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1.
Nat Commun ; 12(1): 961, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574281

RESUMO

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.


Assuntos
/virologia , Sistemas CRISPR-Cas , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , Internalização do Vírus , Células A549 , /metabolismo , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Endossomos/virologia , Células HeLa , Humanos , Mesocricetus , Serina Endopeptidases , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
2.
JCI Insight ; 6(1)2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33427208

RESUMO

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross-SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection.


Assuntos
Anticorpos Neutralizantes/farmacologia , Imunidade Inata/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/uso terapêutico , /fisiopatologia , Cricetinae , Reações Cruzadas , Epitopos , Humanos , Imunidade Inata/imunologia , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Mesocricetus , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Engenharia de Proteínas , Receptores Fc/imunologia , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Células THP-1 , Carga Viral/efeitos dos fármacos , Perda de Peso/efeitos dos fármacos
3.
Nat Commun ; 12(1): 288, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436577

RESUMO

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.


Assuntos
Anticorpos Neutralizantes/farmacologia , Ligação Proteica/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacos , /química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Furões , Humanos , Leucócitos Mononucleares , Macaca mulatta , Masculino , Mesocricetus , Modelos Moleculares , Conformação Proteica , Glicoproteína da Espícula de Coronavírus/química , Células Vero
4.
J Exp Med ; 218(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33211088

RESUMO

SARS-CoV-2, the causative agent of COVID-19, has been responsible for over 42 million infections and 1 million deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here, we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a Syrian hamster model of SARS-CoV-2 and in a mouse-adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). Antibody combinations were effective for prevention and in therapy when administered early. However, in vitro antibody neutralization potency did not uniformly correlate with in vivo protection, and some hu-mAbs were more protective in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors contributes to optimal protection against SARS-CoV-2 MA. The data indicate that intact effector function can affect hu-mAb protective activity and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention.


Assuntos
Anticorpos Monoclonais Murinos , Anticorpos Neutralizantes , Anticorpos Antivirais , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Linhagem Celular , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Feminino , Humanos , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Viral/imunologia , Pneumonia Viral/terapia
5.
Viruses ; 12(12)2020 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371200

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3' untranslated region (UTR) of this ß-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3' UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3' UTRs in the infectious virions and the minigene-transfected cells are almost identical.


Assuntos
Regiões 3' não Traduzidas/genética , Conformação de Ácido Nucleico , Pandemias , RNA Viral/genética , /genética , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , Cricetinae , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mesocricetus , Modelos Moleculares , Plasmídeos , Mutação Puntual , Genética Reversa/métodos , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Ésteres do Ácido Sulfúrico , Transcrição Genética , Vírion/genética , Vírion/fisiologia
7.
Nat Microbiol ; 5(11): 1439-1448, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33028965

RESUMO

SARS-CoV-2 is causing a pandemic of COVID-19, with high infectivity and significant mortality1. Currently, therapeutic options for COVID-19 are limited. Historically, metal compounds have found use as antimicrobial agents, but their antiviral activities have rarely been explored. Here, we test a set of metallodrugs and related compounds, and identify ranitidine bismuth citrate, a commonly used drug for the treatment of Helicobacter pylori infection, as a potent anti-SARS-CoV-2 agent, both in vitro and in vivo. Ranitidine bismuth citrate exhibited low cytotoxicity and protected SARS-CoV-2-infected cells with a high selectivity index of 975. Importantly, ranitidine bismuth citrate suppressed SARS-CoV-2 replication, leading to decreased viral loads in both upper and lower respiratory tracts, and relieved virus-associated pneumonia in a golden Syrian hamster model. In vitro studies showed that ranitidine bismuth citrate and its related compounds exhibited inhibition towards both the ATPase (IC50 = 0.69 µM) and DNA-unwinding (IC50 = 0.70 µM) activities of the SARS-CoV-2 helicase via an irreversible displacement of zinc(II) ions from the enzyme by bismuth(III) ions. Our findings highlight viral helicase as a druggable target and the clinical potential of bismuth(III) drugs or other metallodrugs for the treatment of SARS-CoV-2 infection.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Bismuto/farmacologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Ranitidina/análogos & derivados , Replicação Viral/efeitos dos fármacos , Animais , Betacoronavirus/fisiologia , Quimiocinas/metabolismo , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Citocinas/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Pulmão/patologia , Pulmão/virologia , Mesocricetus , Pandemias , Pneumonia Viral/tratamento farmacológico , RNA Helicases/metabolismo , Ranitidina/farmacologia , Células Vero , Carga Viral
8.
PLoS Pathog ; 16(9): e1008875, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898162

RESUMO

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion.


