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1.
Ann Lab Med ; 41(2): 225-229, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33063685

RESUMO

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic, an online laboratory surveillance system was established to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time reverse transcription-PCR (rRT-PCR) testing capacities and results. SARS-CoV-2 rRT-PCR testing data were collected from 97 clinical laboratories, including 84 medical institutions and 13 independent clinical laboratories in Korea. We assessed the testing capacities to utilize SARS-CoV-2 rRT-PCR based on surveillance data obtained from February 7th to June 4th, 2020 and evaluated positive result characteristics according to the reagents used and sample types. A total of 1,890,319 SARS-CoV-2 rRT-PCR testing were performed, 2.3% of which were positive. Strong correlations were observed between the envelope (E) gene and RNA-dependent RNA polymerase (RdRp)/nucleocapsid (N) genes threshold cycle (Ct) values for each reagent. No statistically significant differences in gene Ct values were observed between the paired upper and lower respiratory tract samples, except in the N gene for nasopharyngeal swab and sputum samples. Our study showed that clinical laboratories in Korea have rapidly expanded their testing capacities in response to the COVID-19 outbreak, with a peak daily capacity of 34,193 tests. Rapid expansion in testing capacity is a critical component of the national response to the ongoing pandemic.


Assuntos
Betacoronavirus/genética , Serviços de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Humanos , Laboratórios Hospitalares , Pandemias , Pneumonia Viral/virologia , RNA Replicase/genética , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , República da Coreia , Proteínas do Envelope Viral/genética , Proteínas Virais/genética
2.
BMC Bioinformatics ; 21(1): 431, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33008363

RESUMO

BACKGROUND: This paper describes a web based tool that uses a combination of sonification and an animated display to inquire into the SARS-CoV-2 genome. The audio data is generated in real time from a variety of RNA motifs that are known to be important in the functioning of RNA. Additionally, metadata relating to RNA translation and transcription has been used to shape the auditory and visual displays. Together these tools provide a unique approach to further understand the metabolism of the viral RNA genome. This audio provides a further means to represent the function of the RNA in addition to traditional written and visual approaches. RESULTS: Sonification of the SARS-CoV-2 genomic RNA sequence results in a complex auditory stream composed of up to 12 individual audio tracks. Each auditory motive is derived from the actual RNA sequence or from metadata. This approach has been used to represent transcription or translation of the viral RNA genome. The display highlights the real-time interaction of functional RNA elements. The sonification of codons derived from all three reading frames of the viral RNA sequence in combination with sonified metadata provide the framework for this display. Functional RNA motifs such as transcription regulatory sequences and stem loop regions have also been sonified. Using the tool, audio can be generated in real-time from either genomic or sub-genomic representations of the RNA. Given the large size of the viral genome, a collection of interactive buttons has been provided to navigate to regions of interest, such as cleavage regions in the polyprotein, untranslated regions or each gene. These tools are available through an internet browser and the user can interact with the data display in real time. CONCLUSION: The auditory display in combination with real-time animation of the process of translation and transcription provide a unique insight into the large body of evidence describing the metabolism of the RNA genome. Furthermore, the tool has been used as an algorithmic based audio generator. These audio tracks can be listened to by the general community without reference to the visual display to encourage further inquiry into the science.


Assuntos
Betacoronavirus/genética , Genoma Viral , Software , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Genômica , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo
3.
Euro Surveill ; 25(39)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33006300

RESUMO

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Nucleoproteínas/genética , Pandemias , Pneumonia Viral/diagnóstico , Mutação Puntual , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Virais/genética , Sequência de Bases , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Busca de Comunicante , Infecções por Coronavirus/virologia , Primers do DNA , Erros de Diagnóstico , Reações Falso-Negativas , Feminino , Genes Virais , Humanos , Pessoa de Meia-Idade , Nasofaringe/virologia , Nucleoproteínas/análise , Filogenia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Romênia , Doença Relacionada a Viagens , Proteínas Virais/análise
4.
Biomed Res Int ; 2020: 1708267, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029489

