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Since its identification in late 2019, SARS-CoV-2 has undergone numerous mutations, resulting in the emergence of several viral variants, which may differ in transmissibility, virulence and/or evasion from host immunity. Particularly, immunity-related changes have been well documented in the Omicron variant, including reports of escaping neutralizing antibodies induced by infection/vaccination with heterologous SARS-CoV-2 or used in serological therapy. These findings may encourage some discussions about the possibility that Omicron is a distinct SARS-CoV-2 serotype. To contribute to this issue, we combined concepts from immunology, virology and evolution and performed an interesting brainstorm on the hypothesis that Omicron is a distinct SARS-CoV-2 serotype. Furthermore, we also discussed the likelihood of emergence of SARS-CoV-2 serotypes over time, which may not necessarily be related to Omicron. Finally, insights into this topic may have direct implications for vaccine formulations, immunodiagnostic platforms and serological therapies, contributing to better management of future outbreaks or waves. • Currently, Omicron is the main target of investigation into the possible emergence of distinct SARS-CoV-2 serotypes. • In addition to the S gene, mutations in non-structural proteins may indirectly influence the emergence of SARS-CoV-2 serotypes. • Recombination events involving the S gene could contribute to the emergence of SARS-CoV-2 serotypes. • SARS-CoV-2 variants must be constantly monitored for the possible emergence of a distinct viral serotype. • The emergence of SARS-CoV-2 serotypes would have direct implications for vaccines, immunodiagnosis and serological therapies. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus causing severe watery diarrhea, vomiting, dehydration, and death in piglets. However, most commercial vaccines are developed based on the GI genotype strains, and have poor immune protection against the currently dominant GII genotype strains. Therefore, four novel replication-deficient human adenovirus 5-vectored vaccines expressing codon-optimized forms of the GIIa and GIIb strain spike and S1 glycoproteins were constructed, and their immunogenicity was evaluated in mice by intramuscular (IM) injection. All the recombinant adenoviruses generated robust immune responses, and the immunogenicity of recombinant adenoviruses against the GIIa strain was stronger than that of recombinant adenoviruses against the GIIb strain. Moreover, Ad-XT-tPA-Sopt-vaccinated mice elicited optimal immune effects. In contrast, mice immunized with Ad-XT-tPA-Sopt by oral gavage did not induce strong immune responses. Overall, IM administration of Ad-XT-tPA-Sopt is a promising strategy against PEDV, and this study provides useful information for developing viral vector-based vaccines. • Four Ad5-vectored vaccines expressing codon-optimized S and S1 proteins of the GIIa and GIIb PEDV strain were constructed. • All recombinant adenoviruses generated robust immune responses in mice. • Ad-XT-tPA-Sopt elicited optimal immune effects. • The neutralizing antibodies showed no significant differences between Ad-XT-tPA-Sopt and inactivated vaccine. • Ad-XT-tPA-Sopt is a promising vaccine against PEDV. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) resemble the parent virus but lack the viral genome, providing a safe and efficient platform for the analysis of virus assembly and budding as well as the development of vaccines and drugs. During the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the formation of SARS-CoV-2 VLPs was investigated as an alternative to authentic virions because the latter requires biosafety level 3 (BSL-3) facilities. This allowed researchers to model its assembly and budding processes, examine the role of mutations in variants of concern, and determine how the structural proteins interact with each other. Also, the absence of viral genome in VLPs circumvents worries of gains in infectivity via mutagenesis. This review summarizes the strengths and limitations of several SARS-CoV-2 VLP systems and details some of the strides that have been made in using these systems to study virus assembly and budding, viral entry, and antibody and vaccine development., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Several viruses have the ability to form large multinucleated cells known as syncytia. Many properties of syncytia and the role they play in the evolution of a viral infection are not well understood. One basic question that has not yet been answered is how quickly syncytia form. We use a novel mathematical model of cell-cell fusion assays and apply it to experimental data from SARS-CoV-2 fusion assays to provide the first estimates of virus-mediated cell fusion rate. We find that for SARS-CoV2, the fusion rate is in the range of 6 × 10−4–12×10−4/h. We also use our model to compare fusion rates when the protease TMPRSS2 is overexpressed (2–4 times larger fusion rate), when the protease furin is removed (one third the original fusion rate), and when the spike protein is altered (1/10th the original fusion rate). The use of mathematical models allows us to provide additional quantitative information about syncytia formation. • A mathematical model is used to analyze SARS-CoV-2 cell-cell fusion assays. • This allows us to estimate the virus-mediated fusion rate for the first time. • Estimates are used to quantify the effect of TMPRSS2 and furin on cell fusion. • Removal of the spike cleavage site causes a ten-fold reduction in fusion rate. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 caused by the novel human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently a major threat to public health worldwide. To deal with the needs of vaccine, we developed four DNA vaccine candidates against SARS-CoV-2, based on the full-length spike (S) or truncated S protein. Following mice vaccination, we measured T-cell response and antigen-specific neutralizing antibody (NAb) titer. All four candidates induced humoral immune responses, including elevated levels of total IgG and NAbs, and cell-mediated immune responses, including multiple cytokine expression. However, the full-length S DNA vaccine enhanced the immune responses most significantly. We then evaluated its appropriate antigen dose and vaccination schedule. Although all immunized groups showed higher immune response than the control group, inoculation with 50 μg antigen led to the highest NAb titer. Immunity was significantly increased after the third inoculation. Thus, the full-length S DNA vaccine can potentially prevent SARS-CoV-2 infection. • We developed four DNA vaccine candidates against SARS-CoV-2 based on spike protein. • They induced humoral and cell-mediated immune responses against SARS-CoV-2. • Inoculation with 50 μg antigen led to the highest neutralizing antibody titer. • Full-length spike DNA vaccine can potentially prevent SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

