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1.
mBio ; 12(2)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33906927

RESUMO

SARS-CoV-2 infection causing the COVID-19 pandemic calls for immediate interventions to avoid viral transmission, disease progression, and subsequent excessive inflammation and tissue destruction. Primary normal human bronchial epithelial cells are among the first targets of SARS-CoV-2 infection. Here, we show that ColdZyme medical device mouth spray efficiently protected against virus entry, excessive inflammation, and tissue damage. Applying ColdZyme to fully differentiated, polarized human epithelium cultured at an air-liquid interphase (ALI) completely blocked binding of SARS-CoV-2 and increased local complement activation mediated by the virus as well as productive infection of the tissue model. While SARS-CoV-2 infection resulted in exaggerated intracellular complement activation immediately following infection and a drop in transepithelial resistance, these parameters were bypassed by single pretreatment of the tissues with ColdZyme mouth spray. Crucially, our study highlights the importance of testing already evaluated and safe drugs such as ColdZyme mouth spray for maintaining epithelial integrity and hindering SARS-CoV-2 entry within standardized three-dimensional (3D) in vitro models mimicking the in vivo human airway epithelium.IMPORTANCE Although our understanding of COVID-19 continuously progresses, essential questions regarding prophylaxis and treatment remain open. A hallmark of severe SARS-CoV-2 infection is a hitherto-undescribed mechanism leading to excessive inflammation and tissue destruction associated with enhanced pathogenicity and mortality. To tackle the problem at the source, transfer of SARS-CoV-2, subsequent binding, infection, and inflammatory responses have to be avoided. In this study, we used fully differentiated, mucus-producing, and ciliated human airway epithelial cultures to test the efficacy of ColdZyme medical device mouth spray in terms of protection from SARS-CoV-2 infection. Importantly, we found that pretreatment of the in vitro airway cultures using ColdZyme mouth spray resulted in significantly shielding the epithelial integrity, hindering virus binding and infection, and blocking excessive intrinsic complement activation within the airway cultures. Our in vitro data suggest that ColdZyme mouth spray may have an impact in prevention of COVID-19.


Assuntos
Antivirais/farmacologia , Mucosa Respiratória/efeitos dos fármacos , /efeitos dos fármacos , Brônquios/citologia , /virologia , Complemento C3/imunologia , Células Epiteliais , Humanos , Imunidade Inata/efeitos dos fármacos , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/imunologia , Mucosa Nasal/virologia , Sprays Orais , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Ligação Viral/efeitos dos fármacos
2.
Pediatr Infect Dis J ; 40(5): e202-e204, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33847302

RESUMO

This cross-sectional study, including children hospitalized for severe acute respiratory syndrome coronavirus 2 infection, demonstrates for the first time that nonhealthcare worker parents perform similarly to healthcare workers in the administration to their children of an unsupervised nasal swab for severe acute respiratory syndrome coronavirus 2 detection by following written instructions and video tutorials.


Assuntos
/virologia , /isolamento & purificação , /diagnóstico , Criança , Pré-Escolar , Estudos Transversais , Feminino , Pessoal de Saúde , Humanos , Lactente , Masculino , Mucosa Nasal/virologia , Pais
3.
Viruses ; 13(2)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33672005

RESUMO

To predict the clinical outcome of coronavirus disease-2019 (COVID-19), we examined relationships among epidemiological data, viral load, and disease severity. We examined viral loads of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) in fatal (15 cases), symptomatic/survived (133 cases), and asymptomatic cases (138 cases) using reverse transcription quantitative real-time PCR (RT-qPCR). We examined 5768 nasopharyngeal swabs (NPS) and attempted to detect the SARS-CoV-2 genome using RT-qPCR. Among them, the viral genome was detected using the method for the 370 NPS samples with a positive rate of 6.4%. A comparison of each age showed that the fatal case was higher than the survived case and asymptomatic patients. Survived cases were older than asymptomatic patients. Notably, the viral load in the fatal cases was significantly higher than in symptomatic or asymptomatic cases (p < 0.05). These results suggested that a high viral load of the SARS-CoV-2 in elderly patients at an early stage of the disease results in a poor outcome. We should, therefore, intervene early to prevent a severe stage of the disease in such cases.