Assuntos
Encéfalo/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Animais , Arvicolinae , Encéfalo/patologia , Cricetinae , Mesocricetus , Camundongos , Camundongos Transgênicos , Proteínas PrPC/genética , Proteínas PrPSc/genética , Doenças Priônicas/genética , Doenças Priônicas/patologia , Domínios Proteicos
9.
J Virol ; 94(22)2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-32900822

RESUMO

Animal models recapitulating human COVID-19 disease, especially severe disease, are urgently needed to understand pathogenesis and to evaluate candidate vaccines and therapeutics. Here, we develop novel severe-disease animal models for COVID-19 involving disruption of adaptive immunity in Syrian hamsters. Cyclophosphamide (CyP) immunosuppressed or RAG2 knockout (KO) hamsters were exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the respiratory route. Both the CyP-treated and RAG2 KO hamsters developed clinical signs of disease that were more severe than those in immunocompetent hamsters, notably weight loss, viral loads, and fatality (RAG2 KO only). Disease was prolonged in transiently immunosuppressed hamsters and was uniformly lethal in RAG2 KO hamsters. We evaluated the protective efficacy of a neutralizing monoclonal antibody and found that pretreatment, even in immunosuppressed animals, limited infection. Our results suggest that functional B and/or T cells are not only important for the clearance of SARS-CoV-2 but also play an early role in protection from acute disease.IMPORTANCE Syrian hamsters are in use as a model of disease caused by SARS-CoV-2. Pathology is pronounced in the upper and lower respiratory tract, and disease signs and endpoints include weight loss and viral RNA and/or infectious virus in swabs and organs (e.g., lungs). However, a high dose of virus is needed to produce disease, and the disease resolves rapidly. Here, we demonstrate that immunosuppressed hamsters are susceptible to low doses of virus and develop more severe and prolonged disease. We demonstrate the efficacy of a novel neutralizing monoclonal antibody using the cyclophosphamide transient suppression model. Furthermore, we demonstrate that RAG2 knockout hamsters develop severe/fatal disease when exposed to SARS-CoV-2. These immunosuppressed hamster models provide researchers with new tools for evaluating therapies and vaccines and understanding COVID-19 pathogenesis.


Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Modelos Animais de Doenças , Mesocricetus , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Imunidade Adaptativa , Animais , Animais Geneticamente Modificados , Betacoronavirus/fisiologia , Ciclofosfamida , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Imunossupressores , Pandemias , Índice de Gravidade de Doença
10.
Nature ; 586(7830): 509-515, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32967005

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Furões/virologia , Humanos , Mesocricetus/virologia , Camundongos , Pneumonia Viral/imunologia , Primatas/virologia , Vacinas Virais/imunologia
11.
Mol Immunol ; 126: 87-94, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32784101

RESUMO

Viral infections can lead to interferon production, which achieves its antiviral function primarily by activating the JAK/STAT pathway and inducing multiple interferon-stimulated genes (ISGs). Although considerable ISGs have been identified in antiviral researches, little is known about ISGs in bluetongue virus (BTV) infection. Viperin is the most highly induced ISG following BTV infection, which suggests that it may play a critical role in the anti-BTV immune response. The aim of this study was to characterize ovine Viperin (oViperin) and explore whether it can inhibit BTV replication. We cloned the coding sequences (CDS) of sheep Viperin, and the sequence analysis showed that oViperin displayed a high similarity with other species. oViperin has a leucine zipper in the N-terminal, a CxxxCxxC motif in the SAM domain, and a conservative C-terminus. We found that oViperin mRNA expression was significantly up-regulated in a time- and multiplicity of infection (MOI)-dependent manner following BTV infection. oViperin overexpression resulted in a significant inhibition in BTV replication, whereas an oViperin knockdown in MDOK cells increased BTV replication. This study shows for the first time, that oViperin has antiviral activity towards BTV infection and provides important information to research the interaction between BTV and oViperin.