The newly discovered SARS-CoV-2 Omicron variant B.1.1.529 is a Variant of Concern (VOC) announced by the World Health Organization (WHO). It's becoming increasingly difficult to keep these variants from spreading over the planet. The fifth wave has begun in several countries because of Omicron variant, and it is posing a threat to human civilization. As a result, we need effective vaccination that can tackle Omicron SARS-CoV-2 variants that are bound to emerge. Therefore, the current study is an initiative to design a peptide-based chimeric vaccine that may potentially battle SARS-CoV-2 Omicron variant. As a result, the most relevant epitopes present in the mutagenic areas of Omicron spike protein were identified using a set of computational tools and immunoinformatic techniques to uncover common MHC-1, MHC-II, and B cell epitopes that may have the ability to influence the host immune mechanism. A final of three epitopes from CD8+ T-cell, CD4+ T-cell epitopes, and B-cell were shortlisted from spike protein, and that are highly antigenic, IFN-γ inducer, as well as overlapping for the construction of twelve vaccine models. As a result, the antigenic epitopes were coupled with a flexible and stable peptide linker, and the adjuvant was added at the N-terminal end to create a unique vaccine candidate. The structure of a 3D vaccine candidate was refined, and its quality was assessed by using web servers. However, the applied immunoinformatic study along with the molecular docking and simulation of 12 modeled vaccines constructs against six distinct HLAs, and TLRs (TLR2, and TLR4) complexes revealed that the V1 construct was non-allergenic, non-toxic, highly immunogenic, antigenic, and most stable. The vaccine candidate's stability was confirmed by molecular dynamics investigations. Finally, we studied the expression of the suggested vaccination using codon optimization and in-silico cloning. The current study proposed V1 Multi-Epitope Vaccine (MEV) as a significant vaccine candidate that may help the scientific community to treat SARS-CoV-2 infections. • Reverse vaccinology approaches was applied for the construction of novel vaccine. • Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy and structural based studies. • V1 vaccine construct was predicted as potent MEV against omicron variant. [ABSTRACT FROM AUTHOR]

Six candidate overlapping genes have been detected in SARS-CoV-2, yet current methods struggle to detect overlapping genes that recently originated. However, such genes might encode proteins beneficial to the virus, and provide a model system to understand gene birth. To complement existing detection methods, I first demonstrated that selection pressure to avoid stop codons in alternative reading frames is a driving force in the origin and retention of overlapping genes. I then built a detection method, CodScr, based on this selection pressure. Finally, I combined CodScr with methods that detect other properties of overlapping genes, such as a biased nucleotide and amino acid composition. I detected two novel ORFs (ORF-Sh and ORF-Mh), overlapping the spike and membrane genes respectively, which are under selection pressure and may be beneficial to SARS-CoV-2. ORF-Sh and ORF-Mh are present, as ORF uninterrupted by stop codons, in 100% and 95% of the SARS-CoV-2 genomes, respectively. • Shaping of synonymous codons is a driving force in the evolution of overlapping genes. • A multi-step computational method to predict overlapping ORFs in viruses is presented. • It predicts two new overlapping ORFs in the spike and membrane genes of SARS-CoV-2. • Both overlapping ORFs (ORF-Sh and ORF-Mh) show a degree of conservation close to 100%. • ORF-Sh and ORF-Mh were found in about 100% of the SARS-CoV-2 genomes in database. [ABSTRACT FROM AUTHOR]

