Insights into the Epidemiology, Phylodynamics, and Evolutionary Changes of Lineage GI-7 Infectious Bronchitis Virus

Infectious bronchitis virus (IBV) is distributed worldwide and causes significant losses in the poultry industry. In recent decades, lineages GI-19 and GI-7 have become the most prevalent IBV strains in China. However, the molecular evolution and phylodynamics of the lineage GI-7 IBV strains remain largely unknown. In this study, we identified 19 IBV strains from clinical samples from January 2021 to June 2022 in China, including 12 strains of GI-19, 3 strains of GI-7, and 1 strain each of GI-1, GI-9, GI-13, and GI-28. These results indicated that lineages GI-19 and GI-7 IBVs are still the most prevalent IBVs in China. Here, we investigated the evolution and transmission dynamics of lineage GI-7 IBVs. Our results revealed that the Taiwan province might be the origin of lineage GI-7 IBVs and that South China plays an important role in the spread of IBV. Furthermore, we found low codon usage bias of the S1 gene in lineage GI-7 IBVs. This allowed IBV to replicate in the host during evolution as a result of reduced competition, mainly driven by natural selection and mutational pressure, where the role of natural selection is more prominent. Collectively, our results reveal the genetic diversity and evolutionary dynamics of lineage GI-7 IBVs, which could assist in the prevention and control of viral infection.

Pathogenicity of GI-23 Avian Infectious Bronchitis Virus Strain Isolated in Brazil.

IBV variants belonging to the GI-23 lineage have circulated since 1998 in the Middle East and have spread to several countries over time. In Brazil, the first report of GI-23 occurred in 2022. The study aimed to evaluate the in vivo pathogenicity of exotic variant GI-23 isolates. Biological samples were screening by real-time RT-PCR and classified in to GI-1 or G1-11 lineages. Interestingly, 47.77% were not classified in these lineages. Nine of the unclassified strains were sequenced and showed a high similarity to the GI-23 strain. All nine were isolated and three, were studied for pathogenicity. At necropsy, the main observations were the presence of mucus in the trachea and congestion in the tracheal mucosa. In addition, lesions on the tracheas showed marked ciliostasis, and the ciliary activity confirmed the high pathogenicity of isolates. This variant is highly pathogenic to the upper respiratory tract and can cause severe kidney lesions. This study confirm a circulation of GI-23 strain in the country and report, to first time, the isolation of an exotic variant of IBV in Brazil.

SARS-Cov-2 Spike-S1 Antigen Test Strip with High Sensitivity Endowed by High-Affinity Antibodies and Brightly Fluorescent QDs/Silica Nanospheres.

The extensive research into developing novel strategies for detecting respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens, especially the sensitive point-of-care testing method, is still urgently needed to reach rapid screening of viral infections. Herein, a new lateral flow immunoassay (LFIA) platform was reported for the detection of SARS-CoV-2 spike-S1 protein antigens, in which four sensitive and specific SARS-CoV-2 mouse monoclonal antibodies (MmAbs) were tailored by using quantum dot (QD)-loaded dendritic mesoporous silica nanoparticles modified further for achieving the -COOH group surface coating (named Q/S-COOH nanospheres). Importantly, compact QD adsorption was achieved in mesoporous channels of silica nanoparticles on account of highly accessible central-radial pores and electrostatic interactions, leading to significant signal amplification. As such, a limit of detection for SARS-CoV-2 spike-S1 testing was found to be 0.03 ng/mL, which is lower compared with those of AuNPs-LFIA (traditional colloidal gold nanoparticles, Au NPs) and enzyme-linked immunosorbent assay methods. These results show that optimizing the affinity of antibody and the intensity of fluorescent nanospheres simultaneously is of great significance to improve the sensitivity of LFIA.

Which strain of the avian coronavirus vaccine will become the prevalent one in China next?

