phage enzyme-linked immunosorbent assay (ELISA). Furthermore, a competitive ELISA (cELISA) was developed based on Nb3 to rapidly and efficiently detect PDCoV antibody levels. The cELISA displayed no cross-reaction with positive sera of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), or porcine circovirus 2 (PCV2), thereby showing good specificity. The cELISA successfully detected positive sera diluted 1:127 (percentage inhibition ≥ 50.02%), indicating high sensitivity. Both the intra- and inter-batch coefficients of variation were less than 10%, indicating good repeatability. The cELISA had a total coincidence rate of 98.33% with the indirect immunofluorescence assay and a significant positive correlation with the virus neutralization test (r = 0.861, P < 0.001), suggesting that the cELISA can be used to measure the neutralizing antibody titers in serum samples. In conclusion, our nanobody-based cELISA showed good performance indicators and can be used to monitor and evaluate antibody levels following clinical infection of PDCoV or vaccine immunization.

This study screened out a high-affinity and specific nanobody Nb3 against porcine deltacoronavirus (PDCoV) S protein and established a nanobody-based competitive ELISA (cELISA) for PDCoV antibody detection. This cELISA is a simple, rapid, and specific method that can effectively measure the neutralizing antibody titers in serum samples.

Avian infectious bronchitis (IB) is one of the major respiratory diseases in poultry. At present, attenuated vaccines are the main commercial vaccines, but they have many defects. We aimed to construct a novel multi-epitope DNA vaccine based on avian infectious bronchitis virus (IBV) S1 and N proteins for the prevention of IBV infection. We screened the dominant B and T cell epitopes of target proteins utilizing epitope prediction tools. A new high-immunogenicity epitope peptide segment named QSN was designed and screened by linking peptide. The physicochemical properties of QSN were analyzed by bioinformatics. The recombinant plasmid pEGFP-QSN was obtained by inserting the synthesized QSN gene into the eukaryotic expression vector pEGFP-N1. On the 7th day of age, chicks were immunized by intramuscular injection of the plasmid, and serum specific antibody IgG, cytokines IFN-γ and IL-2, and T lymphocyte subsets were detected after booster immunization. Bioinformatics analysis showed that QSN had high hydrophilicity without transmembrane region and stable structure after binding to receptor. The recombinant eukaryotic vector was successfully constructed. Two weeks after booster immunization, compared with NS group and pEGFP-N1 group, serum IgG level, concentrations of cytokines IFN-γ and IL-2, and proportion of CD4+ T lymphocytes in pEGFP-QSN group were significantly increased (P < 0.01 or P < 0.05). Collectively, the multi-epitope DNA could stimulate humoral and cellular immune responses in chickens and is expected to be a potential vaccine candidate against IBV infection.

Avian carcasses collected from 103 flocks on 14 quail farms in Korea between 2022 and 2023 were diagnosed with viral diseases (22 flocks), bacterial disease (58 flocks), parasitic diseases (28 flocks) and non-infectious diseases (60 flocks). The only viral disease identified was viral enteritis in quails that showed pathological lesions in duodenum and appeared to be caused by quail coronavirus (QcoV) through viral metagenomics and RT-PCR assay. Two complete genomes of QCoV from samples diagnosed as viral enteritis were obtained using amplicon-based whole genome sequencing. The two QcoVs were gammacoronavirus, but were distinct from other avian coronaviruses. The spike genes of QCoV have 86.2 ∼ 87.1 % identity with that of American turkey coronavirus, but other gene sequences of QcoV was found to be similar to those of Korean infectious bronchitis virus. Genetic analysis based on the complete genomic sequences found QCoVs had a genetic structure similar to avian coronaviruses, yet it seems to be a unique pathogen specific to quail. This is the first report about the complete genome and genetic analysis of QCoV and the result of disease surveillance in quail in South Korea.

To address the increasing demand for high-quality pork protein, it is essential to implement strategies that enhance diets and produce pigs with excellent production traits. Selective breeding and crossbreeding are the primary methods used for genetic improvement in modern agriculture. However, these methods face challenges due to long breeding cycles and the necessity for beneficial genetic variation associated with high-quality traits within the population. This limitation restricts the transfer of desirable alleles across different genera and species. This article systematically reviews past and current research advancements in porcine molecular breeding. It discusses the screening of clustered regularly interspaced short palindromic repeats (CRISPR) to identify resistance loci in swine and the challenges and future applications of genetically modified pigs.

The emergence of transgenic and gene editing technologies has prompted researchers to apply these methods to pig breeding. These advancements allow for alterations in the pig genome through various techniques, ranging from random integration into the genome to site-specific insertion and from target gene knockout (KO) to precise base and prime editing. As a result, numerous desirable traits, such as disease resistance, high meat yield, improved feed efficiency, reduced fat deposition, and lower environmental waste, can be achieved easily and effectively by genetic modification. These traits can serve as valuable resources to enhance swine breeding programmes.

In the era of genome editing, molecular breeding of pigs is critical to the future of agriculture. Long-term and multidomain analyses of genetically modified pigs by researchers, related policy development by regulatory agencies, and public awareness and acceptance of their safety are the keys to realizing the transition of genetically modified products from the laboratory to the market.

