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1.
Sci Adv ; 7(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33523846

RESUMO

Here, we report the topology-matched design of heteromultivalent nanostructures as potent and broad-spectrum virus entry inhibitors based on the host cell membrane. Initially, we investigate the virus binding dynamics to validate the better binding performance of the heteromultivalent moieties as compared to homomultivalent ones. The heteromultivalent binding moieties are transferred to nanostructures with a bowl-like shape matching the viral spherical surface. Unlike the conventional homomultivalent inhibitors, the heteromultivalent ones exhibit a half maximal inhibitory concentration of 32.4 ± 13.7 µg/ml due to the synergistic multivalent effects and the topology-matched shape. At a dose without causing cellular toxicity, >99.99% reduction of virus propagation has been achieved. Since multiple binding sites have also been identified on the S protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), we envision that the use of heteromultivalent nanostructures may also be applied to develop a potent inhibitor to prevent coronavirus infection.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/virologia , Nanopartículas/química , Neuraminidase/química , Animais , Antivirais/farmacologia , Sítios de Ligação , Membrana Celular/metabolismo , Cães , Membrana Eritrocítica/virologia , Humanos , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Ligação Proteica , Glicoproteína da Espícula de Coronavírus , Vírion , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
2.
Nat Commun ; 12(1): 98, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397935

RESUMO

Glucose metabolism and innate immunity evolved side-by-side. It is unclear if and how the two systems interact with each other during hepatitis B virus (HBV) infections and, if so, which mechanisms are involved. Here, we report that HBV activates glycolysis to impede retinoic acid-inducible gene I (RIG-I)-induced interferon production. We demonstrate that HBV sequesters MAVS from RIG-I by forming a ternary complex including hexokinase (HK). Using a series of pharmacological and genetic approaches, we provide in vitro and in vivo evidence indicating that HBV suppresses RLR signaling via lactate dehydrogenase-A-dependent lactate production. Lactate directly binds MAVS preventing its aggregation and mitochondrial localization during HBV infection. Therefore, we show that HK2 and glycolysis-derived lactate have important functions in the immune escape of HBV and that energy metabolism regulates innate immunity during HBV infection.


Assuntos
Vírus da Hepatite B/fisiologia , Imunidade Inata , Metaboloma , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anaerobiose , Animais , Células Cultivadas , Proteína DEAD-box 58/metabolismo , Glucose/metabolismo , Glicólise , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Evasão da Resposta Imune , Interferons/metabolismo , Ácido Láctico/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transdução de Sinais , Vírion/metabolismo
3.
Virol J ; 18(1): 1, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397387

RESUMO

BACKGROUND: Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. RESULTS: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). CONCLUSIONS: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.


Assuntos
/imunologia , Leucemia/imunologia , Testes de Neutralização/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Vírion/imunologia , /imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Células HEK293 , Humanos , Imunoglobulina G/sangue , Camundongos
4.
Anal Chem ; 93(5): 2950-2958, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33481583

RESUMO

There is an urgent need for ultrarapid testing regimens to detect the severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] infections in real-time within seconds to stop its spread. Current testing approaches for this RNA virus focus primarily on diagnosis by RT-qPCR, which is time-consuming, costly, often inaccurate, and impractical for general population rollout due to the need for laboratory processing. The latency until the test result arrives with the patient has led to further virus spread. Furthermore, latest antigen rapid tests still require 15-30 min processing time and are challenging to handle. Despite increased polymerase chain reaction (PCR)-test and antigen-test efforts, the pandemic continues to evolve worldwide. Herein, we developed a superfast, reagent-free, and nondestructive approach of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy with subsequent chemometric analysis toward the prescreening of virus-infected samples. Contrived saliva samples spiked with inactivated γ-irradiated COVID-19 virus particles at levels down to 1582 copies/mL generated infrared (IR) spectra with a good signal-to-noise ratio. Predominant virus spectral peaks are tentatively associated with nucleic acid bands, including RNA. At low copy numbers, the presence of a virus particle was found to be capable of modifying the IR spectral signature of saliva, again with discriminating wavenumbers primarily associated with RNA. Discrimination was also achievable following ATR-FTIR spectral analysis of swabs immersed in saliva variously spiked with virus. Next, we nested our test system in a clinical setting wherein participants were recruited to provide demographic details, symptoms, parallel RT-qPCR testing, and the acquisition of pharyngeal swabs for ATR-FTIR spectral analysis. Initial categorization of swab samples into negative versus positive COVID-19 infection was based on symptoms and PCR results (n = 111 negatives and 70 positives). Following training and validation (using n = 61 negatives and 20 positives) of a genetic algorithm-linear discriminant analysis (GA-LDA) algorithm, a blind sensitivity of 95% and specificity of 89% was achieved. This prompt approach generates results within 2 min and is applicable in areas with increased people traffic that require sudden test results such as airports, events, or gate controls.


