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1.
Nat Commun ; 12(1): 944, 2021 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-33574228

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterize and further evaluate the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection, while administration of the MD65 antibody as late as 3 days after exposure rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data demonstrate the therapeutic value of human monoclonal antibodies as a life-saving treatment for severe COVID-19 infection.


Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , /inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Femenino , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Pulmón/patología , Pulmón/virología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , /fisiología , Seroconversión , Células Vero , Carga Viral
2.
Nat Commun ; 12(1): 961, 2021 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-33574281

RESUMEN

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.


Asunto(s)
/virología , Sistemas CRISPR-Cas , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , Internalización del Virus , Células A549 , /metabolismo , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Endosomas/virología , Células HeLa , Humanos , Mesocricetus , Serina Endopeptidasas , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero
3.
Nat Commun ; 12(1): 856, 2021 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-33558528

RESUMEN

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein-protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.


Asunto(s)
Proteínas Luminiscentes/metabolismo , Acetilcolina/metabolismo , Animales , Células COS , Calcio/metabolismo , Chlorocebus aethiops , Fluorescencia , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/química , Modelos Moleculares
4.
Mem Inst Oswaldo Cruz ; 115: e200278, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33566939

RESUMEN

BACKGROUND: The impact of arbovirus cocirculation in Brazil is unknown. Dengue virus (DENV) reinfection may result in more intense viraemia or immunopathology, leading to more severe disease. The Zika virus (ZIKV) epidemic in the Americas provided pathogenicity evidence that had not been previously observed in flavivirus infections. In contrast to other flaviviruses, electron microscopy studies have shown that ZIKV may replicate in viroplasm-like structures. Flaviviruses produce an ensemble of structurally different virions, collectively contributing to tissue tropism and virus dissemination. OBJECTIVES AND METHODS: In this work, the Aedes albopictus mosquito cell lineage (C6/36 cells) and kidney epithelial cells from African green monkeys (Vero cells) were infected with samples of the main circulating arboviruses in Brazil [DENV-1, DENV-2, DENV-3, DENV-4, ZIKV, Yellow Fever virus (YFV) and Chikungunya virus (CHIKV)], and ultrastructural studies by transmission electron microscopy were performed. FINDINGS: We observed that ZIKV, the DENV serotypes, YFV and CHIKV particles are spherical. ZIKV, DENV-1, -2, -3 and -4 presented diameters of 40-50 nm, and CHIKV presented approximate diameters of 50-60 nm. Viroplasm-like structures was observed in ZIKV replication cycle. MAIN CONCLUSIONS: The morphogenesis of these arboviruses is similar to what has been presented in previous studies. However, we understand that further studies are needed to investigate the relationship between viroplasm-like structures and ZIKV replication dynamics.


Asunto(s)
Arbovirus , Fiebre Chikungunya , Dengue , Epidemias , Fiebre Amarilla , Infección por el Virus Zika , Virus Zika , Animales , Brasil/epidemiología , Fiebre Chikungunya/epidemiología , Chlorocebus aethiops , Dengue/epidemiología , Células Vero , Infección por el Virus Zika/epidemiología
5.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33557278

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. The virus still spreads globally through human-to-human transmission. Nevertheless, there are no specific treatments clinically approved. This study aimed to compare antiviral activity of gemcitabine and its analogue 2'-fluoro-2'-deoxycytidine (2FdC) against SARS-CoV-2 as well as cytotoxicity in vitro. Fluorescent image-based antiviral assays revealed that gemcitabine was highly potent, with a 50% effective concentration (EC50) of 1.2 µM, more active than the well-known nucleoside monophosphate remdesivir (EC50 = 35.4 µM). In contrast, 2FdC was marginally active (EC50 = 175.2 µM). For all three compounds, the 50% cytotoxic concentration (CC50) values were over 300 µM toward Vero CCL-81 cells. Western blot and quantitative reverse-transcription polymerase chain reaction analyses verified that gemcitabine blocked viral protein expression in virus-infected cells, not only Vero CCL-81 cells but also Calu-3 human lung epithelial cells in a dose-dependent manner. It was found that gemcitabine has a synergistic effect when combined with remdesivir. This report suggests that the difluoro group of gemcitabine is critical for the antiviral activity and that its combination with other evaluated antiviral drugs, such as remdesivir, could be a desirable option to treat SARS-CoV-2 infection.


Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Desoxicitidina/análogos & derivados , /efectos de los fármacos , Adenosina Monofosfato/farmacología , Alanina/farmacología , Animales , Antivirales/farmacología , /virología , Línea Celular , Chlorocebus aethiops , Desoxicitidina/farmacología , Quimioterapia Combinada , Humanos , Concentración 50 Inhibidora , Células Vero , Replicación Viral/efectos de los fármacos
6.
PLoS One ; 16(2): e0246366, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33529233

RESUMEN

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.


Asunto(s)
/fisiopatología , Modelos Animales de Enfermedad , Macaca mulatta , /fisiología , Animales , /transmisión , Chlorocebus aethiops , Transmisión de Enfermedad Infecciosa , Femenino , Pulmón/patología , Macaca fascicularis , Masculino , Esparcimiento de Virus
7.
Virulence ; 12(1): 615-629, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-33538234

RESUMEN

It is now clear that the intercellular transport on microtubules by dynein and kinesin-1 motors has an important role in the replication and spread of many viruses. Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded RNA virus of the Coronavirus family, which can infect swine of all ages and cause severe economic losses in the swine industry. Elucidating the molecular mechanisms of the intercellular transport of PEDV through microtubule, dynein and kinesin-1 will be crucial for understanding its pathogenesis. Here, we demonstrate that microtubule, dynein, and kinesin-1 are involved in PEDV infection and can influence PEDV fusion and accumulation in the perinuclear region but cannot affect PEDV attachment or internalization. Furthermore, we adopted a single-virus tracking technique to dynamically observe PEDV intracellular transport with five different types: unidirectional movement toward microtubule plus ends; unidirectional movement toward microtubule minus ends; bidirectional movement along the same microtubule; bidirectional movement along different microtubules and motionless state. Among these types, the functions of dynein and kinesin-1 in PEDV intercellular transport were further analyzed by single-virus tracking and found that dynein and kinesin-1 mainly transport PEDV to the minus and plus ends of the microtubules, respectively; meanwhile, they also can transport PEDV to the opposite ends of the microtubules different from their conventional transport directions and also coordinate the bidirectional movement of PEDV along the same or different microtubules through their cooperation. These results provided deep insights and references to understand the pathogenesis of PEDV as well as to develop vaccines and treatments.


Asunto(s)
Dineínas/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Virus de la Diarrea Epidémica Porcina/fisiología , Animales , Transporte Biológico , Chlorocebus aethiops , Citoplasma/metabolismo , Dineínas/antagonistas & inhibidores , Cinesina/genética , Fusión de Membrana , Microscopía Fluorescente , ARN Interferente Pequeño , Células Vero
8.
Sci Adv ; 7(6)2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33547084

RESUMEN

The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.


Asunto(s)
/metabolismo , Expresión Génica , Interacciones Microbiota-Huesped/genética , ARN Mensajero/metabolismo , Proteínas no Estructurales Virales/metabolismo , Factores de Virulencia/metabolismo , Transporte Activo de Núcleo Celular/genética , Animales , Chlorocebus aethiops , Células HEK293 , Humanos , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transfección , Células Vero , Proteínas no Estructurales Virales/genética
9.
Mem Inst Oswaldo Cruz ; 116: e200443, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33566951

RESUMEN

BACKGROUND: The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE: The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS: In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS: The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS: Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.


Asunto(s)
Efecto Citopatogénico Viral , Vesículas Citoplasmáticas/virología , Células Vero/virología , Animales , Chlorocebus aethiops , Endocitosis , Retículo Endoplásmico/virología , Humanos , Microscopía Electrónica de Transmisión , Nucleocápside , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Internalización del Virus
10.
Sci Adv ; 7(7)2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33579701

RESUMEN

Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19-related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.


Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Mutación Missense , Glicoproteína de la Espiga del Coronavirus , Sustitución de Aminoácidos , Animales , /genética , Chile , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Masculino , /metabolismo , Glicoproteína de la Espiga del Coronavirus/sangre , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero
11.
Viruses ; 13(2)2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33540713

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.


Asunto(s)
Antivirales/farmacología , Olea , Alcohol Feniletílico/análogos & derivados , Extractos Vegetales/farmacología , /efectos de los fármacos , Administración Tópica , Animales , Antivirales/química , Carbohidratos/química , Chlorocebus aethiops , Genoma Viral/efectos de los fármacos , Glicosilación , Pruebas de Sensibilidad Microbiana , Alcohol Feniletílico/administración & dosificación , Alcohol Feniletílico/farmacología , Fosfoproteínas/química , Extractos Vegetales/química , /fisiología , Crema para la Piel , Glicoproteína de la Espiga del Coronavirus/química , Células Vero , Inactivación de Virus/efectos de los fármacos
12.
PLoS Pathog ; 17(2): e1009165, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33571304

RESUMEN

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Convalecencia , Glicoproteína de la Espiga del Coronavirus , Adulto , Anciano , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , /inmunología , Chlorocebus aethiops , Clonación Molecular , Mapeo Epitopo , Epítopos/genética , Epítopos/inmunología , Femenino , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células Vero
13.
Nutrients ; 13(2)2021 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-33572045

RESUMEN

BACKGROUND: Zinc is an essential micronutrient that impacts host-pathogen interplay at infection. Zinc balances immune responses, and also has a proven direct antiviral action against some viruses. Importantly, zinc deficiency (ZD) is a common condition in elderly and individuals with chronic diseases, two groups with an increased risk for severe severe coronavirus disease 2019 (COVID-19) outcomes. We hypothesize that serum zinc content (SZC) influences COVID-19 disease progression, and thus might represent a useful biomarker. METHODS: We ran an observational cohort study with 249 COVID-19 patients admitted in Hospital del Mar. We have studied COVID-19 severity and progression attending to SZC at admission. In parallel, we have studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) replication in the Vero E6 cell line modifying zinc concentrations. FINDINGS: Our study demonstrates a correlation between serum zinc levels and COVID-19 outcome. Serum zinc levels lower than 50 µg/dL at admission correlated with worse clinical presentation, longer time to reach stability, and higher mortality. Our in vitro results indicate that low zinc levels favor viral expansion in SARS-CoV-2 infected cells. INTERPRETATION: Low SZC is a risk factor that determines COVID-19 outcome. We encourage performing randomized clinical trials to study zinc supplementation as potential prophylaxis and treatment with people at risk of zinc deficiency.


Asunto(s)
/sangre , Zinc/sangre , Anciano , Animales , Supervivencia Celular , Chlorocebus aethiops , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Vero , Zinc/administración & dosificación , Zinc/farmacología
14.
Genes (Basel) ; 12(2)2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33572725

RESUMEN

SARS-CoV-2 is a recently emerged, novel human coronavirus responsible for the currently ongoing COVID-19 pandemic. Recombination is a well-known evolutionary strategy of coronaviruses, which may frequently result in significant genetic alterations, such as deletions throughout the genome. In this study we identified a co-infection with two genetically different SARS-CoV-2 viruses within a single patient sample via amplicon-based next generation sequencing in Hungary. The recessive strain contained an 84 base pair deletion in the receptor binding domain of the spike protein gene and was found to be gradually displaced by a dominant non-deleterious variant over-time. We have identified the region of the receptor-binding domain (RBD) that is affected by the mutation, created homology models of the RBDΔ84 mutant, and based on the available experimental data and calculations, we propose that the mutation has a deteriorating effect on the binding of RBD to the angiotensin-converting enzyme 2 (ACE2) receptor, which results in the negative selection of this variant. Extending the sequencing capacity toward the discovery of emerging recombinant or deleterious strains may facilitate the early recognition of novel strains with altered phenotypic attributes and understanding of key elements of spike protein evolution. Such studies may greatly contribute to future therapeutic research and general understanding of genomic processes of the virus.