COVID-19, caused by the SARS-CoV-2 virus, is not only characterized by respiratory symptoms but is also associated with a wide range of systemic complications, including significant hematologic abnormalities. This is a comprehensive review of the current literature, using PubMed and Google Scholar, on the pathophysiology and incidence of thromboembolic events in COVID-19 patients and thromboprophylaxis. COVID-19 infection induces a prothrombotic state in patients through the dysregulation of the renin-angiotensin-aldosterone system (RAAS), endothelial dysfunction, elevated von Willebrand factor (vWF), and a dysregulated immune response involving the complement system and neutrophil extracellular traps (NETs). As a result, thromboembolic complications have emerged in COVID-19 cases, occurring more frequently in severe cases and hospitalized patients. These thrombotic events affect both venous and arterial circulation, with increased incidences of deep venous thrombosis (DVT), pulmonary embolism (PE), systemic arterial thrombosis, and myocardial infarction (MI). While DVT and PE are more common, the literature highlights the potential lethal consequences of arterial thromboembolism (ATE). This review also briefly examines the ongoing discussions regarding the use of anticoagulants for the prevention of thrombotic events in COVID-19 patients. While theoretically promising, current studies have yielded varied outcomes: Some suggest potential benefits, whereas others report an increased risk of bleeding events among hospitalized patients. Therefore, further large-scale studies are needed to assess the efficacy and safety of anticoagulants for thromboprophylaxis in COVID-19 patients.

Infectious bronchitis (IB), caused by the infectious bronchitis virus (IBV), is a highly contagious chicken disease, causing economic losses worldwide. New IBV strains and variants continue to emerge despite using inactivated and live-attenuated vaccines to prevent or control IB. In this study, the S1 genes of 46 IBV strains, isolated from commercial chicken flocks between 2003 and 2024 in Korea were sequenced and genetically characterized. The IBV isolates belonged to Korean group II (K-II), which was included in the GI-19 lineage. The K-II was divided into five sub-genogroups (a-e) based on phylogenetic tree analysis results and nucleotide identification of the S1 gene. Of these, K-IId was the most common genotype in Korea; however, eight novel isolates belonging to the K-IIe sub-genotype were discovered. The nucleotide and amino acid identities of the other four K-II sub-genotypes and the eight isolates were 84.42-95.89 % and 84.02-95.86 %, respectively. The complete genomes of the eight K-IIe isolates were obtained using next-generation sequencing. Various recombination patterns were observed despite the high homology of the S1 gene among the eight IBV strains. Among the eight K-IIe isolates, six were recombinants, exhibiting recombinations between K-IIe and K-IIc, K-IIe and K-IIa, and with the live vaccine strain. Most recombination breakpoints were detected in the nsp2 region of the ORF1a, S2, and M genes. The present study proposed new classification criteria for the K-II belonged to the GI-19 lineage prevalent in South Korea and revealed the recombination patterns of recently identified novel isolates, providing important information on novel viral sub-genotype strains and IBV evolution.

COVID-19 pandemic had unprecedented global impact on health and society, highlighting the need for a detailed understanding of SARS-CoV-2 evolution in response to host and environmental factors. This study investigates the evolution of SARS-CoV-2 via mutation dynamics, focusing on distinct age cohorts, geographical location, and vaccination status within the Indian population, one of the nations most affected by COVID-19.

Comprehensive dataset, across diverse time points during the Alpha, Delta, and Omicron variant waves, captured essential phases of the pandemic's footprint in India. By leveraging genomic data from Global Initiative on Sharing Avian Influenza Data (GISAID), we examined the substitution mutation landscape of SARS-CoV-2 in three demographic segments: children (1-17 years), working-age adults (18-64 years), and elderly individuals (65+ years). A balanced dataset of 69,975 samples was used for the study, comprising 23,325 samples from each group. This design ensured high statistical power, as confirmed by power analysis. We employed bioinformatics and statistical analyses, to explore genetic diversity patterns and substitution frequencies across the age groups.

The working-age group exhibited a notably high frequency of unique substitutions, suggesting that immune pressures within highly interactive populations may accelerate viral adaptation. Geographic analysis emphasizes notable regional variation in substitution rates, potentially driven by population density and local transmission dynamics, while regions with more homogeneous strain circulation show relatively lower substitution rates. The analysis also revealed a significant surge in unique substitutions across all age groups during the vaccination period, with substitution rates remaining elevated even after widespread vaccination, compared to pre-vaccination levels. This trend supports the virus's adaptive response to heightened immune pressures from vaccination, as observed through the increased prevalence of substitutions in important regions of SARS-CoV-2 genome like ORF1ab and Spike, potentially contributing to immune escape and transmissibility.

Our findings affirm the importance of continuous surveillance on viral evolution, particularly in countries with high transmission rates. This research provides insights for anticipating future viral outbreaks and refining pandemic preparedness strategies, thus enhancing our capacity for proactive global health responses.

Infectious bronchitis virus (IBV) is the first coronavirus discovered in the world in the early 1930s and despite decades of extensive immunoprophylaxis efforts, it remains a major health concern to poultry producers worldwide. Rapid evolution due to large poultry population sizes coupled with high mutation and recombination events and the reliance of the antiviral immune response on specific antibodies against the epitopes of the S1 glycoprotein, render the control of IBV extremely challenging. The numerous and rapidly evolving genetic and antigenic IBV types are currently classified based on the whole S1 gene sequence, into 36 lineages clustered in eight genotypes. Most lineages (29) are grouped in genotype I (GI). "Variant 2" (Israel/Variant 2/1998) is the prototype strain of lineage GI-23 and, since this lineage emerged during the mid-1990s in the Middle East, it has evolved into numerous genetically related strains and disseminated to five continents. The hallmarks of IBV Variant 2-like strain infections are high virulence and remarkable nephrotropism and nephropathogenicity; however, the molecular mechanisms of these traits remain to be elucidated. Limited protection from previously utilized vaccine strains and accumulated losses to poultry producers have urged the development and implementation of homologous Variant 2-like vaccine strains. The latest avian coronavirus biology with specific emphasis on the cumulative knowledge about IBV "Variant 2" and emergence of related strains, characteristics and control are reviewed.