RESUMO

We aimed to summarize reliable medical evidence by the meta-analysis of all published retrospective studies that examined data based on the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by clinical symptoms, molecular (RT-PCR) diagnosis, and characteristic CT imaging features in pregnant women. The MEDLINE, PubMed, Scopus, ISI Web of Science, ClinicalKey, and CINAHL databases were used to select the studies. Then, 384 articles were received, including the studies until 01/May/2020. As a result of the full-text evaluation, 12 retrospective articles covering all the data related were selected. A total of 181 pregnant cases with SARS-CoV-2 infections were included in the meta-analysis within the scope of these articles. According to the results, the incidence of fever was 38.1% (95% CI: 14.2-65%) and cough was 22% (95% CI: 10.8-35.2%) among all clinical features of pregnant cases with SARS-CoV-2 infection. So, fever and cough are the most common symptoms in pregnant cases with SARS-CoV-2 infection, and 91.8% (95% CI: 76.7-99.9%) of RT-PCR results are positive. Moreover, abnormal CT incidence is 97.9% (95% CI: 94.2-99.9%) positive. No case was death. However, as this virus spreads globally, it should not be overlooked that the incidence will increase in pregnant women and maybe in the risky group. RT-PCR and CT can be used together in an accurate and safe diagnosis. In conclusion, these findings will provide important guidance for current studies regarding the clinical features and correct detection of SARS-CoV-2 infection in pregnant women, as well as whether it will create emergency tables that will require the use of a viral drug.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/complicações , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/complicações , Pneumonia Viral/diagnóstico , Complicações Infecciosas na Gravidez/diagnóstico , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Feminino , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Gravidez , RNA Viral/análise , RNA Viral/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tomografia Computadorizada por Raios X
5.
Biomed Res Int ; 2020: 7610678, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029522

RESUMO

Background: There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource settings. Therefore, the combination of an optimal assay kit is necessary. Methods: We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. Results: The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. Conclusions: Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Primers do DNA/genética , Pneumonia Viral/diagnóstico , Sondas RNA/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade
6.
PLoS One ; 15(10): e0237689, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33006981

RESUMO

Genomes of tens of thousands of SARS-CoV2 isolates have been sequenced across the world and the total number of changes (predominantly single base substitutions) in these isolates exceeds ten thousand. We compared the mutational spectrum in the new SARS-CoV-2 mutation dataset with the previously published mutation spectrum in hypermutated genomes of rubella-another positive single stranded (ss) RNA virus. Each of the rubella virus isolates arose by accumulation of hundreds of mutations during propagation in a single subject, while SARS-CoV-2 mutation spectrum represents a collection events in multiple virus isolates from individuals across the world. We found a clear similarity between the spectra of single base substitutions in rubella and in SARS-CoV-2, with C to U as well as A to G and U to C being the most prominent in plus strand genomic RNA of each virus. Of those, U to C changes universally showed preference for loops versus stems in predicted RNA secondary structure. Similarly, to what was previously reported for rubella virus, C to U changes showed enrichment in the uCn motif, which suggested a subclass of APOBEC cytidine deaminase being a source of these substitutions. We also found enrichment of several other trinucleotide-centered mutation motifs only in SARS-CoV-2-likely indicative of a mutation process characteristic to this virus. Altogether, the results of this analysis suggest that the mutation mechanisms that lead to hypermutation of the rubella vaccine virus in a rare pathological condition may also operate in the background of the SARS-CoV-2 viruses currently propagating in the human population.