Assuntos
Algoritmos , /fisiologia , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Vírion/química , /virologia , Análise Discriminante , Raios gama , Humanos , Testes Imediatos , Análise de Componente Principal , Saliva/virologia , Sensibilidade e Especificidade , Razão Sinal-Ruído , Vírion/efeitos da radiação , Inativação de Vírus
5.
J Am Chem Soc ; 143(4): 1722-1727, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33481575

RESUMO

The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combatting the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold standard nucleic acid-based approaches require enzymatic amplification to achieve clinically relevant levels of sensitivity and are not typically used outside of a laboratory setting. Here, we report reagent-free viral sensing that directly reads out the presence of viral particles in 5 minutes using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a negatively charged DNA linker that also features a tethered redox probe. When a positive potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the first virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.


Assuntos
Técnicas Biossensoriais/métodos , /virologia , Técnicas Eletroquímicas/métodos , Vírion/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas Eletroquímicas/instrumentação , Eletrodos , Humanos , Testes Imediatos , Saliva/virologia
6.
J Chromatogr A ; 1638: 461879, 2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33465583

RESUMO

Two commercially available agarose ion exchange media, DEAE-Capto and DEAE-Sepharose FF (DEAE-FF), and two gigaporous media DEAE -AP-120 nm and DEAE-AP-280 nm were evaluated for their applicability in adsorption of five proteins with large span of radius ranges from 2.9 nm to 14.1 nm, which include ovalbumin, bovine serum albumin (BSA), haptoglobin, thyroglobulin and hepatitis B surface antigen (HBsAg) virus like particle. The average pore radius of the four media was determined to be 6.9 nm, 18.5 nm, 59.4 nm and 139.3 nm, respectively, which was obtained by log normal distribution for DEAE-Capto and DEAE-FF and by bimodal Gaussian distribution for the two DEAE-AP media. The performance of these four media including phase ratio, static and dynamic binding capacity, and transport properties for the adsorption of these five model proteins as function of pore-to-adsorbate size ratio were investigated and compared. The best ratio of pore-to-adsorbate size was found dependent on the protein size. For protein with radius from 2.9 nm (ovalbumin) to 5.4 nm (BSA), the agarose media was superior to gigaporous media. Both the static and dynamic adsorption capacities reduced with the increase of pore size, and the highest values were obtained at the smallest pore-to-adsorbate size of about 2 times in this study, although the highest accessible surface area was obtained at pore-to-adsorbate size ratio about 16 to 20. For proteins with radius of 5.4 nm or larger than that, their adsorption capacities decreased firstly and then increased with the increase of ratio of pore-to-adsorbate size, and the highest values were obtained on the gigaporous media DEAE-AP-280 nm, which could provide faster diffusivity and larger accessible surface area. However, protein with radius of 14.1 nm (HBsAg) had much lower capacities compared to other proteins at the same pore-to-adsorbate size ratio, implying large protein needs greater pore-to-adsorbate size ratio to achieve a satisfactory capacity. For all the five tested proteins, the DEAE-Capto media having the smallest pore radius and branched dextran chains, was found superior to DEAE-FF in terms of both higher adsorption capacities and uptake kinetics, which suggested that the "chain delivery effect" took place on proteins over large size span from ovalbumin to HBsAg, though the effect on the larger proteins was much less significant than that on the smaller ones. Results from the present work provided more information on how do the relationships of pore size of chromatography media and adsorbate size interactively affect the chromatography behaviors, thus will provide general guidance for selection of suitable adsorbent for biologics of a given size.


