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1.
Arch Virol ; 166(2): 645-649, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33386489

RESUMO

Pleioblastus mosaic virus (PleMV) is a tentative member of the genus Potyvirus in the family Potyviridae and was discovered in bamboo with mosaic symptoms in Tokyo, Japan. Since no information on the genome sequence of PleMV has been reported, its taxonomic position has long been uncertain. Here, we report the first complete genome sequences of two distinct PleMV isolates. Excluding the 3'-terminal poly(A) tail, their genomic RNA sequences consist of 9,634 and 9,643 nucleotides (nt); both contain a large open reading frame (ORF) encoding a polyprotein and a small ORF termed PIPO. The large ORFs of the two isolates share 79.2% and 87.6% sequence identity at the nucleotide (nt) and amino acid (aa) level, respectively, and were found to have the highest nt and aa sequence identity (69.0% and 69.9%) to the potyvirus johnsongrass mosaic virus (JGMV). Phylogenetic analysis showed that PleMV is most closely related to JGMV but forms its own clade. These results suggest that PleMV is a distinct member of the genus Potyvirus.


Assuntos
Genoma Viral/genética , Potyvirus/genética , Sequência de Aminoácidos , Sequência de Bases , Genômica/métodos , Japão , Fases de Leitura Aberta/genética , Filogenia , Poliproteínas/genética , RNA Viral/genética , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/métodos
2.
Exp Eye Res ; 203: 108433, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33400927

RESUMO

Although severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection have emerged globally, findings related to ocular involvement and reported cases are quite limited. Immune reactions against viral infections are closely related to viral and host proteins sequence similarity. Molecular Mimicry has been described for many different viruses; sequence similarities of viral and human tissue proteins may trigger autoimmune reactions after viral infections due to similarities between viral and human structures. With this study, we aimed to investigate the protein sequence similarity of SARS CoV-2 with retinal proteins and retinal pigment epithelium (RPE) surface proteins. Retinal proteins involved in autoimmune retinopathy and retinal pigment epithelium surface transport proteins were analyzed in order to infer their structural similarity to surface glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), envelope protein (E), ORF1ab polyprotein (orf1ab) proteins of SARS CoV-2. Protein similarity comparisons, 3D protein structure prediction, T cell epitopes-MHC binding prediction, B cell epitopes-MHC binding prediction and the evaluation of the antigenicity of peptides assessments were performed. The protein sequence analysis was made using the Pairwise Sequence Alignment and the LALIGN program. 3D protein structure estimates were made using Swiss Model with default settings and analyzed with TM-align web server. T-cell epitope identification was performed using the Immune Epitope Database and Analysis (IEDB) resource Tepitool. B cell epitopes based on sequence characteristics of the antigen was performed using amino acid scales and HMMs with the BepiPred 2.0 web server. The predicted peptides/epitopes in terms of antigenicity were examined using the default settings with the VaxiJen v2.0 server. Analyses showed that, there is a meaningful similarities between 6 retinal pigment epithelium surface transport proteins (MRP-4, MRP-5, RFC1, SNAT7, TAUT and MATE) and the SARS CoV-2 E protein. Immunoreactive epitopic sites of these proteins which are similar to protein E epitope can create an immune stimulation on T cytotoxic and T helper cells and 6 of these 9 epitopic sites are also vaxiJen. These result imply that autoimmune cross-reaction is likely between the studied RPE proteins and SARS CoV-2 E protein. The structure of SARS CoV-2, its proteins and immunologic reactions against these proteins remain largely unknown. Understanding the structure of SARS CoV-2 proteins and demonstration of similarity with human proteins are crucial to predict an autoimmune response associated with immunity against host proteins and its clinical manifestations as well as possible adverse effects of vaccination.