Assuntos
Betacoronavirus/genética , Genoma Viral , RNA Viral/genética , Vírus da Rubéola/genética , Infecções por Coronavirus/virologia , Citidina Desaminase/genética , Bases de Dados Genéticas , Evolução Molecular , Humanos , Mutação , Pandemias , Pneumonia Viral/virologia
7.
Front Immunol ; 11: 2063, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013872

RESUMO

Background: Cases of excessive neutrophil counts in the blood in severe coronavirus disease (COVID-19) patients have drawn significant attention. Neutrophil infiltration was also noted on the pathological findings from autopsies. It is urgent to clarify the pathogenesis of neutrophils leading to severe pneumonia in COVID-19. Methods: A retrospective analysis was performed on 55 COVID-19 patients classified as mild (n = 22), moderate (n = 25), and severe (n = 8) according to the Guidelines released by the National Health Commission of China. Trends relating leukocyte counts and lungs examined by chest CT scan were quantified by Bayesian inference. Transcriptional signatures of host immune cells of four COVID19 patients were analyzed by RNA sequencing of lung specimens and BALF. Results: Neutrophilia occurred in 6 of 8 severe patients at 7-19 days after symptom onset, coinciding with lesion progression. Increasing neutrophil counts paralleled lesion CT values (slope: 0.8 and 0.3-1.2), reflecting neutrophilia-induced lung injury in severe patients. Transcriptome analysis revealed that neutrophil activation was correlated with 17 neutrophil extracellular trap (NET)-associated genes in COVID-19 patients, which was related to innate immunity and interacted with T/NK/B cells, as supported by a protein-protein interaction network analysis. Conclusion: Excessive neutrophils and associated NETs could explain the pathogenesis of lung injury in COVID-19 pneumonia.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/imunologia , Armadilhas Extracelulares/genética , Ativação de Neutrófilo/genética , Neutrófilos/imunologia , Pneumonia Viral/imunologia , Adulto , Idoso , Teorema de Bayes , Infecções por Coronavirus/virologia , Feminino , Humanos , Contagem de Leucócitos , Lesão Pulmonar/imunologia , Lesão Pulmonar/patologia , Masculino , Pessoa de Meia-Idade , Infiltração de Neutrófilos/imunologia , Pandemias , Pneumonia Viral/virologia , Mapas de Interação de Proteínas/imunologia , RNA Viral/genética , Estudos Retrospectivos , Transcriptoma
8.
Viruses ; 12(10)2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33050264

RESUMO

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for the ongoing coronavirus disease (COVID-19) pandemic, is frequently shed in faeces during infection, and viral RNA has recently been detected in sewage in some countries. We have investigated the presence of SARS-CoV-2 RNA in wastewater samples from South-East England between 14th January and 12th May 2020. A novel nested RT-PCR approach targeting five different regions of the viral genome improved the sensitivity of RT-qPCR assays and generated nucleotide sequences at sites with known sequence polymorphisms among SARS-CoV-2 isolates. We were able to detect co-circulating virus variants, some specifically prevalent in England, and to identify changes in viral RNA sequences with time consistent with the recently reported increasing global dominance of Spike protein G614 pandemic variant. Low levels of viral RNA were detected in a sample from 11th February, 3 days before the first case was reported in the sewage plant catchment area. SARS-CoV-2 RNA concentration increased in March and April, and a sharp reduction was observed in May, showing the effects of lockdown measures. We conclude that viral RNA sequences found in sewage closely resemble those from clinical samples and that environmental surveillance can be used to monitor SARS-CoV-2 transmission, tracing virus variants and detecting virus importations.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Esgotos/virologia , Betacoronavirus/isolamento & purificação , Inglaterra/epidemiologia , Monitoramento Ambiental , Variação Genética , Genoma Viral/genética , Humanos , Pandemias , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Águas Residuárias/virologia
9.
PLoS One ; 15(10): e0240502, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33035234