Assuntos
Vírus Bluetongue/fisiologia , Bluetongue/imunologia , Proteínas com Ferro-Enxofre/imunologia , Carneiro Doméstico/imunologia , Replicação Viral/imunologia , Animais , Bluetongue/virologia , Vírus Bluetongue/isolamento & purificação , Linhagem Celular , Clonagem Molecular , Técnicas de Silenciamento de Genes , Imunidade Inata , Proteínas com Ferro-Enxofre/genética , Mesocricetus , RNA Mensageiro/metabolismo , Carneiro Doméstico/genética , Carneiro Doméstico/virologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/imunologia
12.
Exp Parasitol ; 218: 107964, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32822697

RESUMO

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.


Assuntos
Leishmania infantum/química , Leishmania mexicana/química , Proteínas de Membrana/análise , Proteômica/métodos , Proteínas de Protozoários/análise , Animais , Membrana Celular/química , Cromatografia Líquida , Biologia Computacional , Cricetinae , Transportador de Glucose Tipo 2/análise , Interações Hospedeiro-Parasita , Leishmania infantum/metabolismo , Leishmania infantum/patogenicidade , Leishmania infantum/ultraestrutura , Leishmania mexicana/ultraestrutura , Macrófagos Peritoneais/parasitologia , Espectrometria de Massas , Mesocricetus , Metaloendopeptidases/análise , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais , Espectrometria de Massas em Tandem , Virulência
13.
Parasitol Res ; 119(9): 3041-3051, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32779021

RESUMO

Our objective was to investigate clinical progression, presence of parasites and DNAs, parasite loads, and histological alterations in BALB/c mice and Syrian golden hamsters after intraperitoneal inoculation with Leishmania (Mundinia) martiniquensis promastigotes with a goal to choosing an appropriate animal model for visceral leishmaniasis. Infections were monitored for 16 weeks. Infected BALB/c mice were asymptomatic during the infection course. Parasite DNAs were detected in the liver at week 8 of infection, followed by clearance in most animals at week 16; whereas in the spleen, parasite DNAs were detected until week 16. These results are correlated to those obtained measuring parasite loads in both organs. No parasite DNA and no alteration in the bone marrow were observed indicating that no dissemination occurred. These results suggest the control of visceralization of L. martiniquensis by BALB/c mice. In hamsters, weight loss, cachexia, and fatigue were observed after week 11. Leishmania martiniquensis parasites were observed in tissue smears of the liver, spleen, and bone marrow by week 16. Parasite loads correlated with those from the presence of parasites and DNAs in the examined tissues. Alterations in the liver with nuclear destruction and cytoplasmic degeneration of infected hepatocytes, presence of inflammatory infiltrates, necrosis of hepatocytes, and changes in splenic architecture and reduction and deformation of white pulp in the spleen were noted. These results indicate a chronic form of visceral leishmaniasis indicating that the hamster is a suitable animal model for the study of pathological features of chronic visceral leishmaniasis caused by L. martiniquensis.


Assuntos
Leishmania/fisiologia , Leishmaniose Visceral/parasitologia , Animais , Cricetinae , Modelos Animais de Doenças , Humanos , Leishmania/genética , Fígado/parasitologia , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Baço/parasitologia
14.
Braz Dent J ; 31(3): 310-318, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32667525