COVID-19 has caused widespread morbidity and mortality, with its effects extending to multiple organ systems. Despite known risk factors for severe disease, including advanced age and underlying comorbidities, patient outcomes can vary significantly. This variability complicates efforts to predict disease progression and tailor treatment strategies. While diagnostic and therapeutic approaches are still under debate, RNA sequencing (RNAseq) has emerged as a promising tool to provide deeper insights into the pathophysiology of COVID-19 and guide personalized treatment. A comprehensive literature review was conducted using PubMed, Scopus, Web of Science, and Google Scholar. We employed Medical Subject Headings (MeSH) terms and relevant keywords to identify studies that explored the role of RNAseq in COVID-19 diagnostics, prognostics, and therapeutics. RNAseq has proven instrumental in identifying molecular biomarkers associated with disease severity in patients with COVID-19. It allows for the differentiation between asymptomatic and symptomatic individuals and sheds light on the immune response mechanisms that contribute to disease progression. In critically ill patients, RNAseq has been crucial for identifying key genes that may predict patient outcomes, guiding therapeutic decisions, and assessing the long-term effects of the virus. Additionally, RNAseq has helped in understanding the persistence of viral RNA after recovery, offering new insights into the management of post-acute sequelae, including long COVID. RNA sequencing significantly improves COVID-19 management, particularly for critically ill patients, by enhancing diagnostic accuracy, personalizing treatment, and predicting therapeutic responses. It refines patient stratification, improving outcomes, and holds promise for targeted interventions in both acute and long COVID.

Developing a broad-spectrum antiviral is imperative in light of the recent emergence of recurring viral infections. The critical role of host-virus attachment and membrane fusion during enveloped virus entry is a suitable target for developing broad-spectrum antivirals. A new class of flavonoid-based fusion inhibitors are designed to alter the membrane's physical properties. These flavonoid-based molecules (MFDA; myristoyl flavonoid di-aspartic acid) are self-assembled in the membrane, creating distinct nanodomains and effectively inhibiting membrane fusion by modulating the membrane's interfacial properties. The broad-spectrum antiviral efficacy of these compounds are established in effectively blocking the entry of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Type A Influenza, Human coronavirus OC43 (HCoV-OC43), and Vesicular stomatitis virus (VSV). A slightly more effectivity of MFDA in coronavirus infection than other enveloped viruses may be attributed to its secondary interaction with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. A membrane nanodomain formation strategy is highlighted with natural-product-based fusion inhibitors, effectively thwarting the infection of several enveloped viruses, entailing their broad-spectrum antiviral functionality.

Porcine deltacoronavirus (PDCoV), also known as HKU15, is a swine enteropathogenic virus that is believed to have originated in birds. PDCoV belongs to the genus Deltacoronavirus (DCoV), the members of which have mostly been identified in diverse avian species. We recently reported that chicken or porcine aminopeptidase N (APN), the major cellular receptor for PDCoV, can mediate cellular entry via three pseudotyped retroviruses displaying spike proteins from three avian DCoVs (HKU11, HKU13, and HKU17). In the present work, to better understand how avian-origin CoVs may be transmitted to pigs, we investigated the unknown DCoV entry pathway in avian cells. We show that clathrin-mediated endocytosis is involved in the entry of these DCoV pseudoviruses into chicken-origin DF-1 cells. Pseudovirus entry was suppressed by means of pharmacological inhibitors, dominant-negative mutants, and siRNAs targeting various cellular proteins and signalling molecules, suggesting that PDCoV and avian DCoV pseudovirus entry into DF-1 cells depends on clathrin, dynamin-2, cathepsins and a low-pH environment but is independent of caveolae and macropinocytosis. Furthermore, we found that DCoV pseudovirus entry was linked to Rab5- and Rab7-dependent pathways. This is the first report demonstrating that these DCoVs utilize clathrin-mediated endocytosis pathways to enter avian-origin cells, providing new insights into interspecies transmission of DCoVs.

The present study explores the conformational dynamics of the membrane protein of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) within the Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC) complex using an all-atomistic molecular dynamics simulation approach. Significant structural changes were observed in the N-terminal, C-terminal, transmembrane, and beta-sheet sandwich domains of the MERS-CoV membrane protein. This study also highlights the structural similarities between the MERS-CoV and the SARS-CoV-2 membrane proteins, particularly in how both exhibit a distinct kink in the transmembrane helix caused by aromatic residue-lipid interactions. A structural expansion below the transmembrane and above the beta-sheet sandwich domain within the dimer was observed in all the M-proteins. This site on the beta-sheet sandwich domains near the C-terminal end could serve as a potential drug-binding site. Notably, a stable helical structure was identified in the C-terminal domain of the MERS-CoV membrane protein, whereas a proper secondary structural conformation was not observed in the SARS-CoV-2 membrane protein. Further, the SARS-CoV-2 membrane protein exhibited stronger binding to the lipid bilayer than the MERS-CoV, indicating its greater structural stability within the ERGIC complex. The structural similarity between the membrane protein of MERS-CoV and SARS-CoV-2 suggests the feasibility of employing a common inhibitor against these beta-coronaviruses. Furthermore, this analysis enhances our understanding of the membrane protein's interactions with proteins and lipids, paving the way for therapeutic developments against these viruses.