Assuntos
Sequência de Aminoácidos , Doenças Autoimunes/virologia , Proteínas do Olho/química , Doenças Retinianas/virologia , Homologia de Sequência , Proteínas Virais/química , /epidemiologia , Biologia Computacional , /química , Infecções Oculares Virais/virologia , Humanos , Glicoproteínas de Membrana/química , Fosfoproteínas/química , Poliproteínas/química , Epitélio Pigmentado da Retina/química , Proteínas da Matriz Viral/química
3.
Viruses ; 13(1)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477885

RESUMO

Since the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the etiological agent of the current COVID-19 pandemic, a rapid and massive effort has been made to obtain the genomic sequences of this virus to monitor (in near real time) the phylodynamic and diversity of this new pathogen. However, less attention has been given to the assessment of intra-host diversity. RNA viruses such as SARS-CoV-2 inhabit the host as a population of variants called quasispecies. We studied the quasispecies diversity in four of the main SARS-CoV-2 genes (ORF1a, ORF1b, S and N genes), using a dataset consisting of 210 next-generation sequencing (NGS) samples collected between January and early April of 2020 in the State of Victoria, Australia. We found evidence of quasispecies diversity in 68% of the samples, 76% of which was nonsynonymous variants with a higher density in the spike (S) glycoprotein and ORF1a genes. About one-third of the nonsynonymous intra-host variants were shared among the samples, suggesting host-to-host transmission. Quasispecies diversity changed over time. Phylogenetic analysis showed that some of the intra-host single-nucleotide variants (iSNVs) were restricted to specific lineages, highlighting their potential importance in the epidemiology of this virus. A greater effort must be made to determine the magnitude of the genetic bottleneck during transmission and the epidemiological and/or evolutionary factors that may play a role in the changes in the diversity of quasispecies over time.


Assuntos
/genética , Genoma Viral/genética , Quase-Espécies/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais/genética , Austrália , Sequência de Bases , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Poliproteínas/genética , Análise de Sequência de RNA , Vitória
4.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498408

RESUMO

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.


Assuntos
/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas Virais/genética , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Poliproteínas/genética , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa
5.
Sci Rep ; 11(1): 1873, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479389

RESUMO

The development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.


Assuntos
/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , /isolamento & purificação , Bioensaio/métodos , /métodos , Técnicas de Laboratório Clínico/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Fosfoproteínas/genética , Poliproteínas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Proteínas Virais/genética
6.
Talanta ; 224: 121726, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33379001

RESUMO

The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 210 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnosis of SARS-CoV-2. However, the sensitivity of RT-qPCR assays of pharyngeal swab samples are reported to vary from 30% to 60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. A reverse transcription digital PCR (RT-dPCR) method was established and evaluated. To explore the feasibility of RT-dPCR in diagnostic of SARS-CoV-2, a total of 196 clinical pharyngeal swab samples from 103 suspected patients, 77 close contacts and 16 supposed convalescents were analyzed by RT-qPCR and then measured by the proposed RT-dPCR. For the 103 fever suspected patients, 19 (19/25) negative and 42 (42/49) equivocal tested by RT-qPCR were positive according to RT-dPCR. The sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For 29 close contacts (confirmed by additional sample and clinical follow up), 16 (16/17) equivocal and 1 negative tested by RT-qPCR were positive according to RT-dPCR, which is implying that the RT-qPCR is missing a lot of asymptomatic patients. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 91%, 100% and 93%, respectively. RT-dPCR is highly accurate method and suitable for detection of pharyngeal swab samples from COVID-19 suspected patients and patients under isolation and observation who may not be exhibiting clinical symptoms.


Assuntos
/métodos , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , /genética , /genética , Humanos , Faringe/virologia , Fosfoproteínas/genética , Poliproteínas/genética , Proteínas Virais/genética
7.
PLoS One ; 15(11): e0240345, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33170902

RESUMO

In late December 2019, an emerging viral infection COVID-19 was identified in Wuhan, China, and became a global pandemic. Characterization of the genetic variants of SARS-CoV-2 is crucial in following and evaluating it spread across countries. In this study, we collected and analyzed 3,067 SARS-CoV-2 genomes isolated from 55 countries during the first three months after the onset of this virus. Using comparative genomics analysis, we traced the profiles of the whole-genome mutations and compared the frequency of each mutation in the studied population. The accumulation of mutations during the epidemic period with their geographic locations was also monitored. The results showed 782 variants sites, of which 512 (65.47%) had a non-synonymous effect. Frequencies of mutated alleles revealed the presence of 68 recurrent mutations, including ten hotspot non-synonymous mutations with a prevalence higher than 0.10 in this population and distributed in six SARS-CoV-2 genes. The distribution of these recurrent mutations on the world map revealed that certain genotypes are specific to geographic locations. We also identified co-occurring mutations resulting in the presence of several haplotypes. Moreover, evolution over time has shown a mechanism of mutation co-accumulation which might affect the severity and spread of the SARS-CoV-2. The phylogentic analysis identified two major Clades C1 and C2 harboring mutations L3606F and G614D, respectively and both emerging for the first time in China. On the other hand, analysis of the selective pressure revealed the presence of negatively selected residues that could be taken into considerations as therapeutic targets. We have also created an inclusive unified database (http://covid-19.medbiotech.ma) that lists all of the genetic variants of the SARS-CoV-2 genomes found in this study with phylogeographic analysis around the world.