RESUMO

Oral mucositis is a common inflammatory complication among patients with cancer. This study evaluated the histopathological, stereological, and antioxidant markers of 2% eucalyptus extract in induced oral mucositis in male golden hamsters. In this animal study, oral mucositis was induced in 30 male golden hamsters by 5-FU (60 mg/kg) on days 0, 5, and 10 of the study. The cheek pouch was scratched with a sterile needle once daily on days 3 and 4. On days 14-17, 2% eucalyptus hydroalcoholic extract gel and Calendula officinalis extract gel groups were treated and then compared with a non-treated control group. The histopathological and stereological scores and the pouch content of malondialdehyde, as well as the activities of glutathione and myeloperoxidase in the pouch tissue, were evaluated. Histopathologic scores of oral mucositis were lower in the eucalyptus gel group than those of the calendula and control groups (p<0.05). Also, a lower malondialdehyde level and higher myeloperoxidase and glutathione activities were detected in the eucalyptus group in comparison to the calendula and control groups (p<0.001). The thickness of the mucosa and submucosa increased in the eucalyptus group. The numerical density of the fibroblast and the volume density of the collagen significantly increased in the eucalyptus group. In conclusion, the use of eucalyptus hydroalcoholic extract may be associated with reduced intensity of oral mucositis, diminished concentration of malondialdehyde, increased activity of myeloperoxidase and glutathione, increased volume of mucosa and submucosa, increased fibroblast and collagen in the induced oral mucositis in golden hamsters undergoing 5-FU consumption.


Assuntos
Eucalyptus , Mucosite , Estomatite , Animais , Cricetinae , Fluoruracila , Masculino , Mesocricetus , Mucosa Bucal , Extratos Vegetais
15.
Brain Behav Immun ; 89: 579-586, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32629042

RESUMO

Anosmia is one of the most prevalent symptoms of SARS-CoV-2 infection during the COVID-19 pandemic. However, the cellular mechanism behind the sudden loss of smell has not yet been investigated. The initial step of odour detection takes place in the pseudostratified olfactory epithelium (OE) mainly composed of olfactory sensory neurons surrounded by supporting cells known as sustentacular cells. The olfactory neurons project their axons to the olfactory bulb in the central nervous system offering a potential pathway for pathogens to enter the central nervous system by bypassing the blood brain barrier. In the present study, we explored the impact of SARS-CoV-2 infection on the olfactory system in golden Syrian hamsters. We observed massive damage of the OE as early as 2 days post nasal instillation of SARS-CoV-2, resulting in a major loss of cilia necessary for odour detection. These damages were associated with infection of a large proportion of sustentacular cells but not of olfactory neurons, and we did not detect any presence of the virus in the olfactory bulbs. We observed massive infiltration of immune cells in the OE and lamina propria of infected animals, which may contribute to the desquamation of the OE. The OE was partially restored 14 days post infection. Anosmia observed in COVID-19 patient is therefore likely to be linked to a massive and fast desquamation of the OE following sustentacular cells infection with SARS-CoV-2 and subsequent recruitment of immune cells in the OE and lamina propria.


Assuntos
Infecções por Coronavirus/patologia , Bulbo Olfatório/patologia , Mucosa Olfatória/patologia , Pneumonia Viral/patologia , Animais , Betacoronavirus , Cílios/patologia , Infecções por Coronavirus/fisiopatologia , Mesocricetus , Transtornos do Olfato/patologia , Transtornos do Olfato/fisiopatologia , Bulbo Olfatório/virologia , Mucosa Olfatória/virologia , Neurônios Receptores Olfatórios/patologia , Neurônios Receptores Olfatórios/virologia , Pandemias , Pneumonia Viral/fisiopatologia
16.
Nature ; 584(7821): 450-456, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32698192

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy1,2. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml-1. Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Epitopos de Linfócito B/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/ultraestrutura , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/análise , Anticorpos Antivirais/química , Anticorpos Antivirais/ultraestrutura , Betacoronavirus/química , Betacoronavirus/ultraestrutura , Infecções por Coronavirus/prevenção & controle , Microscopia Crioeletrônica , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/ultraestrutura , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Pulmão/patologia , Pulmão/virologia , Masculino , Mesocricetus , Modelos Moleculares , Testes de Neutralização , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/ultraestrutura
17.
Viruses ; 12(7)2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32698441