Infectious bronchitis virus (IBV) is a vital pathogen in poultry farms, which can induce respiratory, nephropathogenic, oviduct, proventriculus, and intestinal diseases. Based on the phylogenetic classification of the full-length S1 gene, IBV isolates have been categorized into nine genotypes comprising 38 lineages. GI (GI-1, GI-2, GI-3, GI-4, GI-5, GI-6, GI-7, GI-13, GI-16, GI-18, GI-19, GI-22, GI-28, and GI-29), GVI-1 and GVII-1 have been reported in China in the past 60 years. In this review, a brief history of IBV in China is described, and the current epidemic strains and licensed IBV vaccine strains, as well as IBV prevention and control strategies, are highlighted. In addition, this article presents unique viewpoints and recommendations for a more effective management of IBV. The recombinant Newcastle Disease virus (NDV) vector vaccine expressed S gene of IBV QX-like and 4/91 strains may be the dominant vaccine strains against NDV and IBV.

A substrate for a cell free in vitro assay system to screen drugs targeting trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein and viral entry.

Host proteases trypsin and trypsin-like proteases have been reported to facilitate the entry of coronavirus SARS-CoV-2 in its host cells. These protease enzymes cleave the viral surface glycoprotein, spike, leading to successful cell surface receptor attachment, fusion and entry of the virus in its host cell. The spike protein has protease cleavage sites between the two domains S1 and S2. Since the cleavage site is recognized by the host proteases, it can be a potential antiviral therapeutic target. Trypsin-like proteases play an important role in virus infectivity and the property of spike protein cleavage by trypsin and trypsin-like proteases can be used to design assays for screening of antiviral candidates against spike protein cleavage. Here, we have documented the development of a proof-of-concept assay system for screening drugs against trypsin/trypsin-like proteases that cleave spike protein between its S1 and S2 domains. The assay system developed uses a fusion substrate protein containing a NanoLuc luciferase reporter protein, the protease cleavage site between S1 and S2 domains of SARS-CoV-2 spike protein and a cellulose binding domain. The substrate protein can be immobilized on cellulose via the cellulose binding domain of the substrate. When trypsin and trypsin-like proteases cleave the substrate, the cellulose binding domain remain bound to the cellulose and the reporter protein is dislodged. Reporter assay using the released reporter protein is the read out of the protease activity. We have demonstrated the proof-of-concept using multiple proteases like trypsin, TMPRSS2, furin, cathepsin B, human airway trypsin and cathepsin L. A significant increment in fold change was observed with increasing enzyme concentration and incubation time. Introduction of increasing amounts of enzyme inhibitors in the reaction reduced the luminescent signal, thus validating the assay. Furthermore, we used SDS-PAGE and immunoblot analyses to study the cleavage band pattern and re-confirm the cleavage for enzymes tested in the assay. Taken together, we have tested an in-vitro assay system using the proposed substrate for screening drugs against trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein. The assay system can also be potentially used for antiviral drug screening against any other enzyme that might cleave the used cleavage site.