Assuntos
Betacoronavirus/genética , Variação Genética , Genoma Viral , Betacoronavirus/classificação , Betacoronavirus/isolamento & purificação , China , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Evolução Molecular , Humanos , Pandemias , Filogenia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Poliproteínas , Estrutura Terciária de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais/química , Proteínas Virais/genética
8.
Biomedica ; 40(Supl. 2): 166-172, 2020 10 30.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-33152200

RESUMO

Introduction: The 2019 coronavirus pandemic (COVID-19) has caused around 25 million cases worldwide. Asymptomatic patients have been described as potential sources of transmission. However, there are difficulties to detect them and to establish their role in the dynamics of virus transmission, which hinders the implementation of prevention strategies. Objective: To describe the behavior of asymptomatic SARS-CoV-2 virus infection in a cohort of workers at the El Dorado "Luis Carlos Galán Sarmiento" International Airport in Bogotá, Colombia. Materials and methods: A prospective cohort of 212 workers from the El Dorado airport was designed. The follow-up began in June, 2020. A survey was used to characterize health and work conditions. Every 21 day, a nasopharyngeal swab was taken to identify the presence of SARS-CoV-2 using RT-PCR. We analyzed the behavior of the cycle threshold (ORF1ab and N genes) according to the day of follow-up. Results: In the first three follow-ups of the cohort, we found an incidence of SARS-CoV-2 infection of 16.51%. The proportion of positive contacts was 14.08%. The median threshold for cycle threshold was 33.53. Conclusion: We characterized the asymptomatic SARS-CoV-2 infection in a cohort of workers. The identification of asymptomatic infected persons continues to be a challenge for epidemiological surveillance systems.


Assuntos
Aeroportos , Infecções Assintomáticas , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Nasofaringe/virologia , Pandemias , Pneumonia Viral/diagnóstico , Adulto , Infecções Assintomáticas/epidemiologia , Betacoronavirus/genética , Betacoronavirus/fisiologia , Colômbia , Busca de Comunicante , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/genética , Fosfoproteínas , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Poliproteínas , Estudos Prospectivos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inquéritos e Questionários , Proteínas Virais/genética , Replicação Viral/genética , Local de Trabalho
9.
Immun Inflamm Dis ; 8(4): 753-762, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33124193

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) is a single-stranded RNA virus responsible for the global pandemic of the coronavirus disease-2019 (COVID-19). To date, there are still no effective approaches for the prevention and treatment of COVID-19. OBJECTIVE: The present study aims to explore the possible mechanisms of SARS-CoV-2 infection in human lung cells. METHODS: Data interpretation was conducted by recruiting bioinformatics analysis, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways analysis using downloaded data from the NCBI Gene Expression Omnibus database. RESULTS: The present study demonstrated that SARS-CoV-2 infection induces the upregulation of 14 interferon-stimulated genes, indicative of immune, and interferon responses to the virus. Notably, genes for pyrimidine metabolism and steroid hormone biosynthesis are selectively enriched in human lung cells after SARS-CoV-2 infection, suggesting that altered pyrimidine metabolism and steroid biosynthesis are remarkable, and perhaps druggable features after SARS-CoV-2 infection. Besides, there is a strong positive correlation between viral ORF1ab, ORF6, and angiotensin-converting enzyme 2 (ACE2) expression in human lung cells, implying that ACE2 facilitates SARS-CoV-2 infection and replication in host cells probably through the induction of ORF1ab and ORF6.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/etiologia , Interferons/metabolismo , Pulmão/patologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/etiologia , Betacoronavirus/metabolismo , Biologia Computacional , Infecções por Coronavirus/patologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Pulmão/citologia , Pulmão/imunologia , Pulmão/virologia , Pandemias , Pneumonia Viral/patologia , Poliproteínas , Pirimidinas/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Transdução de Sinais/imunologia , Esteroides/biossíntese , Regulação para Cima/imunologia , Proteínas Virais/metabolismo
10.
Immunity ; 53(5): 1095-1107.e3, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33128877

RESUMO

Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19 patients. In total, we identified 3-8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8+ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8+ T cell immunity to SARS-CoV-2.