Assuntos
Cromatografia por Troca Iônica/métodos , Ovalbumina/química , Sefarose/química , Vírion/química , Adsorção , Animais , Bovinos , Cromatografia em Gel , Simulação por Computador , Meios de Cultura , Dextranos/química , Antígenos de Superfície da Hepatite B/química , Cinética , Ligantes , Tamanho da Partícula , Porosidade , Soroalbumina Bovina/química , Temperatura
7.
Sci Rep ; 11(1): 2229, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500537

RESUMO

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral evaluation. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3000 times higher luminescence than MOCK control cells at 24 h post-electroporation, indicating robust translation and RNA replication of the replicon. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 µM, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 µM). Taken together, this system could be applied to the safe and effective antiviral evaluation without using infectious SARS-CoV-2. Because this is a PCR-based and transient replicon system, further improvement including the establishment of stable cell line must be achieved.


Assuntos
Antivirais/farmacologia , Desenho de Fármacos , /efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Células CHO , Chlorocebus aethiops , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Eletroporação , Genoma Viral , Células HEK293 , Humanos , Concentração Inibidora 50 , Cinética , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , RNA Viral , Regiões não Traduzidas , Células Vero , Vírion , Replicação Viral/efeitos dos fármacos
8.
Biosens Bioelectron ; 171: 112685, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33113383

RESUMO

The spread of SARS-CoV-2 virus in the ongoing global pandemic has led to infections of millions of people and losses of many lives. The rapid, accurate and convenient SARS-CoV-2 virus detection is crucial for controlling and stopping the pandemic. Diagnosis of patients in the early stage infection are so far limited to viral nucleic acid or antigen detection in human nasopharyngeal swab or saliva samples. Here we developed a method for rapid and direct optical measurement of SARS-CoV-2 virus particles in one step nearly without any sample preparation using a spike protein specific nanoplasmonic resonance sensor. As low as 370 vp/mL were detected in one step within 15 min and the virus concentration can be quantified linearly in the range of 0 to 107 vp/mL. Measurements shown on both generic microplate reader and a handheld smartphone connected device suggest that our low-cost and rapid detection method may be adopted quickly under both regular clinical environment and resource-limited settings.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Imediatos , Vírion/isolamento & purificação , Anticorpos Imobilizados/química , Técnicas Biossensoriais/economia , Técnicas de Laboratório Clínico/economia , Infecções por Coronavirus/economia , Desenho de Equipamento , Humanos , Limite de Detecção , Modelos Moleculares , Pandemias , Glicoproteína da Espícula de Coronavírus/análise , Fatores de Tempo
9.
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
10.
PLoS Biol ; 18(12): e3001015, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33332391

RESUMO

Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.


Assuntos
Capsídeo/metabolismo , HIV-1/metabolismo , Replicação Viral/fisiologia , Capsídeo/química , Capsídeo/fisiologia , Proteínas do Capsídeo/genética , Replicação do DNA/fisiologia , DNA Viral/metabolismo , Células HEK293 , HIV-1/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Simulação de Dinâmica Molecular , Nucleotídeos/metabolismo , Permeabilidade , Ácido Fítico/análise , Ácido Fítico/metabolismo , Vírion/genética , Montagem de Vírus/fisiologia , Replicação Viral/genética
11.
Viruses ; 12(12)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33352888

RESUMO

The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics.


Assuntos
Biotecnologia , Proteínas do Capsídeo/fisiologia , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Epitopos Imunodominantes , Sinais de Localização Nuclear , Parvovirus B19 Humano/fisiologia , Fosfolipases A2/química , Receptores Virais , Tropismo Viral , Vírion/fisiologia
12.
Sci Rep ; 10(1): 21877, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318562

RESUMO

SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 ± 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN.


Assuntos
/virologia , Vírion/metabolismo , Montagem de Vírus , Células HEK293 , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/metabolismo
13.
Cell Rep ; 33(12): 108528, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33326798

RESUMO

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of ß coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.


