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1.
Viruses ; 13(2)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578722

RESUMO

Canine distemper virus (CDV) is a highly lethal contagious viral pathogen mainly found in domestic and wild canids and mustelids. Although, in Italy, circulating strains of Europe 1, Europe wildlife and Arctic type are reported, data relating to Latium and Tuscany regions are limited. In view of this, through passive surveillance, we investigated the presence of CDV and which strains were circulating in these Regions. From March 2017 to October 2019, a group of 122 subjects were tested for CDV using a PCR protocol described in the literature, with 12 detected positive; analyses were carried out on a set of target samples (brain and lung, conjunctival, nasal and rectal swabs, urine or swab from bladder and intracardiac clot) that was defined for the detection of CDV in both live and dead animals. The rectal swab, easily collected also from live animals, represented the most suitable sample for CDV diagnosis, with 9 positive of the 11 (81.82%) tested. In addition, brain and lung of 15 subjects out of 181 susceptible animals collected between 2011 and 2018, during post mortem investigations in routine diagnostic activity, were CDV positive. Molecular analyses of all positive samples, using a 287 bp fragment located within the conserved N terminus of the morbillivirus nucleoprotein gene, detected the circulation of strain CDV599/2016 (KX545421.1) belonging to the "Europe wildlife" lineage, and of strain CDV12254/2015 (KX024709.1), belonging to the Arctic-lineage, thus confirming the co-circulation of the two lineages, as already noted in previous studies.


Assuntos
Vírus da Cinomose Canina/isolamento & purificação , Cinomose/epidemiologia , Cinomose/virologia , Animais , Autopsia/veterinária , Cinomose/patologia , Vírus da Cinomose Canina/genética , Itália/epidemiologia , Proteínas do Nucleocapsídeo/genética , RNA Viral/genética , Estudos Retrospectivos , Vacinas Virais/genética
2.
BMC Microbiol ; 21(1): 58, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33618668

RESUMO

BACKGROUND: A severe form of pneumonia, named coronavirus disease 2019 (COVID-19) by the World Health Organization is widespread on the whole world. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was proved to be the main agent of COVID-19. In the present study, we conducted an in depth analysis of the SARS-COV-2 nucleocapsid to identify potential targets that may allow identification of therapeutic targets. METHODS: The SARS-COV-2 N protein subcellular localization and physicochemical property was analyzed by PSORT II Prediction and ProtParam tool. Then SOPMA tool and swiss-model was applied to analyze the structure of N protein. Next, the biological function was explored by mass spectrometry analysis and flow cytometry. At last, its potential phosphorylation sites were analyzed by NetPhos3.1 Server and PROVEAN PROTEIN. RESULTS: SARS-COV-2 N protein composed of 419 aa, is a 45.6 kDa positively charged unstable hydrophobic protein. It has 91 and 49% similarity to SARS-CoV and MERS-CoV and is predicted to be predominantly a nuclear protein. It mainly contains random coil (55.13%) of which the tertiary structure was further determined with high reliability (95.76%). Cells transfected with SARS-COV-2 N protein usually show a G1/S phase block company with an increased expression of TUBA1C, TUBB6. At last, our analysis of SARS-COV-2 N protein predicted a total number of 12 phosphorylated sites and 9 potential protein kinases which would significantly affect SARS-COV-2 N protein function. CONCLUSION: In this study, we report the physicochemical properties, subcellular localization, and biological function of SARS-COV-2 N protein. The 12 phosphorylated sites and 9 potential protein kinase sites in SARS-COV-2 N protein may serve as promising targets for drug discovery and development for of a recombinant virus vaccine.


Assuntos
/virologia , Proteínas do Nucleocapsídeo/metabolismo , /patogenicidade , Sequência de Aminoácidos , /imunologia , Genoma Viral/genética , Células HCT116 , Humanos , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Fosforilação , Reprodutibilidade dos Testes , Vacinas Virais/uso terapêutico
3.
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
4.
Sci Rep ; 10(1): 18289, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106569

RESUMO

The World Health Organization characterized COVID-19 as a pandemic in March 2020, the second pandemic of the twenty-first century. Expanding virus populations, such as that of SARS-CoV-2, accumulate a number of narrowly shared polymorphisms, imposing a confounding effect on traditional clustering methods. In this context, approaches that reduce the complexity of the sequence space occupied by the SARS-CoV-2 population are necessary for robust clustering. Here, we propose subdividing the global SARS-CoV-2 population into six well-defined subtypes and 10 poorly represented genotypes named tentative subtypes by focusing on the widely shared polymorphisms in nonstructural (nsp3, nsp4, nsp6, nsp12, nsp13 and nsp14) cistrons and structural (spike and nucleocapsid) and accessory (ORF8) genes. The six subtypes and the additional genotypes showed amino acid replacements that might have phenotypic implications. Notably, three mutations (one of them in the Spike protein) were responsible for the geographical segregation of subtypes. We hypothesize that the virus subtypes detected in this study are records of the early stages of SARS-CoV-2 diversification that were randomly sampled to compose the virus populations around the world. The genetic structure determined for the SARS-CoV-2 population provides substantial guidelines for maximizing the effectiveness of trials for testing candidate vaccines or drugs.