Myocarditis is a potentially fatal complication of coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. COVID-19 myocarditis appears to have distinct inflammatory characteristics that distinguish it from other viral etiologies. COVID-19 myocarditis can present with symptoms ranging from dyspnea and chest pain to acute heart failure and death. It is critical to detect any cases of myocarditis, especially fulminant myocarditis, which can be characterized by signs of heart failure and arrhythmias. Serial troponins, echocardiography, and electrocardiograms should be performed as part of the initial workup for suspected myocarditis. The second step in detecting myocarditis is cardiac magnetic resonance imaging and endomyocardial biopsy. Treatment for COVID-19 myocarditis is still debatable; however, combining intravenous immunoglobulins and corticosteroids may be effective, especially in cases of fulminant myocarditis. Overall, more research is needed to determine the incidence of COVID-19 myocarditis, and the use of intravenous immunoglobulins and corticosteroids in combination requires large randomized controlled trials to determine efficacy. The purpose of this review is to summarize current evidence on the subject.

The global COVID-19 pandemic, caused by SARS-CoV-2, has led to significant morbidity and mortality, with a profound impact on cardiovascular health. This review investigates the mechanisms of SARS-CoV-2's interaction with cardiac tissue, particularly emphasizing the role of the Spike protein and ACE2 receptor in facilitating viral entry and subsequent cardiac complications. We dissect the structural features of the virus, its interactions with host cell receptors, and the resulting pathophysiological changes in the heart. Highlighting SARS-CoV-2's broad organ tropism, especially its effects on cardiomyocytes via ACE2 and TMPRSS2, the review addresses how these interactions exacerbate cardiovascular issues in patients with pre-existing conditions such as diabetes and hypertension. Additionally, we assess both direct and indirect mechanisms of virus-induced cardiac damage, including myocarditis, arrhythmias, and long-term complications such as 'long COVID'. This review underscores the complexity of SARS-CoV-2's impact on the heart, emphasizing the need for ongoing research to fully understand its long-term effects on cardiovascular health. Key words: COVID-19, Heart, ACE2, Spike protein, Cardiomyocytes, Myocarditis, Long COVID.

Background: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for causing the Coronavirus disease 2019 (COVID-19) outbreak. While mutations cause the emergence of new variants, the ancestral SARS-CoV-2 strain is unique among other strains. Methods: Various clinical parameters, the activity of cathepsin proteases, and the concentration of various proteins were measured in urine samples from COVID-19-negative participants and COVID-19-positive participants. Urinary extracellular vesicles (uEVs) were isolated from urine samples from the two groups and used for proteomic analysis and subsequent pathway analyses. Results: Activity levels of cathepsin S and L were greater in the urine of COVID-19-positive participants. The concentration of C-reactive protein, transmembrane serine protease 2, and klotho protein were significantly greater in the urine of COVID-19-positive participants. There was a greater amount of uEVs in the COVID-19 group and klotho protein was found to be enriched in uEVs from the COVID-19 group. Pathway analyses of the proteomics data showed most of the identified proteins were involved in signal transduction, stress response, protein metabolism, and transport. The identified proteins were predominantly associated with cellular membranes and with function of the cytoskeleton, enzyme regulation, and signal transduction. Conclusions: Taken together, our data identify novel urinary biomarkers that could be used to further investigate the long-term effects of SARS-CoV-2 infection.

Membrane fusion is an essential component of the viral lifecycle that allows the delivery of the genetic information of the virus into the host cell. Specialized viral glycoproteins exist on the surface of mature virions where they facilitate fusion through significant conformational changes, ultimately bringing opposing membranes into proximity until they eventually coalesce. This process can be positively influenced by a number of specific cellular factors such as pH, enzymatic cleavage, divalent ions, and the composition of the host cell membrane. In this review, we have summarized how anionic lipids have come to be involved in viral fusion and how the endosomal resident anionic lipid BMP has become increasingly implicated as an important cofactor for those viruses that fuse via the endocytic pathway.

Following COVID-19 outbreak with its unprecedented effect on the entire world, the interest to the coronaviruses increased. The causative agent of the COVID-19, severe acute respiratory syndrome coronavirus - 2 (SARS-CoV-2) is one of seven coronaviruses that is pathogenic to humans. Others include SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63 and HCoV-229E. The viruses differ in their pathogenicity. SARS-CoV, MERS-CoV, and SARS-CoV-2 are capable to spread rapidly and cause epidemic, while HCoV-HKU1, HCoV-OC43, HCoV-NL63 and HCoV-229E cause mild respiratory disease. The difference in the viral behavior is due to structural and functional differences. All seven human coronaviruses possess four structural proteins: spike, envelope, membrane, and nucleocapsid. Spike protein with its receptor binding domain is crucial for the entry to the host cell, where different receptors on the host cell are recruited by different viruses. Envelope protein plays important role in viral assembly, and following cellular entry, contributes to immune response. Membrane protein is an abundant viral protein, contributing to the assembly and pathogenicity of the virus. Nucleocapsid protein encompasses the viral RNA into ribonucleocapsid, playing important role in viral replication. The present review provides detailed summary of structural and functional characteristics of structural proteins from seven human coronaviruses, and could serve as a practical reference when pathogenic human coronaviruses are compared, and novel treatments are proposed.