Assuntos
/metabolismo , Antivirais/farmacologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Microcorpos/metabolismo , /efeitos dos fármacos , Sequência de Aminoácidos , Animais , /virologia , Modelos Animais de Doenças , Dissulfetos/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Domínios Proteicos , Multimerização Proteica , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacos
14.
Front Public Health ; 8: 590041, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33330334

RESUMO

Evidence has emerged that SARS-CoV-2, the coronavirus that causes COVID-19, can be transmitted airborne in aerosol particles as well as in larger droplets or by surface deposits. This minireview outlines the underlying aerosol science, making links to aerosol research in other disciplines. SARS-CoV-2 is emitted in aerosol form during normal breathing by both asymptomatic and symptomatic people, remaining viable with a half-life of up to about an hour during which air movement can carry it considerable distances, although it simultaneously disperses. The proportion of the droplet size distribution within the aerosol range depends on the sites of origin within the respiratory tract and on whether the distribution is presented on a number or volume basis. Evaporation and fragmentation reduce the size of the droplets, whereas coalescence increases the mean droplet size. Aerosol particles containing SARS-CoV-2 can also coalesce with pollution particulates, and infection rates correlate with pollution. The operation of ventilation systems in public buildings and transportation can create infection hazards via aerosols, but provides opportunities for reducing the risk of transmission in ways as simple as switching from recirculated to outside air. There are also opportunities to inactivate SARS-CoV-2 in aerosol form with sunlight or UV lamps. The efficiency of masks for blocking aerosol transmission depends strongly on how well they fit. Research areas that urgently need further experimentation include the basis for variation in droplet size distribution and viral load, including droplets emitted by "superspreader" individuals; the evolution of droplet sizes after emission, their interaction with pollutant aerosols and their dispersal by turbulence, which gives a different basis for social distancing.


Assuntos
Microbiologia do Ar , Transmissão de Doença Infecciosa , Vírion , Aerossóis , Humanos
15.
BMC Pulm Med ; 20(1): 301, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198751

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19. METHODS: Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (n = 28) and to the Intermediate Medicine Ward (IMW) (n = 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels. RESULTS: ICU patients showed a marked increase in neutrophils (1.24 × 105 ml- 1, 0.85-2.07), lower lymphocyte (0.97 × 105 ml- 1, 0.024-0.34) and macrophages fractions (0.43 × 105 ml- 1, 0.34-1.62) compared to IMW patients (0.095 × 105 ml- 1, 0.05-0.73; 0.47 × 105 ml- 1, 0.28-1.01 and 2.14 × 105 ml- 1, 1.17-3.01, respectively) (p < 0.01). Study of ICU patients BAL by electron transmission microscopy showed viral particles inside mononuclear cells confirmed by immunostaining with anti-viral capsid and spike antibodies. IL6 and IL8 were significantly higher in ICU patients than in IMW (IL6 p < 0.01, IL8 p < 0.0001), and also in patients who did not survive (IL6 p < 0.05, IL8 p = 0.05 vs. survivors). IL10 did not show a significant variation between groups. Dividing patients by treatment received, lower BAL concentrations of IL6 were found in patients treated with steroids as compared to those treated with tocilizumab (p < 0.1) or antivirals (p < 0.05). CONCLUSIONS: Alveolitis, associated with COVID-19, is mainly sustained by innate effectors which showed features of extensive activation. The burden of pro-inflammatory cytokines IL6 and IL8 in the broncho-alveolar environment is associated with clinical outcome.


Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Infecções por Coronavirus/imunologia , Inflamação/imunologia , Interleucina-6/imunologia , Interleucina-8/imunologia , Leucócitos/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Pneumonia Viral/imunologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/uso terapêutico , Corticosteroides/uso terapêutico , Idoso , Alanina/análogos & derivados , Alanina/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/virologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/terapia , Combinação de Medicamentos , Feminino , Humanos , Hidroxicloroquina/uso terapêutico , Unidades de Terapia Intensiva , Interleucina-10/imunologia , Itália , Leucócitos Mononucleares/virologia , Lopinavir/uso terapêutico , Pulmão/citologia , Pulmão/virologia , Linfócitos/imunologia , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Neutrófilos/imunologia , Pandemias , Pneumonia Viral/terapia , Prognóstico , Estudos Prospectivos , Respiração Artificial/métodos , Ritonavir/uso terapêutico , Glicoproteína da Espícula de Coronavírus/metabolismo , Taxa de Sobrevida , Vírion/metabolismo , Vírion/ultraestrutura
16.
Elife ; 92020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33200986

RESUMO

Interactions between viral RNA and the integrase enzyme are required for HIV-1 particles to become infectious, a process that can be disrupted through multiple mechanisms.