SARS-CoV-2 S1 Subunit Booster Vaccination Elicits Robust Humoral Immune Responses in Aged Mice.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has raised concerns about reduced vaccine effectiveness and the increased risk of infection, and while repeated homologous booster shots are recommended for elderly and immunocompromised individuals, they cannot completely protect against breakthrough infections. In our previous study, we assessed the immunogenicity of an adenovirus-based vaccine expressing SARS-CoV-2 S1 (Ad5.S1) in mice, which induced robust humoral and cellular immune responses (E. Kim, F. J. Weisel, S. C. Balmert, M. S. Khan, et al., Eur J Immunol 51:1774-1784, 2021, https://doi.org/10.1002/eji.202149167). In this follow-up study, we found that the mice had high titers of anti-S1 antibodies 1 year after vaccination, and one booster dose of the nonadjuvanted rS1Beta (recombinant S1 protein of SARS-CoV-2 Beta [B.1.351]) subunit vaccine was effective at stimulating strong long-lived S1-specific immune responses and inducing significantly high neutralizing antibodies against Wuhan, Beta, and Delta strains, with 3.6- to 19.5-fold increases. Importantly, the booster dose also elicited cross-reactive antibodies, resulting in angiotensin-converting enzyme 2 (ACE2) binding inhibition against spikes of SARS-CoV-2, including Omicron variants, persisting for >28 weeks after booster vaccination. Interestingly, the levels of neutralizing antibodies were correlated not only with the level of S1 binding IgG but also with ACE2 inhibition. Our findings suggest that the rS1Beta subunit vaccine candidate as a booster has the potential to offer cross-neutralization against broad variants and has important implications for the vaccine control of newly emerging breakthrough SARS-CoV-2 variants in elderly individuals primed with adenovirus-based vaccines like AZD1222 and Ad26.COV2.S. IMPORTANCE Vaccines have significantly reduced the incidences of severe coronavirus disease 2019 (COVID-19) cases and deaths. However, the emergence of SARS-CoV-2 variants has raised concerns about their increased transmissibility and ability to evade neutralizing antibodies, especially among elderly individuals who are at higher risks of mortality and reductions of vaccine effectiveness. To address this, a heterologous booster vaccination strategy has been considered as a solution to protect the elderly population against breakthrough infections caused by emerging variants. This study evaluated the booster effect of an S1 subunit vaccine in aged mice that had been previously primed with adenoviral vaccines, providing valuable preclinical evidence for elderly people vaccinated with the currently approved COVID-19 vaccines. This study confirms the potential for using the S1 subunit vaccine as a booster to enhance cross-neutralizing antibodies against emerging variants of concern.

Evaluation of Antibody Response and Side Effects Related to the Number of Inactive and mRNA Vaccine Doses Against COVID-19 in Healthcare Workers

Introduction: Studies are showing that a high antibody response increases the protection against variants in the fight against the COVID-19 pandemic. In this study, we aimed to investigate the relationship between antibody response and side effects based on the number of doses administered to healthcare workers who were vaccinated against COVID-19. Material(s) and Method(s): Healthcare workers, who were vaccinated with two doses of BNT162b2 (Group 1), a single dose of BNT162b2 following two doses of CoronaVac (Group 2), or two doses of BNT162b2 following two doses of CoronaVac (Group 3), were randomly assigned to this study. Serum samples were taken from the participants 30 +/- 2 days after the last vaccination date, and the SARSCoV- 2 anti-spike S1 RBD IgG test was administered to these samples. A questionnaire was conducted detailing the demographics of the patients as well as their post-vaccination complaints. The results were analyzed statistically. Analysis results with a p-value of <0.05 were considered significant. Result(s): A total of 179 healthcare professionals with a mean age of 41.7 +/- 10.6 years were included in our study. Of the studied samples, 95.5% (n= 171) were interpreted as anti-spike S1 RBD IgG seropositive. Positivity rates and mean antibody levels were 93.2%, 95.9%, 97.8%, and 107.4 +/- 117.1, 152.7 +/- 108.5, 201.4 +/- 114.9 (AU/mL) for Group 1, Group 2, and Group 3, respectively (p< 0.05). In general, no significant differences in antibody response were seen based on gender or age. However, a significant correlation was found between the occurrence of vaccine-related side effects and antibody titer (p< 0.001). The most common side effect was pain in the area where the vaccine was administered, with a rate of 77.4% (n= 48). More vaccine-related side effects were reported in participants under the age of 40 and in female healthcare workers. Conclusion(s): We believe that booster doses are effective for increasing the immune response and thus protecting against COVID-19. More extensive research should be conducted to confirm the link between the occurrence of vaccine-related side effects and antibody titer. Furthermore, studies on the safety of increasing the number of vaccine doses are required.Copyright © 2023 Bilimsel Tip Yayinevi. All rights reserved.

