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    Ultrastructural insight into SARS-CoV-2 attachment, entry and budding in human airway epithelium

    Authors: Andreia L Pinto; Ranjit K Rai; Jonathan C Brown; Paul Griffin; James R Edgar; Anand Shah; Aran Singanayagam; Claire Hogg; Wendy S Barclay; Clare E Futter; Thomas Burgoyne

    doi:10.1101/2021.04.10.439279 Date: 2021-04-11 Source: bioRxiv

    Ultrastructural studies of SARS-CoV-2 infected MESHD cells are crucial to better understand the mechanisms of viral entry and budding within host cells. Many studies are limited by the lack of access to appropriate cellular models. As the airway epithelium is the primary site of infection it is essential to study SARS-CoV-2 infection MESHD of these cells. Here, we examined human airway epithelium, grown as highly differentiated air-liquid interface cultures and infected with three different isolates of SARS-CoV-2 including the B.1.1.7 variant (Variant of Concern 202012/01) by transmission electron microscopy and tomography. For all isolates, the virus infected ciliated but not goblet epithelial cells. Two key SARS-CoV-2 entry molecules, ACE2 HGNC and TMPRSS2 HGNC, were found to be localised to the plasma membrane including microvilli but excluded from cilia. Consistent with these observations, extracellular virions were frequently seen associated with microvilli and the apical plasma membrane but rarely with ciliary membranes. Profiles indicative of viral fusion at the apical plasma membrane demonstrate that the plasma membrane is one site of entry where direct fusion releasing the nucleoprotein PROTEIN-encapsidated genome occurs. Intact intracellular virions were found within ciliated cells in compartments with a single membrane bearing S glycoprotein PROTEIN. Profiles strongly suggesting viral budding from the membrane was observed in these compartments and this may explain how virions gain their S glycoprotein PROTEIN containing envelope.

    Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike PROTEIN glycoprotein

    Authors: Shijian Zhang; Eden P. Go; Haitao Ding; Saumya Anang; John C. Kappes; Heather Desaire; Joseph G. Sodroski

    doi:10.1101/2021.04.01.438120 Date: 2021-04-01 Source: bioRxiv

    The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19 MESHD, uses its spike ( S) glycoprotein PROTEIN anchored in the viral membrane to enter host cells. The S glycoprotein PROTEIN is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein PROTEIN consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S MESHD, M, E and N proteins PROTEIN contained spike glycoproteins PROTEIN that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to enrich the Golgi-resident fraction of a wild-type SARS-CoV-2 S glycoprotein PROTEIN trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins PROTEIN modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein PROTEIN N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on soluble and virion S trimers, is predominantly modified in the Golgi by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 HGNC and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein PROTEIN carbohydrates and could assist the design of interventions.

    Weak humoral immune reactivity among residents of long-term care facilities following one dose of the BNT162b2 mRNA COVID-19 MESHD vaccine

    Authors: Mark A Brockman; Francis Mwimanzi; Yurous Sang; Kurtis Ng; Olga Agafitei; Siobhan Ennis; Hope Lapointe; Landon Young; Gisele Umviligihozo; Laura Burns; Chanson J Brumme; Victor Leung; Julio S G Montaner; Daniel Holmes; Mari DeMarco; Janet Simons; Masahiro Niikura; Ralph Pantophlet; Marc G Romney; Zabrina L Brumme

    doi:10.1101/2021.03.17.21253773 Date: 2021-03-24 Source: medRxiv

    Background. Several Canadian provinces are extending the interval between COVID-19 MESHD vaccine doses to increase population vaccine coverage more rapidly. However, immunogenicity of these vaccines after one dose is incompletely characterized, particularly among the elderly, who are at greatest risk of severe COVID-19 MESHD. Methods. We assessed SARS-CoV-2 humoral responses pre-vaccine and one month following the first dose of BNT162b2 mRNA vaccine, in 12 COVID-19 MESHD seronegative residents of long-term care facilities (median age, 82 years), 18 seronegative healthcare workers (HCW; median age, 36 years) and 4 convalescent HCW. Total antibody responses to SARS-CoV-2 nucleocapsid (N PROTEIN) and spike protein PROTEIN receptor binding domain (S/RBD) were assessed using commercial immunoassays. We quantified IgG and IgM responses to S/RBD and determined the ability of antibodies to block S/RBD binding to ACE2 HGNC receptor using ELISA. Neutralizing antibody activity was also assessed using pseudovirus and live SARS-CoV-2. Results. After one vaccine dose, binding antibodies against S/RBD were ~4-fold lower in residents compared to HCW (p<0.001). Inhibition of ACE2 HGNC binding was 3-fold lower in residents compared to HCW (p=0.01) and pseudovirus neutralizing activity was 2-fold lower (p=0.003). While six (33%) seronegative HCW neutralized live SARS-CoV-2, only one (8%) resident did (p=0.19). In contrast, convalescent HCW displayed 7- to 20-fold higher levels of binding antibodies and substantial ability to neutralize live virus after one dose. Interpretation. Extending the interval between COVID-19 MESHD vaccine doses may pose a risk to the elderly due to lower vaccine immunogenicity in this group. We recommend that second doses not be delayed in elderly individuals.