The fall of 2020 brought several new variants of SARS-CoV-2 circulating across the globe, and the steadily increasing COVID-19 cases are responsible for the emergence of these variants. All the SARS-CoV-2 variants reported to date have multiple mutations in the spike (S) protein, specifically in the receptor-binding domain (RBD). Here, we employed an integrated computational approach involving structure and sequence based predictions to study the effect of naturally occurring variations in the S-RBD on its stability and ACE2 binding affinity. The hotspot stabilizing residue mutations N501I, N501Y, Q493L, Q493H and K417R, strengthen the RBD-ACE2 complex by modulating the interaction statistics at the interface. Thus, we report here some critical mutations that could increase the binding affinity of the SARS-CoV-2 RBD with ACE2, increasing the viral infectivity and pathogenicity. Understanding the effect of these mutations will help in developing potential vaccines and therapeutics. [Display omitted] • Effect of mutations in RBD hotspot region involved in ACE2 receptor binding. • Mutation N501I, N501Y, Q493L, Q493H and K417R, strengthen the RBD-ACE2 complex. • Y at position 501 stabilize the hotspot-353 of ACE2. [ABSTRACT FROM AUTHOR]

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the global swine industry, demanding a thorough understanding of its cellular invasion mechanism for effective interventions. This study meticulously investigates the impact of O- and N -linked glycans on PEDV proteins and host cell interaction, shedding light on their influence on the virus's invasion process. Utilizing CRISPR-Cas9 technology to inhibit cell surface O- and N -linked glycan synthesis demonstrated no discernible impact on virus infection. However, progeny PEDV strains lacking these glycans exhibited a minor effect of O-linked glycans on virus infection. Conversely, a notable 40% reduction in infectivity was observed when the virus surface lacked N -linked glycans, emphasizing their pivotal role in facilitating virus recognition and binding to host cells. Additionally, inhibition studies utilizing kifunensine, a natural glycosidase I inhibitor, reaffirmed the significant role of N -linked glycans in virus infection. Inhibiting N -linked glycan synthesis with kifunensine substantially decreased virus entry into cells and potentially influenced spike protein expression. Assessment of the stability and recovery potential of N -linked glycan-deficient strains underscored the critical importance of N -glycans at various stages of the virus lifecycle. In vivo experiments infecting piglets with N -glycan-deficient strains exhibited milder clinical symptoms, reduced virus excretion, and less severe pathological lesions compared to conventional strains. These findings offer promising translational applications, proposing N -glycosylation inhibitors as potential therapeutic interventions against PEDV. The utilization of these inhibitors might mitigate virus invasion and disease transmission, providing avenues for effective antiviral strategies and vaccine development. Nonetheless, further research is warranted to elucidate the precise mechanisms of N -linked glycans in PEDV infection for comprehensive clinical applications. • Research Summary: Highlighted the pivotal role of N -linked glycans in PEDV binding to cells, shedding light on the virus's invasion mechanism. • Research Methods: Used CRISPR-Cas9 to create N - and O-linked glycan-deficient IPEC-J2 cells, employing kifunensine to study PEDV invasion. • Key Findings: Blocking N -linked glycan synthesis significantly reduced PEDV infectivity, while O-linked glycan deficiency had minimal impact on virus invasion. • Innovation: Revealed the crucial involvement of N -linked glycans in PEDV infection, guiding future antiviral therapy research. • Research Significance: Uncovered the essential role of N -linked glycans, paving the way for targeted antiviral treatments and advancements in virus therapeutics. [ABSTRACT FROM AUTHOR]