Assuntos
Betacoronavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Betacoronavirus/isolamento & purificação , Convalescença , Coronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Mapeamento de Epitopos , Epitopos de Linfócito T , Feminino , Humanos , Epitopos Imunodominantes , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Poliproteínas , Proteínas Virais/imunologia , Adulto Jovem
11.
EBioMedicine ; 61: 103036, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33045467

RESUMO

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.


Assuntos
Proteínas de Bactérias/metabolismo , Betacoronavirus/genética , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endodesoxirribonucleases/metabolismo , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Cavidade Nasal/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Poliproteínas , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(37): 23165-23173, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868448

RESUMO

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.


Assuntos
Genes Bacterianos/genética , Mitocôndrias/genética , Mitocôndrias/fisiologia , Proteínas de Plantas/genética , Plantas/genética , Substituição de Aminoácidos/genética , Escherichia coli/genética , Fixação de Nitrogênio/genética , Nitrogenase/genética , Poliproteínas/genética , Proteômica/instrumentação
13.
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 , Poliproteínas , 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
14.
Chem Biol Interact ; 331: 109226, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971122

RESUMO

Presence of Simple Sequence Repeats (SSRs), both in genic and intergenic regions, have been widely studied in eukaryotes, prokaryotes, and viruses. In the current study, we undertook a survey to analyze the frequency and distribution of microsatellites or SSRs in multiple genomes of Coronaviridae members. We successfully identified 919 SSRs with length ≥12 bp across 55 reference genomes majority of which (838 SSRs) were found abundant in genic regions. The in-silico analysis further identified the preferential abundance of hexameric SSRs than any other size-based motif class. Our analysis shows that the genome size and GC content of the genome had a weak influence on SSR frequency and density. However, we find a positive correlation of SSRs GC content with genomic GC content. We also report relatively low abundances of all theoretically possible 501 repeat motif classes in all the genomes of Coronaviridae. The majority of SSRs were AT-rich. Overall, we see an underrepresentation of SSRs across the genomes of Coronaviridae. Besides, our integrative study highlights the presence of SSRs in ORF1ab (nsp3, nsp4, nsp5A_3CLpro and nsp5B_3CLpro, nsp6, nsp10, nsp12, nsp13, & nsp15 domains), S, ORF3a, ORF7a, N & 3' UTR regions of SARS-CoV-2 and harbours multiple mutations (3'UTR and ORF1ab SSRs serving as major mutational hotspots). This indicates the genic SSRs are under selection pressure against mutations that might alter the reading frame and at the same time responsible for rapid protein evolution. Our preliminary results indicate the significance of the limited repertoire of SSRs in the genomes of Coronaviridae.


Assuntos
Coronaviridae/genética , Repetições de Microssatélites/genética , Regiões 3' não Traduzidas , Composição de Bases , Sequência de Bases , Betacoronavirus/genética , Evolução Molecular , Genoma Viral , Humanos , Mutação , Poliproteínas , Proteínas Virais/genética
15.
Proc Natl Acad Sci U S A ; 117(39): 24384-24391, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32913053

RESUMO

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269-277 and A2/Orf1ab3183-3191 Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S269 +CD8+ and A2/Orf1ab3183 +CD8+ populations indicated that A2/S269 +CD8+ T cells were detected at comparable frequencies (∼1.3 × 10-5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10-6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein-Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10-4) populations. Phenotyping A2/S269 +CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269 +CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269 +CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19.