    Inhibiting SARS-CoV-2 infection MESHD in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor HGNC signal

    Authors: Keiji Tanimoto; Kiichi Hirota; Takahiro Fukazawa; Yoshiyuki Matsuo; Toshihito Nomura; Nazmul Tanuza; Nobuyuki Hirohashi; Hidemasa Bono; Takemasa Sakakuchi

    doi:10.1101/2021.03.04.433658 Date: 2021-03-04 Source: bioRxiv

    Since understanding molecular mechanisms of SARS-CoV-2 infection MESHD is extremely important for developing effective therapies against COVID-19 MESHD, we focused on the internalization mechanism of SARS-CoV-2 via ACE2 HGNC. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19 MESHD, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 HGNC in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 HGNC in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 HGNC might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor HGNC ( AHR MESHD AHR HGNC), the AHR MESHD AHR HGNC agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole ( OMP HGNC) were tested to assess whether those treatments affected ACE2 HGNC expression. Both FICZ and OMP HGNC clearly suppressed ACE2 HGNC expression in a dose-dependent manner along with inducing CYP1A1 HGNC. Knock-down experiments indicated a reduction of ACE2 HGNC by FICZ treatment in an AHR HGNC-dependent manner. Finally, treatments of AHR MESHD agonists inhibited SARS-CoV-2 infection MESHD into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein PROTEIN. We here demonstrate that treatment with AHR HGNC AHR MESHD agonists, including CSE, FICZ, and OMP HGNC, decreases expression of ACE2 HGNC via AHR MESHD AHR HGNC activation, resulting in suppression of SARS-CoV-2 infection MESHD in mammalian cells.

    Modeling SARS-CoV-2 infection MESHD and its individual differences with ACE2 HGNC-expressing human iPS cells

    Authors: Emi Sano; Ayaka Sakamoto; Natsumi Mimura; Ai Hirabayashi; Yukiko Muramoto; Takeshi Noda; Takuya Yamamoto; Kazuo Takayama

    doi:10.1101/2021.02.22.432218 Date: 2021-02-22 Source: bioRxiv

    Genetic differences are a primary reason for differences in the susceptibility and severity of coronavirus disease 2019 MESHD ( COVID-19 MESHD). Because induced pluripotent stem MESHD (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection MESHD in vitro. Notably, undifferentiated MESHD human iPS cells themselves cannot be infected bySARS-CoV-2. Using adenovirus vectors, here we found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 ( ACE2 HGNC) ( ACE2 HGNC-iPS cells) can be infected with SARS-CoV-2. In infected ACE2 HGNC-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein PROTEIN, the budding of viral particles, the production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We also evaluated COVID-19 MESHD therapeutic drugs in ACE2 HGNC-iPS cells and confirmed the strong antiviral effects of Remdesivir, EIDD-2801, and interferon-beta HGNC. In addition, we performed SARS-CoV-2 infection MESHD experiments on ACE2 HGNC-iPS/ES cells from 8 individuals. Male iPS/ES cells were more capable of producing the virus as compared with female iPS/ES cells. These findings suggest that ACE2 HGNC-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection MESHD in vitro, but they are also a useful resource to clarify the causes of individual differences in COVID-19 MESHD due to genetic differences.