Prediction of Post-Acute-Sequelae of COVID-19 by Cargo Protein Biomarkers of Blood Total Extracellular Vesicles in Acute COVID-19.

BACKGROUND: SARS-CoV-2 invades mitochondria of infected cells resulting in disordered metabolism, mitophagy, and abnormal levels of mitochondrial proteins in extracellular vesicles. Blood extracellular vesicle SARS-CoV-2 proteins and mitochondrial proteins were quantified in COVID-19 to assess possible roles as biomarkers. METHODS: Total extracellular vesicles were precipitated from blood of age- and gender-matched participants with no infection (n=10), acute COVID-19 (n=16), post-acute sequelae of COVID-19 (PASC or long COVID) (n=30), or post-acute COVID without PASC (n=8) and their extracted proteins quantified by enzyme-linked immunosorbent assays (ELISAs). RESULTS: Total extracellular vesicle levels of S1 (receptor-binding domain [RBD]) protein were significantly higher in acute infections than in uninfected controls, post-acute infection without PASC, and PASC. Total extracellular vesicle levels of nucleocapsid (N) protein were significantly higher in PASC than in uninfected controls, acute infections, and post-acute infection without PASC. Neither acute levels of S1(RBD) or N proteins predicted progression to PASC. Levels of neither SARS-CoV-2 protein in established PASC correlated with neuropsychiatric manifestations. Significant decreases in total extracellular vesicle levels of the mitochondrial proteins MOTS-c, VDAC-1, and humanin, and elevations of levels of SARM-1 were observed in acutely infected patients who would develop PASC. Significant decreases in total extracellular vesicle levels of MOTS-c and humanin, but not VDAC-1, and elevations of total extracellular vesicle levels of SARM-1 were characteristic of PASC patients with neuropsychiatric manifestations. CONCLUSIONS: Total extracellular vesicle levels of SARS-CoV-2 proteins in COVID-19 indicate intracellular presence of SARS-CoV-2. Abnormal total extracellular vesicles levels of mitochondrial proteins in acute infections predict a high risk of PASC and later in established PASC are indicative of neuropsychiatric manifestations.

Deletion of pentad residues in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets.

Our previous study revealed that tissue culture-adapted porcine epidemic diarrhea virus (PEDV) strains, namely KNU-141112-S DEL2/ORF3 and -S DEL5/ORF3, were attenuated to different extents in vivo, suggesting that their independent deletion (DEL) signatures, including 2-amino acid (aa; residues 56-57) or 5-aa (residues 56-60) DEL in the N-terminal domain (NTD) of the spike (S) protein, may contribute to the reduced virulence of each strain. To investigate whether each DEL in the NTD of the S1 subunit is a determinant for the virulence of PEDV, we generated two mutant viruses, named icS DEL2 and icS DEL5, by introducing the identical double or quintuple aa DEL into S1 using reverse genetics with an infectious cDNA clone of KNU-141112 (icKNU-141112). We then orally inoculated conventional suckling piglets with icKNU-141112, icS DEL2, or icS DEL5 to compare their pathogenicities. The virulence of both DEL mutant viruses was significantly diminished compared to that of icKNU-141112, which causes severe clinical signs and 100 % mortality. Interestingly, the degree of attenuation differed between the two mutant viruses: icS DEL5 caused neither diarrhea nor mortality, whereas icS DEL2 caused mild to moderate diarrhea, higher viral titers in feces and intestinal tissues, and 25 % mortality. Furthermore, the icS DEL5-infected piglets displayed no remarkable macroscopic and microscopic intestinal lesions, while the icS DEL2-infected piglets showed histopathological changes in small intestine tissues, including moderate-to-severe villous atrophy. Our data indicate that the loss of the pentad (56GENQG60) residues in S alone can be sufficient to give rise to an attenuated phenotype of PEDV.