Cell entry of severe acute respiratory coronavirus-2 (SARS-CoV-2) and other CoVs can occur via two distinct routes. Following receptor binding by the spike glycoprotein, membrane fusion can be triggered by spike cleavage either at the cell surface in a transmembrane serine protease 2 (TMPRSS2)-dependent manner or within endosomes in a cathepsin-dependent manner. Cellular sialoglycans have been proposed to aid in CoV attachment and entry, although their functional contributions to each entry pathway are unknown. In this study, we used genetic and enzymatic approaches to deplete sialic acid from cell surfaces and compared the requirement for sialoglycans during endosomal and cell-surface CoV entry using lentiviral particles pseudotyped with the spike proteins of different sarbecoviruses. We show that entry of SARS-CoV-1, WIV1-CoV and WIV16-CoV, like the SARS-CoV-2 omicron variant, depends on endosomal cathepsins and requires cellular sialoglycans for entry. Ancestral SARS-CoV-2 and the delta variant can use either pathway for entry, but only require sialic acid for endosomal entry in cells lacking TMPRSS2. Binding of SARS-CoV-2 spike protein to cells did not require sialic acid, nor was sialic acid required for SARS-CoV-2 entry in TMRPSS2-expressing cells. These findings suggest that cellular sialoglycans are not strictly required for SARS-CoV-2 attachment, receptor binding or fusion, but rather promote endocytic entry of SARS-CoV-2 and related sarbecoviruses. In contrast, the requirement for sialic acid during entry of MERS-CoV pseudoparticles and authentic HCoV-OC43 was not affected by TMPRSS2 expression, consistent with a described role for sialic acid in merbecovirus and embecovirus cell attachment. Overall, these findings clarify the role of sialoglycans in SARS-CoV-2 entry and suggest that cellular sialoglycans mediate endosomal, but not cell-surface, SARS-CoV-2 entry.

Coronaviruses (CoVs) pose a major risk to global public health due to their ability to infect diverse animal species and potential for emergence in humans. The CoV spike protein mediates viral entry into the cell and plays a crucial role in determining the binding affinity to host cell receptors. With particular emphasis on α- and β-coronaviruses that infect humans and domestic animals, current research on CoV receptor use suggests that the exploitation of the angiotensin-converting enzyme 2 (ACE2) receptor poses a significant threat for viral emergence with pandemic potential. This review summarizes the approaches used to study binding interactions between CoV spike proteins and the human ACE2 (hACE2) receptor. Solid-phase enzyme immunoassays and cell binding assays allow qualitative assessment of binding but lack quantitative evaluation of affinity. Surface plasmon resonance, Bio-layer interferometry, and Microscale Thermophoresis on the other hand, provide accurate affinity measurement through equilibrium dissociation constants (KD). In silico modeling predicts affinity through binding structure modeling, protein-protein docking simulations, and binding energy calculations but reveals inconsistent results due to the lack of a standardized approach. Machine learning and deep learning models utilize simulated and experimental protein-protein interaction data to elucidate the critical residues associated with CoV binding affinity to hACE2. Further optimization and standardization of existing approaches for studying binding affinity could aid pandemic preparedness. Specifically, prioritizing surveillance of CoVs that can bind to human receptors stands to mitigate the risk of zoonotic spillover.

Porcine deltacoronavirus (PDCoV) is an emerging pathogen that can cause severe diarrhoea and high mortality in suckling piglets. Moreover, evidence of PDCoV infection in humans has raised concerns regarding potential public health risks. To identify potential therapeutic targets for PDCoV, we performed a genome-wide CRISPR/Cas9 library screening to find key host factors important to PDCoV infection. Several host genes in this screen were enriched, including ANPEP, which encodes the PDCoV receptor aminopeptidase N (APN). Furthermore, we discovered C16orf62, also known as the VPS35 endosomal protein sorting factor like (VPS35L), as an important host factor required for PDCoV infection. C16orf62 is an important component of the multiprotein retriever complex involved in protein recycling in the endosomal compartment and its gene knockout led to a remarkable decrease in the binding and internalization of PDCoV into host cells. While we did not find evidence for direct interaction between C16orf62 and the viral s (spike) protein, C16orf62 gene knockout was shown to downregulate APN expression at the cell surface. This study marks the first instance of a genome-wide CRISPR/Cas9-based screen tailored for PDCoV, revealing C16orf62 as a host factor required for PDCoV replication. These insights may provide promising avenues for the development of antiviral drugs against PDCoV infection.

The nucleocapsid (N) protein of coronaviruses is a structural protein that binds viral RNA for assembly into the mature virion, a process that occurs in the cytoplasm. Several coronavirus N proteins also localize to the nucleus. Herein, we identify that two sequences (NLSs) are required for nuclear localization of the SARS-CoV-2 N protein. Deletion or mutation of these two sequences creates an N protein that does not localize to the nucleus in HEK293T cells. Overexpression of both wild-type and NLS-mutated N proteins dysregulate a largely overlapping set of mRNAs in HEK293T cells, suggesting that these N proteins do not have direct nuclear effects on transcription. Consistent with that hypothesis, both N proteins induce nuclear localization of NF-κB p65 and dysregulate a set of previously identified NF-κB-dependent genes. The effects of N on nuclear properties are proposed to alter host cell functions that contribute to viral pathogenesis or replication.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that infect pigs' intestinal epithelial cells, causing high morbidity and mortality. Due to the rapid mutation of PEDV, vaccine efficacy is uncertain, prompting exploration of alternative treatments. Nanobodies, also known as variable heavy chain domains of heavy chain-only antibodies (VHHs), offer significant potential in biomedical applications due to their small size and high specificity. In this study, yeast two-hybrid technology was employed to screen for eight specific VHH sequences targeting the PEDV S protein from a synthetically constructed nanobody yeast library. The VHH genes were then cloned into expression plasmids for recombinant protein production, and the resulting VHHs (termed PEDV S-VHHs) were purified. Indirect immunofluorescence assay (IFA) and Western blotting analysis confirmed that these VHHs specifically bind to both PEDV and its S protein. Neutralization assays demonstrated that seven PEDV S-VHHs exhibited potent neutralizing activity against PEDV. Additionally, a combination of these seven antibodies showed enhanced antiviral effects. Preliminary predictions were also made regarding the binding sites between these VHHs and PEDV. The PEDV S-VHHs described in this study hold potential as candidates for the prevention and treatment of PEDV infection.