Assuntos
Betacoronavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Antígeno HLA-A2/imunologia , Pneumonia Viral/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T , Feminino , Humanos , Memória Imunológica , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Pandemias , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Poliproteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas Virais/química , Proteínas Virais/imunologia
16.
Nat Commun ; 11(1): 4032, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788575

RESUMO

Hydrogel-based devices are widely used as flexible electronics, biosensors, soft robots, and intelligent human-machine interfaces. In these applications, high stretchability, low hysteresis, and anti-fatigue fracture are essential but can be rarely met in the same hydrogels simultaneously. Here, we demonstrate a hydrogel design using tandem-repeat proteins as the cross-linkers and random coiled polymers as the percolating network. Such a design allows the polyprotein cross-linkers only to experience considerable forces at the fracture zone and unfold to prevent crack propagation. Thus, we are able to decouple the hysteresis-toughness correlation and create hydrogels of high stretchability (~1100%), low hysteresis (< 5%), and high fracture toughness (~900 J m-2). Moreover, the hydrogels show a high fatigue threshold of ~126 J m-2 and can undergo 5000 load-unload cycles up to 500% strain without noticeable mechanical changes. Our study provides a general route to decouple network elasticity and local mechanical response in synthetic hydrogels.


Assuntos
Reagentes para Ligações Cruzadas/química , Hidrogéis/química , Poliproteínas/química , Estresse Mecânico , Resinas Acrílicas/química , Fluorescência , Fenômenos Mecânicos
17.
Arch Virol ; 165(10): 2373-2377, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761270
18.
J Enzyme Inhib Med Chem ; 35(1): 1539-1544, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32746637

RESUMO

Coronavirus disease 2019 (COVID-19) has been a pandemic disease of which the termination is not yet predictable. Currently, researches to develop vaccines and treatments is going on globally to cope with this disastrous disease. Main protease (3CLpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the good targets to find antiviral agents before vaccines are available. Some flavonoids are known to inhibit 3CLpro from SARS-CoV which causes SARS. Since their sequence identity is 96%, a similar approach was performed with a flavonoid library. Baicalin, herbacetin, and pectolinarin have been discovered to block the proteolytic activity of SARS-CoV-2 3CLpro. An in silico docking study showed that the binding modes of herbacetin and pectolinarin are similar to those obtained from the catalytic domain of SARS-CoV 3CLpro. However, their binding affinities are different due to the usage of whole SARS-CoV-2 3CLpro in this study. Baicalin showed an effective inhibitory activity against SARS-CoV-2 3CLpro and its docking mode is different from those of herbacetin and pectolinarin. This study suggests important scaffolds to design 3CLpro inhibitors to develop antiviral agents or health-foods and dietary supplements to cope with SARS-CoV-2.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Flavonoides/química , Pneumonia Viral/tratamento farmacológico , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Antivirais/química , Betacoronavirus , Desenho de Fármacos , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Acoplamento Molecular , Pandemias , Poliproteínas , Inibidores de Proteases/química , Ligação Proteica , Conformação Proteica , Espectrofotometria , Triptofano/química
19.
Arch Virol ; 165(10): 2385-2388, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32647930

RESUMO

Spartina mottle virus (SpMV), an unassigned member of the family Potyviridae, has been known since 1980, when it was first described in England and Wales in symptomatic plants of the genus Spartina. In infected cells, flexuous particles and pinwheel inclusion bodies were found that resemble those of potyvirids. To date, the NCBI database contains only two partial sequences of a German (Nessmersiel) and an Italian (Assisi) isolate, suggesting that SpMV could be the first member of a new genus, called "Sparmovirus", in the family Potyviridae. In this study, the first complete genome sequence of the German SpMV isolate (SpMV Ger) was determined. The genome of SpMV is a single-stranded, monopartite, polyadenylated RNA consisting of 9376 nucleotides. Sequence analysis revealed a genome organization similar to that of classical potyviruses, including many conserved features. In phylogenetic analysis, SpMV could not be assigned to any of the known genera, but it showed the closest relationship to rymoviruses and common reed chlorotic stripe virus (CRCSV, unassigned). Sequence comparisons confirmed that a new genus should be established containing SpMV, CRCSV, and three Bermuda grass mosaic virus isolates, which are considered divergent strains of SpMV.


Assuntos
Genoma Viral , Poaceae/virologia , Poliproteínas/genética , Potyviridae/genética , RNA Viral/genética , Alemanha , Fases de Leitura Aberta , Filogenia , Doenças das Plantas/virologia , Potyviridae/classificação , Potyviridae/isolamento & purificação , Sequenciamento Completo do Genoma
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