SARS-CoV-2 S Protein Subunit 1 Elicits Ca2+ Influx - Dependent Ca2+ Signals in Pancreatic Stellate Cells and Macrophages In Situ.

The S protein subunit 1 (S1) of SARS-CoV-2 is known to be responsible for the binding of the virus to host cell receptors, but the initial intracellular signalling steps following receptor activation of cells in the exocrine pancreas are unknown. Using an intact live mouse pancreatic lobule preparation, we observed that S1 elicited Ca2+ signals in stellate cells and macrophages, but not in the dominant acinar cells. The Ca2+ signals occurred mostly in the form of repetitive Ca2+ spikes. The probability of observing Ca2+ signals depended on the S1 concentration. The threshold was close to 70 nM, whereas at 600 nM, all cells responded. The SARS-Cov-2 nucleocapsid protein did not elicit any Ca2+ signals in any of the three cell types tested. The S1-induced Ca2+ signals in stellate cells started much faster (122 ± 37s) than those in macrophages (468 ± 68s). Furthermore, the interleukin-18 binding protein (IL-18BP) abolished the responses in macrophages without affecting the Ca2+ signals in stellate cells. The S1-elicited Ca2+ signals were completely dependent on the presence of external Ca2+ and were abolished by a selective inhibitor (CM4620) of Orai1 Ca2+ Release Activated Ca2+ channels. SARS-CoV-2 may contribute to acute pancreatitis, an often fatal inflammatory human disease. The S1-elicited Ca2+ signals we have observed in the pancreatic stellate cells and endogenous macrophages may play an important part in the development of the inflammatory process.

Construction and immunogenicity of recombinant rabies virus expressing S gene of SARS-CoV-2

[Background] The coronavirus disease 2019（COVID-19） pandemic has lasted for nearly three years in the globe, which has not only caused serious harm to humans but also affected companion animals. The COVID-19 vaccines for human have been used globally, while those for animals are rarely reported. [Objective] To develop a bivalent vaccine against both severe acute respiratory syndrome coronavirus 2（SARS-CoV-2） and rabies virus（RABV） for animal use. [Methods] We cloned the S and S1 genes of SARS-CoV-2 into the region between G and L genes of the attenuated RABV vaccine strain rHEP-Flury to construct the recombinant plasmids pHEP-nCOV-S and pHEP-nCOV-S1, respectively.The two plasmids were respectively co-transfected into BHK-21 cells with the helper plasmids and finally the recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 were rescued. The recombinant viruses were confirmed by RT-PCR and direct fluorescent antibody staining against RABV N protein.Western blotting was employed to detect the expression of S and S1 proteins in the cells infected with the recombinant viruses. The growth curves, pathogenicity, and immunogenicity of recombinant viruses were confirmed in NA cells and mice. [Results] The rescued recombinant viruses rHEP-nCOV-S and rHEP-nCOV-S1 respectively carrying the S and S1 genes of SARS-CoV-2 were confirmed by direct fluorescent antibody assay based on the green fluorescence from the supernatants 7 days post infection.rHEP-nCOV-S1 rather than rHEP-nCOV-S showed stronger proliferation and diffusion abilities than the parental virus rHEP-Flury in NA cells. The specific bands at 72 kDa and 144 kDa in the Western blotting confirmed the efficient expression of S and S1 in the recombinant viruses, respectively. The mice vaccinated with the recombinant viruses did not show significant changes in the body weight compared with those vaccinated with rHEP-Flury, and the recombinant viruses induced the production of neutralizing antibody against RABV in mice. [Conclusion] The production of the recombinant RABV carrying the S/S1 gene of SARS-CoV-2 provides a foundation for the development of the bivalent vaccine against both SARS-CoV-2 and rabies virus for animal use.

SARS-CoV-2 Spike-Mediated Entry and Its Regulation by Host Innate Immunity.