Asunto(s)
/metabolismo , /metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , /virología , Línea Celular , Chlorocebus aethiops , Simulación por Computador , Humanos , Pandemias , Unión Proteica , Dominios Proteicos , Eliminación de Secuencia , Células Vero
15.
Nat Commun ; 12(1): 263, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431828

RESUMEN

Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals. While SVs are mobile within the clusters, the clusters have no boundaries consistent with a liquid phase. We previously found that purified synapsin, a peripheral SV protein, can assemble into liquid condensates and trap liposomes into them. How this finding relates to the physiological formation of SV clusters in living cells remains unclear. Here, we report that synapsin alone, when expressed in fibroblasts, has a diffuse cytosolic distribution. However, when expressed together with synaptophysin, an integral SV membrane protein previously shown to be localized on small synaptic-like microvesicles when expressed in non-neuronal cells, is sufficient to organize such vesicles in clusters highly reminiscent of SV clusters and with liquid-like properties. This minimal reconstitution system can be a powerful model to gain mechanistic insight into the assembly of structures which are of fundamental importance in synaptic transmission.


Asunto(s)
Neuronas/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo , Animales , Células COS , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citosol/metabolismo , Endocitosis , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Ratones , Electricidad Estática , Vesículas Sinápticas/ultraestructura
16.
Nat Commun ; 12(1): 134, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33420022

RESUMEN

Understanding the factors that contribute to efficient SARS-CoV-2 infection of human cells may provide insights on SARS-CoV-2 transmissibility and pathogenesis, and reveal targets of intervention. Here, we analyze host and viral determinants essential for efficient SARS-CoV-2 infection in both human lung epithelial cells and ex vivo human lung tissues. We identify heparan sulfate as an important attachment factor for SARS-CoV-2 infection. Next, we show that sialic acids present on ACE2 prevent efficient spike/ACE2-interaction. While SARS-CoV infection is substantially limited by the sialic acid-mediated restriction in both human lung epithelial cells and ex vivo human lung tissues, infection by SARS-CoV-2 is limited to a lesser extent. We further demonstrate that the furin-like cleavage site in SARS-CoV-2 spike is required for efficient virus replication in human lung but not intestinal tissues. These findings provide insights on the efficient SARS-CoV-2 infection of human lungs.


Asunto(s)
/metabolismo , /transmisión , Ácidos Siálicos/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Acoplamiento Viral , Animales , Células CACO-2 , Línea Celular Tumoral , Chlorocebus aethiops , Cricetinae , Furina/metabolismo , Células HEK293 , Heparitina Sulfato/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/virología , Pulmón/patología , Pulmón/virología , Síndrome Respiratorio Agudo Grave/patología , Células Vero , Internalización del Virus , Replicación Viral/fisiología
17.
Nat Commun ; 12(1): 288, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436577

RESUMEN

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.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Unión Proteica/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/efectos de los fármacos , /química , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Hurones , Humanos , Leucocitos Mononucleares , Macaca mulatta , Masculino , Mesocricetus , Modelos Moleculares , Conformación Proteica , Glicoproteína de la Espiga del Coronavirus/química , Células Vero
18.
Nat Commun ; 12(1): 63, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397909

RESUMEN

The SARS-CoV-2 pandemic poses the greatest global public health challenge in a century. Neutralizing antibody is a correlate of protection and data on kinetics of virus neutralizing antibody responses are needed. We tested 293 sera from an observational cohort of 195 reverse transcription polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections collected from 0 to 209 days after onset of symptoms. Of 115 sera collected ≥61 days after onset of illness tested using plaque reduction neutralization (PRNT) assays, 99.1% remained seropositive for both 90% (PRNT90) and 50% (PRNT50) neutralization endpoints. We estimate that it takes at least 372, 416 and 133 days for PRNT50 titres to drop to the detection limit of a titre of 1:10 for severe, mild and asymptomatic patients, respectively. At day 90 after onset of symptoms (or initial RT-PCR detection in asymptomatic infections), it took 69, 87 and 31 days for PRNT50 antibody titres to decrease by half (T1/2) in severe, mild and asymptomatic infections, respectively. Patients with severe disease had higher peak PRNT90 and PRNT50 antibody titres than patients with mild or asymptomatic infections. Age did not appear to compromise antibody responses, even after accounting for severity. We conclude that SARS-CoV-2 infection elicits robust neutralizing antibody titres in most individuals.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , /inmunología , Adolescente , Adulto , Animales , Anticuerpos Antivirales/sangre , /epidemiología , Chlorocebus aethiops , Estudios de Cohortes , Femenino , Hong Kong/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Pandemias , Células Vero , Adulto Joven
19.
Vet Res ; 52(1): 2, 2021 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397461