Dynamic tracking of variant frequencies among viruses circulating in the global pandemic has revealed the emergence and dominance of a D614G mutation in the SARS-CoV-2 spike protein. To address whether pandemic SARS-CoV-2 G614 variant has evolved to become more pathogenic, we infected adult hamsters (>10 months old) with two natural SARS-CoV-2 variants carrying either D614 or G614 spike protein to mimic infection of the adult/elderly human population. Hamsters infected by the two variants exhibited comparable viral loads and pathology in lung tissues as well as similar amounts of virus shed in nasal washes. Altogether, our study does not find that naturally circulating D614 and G614 SARS-CoV-2 variants differ significantly in pathogenicity in hamsters. • Adult hamsters support SARS-CoV-2 replication in upper and lower respiratory tracts. • Adult hamsters suffer severe pneumonia and weight loss after SARS-CoV-2 infection. • Viral loads were similar for older (614D) and newer (614G) SARS-CoV-2 in hamsters. • Lung pathogenesis was similar for an older (614D) and newer (614G) clinical isolate. [ABSTRACT FROM AUTHOR]

Here we investigated the virulence properties of a unique cell-adapted SARS-CoV-2 mutant showing a ten-amino acid deletion encompassing the furin cleavage site of the spike protein (Δ 680 SPRAARSVAS 689 ; Δ680-689-B.1) in comparison to its parental strain (wt-B.1) and two Delta variants (AY.122 and AY.21) of concern. After intranasal inoculation, transgenic K18-hACE2 mice were monitored for 14 days for weight change, lethality, and clinical score; oral swabs were daily collected and tested for the presence of N protein subgenomic RNA. At 3 and 7 dpi mice were also sacrificed and organs collected for molecular, histopathological, and immune response profile investigations. The Δ680-689-B.1-infected mice exhibited reduced shedding, lower virulence at the lung level, and milder pulmonary lesions. In the lung, infection with Δ680-689-B.1 was associated with a significant lower expression of some cytokines at 3 dpi (IL-4, IL-27, and IL-28) and 7 dpi (IL-4, IL-27, IL-28, IFN-γ and IL-1α). • The virulence properties of a SARS-CoV-2 mutant showing a deletion encompassing the S FCS (Δ680-689-B.1) were investigated. • Δ680-689-B.1- infected mice showed reduced shedding and milder pulmonary lesions. • Infection with Δ680-689-B.1 was associated with a significant lower expression of some cytokines at 3 dpi and 7 dpi. • IL-6, GRO-α, G-CSF significantly increased in mice infected with Delta VOC isolates. [ABSTRACT FROM AUTHOR]

Feline coronavirus (FCoV) is an etiological agent that causes a benign enteric illness and the fatal systemic disease feline infectious peritonitis (FIP). The FCoV spike (S) protein is considered the viral regulator for binding and entry to the cell. This protein is also involved in FCoV tropism and virulence, as well as in the switch from enteric disease to FIP. This regulation is carried out by spike's major functions: receptor binding and virus-cell membrane fusion. In this review, we address important aspects in FCoV genetics, replication and pathogenesis, focusing on the role of S. To better understand this, FCoV S protein models were constructed, based on the human coronavirus NL63 (HCoV-NL63) S structure. We describe the specific structural characteristics of the FCoV S, in comparison with other coronavirus spikes. We also revise the biochemical events needed for FCoV S activation and its relation to the structural features of the protein. [ABSTRACT FROM AUTHOR]

We recently established a mouse model (288–330 +/+ ) that developed acute respiratory disease resembling human pathology following infection with a high dose (5 × 10 6 PFU) of mouse-adapted MERS-CoV (icMERSma1). Although this high dose conferred fatal respiratory disease in mice, achieving similar pathology at lower viral doses may more closely reflect naturally acquired infections. Through continued adaptive evolution of icMERSma1 we generated a novel mouse-adapted MERS-CoV (maM35c4) capable of achieving severe respiratory disease at doses between 10 3 and 10 5 PFU. Novel mutations were identified in the maM35c4 genome that may be responsible for eliciting etiologies of acute respiratory distress syndrome at 10–1000 fold lower viral doses. Importantly, comparative genetics of the two mouse-adapted MERS strains allowed us to identify specific mutations that remained fixed through an additional 20 cycles of adaptive evolution. Our data indicate that the extent of MERS-CoV adaptation determines the minimal infectious dose required to achieve severe respiratory disease. [ABSTRACT FROM AUTHOR]