    SARS CoV-2 nucleoprotein PROTEIN enhances the infectivity of lentiviral spike particles

    Authors: Tarun Mishra; Sreepadmanabh M; Pavitra Ramdas; Amit Kumar Sahu; Atul Kumar; Ajit Chande

    doi:10.1101/2021.02.11.430757 Date: 2021-02-15 Source: bioRxiv

    The establishment of SARS CoV-2 spike PROTEIN-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2 HGNC+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N PROTEIN) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that this augmentation by N renders LV spike particles less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 HGNC microbody. Biochemical analysis revealed that the spike protein PROTEIN is better enriched in virions when the particles are produced in the presence of SARS CoV-2 nucleoprotein PROTEIN. Importantly, this improvement in infectivity is achieved without a concomitant increase in sensitivity towards RBD binding-based neutralization. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.

    Expression of human ACE2 HGNC N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E PROTEIN. coli ribonuclease I and human RNase A

    Authors: Shuang-yong Xu; Alexey Fomenkov; Tien-Hao Chen; Erbay Yigit; Yinhui Lu; Karl E Kadler

    doi:10.1101/2021.01.31.429007 Date: 2021-02-01 Source: bioRxiv

    The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ~30 kb. Human ACE2 HGNC protein is the receptor for SARS-CoV-2 virus attachment MESHD and initiation of infection MESHD. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions the RNA is protected by viral capsid proteins, membrane proteins and nucleocapsid PROTEIN proteins. To overcome this protection we set out to construct RNase fusion with human ACE2 HGNC receptor N-terminal domain (ACE2NTD). We constructed six proteins expressed in E. coli cells: 1) MBP-ACE2NTD, 2) ACE2NTD-GFP, 3) RNase I (6xHis), 4) RNase III (6xHis), 5) RNase I-ACE2NTD (6xHis), and 6) human RNase A HGNC-ACE2NTD150 (6xHis). We evaluated fusion expression in different E. coli strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6xHis) and hRNase A-ACE2NTD (6xHis) fusions are active in cleaving COVID-19 MESHD RNA in vitro. The recombinant RNase I (6xHis) and RNase III (6xHis) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human cell receptor and nuclease that may be used to degrade viral nucleic acids in our environment.

    Evaluation of vertical transmission of SARS-CoV-2 in utero: nine pregnant women and their newborns

    Authors: Liang Dong; Shiyao Pei; Qin Ren; Shuxiang Fu; Liang Yu; Hui Chen; Xiang Chen; Mingzhu Yin

    doi:10.1101/2020.12.28.20248874 Date: 2021-01-08 Source: medRxiv

    BackgroundSevere acute respiratory syndrome coronavirus 2 MESHD (SARS-CoV-2), mainly transmitted by droplets and close contact, has caused a pandemic worldwide as of November 2020. According to the current case reports and cohort studies, the symptoms of pregnant women infected with SARS-CoV-2 were similar to normal adults and may cause a series of adverse consequences of pregnancy (placental abruption, fetal distress, epilepsy MESHD during pregnancy, etc.). However, whether SARS-CoV-2 can be transmitted to the fetus through the placental barrier is still a focus of debate. MethodsIn this study, in order to find out whether SARS-CoV-2 infect MESHD fetus through placental barrier, we performed qualitative detection of virus structural protein (spike PROTEIN protein and nucleoprotein PROTEIN) and targeted receptor protein ( ACE2 HGNC, CD147 HGNC and GRP78 HGNC) expression on the placental tissue of seven pregnant women diagnosed with COVID-19 MESHD through immunohistochemistry. Amniotic fluid, neonatal throat, anal swab and breastmilk samples were collected immediately in the operating room for verification after delivery, which were all tested for SARS-CoV-2 by reverse transcriptionpolymerase chain reaction (RT-PCR). Results: The result showed that CD147 HGNC was expressed on the basal side of the chorionic trophoblast cell membrane and ACE2 HGNC was expressed on the maternal side, while GRP78 HGNC was strongly expressed in the cell membrane and cytoplasm. The RT-PCR results of Amniotic fluid, neonatal throat, anal swab and breastmilk samples were all negative. Conclusions: We believed that despite the detection of viral structural proteins in the placenta, SARS-CoV-2 cannot be transmitted to infants due to the presence of the placental barrier.