Assuntos
Integrase de HIV , HIV-1 , Integrase de HIV/genética , HIV-1/genética , Morfogênese , RNA Viral/genética , Vírion
17.
Nat Commun ; 11(1): 5920, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219228

RESUMO

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Humanos , RNA/metabolismo , RNA Viral/genética , RNA Viral/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Recombinases/metabolismo , Saliva/virologia , Vírion/genética
18.
Biomedica ; 40(Supl. 2): 148-158, 2020 10 30.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-33152198

RESUMO

Introduction: SARS-CoV-2 has been identified as the new coronavirus causing an outbreak of acute respiratory disease in China in December, 2019. This disease, currently named COVID-19, has been declared as a pandemic by the World Health Organization (WHO). The first case of COVID-19 in Colombia was reported on March 6, 2020. Here we characterize an early SARS-CoV-2 isolate from the pandemic recovered in April, 2020. Objective: To describe the isolation and characterization of an early SARS-CoV-2 isolate from the epidemic in Colombia. Materials and methods: A nasopharyngeal specimen from a COVID-19 positive patient was inoculated on different cell lines. To confirm the presence of SARS-CoV-2 on cultures we used qRT-PCR, indirect immunofluorescence assay, transmission and scanning electron microscopy, and next-generation sequencing. Results: We determined the isolation of SARS-CoV-2 in Vero-E6 cells by the appearance of the cytopathic effect three days post-infection and confirmed it by the positive results in the qRT-PCR and the immunofluorescence with convalescent serum. Transmission and scanning electron microscopy images obtained from infected cells showed the presence of structures compatible with SARS-CoV-2. Finally, a complete genome sequence obtained by next-generation sequencing allowed classifying the isolate as B.1.5 lineage. Conclusion: The evidence presented in this article confirms the first isolation of SARSCoV-2 in Colombia. In addition, it shows that this strain behaves in cell culture in a similar way to that reported in the literature for other isolates and that its genetic composition is consistent with the predominant variant in the world. Finally, points out the importance of viral isolation for the detection of neutralizing antibodies, for the genotypic and phenotypic characterization of the strain and for testing compounds with antiviral potential.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Animais , Betacoronavirus/genética , Betacoronavirus/fisiologia , Betacoronavirus/ultraestrutura , Chlorocebus aethiops , Colômbia/epidemiologia , Convalescença , Infecções por Coronavirus/epidemiologia , Efeito Citopatogênico Viral , Técnica Indireta de Fluorescência para Anticorpo , Genoma Viral , Humanos , Microscopia Eletrônica , Tipagem Molecular , Nasofaringe/virologia , Pneumonia Viral/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Especificidade da Espécie , Células Vero , Vírion/ultraestrutura , Cultura de Vírus
19.
Nat Commun ; 11(1): 5885, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208793

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic ß-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Replicação Viral , Células A549 , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Vesículas Citoplasmáticas/virologia , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/metabolismo , Células Vero , Vírion/química , Vírion/metabolismo , Montagem de Vírus
20.
Biointerphases ; 15(6): 061005, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203214

RESUMO

The emergence of SARS-CoV-2 highlights the global need for platform technologies to enable the rapid development of diagnostics, vaccines, treatments, and personal protective equipment (PPE). However, many current technologies require the detailed mechanistic knowledge of specific material-virion interactions before they can be employed, for example, to aid in the purification of vaccine components or in the design of a more effective PPE. Here, we show that an adaption of a polymer microarray method for screening bacterial-surface interactions allows for the screening of polymers for desirable material-virion interactions. Nonpathogenic virus-like particles including fluorophores are exposed to the arrays in an aqueous buffer as a simple model of virions carried to the surface in saliva/sputum. Competitive binding of Lassa and Rubella virus-like particles is measured to probe the relative binding properties of a selection of copolymers. This provides the first step in the development of a method for the discovery of novel materials with promise for viral binding, with the next being development of this method to assess absolute viral adsorption and assessment of the attenuation of the activity of live virus, which we propose would be part of a material scale up step carried out in high containment facilities, alongside the use of more complex media to represent biological fluids.


Assuntos
Análise em Microsséries , Polímeros/química , Vírion/isolamento & purificação , Adsorção , Infecções por Coronavirus/diagnóstico , Pandemias , Pneumonia Viral/diagnóstico , Raios Ultravioleta
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