During viral entry, enveloped viruses require the fusion of their lipid envelope with host cell membranes. For coronaviruses, this critical step is governed by the virally-encoded spike (S) protein, a class I viral fusion protein that has several unique features. Coronavirus entry is unusual in that it is often biphasic in nature, and can occur at or near the cell surface or in late endosomes. Recent advances in structural, biochemical and molecular biology of the coronavirus S protein has shed light on the intricacies of coronavirus entry, in particular the molecular triggers of coronavirus S-mediated membrane fusion. Furthermore, characterization of the coronavirus fusion peptide (FP), the segment of the fusion protein that inserts to a target lipid bilayer during membrane fusion, has revealed its particular attributes which imparts some of the unusual properties of the S protein, such as Ca 2+ -dependency. These unusual characteristics can explain at least in part the biphasic nature of coronavirus entry. In this review, using severe acute respiratory syndrome coronavirus (SARS-CoV) as model virus, we give an overview of advances in research on the coronavirus fusion peptide with an emphasis on its role and properties within the biological context of host cell entry. [ABSTRACT FROM AUTHOR]

The study analyzed the molecular dynamics of the circulating SARS-CoV-2 Omicron variant from its identification in November 2021 to January 2023. The SARS-CoV-2 sequences from Bangladesh revealed three distinct waves of the Omicron variant. More than 50 sub-lineages of Omicron variant were introduced into the country, with the majority belonging to the major lineages of BA.1-like (24.91%), BA.2-like (43.35%), BA.5-like (5.76%), XBB (10.47%), and "Others and Unassigned" (18.64%). Furthermore, the relative frequencies over time revealed that Omicron lineages existed for a short period of time before being replaced by other sub-lineages. Many potential mutations were found in the receptor binding domain of the Spike protein including G339D/H, S371 L/F, K417 N, T478K, E484A, Q493R, Q498R, and N501Y. In conclusion, the SARS-CoV-2 Omicron variant from Bangladesh showed diverse genetic features and continuous evolution. Therefore, the choice of vaccine and monitoring of hospitalized patients is important alongside genetic characterization of the circulating SARS-CoV-2. • Three waves of Omicron variants circulated in Bangladesh. • Multiple lineages and sub-lineages are prevalent in the country from late 2021 to early 2023. • Sub-lineages XBB.1 and QR1.1 have been introduced in early 2023 and showed continuing evolution. • Mutations in the spike protein's receptor binding site are particularly important for pathogenicity and immune escape. [ABSTRACT FROM AUTHOR]

Spike (S) glycoprotein on the viral envelope is the main determinant of infectivity. The S protein of coronavirus infectious bronchitis virus (IBV) contains 29 putative asparagine(N)-linked glycosylation sites. These post-translational modifications may assist in protein folding and play important roles in the functionality of S protein. In this study, we used bioinformatics tools to predict N-linked glycosylation sites and to analyze their distribution in IBV strains and variants. Among these sites, 8 sites were confirmed in the S protein extracted from partially purified virus particles by proteomics approaches. N-D and N-Q substitutions at 13 predicted sites were introduced into an infectious clone system. The impact on S protein-mediated cell-cell fusion, viral recovery and infectivity was assessed, leading to the identification of sites essential for the functions of IBV S protein. Further characterization of these and other uncharacterized sites may reveal novel aspects of N-linked glycosylation in coronavirus replication and pathogenesis. [ABSTRACT FROM AUTHOR]

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) was first identified in 2012, and it continues to threaten human health worldwide. No MERS vaccines are licensed for human use, reinforcing the urgency to develop safe and efficacious vaccines to prevent MERS. MERS-CoV spike protein forms a trimer, and its receptor-binding domain (RBD) serves as a vaccine target. Nevertheless, the protective efficacy of RBD in its native trimeric form has never been evaluated. In this study, a trimeric protein, RBD-Fd, was generated by fusing RBD with foldon trimerization motif. It bound strongly to the receptor of MERS-CoV, dipeptidyl peptidase 4 (DPP4), and elicited robust RBD-specific neutralizing antibodies in mice, maintaining long-term neutralizing activity against MERS-CoV infection. RBD-Fd potently protected hDPP4 transgenic mice from lethal MERS-CoV challenge. These results suggest that MERS-CoV RBD in its trimeric form maintains native conformation and induces protective neutralizing antibodies, making it a candidate for further therapeutic development. [ABSTRACT FROM AUTHOR]