The constantly evolving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) fuel the worldwide coronavirus disease (COVID-19) pandemic. The spike protein is essential for the SARS-CoV-2 viral entry and thus has been extensively targeted by therapeutic antibodies. However, mutations along the spike in SARS-CoV-2 VOC and Omicron subvariants have caused more rapid spread and strong antigenic drifts, rendering most of the current antibodies ineffective. Hence, understanding and targeting the molecular mechanism of spike activation is of great interest in curbing the spread and development of new therapeutic approaches. In this review, we summarize the conserved features of spike-mediated viral entry in various SARS-CoV-2 VOC and highlight the converging proteolytic processes involved in priming and activating the spike. We also summarize the roles of innate immune factors in preventing spike-driven membrane fusion and provide outlines for the identification of novel therapeutics against coronavirus infections.

Virtual Screening-Based Peptides Targeting Spike Protein to Inhibit Porcine Epidemic Diarrhea Virus (PEDV) Infection.

Due to the rapid mutation of porcine epidemic diarrhea virus (PEDV), existing vaccines cannot provide sufficient immune protection for pigs. Therefore, it is urgent to design the affinity peptides for the prevention and control of this disease. In this study, we made use of a molecular docking technology for virtual screening of affinity peptides that specifically recognized the PEDV S1 C-terminal domain (CTD) protein for the first time. Experimentally, the affinity, cross-reactivity and sensitivity of the peptides were identified by an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR) test, separately. Subsequently, Cell Counting Kit-8 (CCK-8), quantitative real-time PCR (qRT-PCR), Western blot and indirect immunofluorescence were used to further study the antiviral effect of different concentrations of peptide 110766 in PEDV. Our results showed that the P/N value of peptide 110766 at 450 nm reached 167, with a KD value of 216 nM. The cytotoxic test indicated that peptide 110766 was not toxic to vero cells. Results of the absolute quantitative PCR revealed that different concentrations (3.125 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM, 200 µM) of peptide 110766 could significantly reduce the viral load of PEDV compared with the virus group (p < 0.0001). Similarly, results of Western blot and indirect immunofluorescence also suggested that the antiviral effect of peptide 110766 at 3.125 is still significant. Based on the above research, high-affinity peptide 110766 binding to the PEDV S1-CTD protein was attained by a molecular docking technology. Therefore, designing, screening, and identifying affinity peptides can provide a new method for the development of antiviral drugs for PEDV.

The Alpha-1 Subunit of the Na+/K+-ATPase (ATP1A1) Is a Host Factor Involved in the Attachment of Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea (PED) is an acute and severe atrophic enteritis caused by porcine epidemic diarrhea virus (PEDV) that infects pigs and makes huge economic losses to the global swine industry. Previously, researchers have believed that porcine aminopeptidase-N (pAPN) was the primary receptor for PEDV, but it has been found that PEDV can infect pAPN knockout pigs. Currently, the functional receptor for PEDV remains unspecified. In the present study, we performed virus overlay protein binding assay (VOPBA), found that ATP1A1 was the highest scoring protein in the mass spectrometry results, and confirmed that the CT structural domain of ATP1A1 interacts with PEDV S1. First, we investigated the effect of ATP1A1 on PEDV replication. Inhibition of hosts ATP1A1 protein expression using small interfering RNA (siRNAs) significantly reduced the cells susceptibility to PEDV. The ATP1A1-specific inhibitors Ouabain (a cardiac steroid) and PST2238 (a digitalis toxin derivative), which specifically bind ATP1A1, could block the ATP1A1 protein internalization and degradation, and consequently reduce the infection rate of host cells by PEDV significantly. Additionally, as expected, overexpression of ATP1A1 notably enhanced PEDV infection. Next, we observed that PEDV infection of target cells resulted in upregulation of ATP1A1 at the mRNA and protein levels. Furthermore, we found that the host protein ATP1A1 was involved in PEDV attachment and co-localized with PEDV S1 protein in the early stage of infection. In addition, pretreatment of IPEC-J2 and Vero-E6 cells with ATP1A1 mAb significantly reduced PEDV attachment. Our observations provided a perspective on identifying key factors in PEDV infection, and may provide valuable targets for PEDV infection, PEDV functional receptor, related pathogenesis, and the development of new antiviral drugs.