Assuntos
/diagnóstico , Mucosa Nasal/virologia , Carga Viral , Adulto , Idoso , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , RNA Viral/análise , Índice de Gravidade de Doença
4.
Emerg Infect Dis ; 27(4): 1193-1195, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33754987

RESUMO

After experimental inoculation, severe acute respiratory syndrome coronavirus 2 infection was confirmed in bank voles by seroconversion within 8 days and detection of viral RNA in nasal tissue for up to 21 days. However, transmission to contact animals was not detected. Thus, bank voles are unlikely to establish effective transmission cycles in nature.


Assuntos
Arvicolinae , Transmissão de Doença Infecciosa , Doenças dos Roedores , Soroconversão , Eliminação de Partículas Virais , Animais , Anticorpos Antivirais , /transmissão , Modelos Animais de Doenças , Suscetibilidade a Doenças , Mucosa Nasal/virologia , Doenças dos Roedores/imunologia , Doenças dos Roedores/transmissão , Doenças dos Roedores/virologia
5.
Nature ; 592(7852): 122-127, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33636719

RESUMO

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.


Assuntos
/transmissão , Mutação , /fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral/genética , /genética , Animais , Brônquios/citologia , Brônquios/virologia , Linhagem Celular , Células Cultivadas , Cricetinae , Modelos Animais de Doenças , Células Epiteliais/virologia , Feminino , Furões/virologia , Efeito Fundador , Técnicas de Introdução de Genes , Aptidão Genética , Humanos , Masculino , Mesocricetus , Camundongos , Mucosa Nasal/citologia , Mucosa Nasal/virologia , Ligação Proteica , RNA Viral/análise , /metabolismo , /patogenicidade
6.
Vet Microbiol ; 255: 109017, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33639390

RESUMO

Bovine coronavirus (BCoV) is one of the agents causing bovine respiratory disease complex (BRDC), with single infection tending to be mild to moderate; the probability of developing pneumonia in BRDC may be affected by viral and bacterial combinations. Previously, we reported that bovine respiratory syncytial virus (BRSV) infection enhances adherence of Pasteurella multocida (PM) to cells derived from the bovine lower respiratory tract but that BRSV infection in cells derived from the upper respiratory tract reduces PM adherence. In this study, we sought to clarify whether the modulation of bacterial adherence to cells derived from the bovine upper and lower respiratory tract is shared by other BRDC-related viruses by infecting bovine epithelial cells from the trachea, bronchus and lung with BCoV and/or PM. The results showed that cells derived from both the upper and lower respiratory tract were susceptible to BCoV infection. Furthermore, all cells infected with BCoV exhibited increased PM adherence via upregulation of two major bacterial adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and platelet-activating factor receptor (PAF-R), suggesting that compared with BRSV infection, BCoV infection differentially modulates bacterial adherence. In summary, we identified distinct interaction between bovine respiratory viruses and bacterial infections.


Assuntos
Aderência Bacteriana/fisiologia , Coronavirus Bovino/fisiologia , Mucosa Respiratória/metabolismo , Animais , Western Blotting , Bovinos , Humanos , Mucosa Nasal/virologia , Receptores de Superfície Celular/metabolismo , Mucosa Respiratória/microbiologia , Mucosa Respiratória/virologia , Células Tumorais Cultivadas , Regulação para Cima
7.
Sci Rep ; 11(1): 3134, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542443

RESUMO

We aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.