Coronavirus spike proteins mediate host-cell-attachment and virus entry. Virus replication takes place within the host cell cytosol, whereas assembly and budding occur at the endoplasmic reticulum-Golgi intermediate compartment. In this study we demonstrated that the last 39 amino acid stretches of Alphacoronavirus spike cytoplasmic domains of the human coronavirus 229E, NL63, and the porcine transmissible gastroenteritis virus TGEV interact with tubulin alpha and beta chains. In addition, a partial co-localization of TGEV spike proteins with authentic host cell β-tubulin was observed. Furthermore, drug-induced microtubule depolymerization led to changes in spike protein distribution, a reduction in the release of infectious virus particles and less amount of spike protein incorporated into virions. These data demonstrate that interaction of Alphacoronavirus spike proteins with tubulin supports S protein transport and incorporation into virus particles. [ABSTRACT FROM AUTHOR]

The spike protein S of coronaviruses contains a highly conserved cytoplasmic cysteine-rich motif adjacent to the transmembrane region. This motif is palmitoylated in the Betacoronaviruses MHV and SARS-CoV. Here, we demonstrate by metabolic labeling with [ 3 H]-palmitic acid that the S protein of transmissible gastroenteritis coronavirus (TGEV), an Alphacoronavirus , is palmitoylated as well. This is relevant for TGEV replication as virus growth was compromised by the general palmitoylation inhibitor 2-bromopalmitate. Mutation of individual cysteine clusters in the cysteine-rich motif of S revealed that all cysteines must be replaced to abolish acylation and incorporation of S into virus-like particles (VLP). Conversely, the interaction of S with the M protein, essential for VLP incorporation of S, was not impaired by lack of palmitoylation. Thus, palmitoylation of the S protein of Alphacoronaviruses is dispensable for S–M interaction, but required for the generation of progeny virions. [ABSTRACT FROM AUTHOR]

Abstract: In previous studies, the Beaudette strain of coronavirus infectious bronchitis virus (IBV) was adapted from chicken embryo to Vero, a monkey kidney cell line, by serial propagation for 65 passages. To characterize the susceptibility of other human and animal cells to IBV, 15 human and animal cell lines were infected with the Vero-adapted IBV and productive infection was observed in four human cell lines: H1299, HepG2, Hep3B and Huh7. In other cell lines, the virus cannot be propagated beyond passage 5. Interestingly, cellular furin abundance in five human cell lines was shown to be strongly correlated with productive IBV infection. Cleavage of IBV spike protein by furin may contribute to the productive IBV infection in these cells. The findings that IBV could productively infect multiple human and animal cells of diverse tissue and organ origins would provide a useful system for studying the pathogenesis of coronavirus. [Copyright &y& Elsevier]

Abstract: Canine alphacoronaviruses (CCoV) exist in two serotypes, type I and II, both of which can cause severe gastroenteritis. Here, we characterize a canine alphacoronavirus, designated CCoV-A76, first isolated in 1976. Serological studies show that CCoV-A76 is distinct from other CCoVs, such as the prototype CCoV-1-71. Efficient replication of CCoV-A76 is restricted to canine cell lines, in contrast to the prototypical type II strain CCoV-1-71 that more efficiently replicates in feline cells. CCoV-A76 can use canine aminopeptidase N (cAPN) receptor for infection of cells, but was unable to use feline APN (fAPN). In contrast, CCoV-1-71 can utilize both. Genomic analysis shows that CCoV-A76 possesses a distinct spike, which is the result of a recombination between type I and type II CCoV, that occurred between the N- and C-terminal domains (NTD and C-domain) of the S1 subunit. These data suggest that CCoV-A76 represents a recombinant coronavirus form, with distinct host cell tropism. [Copyright &y& Elsevier]

Abstract: Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus–cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus–virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell–cell fusion was independent of cathepsin L, a protease essential for virus–cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus–cell and cell–cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. [Copyright &y& Elsevier]

Abstract: Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we have further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV. [Copyright &y& Elsevier]

Abstract: The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1–S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822–C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell–cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion. [Copyright &y& Elsevier]