Monitoring of SARS-CoV-2 Infection in Ragusa Area: Next Generation Sequencing and Serological Analysis.

The coronavirus disease 19 (COVID-19) post pandemic evolution is correlated to the development of new variants. Viral genomic and immune response monitoring are fundamental to the surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since 1 January to 31 July 2022, we monitored the SARS-CoV-2 variants trend in Ragusa area sequencing n.600 samples by next generation sequencing (NGS) technology: n.300 were healthcare workers (HCWs) of ASP Ragusa. The evaluation of anti-Nucleocapside (N), receptor-binding domain (RBD), the two subunit of S protein (S1 and S2) IgG levels in 300 exposed vs. 300 unexposed HCWs to SARS-CoV-2 was performed. Differences in immune response and clinical symptoms related to the different variants were investigated. The SARS-CoV-2 variants trend in Ragusa area and in Sicily region were comparable. BA.1 and BA.2 were the most representative variants, whereas the diffusion of BA.3 and BA.4 affected some places of the region. Although no correlation was found between variants and clinical manifestations, anti-N and anti-S2 levels were positively correlated with an increase in the symptoms number. SARS-CoV-2 infection induced a statistically significant enhancement in antibody titers compared to that produced by SARS-CoV-2 vaccine administration. In post-pandemic period, the evaluation of anti-N IgG could be used as an early marker to identify asymptomatic subjects.

Proinflammatory Responses in SARS-CoV-2 and Soluble Spike Glycoprotein S1 Subunit Activated Human Macrophages.

Critically ill COVID-19 patients display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We inoculated and treated human macrophage cell line THP-1 with SARS-CoV-2 and purified, glycosylated, soluble SARS-CoV-2 spike protein S1 subunit (S1) to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-α and CXCL10 mRNAs, and induced secretion of TNF-α from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication or viral entry, virus exposure resulted in upregulation of both TNF-α and CXCL10 genes. Our study shows that extracellular soluble S1 protein is a key viral component inducing pro-inflammatory responses in macrophages, independent of virus replication. Thus, virus- or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.

Bioaccumulation Pattern of the SARS-CoV-2 Spike Proteins in Pacific Oyster Tissues.

There is mounting evidence of the contamination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the sewage, surface water, and even marine environment. Various studies have confirmed that bivalve mollusks can bioaccumulate SARS-CoV-2 RNA to detectable levels. However, these results do not provide sufficient evidence for the presence of infectious viral particles. To verify whether oysters can bind the viral capsid and bioaccumulate the viral particles, Pacific oysters were artificially contaminated with the recombinant SARS-CoV-2 spike protein S1 subunit (rS1). The bioaccumulation pattern of the rS1 in different tissues was investigated by immunohistological assays. The results revealed that the rS1 was bioaccumulated predominately in the digestive diverticula. The rS1 was also present in the epithelium of the nondigestive tract tissues, including the gills, mantle, and heart. In addition, three potential binding ligands, including angiotensin-converting enzyme 2 (ACE 2)-like substances, A-type histo-blood group antigen (HBGA)-like substances, and oyster heat shock protein 70 (oHSP 70), were confirmed to bind rS1 and were distributed in tissues with various patterns. The colocalization analysis of rS1 and those potential ligands indicated that the distributions of rS1 are highly consistent with those of ACE 2-like substances and oHSP 70. Both ligands are distributed predominantly in the secretory absorptive cells of the digestive diverticula and may serve as the primary ligands to bind rS1. Therefore, oysters are capable of bioaccumulating the SARS-CoV-2 capsid readily by filter-feeding behavior assisted by specific binding ligands, especially in digestive diverticula. IMPORTANCE This is the first article to investigate the SARS-CoV-2 spike protein bioaccumulation pattern and mechanism in Pacific oysters by the histochemical method. Oysters can bioaccumulate SARS-CoV-2 capsid readily by filter-feeding behavior assisted by specific binding ligands. The new possible foodborne transmission route may change the epidemic prevention strategies and reveal some outbreaks that current conventional epidemic transmission routes cannot explain. This original and interdisciplinary paper advances a mechanistic understanding of the bioaccumulation of SARS-CoV-2 in oysters inhabiting contaminated surface water.