RESUMEN

Porcine epidemic diarrhea (PED) is a coronavirus disease characterized by the rapid spread of severe diarrhea among pigs. PED virus (PEDV) infects and replicates mainly in the epithelial cells of the duodenum, jejunum, ileum and colon. Serum or mucosal IgA antibody levels have been used to predict both vaccine efficacy and the level of protective immunity to enteric infectious diseases in individuals or herds. Details of the B-cell immune response upon PEDV infection, such as the systemic and mucosal PEDV IgA antibody response, the distribution of IgA antibody-secreting cells (ASCs), and their role in virus clearance are not yet clear. In this experimental infection study, we observed similar fluctuations in PEDV IgA antibody levels in serum and intestinal contents of the upper and lower jejunum and ileum, but not fecal samples, over the 4-week experimental course. ASCs that actively secrete PEDV IgA antibody without in vitro stimulation were distributed mainly in the upper jejunum, whereas memory B cells that showed enhanced PEDV IgA antibody production upon in vitro stimulation were observed in mesenteric lymph nodes and the ileum. Our findings will contribute to the development of effective vaccines and diagnostic methods for PEDV.


Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Coronavirus/veterinaria , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos/virología , Animales , Chlorocebus aethiops , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Heces/química , Heces/virología , Inmunoglobulina A/sangre , Inmunoglobulina A/química , Inmunoglobulina A/metabolismo , Inmunoglobulina G/sangre , Mucosa Intestinal/metabolismo , ARN Viral , Porcinos , Enfermedades de los Porcinos/sangre , Enfermedades de los Porcinos/inmunología , Células Vero
20.
Vet Res ; 52(1): 7, 2021 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431056

RESUMEN

Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis during many viral infections, but its role during coronavirus infection is undetermined. Infectious bronchitis virus is the representative strain of Gammacoronavirus, which causes acute and highly contagious diseases in the poultry farm. In this study, we investigated the role of ERK1/2 signaling pathway in IBV infection. We found that IBV infection activated ERK1/2 signaling and the up-regulation of phosphatase DUSP6 formed a negative regulation loop. Pharmacological inhibition of MEK1/2-ERK1/2 signaling suppressed the expression of DUSP6, promoted cell death, and restricted virus replication. In contrast, suppression of DUSP6 by chemical inhibitor or siRNA increased the phosphorylation of ERK1/2, protected cells from apoptosis, and facilitated IBV replication. Overexpression of DUSP6 decreased the level of phospho-ERK1/2, promoted apoptosis, while dominant negative mutant DUSP6-DN lost the regulation function on ERK1/2 signaling and apoptosis. In conclusion, these data suggest that MEK-ERK1/2 signaling pathway facilitates IBV infection, probably by promoting cell survival; meanwhile, induction of DUSP6 forms a negative regulation loop to restrict ERK1/2 signaling, correlated with increased apoptosis and reduced viral load. Consequently, components of the ERK pathway, such as MEK1/2 and DUSP6, represent excellent targets for the development of antiviral drugs.


Asunto(s)
Apoptosis/fisiología , Fosfatasas de Especificidad Dual/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Virus de la Bronquitis Infecciosa/fisiología , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Animales , Butadienos/farmacología , Línea Celular , Pollos , Chlorocebus aethiops , Fosfatasas de Especificidad Dual/antagonistas & inhibidores , Fosfatasas de Especificidad Dual/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/antagonistas & inhibidores , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Nitrilos/farmacología , Regulación hacia Arriba , Replicación Viral
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