Abstract: We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by the diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level. [Copyright &y& Elsevier]

Abstract: Seven-mer phage random peptide libraries were panned against 2C10, a monoclonal antibody that showed neutralizing activities against PEDV. Recombinant M13 phages displaying the peptides SHRLP(Y/Q)(P/V) or GPRPVTH on the g3p minor coat protein showed strong binding affinity with 2C10 (70% and 30% of recovered phages, respectively) after multiple panning. Sequence analysis suggested that these peptides are similar with 1368GPRLQPY1374 found at the carboxy-terminal of the S protein. In neutralization inhibition assays, the two peptide motifs and a 24-mer synthetic peptide corresponding to the C-terminal endodomain of PEDV S protein were observed to compete for the antigen binding site of 2C10, as demonstrated by the loss or reduction of neutralizing activity of the monoclonal antibody. This new finding suggests that the newly discovered peptide motifs mimic a neutralizing epitope PEDV. [Copyright &y& Elsevier]

Abstract: Development of an effective vaccine for severe acute respiratory syndrome (SARS) remains to be a priority to prevent possible re-emergence of SARS coronavirus (SARS-CoV). We previously demonstrated that the receptor-binding domain (RBD) of SARS-CoV S protein is a major target of neutralizing antibodies. This suggests that the RBD may serve as an ideal vaccine candidate. Recombinant adeno-associated virus (rAAV) has been proven to be an effective system for gene delivery and vaccine development. In this study, a novel vaccine against SARS-CoV was developed based on the rAAV delivery system. The gene encoding RBD was cloned into a pAAV-IRES-hrGFP plasmid. The immunogenicity induced by the resulting recombinant RBD-rAAV was evaluated in BALB/c mice. The results demonstrated that (1) a single dose of RBD-rAAV vaccination could induce sufficient neutralizing antibody against SARS-CoV infection; (2) two more repeated doses of the vaccination boosted the neutralizing antibody to about 5 times of the level achieved by a single dose of the immunization and (3) the level of the antibody continued to increase for the entire duration of the experiment of 5.5 months. These results suggested that RBD-rAAV is a promising SARS candidate vaccine. [Copyright &y& Elsevier]

Abstract: The coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 Å) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding “zippers” that strongly constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 α-helix. [Copyright &y& Elsevier]

Abstract: The spike (S) protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is not only responsible for receptor binding, but also a major antigenic determinant capable of inducing protective immunity. In this study, we demonstrated that the receptor-binding domain (RBD) of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS-CoV. Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein. Depletion of RBD-specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum-mediated neutralizing activity, while affinity-purified anti-RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus, indicating that the RBD of S protein is a critical neutralization determinant of SARS-CoV during viral infection and immunization. Two monoclonal antibodies (1A5 and 2C5) targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS-CoV. Both 1A5 and 2C5 possessed potent neutralizing activities, although they directed against distinct conformation-dependant epitopes as shown by ELISA and binding competition assay. We further demonstrated that 2C5, but not 1A5, was able to block binding of the RBD to angiotensin-converting enzyme 2 (ACE2), the functional receptor on targeted cells. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV and for designing SARS vaccines. [Copyright &y& Elsevier]

Emerging porcine epidemic diarrhea viruses (PEDVs) have caused large economic losses since 2010, and G2b is the prevalent globally epidemic genotype. Given the fastidious isolation of emerging PEDV in cell culture and difficulties in retaining the isolate infectivity upon further in vitro passage, highly attenuated recombinant vesicular stomatitis virus (rVSV MT ) was used as a vector to express the PEDV spike (S) protein, aiming to develop a subunit vaccine against G2b viruses. An S protein with 19 of its cytoplasmic domain amino acids deleted could be incorporated into VSV particles, generating rVSV MT (VSV MT -S Δ19 ) with high efficiency. Our results suggest that VSV MT -S Δ19 could effectively induce PEDV-specific immunity in pigs via intramuscular, but not intranasal, immunization. Notably, immunizations of sows with VSV MT -S Δ19 provided protective lactogenic immunity against a virulent G2b PEDV challenge in piglets. Consequently, recombinant VSV MT may be a promising platform for preparing a subunit vaccine against PEDV., (Copyright © 2019 Elsevier Inc. All rights reserved.)