RESUMO

BACKGROUND: A greater understanding of the antibody response to SARS-CoV-2 in an infected population is important for the development of a vaccination. AIM: To investigate SARS-CoV-2 IgA and IgG antibodies in Thai patients with differing severities of COVID-19. METHODS: Plasma from the following patient groups was examined: 118 adult patients with confirmed SARS-CoV-2 infections, 49 patients under investigation (without confirmed infections), 20 patients with other respiratory infections, and 102 healthy control patients. Anti-SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA) from EUROIMMUN was performed to assess for IgA and IgG antibodies. The optical density (OD) ratio cutoff for a positive result was 1.1 for IgA and 0.8 for IgG. Additionally, the association of the antibody response with both the severity of disease and the date after onset of symptoms was analyzed. RESULTS: A total of 289 participants were enrolled and 384 samples analyzed from March 10 to May 31, 2020. Patients were categorized, based on their clinical manifestations, as mild (n = 59), moderate (n = 27), or severe (n = 32). The overall sensitivity of IgA and IgG from the samples collected after day 7 of the symptoms was 87.9% (95% CI: 79.8-93.6) and 84.8% (95% CI: 76.2-91.3), respectively. Compared to the mild group, the severe group had significantly higher levels of spike 1 (S1) antigen-specific IgA and IgG. All patients in the moderate and severe groups had S1-specific IgG, while 20% of the patients in the mild group did not have any IgG detected after two weeks after the onset of symptoms. Interestingly, in the severe group, the SARS-CoV-2 IgG level was significantly higher in males than females (p = 0.003). CONCLUSION: The serological test for SARS-CoV-2 has a high sensitivity more than two weeks after the onset of illness. Additionally, the serological response differs among patients based on sex as well as the severity of infection.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Adulto , Idoso , Formação de Anticorpos , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Estudos de Casos e Controles , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Índice de Gravidade de Doença , Fatores Sexuais , Glicoproteína da Espícula de Coronavírus/imunologia
10.
J Korean Med Sci ; 35(39): e358, 2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33045775

RESUMO

Although some comorbidities, such as diabetes mellitus, lung disease, and chronic kidney disease, are known as risk factors for poor clinical outcome in patients with coronavirus disease 2019 (COVID-19), it is unknown if human immunodeficiency virus (HIV) patients with COVID-19 would have poor prognosis than others. Rare cases of HIV patients with COVID-19 have been reported. As of May 25th, 2020, over 11,000 patients have been diagnosed with COVID-19 and over 13,000 are living with HIV in Korea. Here, we present the first HIV patient with COVID-19 in Korea. The 29-year-old Korean man had been taking Genvoya® regularly for seven years and HIV was well suppressed with CD4 counts of 555/mm³. He had mild symptoms of sore throat, dry cough, loss of taste and smell. He received hydroxychloroquine while Genvoya® was continued. Pneumonia diagnosed in chest computed tomography improved without oxygen supplementation. He was discharged on hospital day 31. HIV patients are considered as immunocompromised, but this case suggests that well controlled HIV patients have satisfactory prognosis following proper medical care.


Assuntos
Infecções por Coronavirus/diagnóstico , Infecções por HIV/patologia , Pneumonia Viral/diagnóstico , Adulto , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Contagem de Linfócito CD4 , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Infecções por HIV/complicações , Humanos , Hidroxicloroquina/uso terapêutico , Masculino , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Prognóstico , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tomografia Computadorizada por Raios X
11.
Nat Commun ; 11(1): 4906, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999292

RESUMO

The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3'- or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40-60 min.


Assuntos
Proteínas de Bactérias/metabolismo , Betacoronavirus/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , Transativadores/metabolismo , Betacoronavirus/isolamento & purificação , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , DNA de Cadeia Simples , Pandemias , Pneumonia Viral , RNA Guia/genética , RNA Viral/genética
12.
Nat Commun ; 11(1): 4693, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943634

RESUMO

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp's top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.