Assuntos
/diagnóstico , Mucosa Nasal/virologia , RNA Viral/isolamento & purificação , Saliva/virologia , Manejo de Espécimes , Adulto , Estudos de Coortes , Feminino , Humanos , Masculino , Nasofaringe/virologia , /isolamento & purificação , Sensibilidade e Especificidade , Singapura/epidemiologia
8.
Sci Rep ; 11(1): 780, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436939

RESUMO

The COVID-19 pandemic caused by the SARS-CoV-2 virus motivates diverse diagnostic approaches due to the novel causative pathogen, incompletely understood clinical sequelae, and limited availability of testing resources. Given the variability in viral load across and within patients, absolute viral load quantification directly from crude lysate is important for diagnosis and surveillance. Here, we investigate the use of digital droplet PCR (ddPCR) for SARS-CoV-2 viral load measurement directly from crude lysate without nucleic acid purification. We demonstrate ddPCR accurately quantifies SARS-CoV-2 standards from purified RNA and multiple sample matrices, including commonly utilized universal transport medium (UTM). In addition, we find ddPCR functions robustly at low input viral copy numbers on nasopharyngeal swab specimens stored in UTM without upfront RNA extraction. We also show ddPCR, but not qPCR, from crude lysate shows high concordance with viral load measurements from purified RNA. Our data suggest ddPCR offers advantages to qPCR for SARS-CoV-2 detection with higher sensitivity and robustness when using crude lysate rather than purified RNA as input. More broadly, digital droplet assays provide a potential method for nucleic acid measurement and infectious disease diagnosis with limited sample processing, underscoring the utility of such techniques in laboratory medicine.


Assuntos
/métodos , Carga Viral , /diagnóstico , Humanos , Mucosa Nasal/virologia , RNA Viral/química , RNA Viral/genética , RNA Viral/normas , /isolamento & purificação , Sensibilidade e Especificidade
9.
Viruses ; 13(1)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467732

RESUMO

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.


Assuntos
Doenças Assintomáticas , Modelos Animais de Doenças , /fisiologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , /patologia , Feminino , Furões , Genoma Viral/genética , Mutação , Mucosa Nasal/virologia , RNA Viral/genética , Carga Viral , Eliminação de Partículas Virais
10.
Otolaryngol Head Neck Surg ; 164(2): 305-307, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33320052

RESUMO

Current COVID-19 vaccine candidates are administered by injection and designed to produce an IgG response, preventing viremia and the COVID-19 syndrome. However, systemic respiratory vaccines generally provide limited protection against viral replication and shedding within the airway, as this requires a local mucosal secretory IgA response. Indeed, preclinical studies of adenovirus and mRNA candidate vaccines demonstrated persistent virus in nasal swabs despite preventing COVID-19. This suggests that systemically vaccinated patients, while asymptomatic, may still be become infected and transmit live virus from the upper airway. COVID-19 is known to spread through respiratory droplets and aerosols. Furthermore, significant evidence has shown that many clinic and surgical endonasal procedures are aerosol generating. Until further knowledge is acquired regarding mucosal immunity following systemic vaccination, otolaryngology providers should maintain precautions against viral transmission to protect the proportion of persistently vulnerable patients who exhibit subtotal vaccine efficacy or waning immunity or who defer vaccination.


Assuntos
/prevenção & controle , Mucosa Nasal/virologia , Aerossóis , Infecções Assintomáticas , Humanos
11.
Front Immunol ; 11: 580867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33133098

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is primarily diagnosed through viral RNA positivity in nasopharyngeal swabs, and it is associated with the early detection of specific immunoglobulins to SARS-CoV-2 proteins. We describe two moderate coronavirus disease 2019 (COVID-19) patients with WHO score 4/5 at the time of hospitalization, pneumonia, and oxygen saturation <94% and with a strong discrepancy between viral RNA and antibodies to SARS-CoV-2. One patient was positive for viral RNA but completely negative for binding and neutralizing antibodies, whereas the second patient was negative for viral RNA but with high levels of both neutralizing and binding antibodies. This observation is relevant to better understand the pathogenesis of this novel infection.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/análise , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Técnicas de Laboratório Clínico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Mucosa Nasal/virologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real , Testes Sorológicos
12.
Science ; 370(6513)2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33033192

RESUMO

The variable outcome of viral exposure is only partially explained by known factors. We administered respiratory syncytial virus (RSV) to 58 volunteers, of whom 57% became infected. Mucosal neutrophil activation before exposure was highly predictive of symptomatic RSV disease. This was associated with a rapid, presymptomatic decline in mucosal interleukin-17A (IL-17A) and other mediators. Conversely, those who resisted infection showed presymptomatic activation of IL-17- and tumor necrosis factor-related pathways. Vulnerability to infection was not associated with baseline microbiome but was reproduced in mice by preinfection chemokine-driven airway recruitment of neutrophils, which caused enhanced disease mediated by pulmonary CD8+ T cell infiltration. Thus, mucosal neutrophilic inflammation at the time of RSV exposure enhances susceptibility, revealing dynamic, time-dependent local immune responses before symptom onset and explaining the as-yet unpredictable outcomes of pathogen exposure.