Porcine epidemic diarrhea virus (PEDV) causes the third most important disease in the pig industry, after African swine fever and porcine reproductive and respiratory syndrome, and leads to illness or death of the entire litter, causing significant economic losses. In this study, three PEDV strains (HN-1, HN-2, and SC2023) were isolated from swine farms with suspected PEDV infections in Sichuan and Henan provinces. Phylogenetic analysis based on complete S gene sequences showed that all three strains belonged to the G2c subgroup. HN-1 adapted readily to cell culture, grew to a viral titer as high as 2 × 108 TCID50/mL in Vero cells, and caused the formation of large syncytia. We analyzed the amino acid sequence of the HN-1 isolate and found that its S1 subunit contained a three-amino-acid insertion (355KRL358). A seven-amino-acid-deletion (1377FEKVHVQ1383) in the S2 subunit resulted in the partial deletion of the endocytosis signal YxxΦ and the complete deletion of the endoplasmic reticulum retrieval signal (ERRS) KVHVQ in the cytoplasmic tail of the S protein. Consequently, HN-1 is predicted to be less pathogenic than its parent strain, an attribute that facilitates rapid cell-to-cell spread by enhancing syncytium formation. In addition, strain HN-1 was found to have the mutation 884-885SG→RR, which may favor adaptation to cell culture by providing new trypsin cleavage sites. These results suggest that HN-1 is a G2c subtype variant that adapts well to cell culture and can be used to study the adaptive mechanisms of PEDV and develop attenuated vaccines.

Equine colitis is a devastating disease with a high mortality rate. Infectious pathogens associated with colitis in the adult horse include Clostridioides difficile, Clostridium perfringens, Salmonella spp., Neorickettsia risticii/findlaynesis, and equine coronavirus. Antimicrobial-associated colitis can be associated with the presence of infectious pathogens. Colitis can also be due to non-infectious causes, including non-steroidal anti-inflammatory drug administration, sand ingestion, and infiltrative bowel disease. Current treatments focus on symptomatic treatment (restoring fluid and electrolyte balance, preventing laminitis and sepsis). Intestinal epithelial ion channels are key regulators of electrolyte (especially sodium and chloride) and water movement into the lumen. Dysfunctional ion channels play a key role in the development of diarrhea. Infectious pathogens, including Salmonella spp. and C. difficile, have been shown to regulate ion channels in a variety of ways. In other species, there has been an increased interest in ion channel manipulation as an anti-diarrheal treatment. While targeting ion channels also represents a promising way to manage diarrhea associated with equine colitis, ion channels have not been well studied in the equine colon. This review provides an overview of what is known about colonic ion channels and their known or putative role in specific types of equine colitis due to various pathogens.

The aim of the present review was to highlight natural product investigations in silico and in vitro to find plants and chemicals that inhibit or stimulate angiotensin-converting enzyme 2 (ACE-2).

The global reduction of incidents and fatalities attributable to infections with SARS-CoV-2 is one of the most public health problems. In the absence of specific therapy for coronavirus disease 2019 (COVID-19), phytocompounds generated from plant extracts may be a promising strategy worth further investigation, motivating researchers to evaluate the safety and anti-SARS-CoV-2 effectiveness of these ingredients.

To review phytochemicals in silico for anti-SARS-CoV-2 activity and to assess their safety and effectiveness in vitro and in vivo.

The present review was conducted using various scientific databases and studies on anti-SARS-CoV-2 phytochemicals were analyzed and summarized. The results obtained from the in silico screening were subjected to extraction, isolation, and purification. The in vitro studies on anti-SarcoV-2 were also included in this review. In addition, the results of this research were interpreted, analyzed, and documented on the basis of the bibliographic information obtained.

This review discusses recent research on using natural remedies to cure or prevent COVID-19 infection. The literature analysis shows that the various herbal preparations (extracts) and purified compounds can block the replication or entrance of the virus directly to carry out their anti-SARS-CoV-2 effects. It is interesting to note that certain items can prevent SARS-CoV-2 from infecting human cells by blocking the ACE-2 receptor or the serine protease TMPRRS2. Moreover, natural substances have been demonstrated to block proteins involved in the SARS-CoV-2 life cycle, such as papain- or chymotrypsin-like proteases.

The natural products may have the potential for use singly or in combination as alternative drugs to treat/prevent COVID-19 infection, including blocking or stimulating ACE-2. In addition, their structures may provide indications for the development of anti-SARS-CoV-2 drugs.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging alpha-coronavirus that causes diarrhea in piglets and results in serious economic losses. During SADS-CoV infection, the spike protein (S) serves as a crucial structural component of the virion, interacting with receptors and eliciting the production of neutralizing antibodies. Due to the potential risk of zoonotic transmission of SADS-CoV, the identification and screening of epitopes on the S glycoproteins will be crucial for development of sensitive and specific diagnostic tools. In this study, we immunized BALB/c mice with recombinant SADS-CoV S trimer protein and generated two S1-specific monoclonal antibodies (mAbs): 8D6 and 6E9, which recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 8D6 was mapped to 311NPDQRD316, the minimal fragment recognized by mAb 6E9 was mapped to 492ARFVDRL498. Homology analysis of the regions corresponding to 13 typical strains of different SADS-CoV subtypes showed high conservation of these two epitopes. These findings contribute to a deeper understanding of the structure of the SADS-CoV S protein, which is valuable for vaccine design and holds potential for developing diagnostic methods to detect SADS-CoV.