Assuntos
Alphavirus/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Genômica , RNA Viral/genética , Alphavirus/genética , Alphavirus/ultraestrutura , Animais , Sítios de Ligação , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Vírus Chikungunya/genética , Chlorocebus aethiops , Modelos Moleculares , Nucleocapsídeo/metabolismo , Ligação Proteica , RNA Viral/química , Vírus da Floresta de Semliki/metabolismo , Células Vero , Montagem de Vírus , Replicação Viral
13.
Nat Commun ; 11(1): 4682, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943628

RESUMO

The ongoing Corona Virus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emphasized the urgent need for antiviral therapeutics. The viral RNA-dependent-RNA-polymerase (RdRp) is a promising target with polymerase inhibitors successfully used for the treatment of several viral diseases. We demonstrate here that Favipiravir predominantly exerts an antiviral effect through lethal mutagenesis. The SARS-CoV RdRp complex is at least 10-fold more active than any other viral RdRp known. It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. The coronavirus RdRp complex represents an Achilles heel for SARS-CoV, supporting nucleoside analogues as promising candidates for the treatment of COVID-19.


Assuntos
Amidas/farmacologia , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Pirazinas/farmacologia , Amidas/farmacocinética , Animais , Antivirais/farmacocinética , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Moleculares , Mutagênese/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Pirazinas/farmacocinética , RNA Replicase/química , RNA Replicase/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Análise de Sequência , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
14.
Cell Mol Immunol ; 17(10): 1098-1100, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32939033
15.
Nat Commun ; 11(1): 4812, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968075

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Benchmarking , Técnicas de Laboratório Clínico/normas , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Primers do DNA/genética , Temperatura Alta , Humanos , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade , Suécia/epidemiologia , Ensaio de Placa Viral/métodos
16.
Viruses ; 12(9)2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32883050

RESUMO

Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of ~200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.


Assuntos
Inteligência Artificial , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/virologia , Animais , Chlorocebus aethiops , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Cães , Humanos , Células Madin Darby de Rim Canino , Pandemias , Pneumonia Viral/diagnóstico , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Células Vero
17.
Proc Natl Acad Sci U S A ; 117(39): 24450-24458, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32900935

RESUMO

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Técnicas de Laboratório Clínico/economia , Colorimetria , Infecções por Coronavirus/economia , Infecções por Coronavirus/virologia , Genoma Viral/genética , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Pandemias , Pneumonia Viral/virologia , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Fatores de Tempo , Proteínas Virais/genética , Inativação de Vírus
18.
Nat Commun ; 11(1): 4464, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900994

RESUMO

The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , Bioensaio , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/normas , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , RNA Viral/análise , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
19.
Nat Biotechnol ; 38(10): 1164-1167, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32948856

RESUMO

We measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in primary sewage sludge in the New Haven, Connecticut, USA, metropolitan area during the Coronavirus Disease 2019 (COVID-19) outbreak in Spring 2020. SARS-CoV-2 RNA was detected throughout the more than 10-week study and, when adjusted for time lags, tracked the rise and fall of cases seen in SARS-CoV-2 clinical test results and local COVID-19 hospital admissions. Relative to these indicators, SARS-CoV-2 RNA concentrations in sludge were 0-2 d ahead of SARS-CoV-2 positive test results by date of specimen collection, 0-2 d ahead of the percentage of positive tests by date of specimen collection, 1-4 d ahead of local hospital admissions and 6-8 d ahead of SARS-CoV-2 positive test results by reporting date. Our data show the utility of viral RNA monitoring in municipal wastewater for SARS-CoV-2 infection surveillance at a population-wide level. In communities facing a delay between specimen collection and the reporting of test results, immediate wastewater results can provide considerable advance notice of infection dynamics.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/análise , Águas Residuárias/virologia , Vigilância Epidemiológica Baseada em Águas Residuárias , Betacoronavirus/genética , Biotecnologia , Connecticut/epidemiologia , Humanos , Prevalência , RNA Viral/genética , Esgotos/virologia , Fatores de Tempo
20.
J Microbiol ; 58(10): 886-891, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32989642

RESUMO

Various treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The results substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful method for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/virologia , Manejo de Espécimes/métodos , Inativação de Vírus , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/virologia , Genoma Viral , Instabilidade Genômica , Temperatura Alta , Humanos , Pandemias , Pneumonia Viral/diagnóstico , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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