RESUMO

In late 2019, an outbreak of a severe respiratory disease caused by an emerging coronavirus, SARS-CoV-2, resulted in high morbidity and mortality in infected humans. Complete understanding of COVID-19, the multi-faceted disease caused by SARS-CoV-2, requires suitable small animal models, as does the development and evaluation of vaccines and antivirals. Since age-dependent differences of COVID-19 were identified in humans, we compared the course of SARS-CoV-2 infection in young and aged Syrian hamsters. We show that virus replication in the upper and lower respiratory tract was independent of the age of the animals. However, older hamsters exhibited more pronounced and consistent weight loss. In situ hybridization in the lungs identified viral RNA in bronchial epithelium, alveolar epithelial cells type I and II, and macrophages. Histopathology revealed clear age-dependent differences, with young hamsters launching earlier and stronger immune cell influx than aged hamsters. The latter developed conspicuous alveolar and perivascular edema, indicating vascular leakage. In contrast, we observed rapid lung recovery at day 14 after infection only in young hamsters. We propose that comparative assessment in young versus aged hamsters of SARS-CoV-2 vaccines and treatments may yield valuable information, as this small-animal model appears to mirror age-dependent differences in human patients.


Assuntos
Betacoronavirus , Infecções por Coronavirus/etiologia , Modelos Animais de Doenças , Pneumonia Viral/etiologia , Fatores Etários , Animais , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Cricetinae , Progressão da Doença , Feminino , Pulmão/virologia , Masculino , Mesocricetus , Pandemias , RNA Viral/análise , Vacinas Virais/imunologia
18.
Proc Natl Acad Sci U S A ; 117(28): 16587-16595, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32571934

RESUMO

At the end of 2019, a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was detected in Wuhan, China, that spread rapidly around the world, with severe consequences for human health and the global economy. Here, we assessed the replicative ability and pathogenesis of SARS-CoV-2 isolates in Syrian hamsters. SARS-CoV-2 isolates replicated efficiently in the lungs of hamsters, causing severe pathological lung lesions following intranasal infection. In addition, microcomputed tomographic imaging revealed severe lung injury that shared characteristics with SARS-CoV-2-infected human lung, including severe, bilateral, peripherally distributed, multilobular ground glass opacity, and regions of lung consolidation. SARS-CoV-2-infected hamsters mounted neutralizing antibody responses and were protected against subsequent rechallenge with SARS-CoV-2. Moreover, passive transfer of convalescent serum to naïve hamsters efficiently suppressed the replication of the virus in the lungs even when the serum was administrated 2 d postinfection of the serum-treated hamsters. Collectively, these findings demonstrate that this Syrian hamster model will be useful for understanding SARS-CoV-2 pathogenesis and testing vaccines and antiviral drugs.


Assuntos
Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Pulmão/patologia , Pneumonia Viral/virologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/patogenicidade , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/terapia , Cricetinae , Humanos , Imunização Passiva , Pulmão/diagnóstico por imagem , Pulmão/virologia , Mesocricetus , Pandemias , Pneumonia Viral/patologia , Ribonucleoproteínas/química , Células Vero , Proteínas Virais/química , Replicação Viral
19.
Science ; 369(6506): 956-963, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32540903

RESUMO

Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-recovered participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained weight and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design.


Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/imunologia , Pneumonia Viral/prevenção & controle , Adulto , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/uso terapêutico , Afinidade de Anticorpos , Especificidade de Anticorpos , Betacoronavirus/fisiologia , Sítios de Ligação , Linhagem Celular , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Epitopos , Feminino , Humanos , Imunização Passiva , Pulmão/virologia , Masculino , Mesocricetus , Pessoa de Meia-Idade , Testes de Neutralização , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Carga Viral , Replicação Viral
20.
Nat Commun ; 11(1): 3112, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561757

RESUMO

Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion.


Assuntos
Anticorpos Antivirais/metabolismo , Vírus da Dengue/ultraestrutura , Proteínas do Envelope Viral/metabolismo , Zika virus/ultraestrutura , Aedes , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/imunologia , Linhagem Celular , Microscopia Crioeletrônica , Dengue/imunologia , Dengue/terapia , Dengue/virologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Vírus da Dengue/metabolismo , Evasão da Resposta Imune , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Mesocricetus , Multimerização Proteica , Propriedades de Superfície , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/ultraestrutura , Ligação Viral , Zika virus/imunologia , Zika virus/metabolismo , Infecção por Zika virus
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