Feline coronavirus (FCoV), as one of the important pathogens of feline viral gastroenteritis, has been attracting great attention. A total of 1869 rectal and nasal swabs, feces, and ascites samples were collected from eight regions in Guangxi province during 2021-2024. The multiplex RT-qPCR established in our laboratory was used to test these samples for FCoV, and 17.66% (330/1869) of the samples were positive for FCoV. The S, M, and N genes of 63 FCoV-positive samples were amplified and sequenced, and the genetic and evolutionary characteristics were analyzed. Similarity analysis showed that the nucleotide and amino acid homologies of S, M, and N genes were 81.2-99.6% and 70.2-99.5%, 89.9-100% and 91.6-100%, and 90.1-100% and 91.5-100%, respectively. Phylogenetic analysis revealed that all 63 FCoV strains, based on S gene sequences, belonged to type I FCoV (FCoV-I), and were clustered with Chinese strains and the Netherlands UU strains. Recombinant signals were detected in the S gene of strains GXLZ03-2022, GXLZ08-2022, and CCoV GD/2020/X9. The results suggest that FCoV is still prevalent in the Guangxi province of southern China, and the prevalent FCoV strains show high genetic diversity and novel epidemic characteristics.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has filled a gap in our knowledge regarding the prevention of CoVs. Swine coronavirus (CoV) is a significant pathogen that causes huge economic losses to the global swine industry. Until now, anti-CoV prevention and control have been challenging due to the rapidly generated variants. Silver nanoparticles (AgNPs) with excellent antimicrobial activity have attracted great interest for biosafety prevention and control applications. In this study, we synthesized chitosan-modified AgNPs (Chi-AgNPs) with good biocompatibility to investigate their antiviral effects on swine CoVs. In vitro assays showed that Chi-AgNPs could significantly impaired viral entry. The direct interaction between Chi-AgNPs and CoVs can destroy the viral surface spike (S) protein secondary structure associated with viral membrane fusion, which is caused by the cleavage of disulfide bonds in the S protein. Moreover, the mechanism showed that Chi-AgNPs reduced the virus-induced apoptosis of Vero cells via the ROS/p53 signaling activation pathway. Our data suggest that Chi-AgNPs can serve as a preventive strategy for CoVs infection and provide a molecular basis for the viricidal effect of Chi-AgNPs on CoVs.

The coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents the most disastrous infectious disease pandemic of the past century. As a member of the Betacoronavirus genus, the SARS-CoV-2 genome encodes a total of 29 proteins. The spike protein, RNA-dependent RNA polymerase, and proteases play crucial roles in the virus replication process and are promising targets for drug development. In recent years, structural studies of these viral proteins and of their complexes with antibodies and inhibitors have provided valuable insights into their functions and laid a solid foundation for drug development. In this review, we summarize the structural features of these proteins and discuss recent progress in research regarding therapeutic development, highlighting mechanistically representative molecules and those that have already been approved or are under clinical investigation.

Transmissible gastroenteritis virus (TGEV) is an etiological agent of enteric disease that results in high mortality rates in piglets. The economic impact of the virus is considerable, causing significant losses to the pig industry. The development of an efficacious subunit vaccine to provide promising protection against TGEV is of the utmost importance. The viral antigen, spike glycoprotein (S), is widely regarded as one of the most effective antigenic components for vaccine research. In this study, we employed immunoinformatics and molecular dynamics approaches to develop an 'ideal' multi-epitope vaccine. Firstly, the dominant, non-toxic, highly antigenic T (Th, CTL) and B cell epitopes predicted from the TGEV S protein were artificially engineered in tandem to design candidate subunit vaccines. Molecular docking and dynamic simulation results demonstrate that it exhibits robust interactions with toll-like receptor 4 (TLR4). Of particular significance was the finding that the vaccine was capable of triggering an immune response in mammals, as evidenced by the immune simulation results. The humoral aspect is typified by elevated levels of IgG and IgM, whereas the cellular immune aspect is capable of eliciting the robust production of interleukins and cytokines (IFN-γ and IL-2). Furthermore, the adoption of E. coli expression systems for the preparation of vaccines will also result in cost savings. This study offers logical guidelines for the development of a secure and efficacious subunit vaccine against TGEV, in addition to providing a novel theoretical foundation and strategy to prevent associated CoV infections.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has underscored the urgent necessity for the development of antiviral compounds that can effectively target coronaviruses. In this study, we present the first evidence of the antiviral efficacy of hyperforin, a major metabolite of St. John's wort, for which safety and bioavailability in humans have already been established.

Antiviral assays were conducted in cell culture with four human coronaviruses: three of high virulence, SARS-CoV-2, SARS-CoV, and MERS-CoV, and one causing mild symptoms, HCoV-229E. The antiviral activity was also evaluated in human primary airway epithelial cells. To ascertain the viral step inhibited by hyperforin, time-of-addition assays were conducted. Subsequently, a combination assay of hyperforin with remdesivir was performed.