Assuntos
Betacoronavirus/genética , Polimorfismo Genético , Betacoronavirus/classificação , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Genótipo , Humanos , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Filogenia , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética
5.
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
6.
Anal Chem ; 92(20): 14139-14144, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32967427

RESUMO

The infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) has threatened public health worldwide. The easy human-to-human transmission of this virus has rapidly evolved into a global pandemic. Therefore, to control the community spread of the virus, it is crucial to identify the infected individuals, including asymptomatic people. Hence, a specific and rapid assay is crucial for the early diagnosis and active monitoring of individuals potentially exposed to SARS-CoV-2 for controlling the COVID-19 outbreak. In this study, we have developed the novel lateral flow strip membrane (LFSM) assay that allows the simultaneous detection of RdRp, ORF3a, and N genes using the PCR product obtained by using the single-tube reverse transcription polymerase chain reaction (RT-PCR). The LFSM assay allows detection of SARS-CoV-2 in 30 min at 25 °C after the RT-PCR with the detection limit of 10 copies/test for each gene. The clinical performance of the LFSM assay for the detection of SARS-Cov-2 was evaluated using 162 clinical samples previously detected by using the commercial assay. The percent positive agreement, percent negative agreement, and overall percent agreement of the LFSM assay with the commercial assay were 100% (94.2-100%), 99.0% (94.6-100%), and 99.4% (96.6-100%), respectively. Therefore, the results of the LFSM assay showed significantly high concordance with the commercial assay for the detection of SARS-CoV-2 in clinical specimens. Therefore, we conclude that the developed LFSM assay can be used alone or complementary to the RT-PCR or other methods for the diagnosis and monitoring of the patients to curb community transmission and the pandemic.


Assuntos
Betacoronavirus/genética , Fluorometria/métodos , Proteínas do Nucleocapsídeo/análise , Proteínas Virais Reguladoras e Acessórias/análise , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Primers do DNA/química , Primers do DNA/metabolismo , Corantes Fluorescentes/química , Fluorometria/instrumentação , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , /metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
7.
Sci Rep ; 10(1): 15643, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973171

RESUMO

As the SARS-CoV-2 pandemic is rapidly progressing, the need for the development of an effective vaccine is critical. A promising approach for vaccine development is to generate, through codon pair deoptimization, an attenuated virus. This approach carries the advantage that it only requires limited knowledge specific to the virus in question, other than its genome sequence. Therefore, it is well suited for emerging viruses, for which we may not have extensive data. We performed comprehensive in silico analyses of several features of SARS-CoV-2 genomic sequence (e.g., codon usage, codon pair usage, dinucleotide/junction dinucleotide usage, RNA structure around the frameshift region) in comparison with other members of the coronaviridae family of viruses, the overall human genome, and the transcriptome of specific human tissues such as lung, which are primarily targeted by the virus. Our analysis identified the spike (S) and nucleocapsid (N) proteins as promising targets for deoptimization and suggests a roadmap for SARS-CoV-2 vaccine development, which can be generalizable to other viruses.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/prevenção & controle , Proteínas do Nucleocapsídeo/genética , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Virais/imunologia , Sequência de Bases , Infecções por Coronavirus/imunologia , Genoma Viral/genética , Humanos , Proteínas do Nucleocapsídeo/imunologia , Fosfoproteínas , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Produtos Inativados/imunologia , Sequenciamento Completo do Genoma
8.
Cell Mol Immunol ; 17(10): 1098-1100, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32939033
9.
Ann Saudi Med ; 40(5): 373-381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32954791

RESUMO

BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nasofaringe/virologia , Pneumonia Viral/diagnóstico , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Automação , Chlorocebus aethiops , Técnicas de Laboratório Clínico , Vírus da Encefalomiocardite/genética , Humanos , Levivirus/genética , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , RNA Viral/análise , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética
10.
PLoS One ; 15(9): e0238344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881907