Assuntos
Mucosa Nasal/imunologia , Mucosa Nasal/virologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios , Adolescente , Adulto , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Quimiocina CXCL1/farmacologia , Humanos , Inflamação/imunologia , Inflamação/virologia , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mucosa Nasal/patologia , Neutrófilos/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/patologia , Fator de Necrose Tumoral alfa/imunologia , Adulto Jovem
13.
Viruses ; 12(10)2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33081421

RESUMO

To address the expression pattern of the SARS-CoV-2 receptor ACE2 and the viral priming protease TMPRSS2 in the respiratory tract, this study investigated RNA sequencing transcriptome profiling of samples of airway and oral mucosa. As shown, ACE2 has medium levels of expression in both small airway epithelium and masticatory mucosa, and high levels of expression in nasal epithelium. The expression of ACE2 is low in mucosal-associated invariant T (MAIT) cells and cannot be detected in alveolar macrophages. TMPRSS2 is highly expressed in small airway epithelium and nasal epithelium and has lower expression in masticatory mucosa. Our results provide the molecular basis that the nasal mucosa is the most susceptible locus in the respiratory tract for SARS-CoV-2 infection and consequently for subsequent droplet transmission and should be the focus for protection against SARS-CoV-2 infection.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/genética , Peptidil Dipeptidase A/biossíntese , Pneumonia Viral/genética , Serina Endopeptidases/biossíntese , Internalização do Vírus , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Epitélio/metabolismo , Epitélio/virologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Mucosa Nasal/metabolismo , Mucosa Nasal/virologia , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , Serina Endopeptidases/genética
15.
ACS Sens ; 5(10): 3043-3048, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-32989986

RESUMO

Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast, and cheap tools to detect viral particles in biological material so to identify the people capable of spreading the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal solution of multiple viral-target gold nanoparticles-AuNPs functionalized with antibodies targeting three surface proteins of SARS-CoV-2 (spike, envelope, and membrane)-is red-shifted in few minutes when mixed with a solution containing the viral particle. The optical density of the mixed solution measured at 560 nm was compared to the threshold cycle (Ct) of a real-time PCR (gold standard for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of the real-time PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open a new perspective not only in the context of the current and possible future pandemics, but also in microbiology, as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concentration.


Assuntos
Betacoronavirus/química , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Mucosa Nasal/virologia , Faringe/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Técnicas Biossensoriais , Ouro , Humanos , Proteínas de Membrana/química , Nanopartículas Metálicas , Pandemias , Fotoquímica , Reação em Cadeia da Polimerase , Manejo de Espécimes , Glicoproteína da Espícula de Coronavírus/química , Níveis Máximos Permitidos , Proteínas do Envelope Viral/química
16.
Cell Rep ; 32(12): 108175, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32946807

RESUMO

To predict the tropism of human coronaviruses, we profile 28 SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) using single-cell transcriptomics across various healthy human tissues. SCARFs include cellular factors both facilitating and restricting viral entry. Intestinal goblet cells, enterocytes, and kidney proximal tubule cells appear highly permissive to SARS-CoV-2, consistent with clinical data. Our analysis also predicts non-canonical entry paths for lung and brain infections. Spermatogonial cells and prostate endocrine cells also appear to be permissive to SARS-CoV-2 infection, suggesting male-specific vulnerabilities. Both pro- and anti-viral factors are highly expressed within the nasal epithelium, with potential age-dependent variation, predicting an important battleground for coronavirus infection. Our analysis also suggests that early embryonic and placental development are at moderate risk of infection. Lastly, SCARF expression appears broadly conserved across a subset of primate organs examined. Our study establishes a resource for investigations of coronavirus biology and pathology.