    Development and Evaluation of Two Rapid Indigenous IgG-ELISA immobilized with ACE-2 HGNC Binding Peptides for Detection Neutralizing Antibodies Against SARS-CoV-2

    Authors: Bijon Kumar Sil; Nihad Adnan; Mumtarin Jannat Oishee; Tamanna Ali; Nowshin Jahan; Shahad Saif Khandker; Eiry Kobatake; Masayasu Mie; Dr. Mohib Ullah Khondoker; Md. Ahsanul Haq; Mohd. Raeed Jamiruddin

    doi:10.1101/2020.12.19.20248535 Date: 2020-12-22 Source: medRxiv

    COVID-19 MESHD COVID-19 MESHD pandemic situation demands effective serological tests with a view to adopting and developing policy for disease management, determining protective immunity as well as for sero-epidemiological study. Our study aims to develop and evaluate two rapid in-house ELISA assays targeting neutralizing antibodies (IgG) against S1 subunit of spike PROTEIN in SARS-CoV-2 and Receptor Binding Domain (RBD), as well as comparative analysis with nucleocapsid ( NCP PROTEIN) ELISA. The assays were conducted with 184 samples in three panels collected from 134 patients. Panel 1 and 2 consist of RT-PCR positive samples collected within two weeks and after two weeks of symptom onset, respectively. Negative samples are included in panel 3 from healthy donors and pre-pandemic dengue patients. The total assay time has been set 30 minutes for both of the ELISA assays. Results show that S1 and RBD ELISA demonstrates 73.68% and 84.21% sensitivities, respectively for samples collected within two weeks, whereas 100% sensitivities were achieved by both for samples that were collected after two weeks of the onset of symptoms. S1-ELISA shows 0% positivity to panel 3 while for RBD-ELISA the figure is 1%. A strong correlation (rs=0.804, p<0.0001)) has been observed between these two assays. When compared with NCP PROTEIN-ELISA, S1 slightly better correlation (rs=0.800, p<0.0001) than RBD (rs=0.740, p<0.0001). Our study suggests S1-ELISA as more sensitive one than the RBD or nucleocapsid ELISA during the later phase of infection, while for overall sero-monitoring RBD specific IgG ELISA is recommended. Moreover, non-reactivity to dengue emphasize the use of these assays for serosurveillance of COVID-19 MESHD in the dengue endemic regions.


    Authors: Luca Elli; Federica Facciotti; Vincenza Lombardo; Alice Scricciolo; David S Sanders; Valentina Vaira; Donatella Barisani; Maurizio Vecchi; Andrea Costantino; Lucia Scaramella; Bernardo Dell'Osso; Luisa Doneda; Leda Roncoroni

    doi:10.1101/2020.12.15.20248039 Date: 2020-12-16 Source: medRxiv

    Objective. The SARS-CoV-2 pandemic has spread across the world causing a dramatic number of infections and deaths MESHD. No data are available about the effects of an infection in patients affected by celiac disease ( CD MESHD) in terms of the development of related symptoms and antibodies. We aimed to investigate the impact of the SARS-CoV-2 pandemic in celiac patients. Design. During a lockdown, the celiac patients living in the Milan area were contacted and interviewed about the development of COVID-19 MESHD symptoms as well as adherence to an anti-virus lifestyle and a gluten-free diet (GFD). They were also given a stress questionnaire to fill in. The development of anti-SARS-CoV-2 IgG and IgA (anti-RBD and N proteins PROTEIN) and the expression of the duodenal ACE2 HGNC receptor were investigated. When available, duodenal histology, anti-tissue transglutaminase IgA (tTGA), presence of immunologic comorbidities and adherence to the GFD were analysed as possible risk factors. Results. 362 celiac patients have been interviewed and 42 (11%) presented with COVID-19 MESHD symptoms. The presence of symptoms was not influenced by tTGA positivity, presence of duodenal atrophy MESHD or adherence to GFD. 37% of the symptomatic patients presented anti-SARS-CoV-2 immunoglobulins (Ig). Globally, 18% of celiac patients showed anti-SARS-CoV-2 Ig vs 25% of the non-celiac control (p=0.18). The values of anti-RBD IgG/IgA and anti-N IgG did not differ from the non-celiac controls. Celiac patients had a significant lower level of anti-N IgA. The ACE2 HGNC receptor was detected in the non-atrophic duodenal mucosa of celiac patients; atrophy MESHD was associated with a lower expression of the ACE2 HGNC receptor. Conclusion. CD MESHD patients have an anti-SARS-CoV-2 Ig positiveness and profile similar to non-celiac controls, except for anti-N IgA. The main celiac parameters and adherence to the GFD do not influence the development of a different Ig profile.

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MeSH Disease
HGNC Genes
SARS-CoV-2 Proteins

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