The results demonstrated that hyperforin exhibited notable antiviral activity against the four tested human coronaviruses, with IC50 values spanning from 0.24 to 2.55 µM. Kinetic studies indicated that the observed activity occur at a post-entry step, potentially during replication. The antiviral efficacy of hyperforin was additionally corroborated in human primary airway epithelial cells. The results demonstrated a reduction in both intracellular and extracellular SARS-CoV-2 viral RNA, confirming that hyperforin targeted the replication step. Finally, an additive antiviral effect on SARS-CoV-2 was observed when hyperforin was combined with remdesivir.

In conclusion, hyperforin has been identified as a novel pan-coronavirus inhibitor with activity in human primary airway epithelial cells, a preclinical model for coronaviruses. These findings collectively suggest that hyperforin has potential as a candidate antiviral agent against current and future human coronaviruses.

More than 3 years into the global pandemic, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a significant threat to public health. Immunities acquired from infection or current vaccines fail to provide long term protection against subsequent infections, mainly due to their fast-waning nature and the emergence of variants of concerns (VOCs) such as Omicron. To overcome these limitations, SARS-CoV-2 Spike protein receptor binding domain (RBD)-based epitopes are investigated as conjugates with a powerful carrier, the mutant bacteriophage Qβ (mQβ). The epitope design is critical to eliciting potent antibody responses with the full length RBD being superior to peptide and glycopeptide antigens. The full length RBD conjugated with mQβ activates both humoral and cellular immune systems in vivo, inducing broad spectrum, persistent, and comprehensive immune responses effective against multiple VOCs including Delta and Omicron variants, rendering it a promising vaccine candidate.

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

The appearance of new respiratory virus infections in humans with epidemic or pandemic potential has underscored the urgent need for effective broad-spectrum antivirals (BSAs). Bioactive compounds derived from plants may provide a natural source of new BSA candidates. Here, we investigated the novel phytocomplex formulation SP4™ as a candidate direct-acting BSA against major current human respiratory viruses, including coronaviruses and influenza viruses. SP4™ inhibited the in vitro replication of SARS-CoV-2, hCoV-OC43, hCoV-229E, Influenza A and B viruses, and respiratory syncytial virus in the low-microgram range. Using hCoV-OC43 as a representative respiratory virus, most of the antiviral activity of SP4™ was observed to stem primarily from its dimeric A-type proanthocyanidin (PAC-A) component. Further investigations of the mechanistic mode of action showed SP4™ and its PAC-A-rich fraction to prevent hCoV-OC43 from attaching to target cells and exert virucidal activity. This occurred through their interaction with the spike protein of hCoV-OC43 and SARS-CoV-2, thereby interfering with spike functions and leading to the loss of virion infectivity. Overall, these findings support the further development of SP4™ as a candidate BSA of a natural origin for the prevention of human respiratory virus infections.

Infectious bronchitis virus (IBV) restricts cell tropism. Except for the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered IBV vaccine development and research on the mechanisms of IBV infection. A novel Vero cell-adapted strain, HV80, has been previously reported. In this study, we constructed recombinants expressing the chimeric S glycoprotein, S1 or S2 subunit of strain H120 and demonstrated that mutations on S2 subunit are associated with the strain HV80 Vero cell adaptation. R687P or P687R substitution recombinants were constructed with the genome backbone of strains HV80 or H120. We found that the RRRR690/S motif at the S2' cleavage site is crucial to the Vero cell adaptation of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that the Q855H mutation induced syncytium formation. A transient transfection assay demonstrated the S glycoprotein with the PRRR690/S motif at the S2' cleavage site induced low-level cell-cell fusion, while H855Q substitution hindered cell-cell fusion and blocked cleavage event with S20 product. This study provides a basis for the construction of IBV recombinants capable of replicating in Vero cells, thus contributing to the advancement in the development of genetically engineered cell-based IBV vaccines.

A novel pangolin-origin MERS-like coronavirus (CoV), MjHKU4r-CoV-1, was recently identified. It is closely related to bat HKU4-CoV, and is infectious in human organs and transgenic mice. MjHKU4r-CoV-1 uses the dipeptidyl peptidase 4 (DPP4 or CD26) receptor for virus entry and has a broad host tropism. However, the molecular mechanism of its receptor binding and determinants of host range are not yet clear. Herein, we determine the structure of the MjHKU4r-CoV-1 spike (S) protein receptor-binding domain (RBD) complexed with human CD26 (hCD26) to reveal the basis for its receptor binding. Measuring binding capacity toward multiple animal receptors for MjHKU4r-CoV-1, mutagenesis analyses, and homology modeling highlight that residue sites 291, 292, 294, 295, 336, and 344 of CD26 are the crucial host range determinants for MjHKU4r-CoV-1. These results broaden our understanding of this potentially high-risk virus and will help us prepare for possible outbreaks in the future.

Highly pathogenic coronaviruses have caused significant outbreaks in humans and animals, posing a serious threat to public health. The rapid global spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in millions of infections and deaths. However, the mechanisms through which coronaviruses evade a host's antiviral immune system are not well understood. Liquid-liquid phase separation (LLPS) is a recently discovered mechanism that can selectively isolate cellular components to regulate biological processes, including host antiviral innate immune signal transduction pathways. This review focuses on the mechanism of coronavirus-induced LLPS and strategies for utilizing LLPS to evade the host antiviral innate immune response, along with potential antiviral therapeutic drugs and methods. It aims to provide a more comprehensive understanding and novel insights for researchers studying LLPS induced by pandemic viruses.