Assuntos
Infecções por Coronavirus/patologia , Mucosa Nasal/metabolismo , Pneumonia Viral/patologia , Receptores Virais/genética , Tropismo Viral/genética , Internalização do Vírus , Células A549 , Animais , Betacoronavirus/crescimento & desenvolvimento , Linhagem Celular , Chlorocebus aethiops , Enterócitos/metabolismo , Perfilação da Expressão Gênica , Células Caliciformes/metabolismo , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Mucosa Nasal/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Análise de Célula Única , Células Vero
17.
Proc Natl Acad Sci U S A ; 117(37): 22727-22735, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868442

RESUMO

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per µL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.


Assuntos
Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Testes Imediatos/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/normas , Mucosa Nasal/virologia , Pandemias , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Smartphone
18.
J Proteome Res ; 19(11): 4393-4397, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32786682

RESUMO

The detection of viral RNA by polymerase chain reaction (PCR) is currently the main diagnostic tool for COVID-19 ( Eurosurveillance 2019, 25 (3), 1). The PCR-based test, however, shows limited sensitivity, especially in the early and late stages of disease development ( Nature 2020, 581, 465-469; J. Formosan Med. Assoc. 2020, 119 (6) 1123), and is relatively time-consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemic conditions. Mass spectrometry is one of such possibilities. We have developed a mass-spectrometry-based method for the detection of the SARS CoV-2 virus in nasopharynx epithelial swabs based on the detection of the viral nucleocapsid N protein. Our approach shows confident identification of the N protein in patient samples, even those with the lowest viral loads, and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and the addition of isopropanol and tryptic digestion of the proteins sedimented from the swabs followed by MS analysis. A set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of SARS-CoV-2, is detected. The obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Espectrometria de Massas/métodos , Nasofaringe/virologia , Proteínas do Nucleocapsídeo/análise , Pneumonia Viral/diagnóstico , Betacoronavirus/química , Infecções por Coronavirus/virologia , Humanos , Mucosa Nasal/virologia , Pandemias , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fosfoproteínas , Pneumonia Viral/virologia , Proteômica , Carga Viral
19.
Virol J ; 17(1): 125, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811514

RESUMO

We recently reported the development of the first African green monkey (AGM) model for COVID-19 based on a combined liquid intranasal (i.n.) and intratracheal (i.t.) exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we followed up on this work by assessing an i.n. particle only route of exposure using the LMA mucosal atomization device (MAD). Six AGMs were infected with SARS-CoV-2; three animals were euthanized near the peak stage of virus replication (day 5) and three animals were euthanized during the early convalescence period (day 34). All six AGMs supported robust SARS-CoV-2 replication and developed respiratory disease. Evidence of coagulation dysfunction as noted by a transient increases in aPTT and circulating levels of fibrinogen was observed in all AGMs. The level of SARS-CoV-2 replication and lung pathology was not quite as pronounced as previously reported with AGMs exposed by the combined i.n. and i.t. routes; however, SARS-CoV-2 RNA was detected in nasal swabs of some animals as late as day 15 and rectal swabs as late as day 28 after virus challenge. Of particular importance to this study, all three AGMs that were followed until the early convalescence stage of COVID-19 showed substantial lung pathology at necropsy as evidenced by multifocal chronic interstitial pneumonia and increased collagen deposition in alveolar walls despite the absence of detectable SARS-CoV-2 in any of the lungs of these animals. These findings are consistent with human COVID-19 further demonstrating that the AGM faithfully reproduces the human condition.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Animais , Betacoronavirus/imunologia , Chlorocebus aethiops , Convalescença , Infecções por Coronavirus/sangue , Modelos Animais de Doenças , Feminino , Lesão Pulmonar/patologia , Lesão Pulmonar/virologia , Mucosa Nasal/virologia , Pandemias , Pneumonia Viral/sangue , Soroconversão , Carga Viral , Eliminação de Partículas Virais
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