Dynamics of SARS-CoV-2 Spike-IgG throughout Three COVID-19 Vaccination Regimens: A 21-Month Longitudinal Study of 82 Norwegian Healthcare Workers.

To facilitate interpretation of clinical SARS-CoV-2 anti-spike IgG analyses post-vaccination, 82 healthcare workers were followed through three vaccination-regimens: two regimens were comprised of two doses of BNT162b2 three or six weeks apart, followed by a dose of mRNA-vaccine, and in the other regimen, the first dose was replaced by ChAdOx1 nCov-19. After each dose, anti-spike IgG was compared between regimens. As many participants became infected, anti-spike IgG persistence was compared between infected and uninfected participants. Thirteen to twenty-one days after the first dose, seroconversion, and the median anti-spike IgG level in the ChAdOx1 group was significantly lower than in the BNT162b2 groups (23 versus 68 and 73 AU/mL). The second dose caused a significant increase in anti-spike IgG, but the median level was lower in the BNT162b2-short-interval group (280 AU/mL), compared to the BNT162b2-long-interval (1075 AU/mL) and ChAdOx1 (1160 AU/mL) group. After the third dose, all groups showed increases to similar anti-spike IgG levels (2075-2390 AU/mL). Over the next half year, anti-spike IgG levels declined significantly in all groups, but appeared to persist longer after post-vaccination infection. This is the first three-dose study with one dose of ChAdOx1. Despite initial differences, all vaccine regimens gave similarly high antibody levels and persistence after the third dose.

Cytoplasmic Tail Truncation Stabilizes S1-S2 Association and Enhances S Protein Incorporation into SARS-CoV-2 Pseudovirions.

Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. IMPORTANCE Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.

Reversal of the unique Q493R mutation increases the affinity of Omicron S1-RBD for ACE2.

The SARS-CoV-2 Omicron variant containing 15 mutations, including the unique Q493R, in the spike protein receptor binding domain (S1-RBD) is highly infectious. While comparison with previously reported mutations provide some insights, the mechanism underlying the increased infections and the impact of the reversal of the unique Q493R mutation seen in BA.4, BA.5, BA.2.75, BQ.1 and XBB lineages is not yet completely understood. Here, using structural modelling and molecular dynamics (MD) simulations, we show that the Omicron mutations increases the affinity of S1-RBD for ACE2, and a reversal of the unique Q493R mutation further increases the ACE2-S1-RBD affinity. Specifically, we performed all atom, explicit solvent MD simulations using a modelled structure of the Omicron S1-RBD-ACE2 and compared the trajectories with the WT complex revealing a substantial reduction in the Cα-atom fluctuation in the Omicron S1-RBD and increased hydrogen bond and other interactions. Residue level analysis revealed an alteration in the interaction between several residues including a switch in the interaction of ACE2 D38 from S1-RBD Y449 in the WT complex to the mutated R residue (Q493R) in Omicron complex. Importantly, simulations with Revertant (Omicron without the Q493R mutation) complex revealed further enhancement of the interaction between S1-RBD and ACE2. Thus, results presented here not only provide insights into the increased infectious potential of the Omicron variant but also a mechanistic basis for the reversal of the Q493R mutation seen in some Omicron lineages and will aid in understanding the impact of mutations in SARS-CoV-2 evolution.