RESUMO

A novel severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) causing COVID-19 pandemic in humans, recently emerged and has exported in more than 200 countries as a result of rapid spread. In this study, we have made an attempt to investigate the SARS-CoV-2 genome reported from 13 different countries, identification of mutations in major coronavirus proteins of these different SARS-CoV-2 genomes and compared with SARS-CoV. These thirteen complete genome sequences of SARS-CoV-2 showed high identity (>99%) to each other, while they shared 82% identity with SARS-CoV. Here, we performed a very systematic mutational analysis of SARS-CoV-2 genomes from different geographical locations, which enabled us to identify numerous unique features of this viral genome. This includes several important country-specific unique mutations in the major proteins of SARS-CoV-2 namely, replicase polyprotein, spike glycoprotein, envelope protein and nucleocapsid protein. Indian strain showed mutation in spike glycoprotein at R408I and in replicase polyprotein at I671T, P2144S and A2798V,. While the spike protein of Spain & South Korea carried F797C and S221W mutation, respectively. Likewise, several important country specific mutations were analyzed. The effect of mutations of these major proteins were also investigated using various in silico approaches. Main protease (Mpro), the therapeutic target protein of SARS with maximum reported inhibitors, was thoroughly investigated and the effect of mutation on the binding affinity and structural dynamics of Mpro was studied. It was found that the R60C mutation in Mpro affects the protein dynamics, thereby, affecting the binding of inhibitor within its active site. The implications of mutation on structural characteristics were determined. The information provided in this manuscript holds great potential in further scientific research towards the design of potential vaccine candidates/small molecular inhibitor against COVID19.


Assuntos
Betacoronavirus/genética , Cisteína Endopeptidases/genética , Genoma Viral , Mutação , Proteínas do Nucleocapsídeo/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Betacoronavirus/classificação , Cisteína Endopeptidases/química , Variação Genética , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/química , Fosfoproteínas , Filogenia , Glicoproteína da Espícula de Coronavírus/química , Proteínas do Envelope Viral/química , Proteínas não Estruturais Virais/química
11.
Biochem Biophys Res Commun ; 532(1): 134-138, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-32829876

RESUMO

SARS-CoV-2 is the etiologic agent of COVID-19. There is currently no effective means of preventing infections by SARS-CoV-2, except through restriction of population movement and contact. An understanding of the origin, evolution and biochemistry (molecular biology) of SARS-CoV-2 is a prerequisite to its control. Mutations in the phosphorylation sites of SARS-CoV-2 encoded nucleocapsid protein isolated from various populations and locations, are described. Mutations occurred in the phosphorylation sites, all located within a stretch which forms a phosphorylation dependent interaction site, including C-TAK1 phosphorylation sites for 14-3-3. The consequences of these mutations are discussed and a structure-based model for the role of protein 14-3-3 in the sequestration and inhibition of SARS-CoV-2 nucleocapsid protein's function is presented. It is proposed that the phosphorylation of SARS-CoV-2 nucleocapsid protein and its sequestration by Protein 14-3-3 is a cellular response mechanism for the control and inhibition of the replication, transcription and packaging of the SARS-CoV-2 genome.


Assuntos
Proteínas 14-3-3/química , Betacoronavirus/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Proteínas do Nucleocapsídeo/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Sítios de Ligação , Infecções por Coronavirus/virologia , Expressão Gênica , Humanos , Simulação de Acoplamento Molecular , Mutação , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosfoproteínas , Fosforilação , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Termodinâmica
12.
Int J Mol Sci ; 21(16)2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32784770

RESUMO

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Humanos , Técnicas de Diagnóstico Molecular/normas , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/normas , Mucosa Respiratória/virologia , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genética
13.
EBioMedicine ; 59: 102951, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32818801

RESUMO

BACKGROUND: . The occurrence of trans-placental transmission of severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection remains highly debated. Placental positivity for SARS-CoV-2 has been reported in selected cases, but infection or virus-associated disease of fetal tissues or newborns remains to be demonstrated. METHODS: We screened for SARS-CoV-2 spike (S) protein expression placentas from 101 women who delivered between February 7 and May 15, 2020, including 15 tested positive for SARS-CoV-2 RNA, 34 tested negative, and 52 not evaluated as they did not meet testing criteria (32), or delivered before COVID-19 pandemic declaration (20). Immunostain for SARS-CoV-2 nucleocapsid (N) was performed in the placentas of all COVID-19 positive women. One placenta resulted positive for the SARS-CoV-2 S and N proteins, which was further studied by RNA-in situ hybridization and RT-PCR for S transcripts, and by electron microscopy. A comprehensive immunohistochemical and immunofluorescence analysis of the placental inflammatory infiltrate completed the investigations. FINDINGS: SARS-CoV-2 S and N proteins were strongly expressed in the placenta of a COVID-19 pregnant woman whose newborn tested positive for viral RNA and developed COVID-19 pneumonia soon after birth. SARS-CoV-2 antigens, RNA and/or particles morphologically consistent with coronavirus were identified in villous syncytiotrophoblast, endothelial cells, fibroblasts, in maternal macrophages, and in Hofbauer cells and fetal intravascular mononuclear cells. The placenta intervillous inflammatory infiltrate consisted of neutrophils and monocyte-macrophages expressing activation markers. Absence of villitis was associated with an increase in the number of Hofbauer cells, which expressed PD-L1. Scattered neutrophil extracellular traps (NETs) were identified by immunofluorescence. INTERPRETATION: We provide first-time evidence for maternal-fetal transmission of SARS-CoV-2, likely propagated by circulating virus-infected fetal mononuclear cells. Placenta infection was associated with recruitment of maternal inflammatory cells in the intervillous space, without villitis. PD-L1 expression in syncytiotrophoblast and Hofbaeur cells, together with limited production of NETs, may have prevented immune cell-driven placental damage, ensuring sufficient maternal-fetus nutrient exchanges.


Assuntos
Infecções por Coronavirus/transmissão , Placenta/virologia , Pneumonia Viral/transmissão , Adulto , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Recém-Nascido , Macrófagos/virologia , Microscopia Eletrônica , Nasofaringe/virologia , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosfoproteínas , Placenta/citologia , Placenta/patologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Gravidez , RNA Viral/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
15.
Virology ; 549: 1-4, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32758712

RESUMO

The current outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China firstly. A rapid, highly sensitive, specific, and simple operational method was needed for the detection of SARS-CoV-2. Here, we established a real-time reverse-transcription recombinase-aided amplification assay (RT-RAA) to detect SARS-CoV-2 rapidly. The primers and probe were designed based on the nucleocapsid protein gene (N gene) sequence of SARS-CoV-2. The detection limit was 10 copies per reaction in this assay, which could be conducted within 15 min at a constant temperature (39 °C), without any cross-reactions with other respiratory tract pathogens, such as other coronaviruses. Furthermore, compared with commercial real-time RT-PCR assay, it showed a kappa value of 0.959 (p < 0.001) from 150 clinical specimens. These results indicated that this real-time RT-RAA assay may be a valuable tool for detecting SARS-CoV-2.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Genes Virais , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas do Nucleocapsídeo/genética , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , China/epidemiologia , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Recombinases , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade
16.
PLoS One ; 15(8): e0237418, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790779

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. By the first week of July, more than 10,000,000 positive cases of COVID-19 have been reported worldwide, although this number could be greatly underestimated. In the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. Here, we demonstrate the use of the miniPCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization (WHO) for targeting three different regions that encode for the N protein. Prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a commercial 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of ~625 to 2×105 DNA copies. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited. The portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Betacoronavirus/química , Infecções por Coronavirus/virologia , DNA Viral/genética , Confiabilidade dos Dados , Humanos , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/economia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Mol Genet Genomics ; 295(6): 1501-1516, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32767127

RESUMO

Encapsidation by nucleocapsid (N) protein is crucial for viral RNA to serve as a functional template for virus replication. However, the potential region that is vital for RNA encapsidation of Nipah virus (NiV) is still unknown. Thus, this study was aimed to identify these regions using a NiV minireplicon system. A series of broad range internal deletion mutations was generated in the 5' non-translated region (NTR) of the N gene mRNA region of NiV leader promoter via site-directed overlapping PCR-mediated mutagenesis. The mutation effects on synthesis and encapsidation of antigenome RNA, transcription, and RNA binding affinity of N protein were evaluated. The deletions of nucleotides 73-108, 79-108, and 85-108 from NiV leader promoter inhibited the encapsidation of antigenome RNA, while the deletion of nucleotides 103-108 suppressed the synthesis and encapsidation of antigenome RNA, implying that these regions are required for genome replication. Surprisingly, none of the mutations had detrimental effect on viral transcription. Using isothermal titration calorimetry, the binding of NiV N protein to genome or antigenome RNA transcript lacking of nucleotides 73-108 was found to be suppressed. Additionally, in silico analysis on secondary structure of genome RNA further supported the plausible cause of inefficient encapsidation of antigenome RNA by the loss of encapsidation signal in genome template. In conclusion, this study suggests that the nucleotides 73-90 within 5' NTR of the N gene mRNA region in NiV leader promoter contain cis-acting RNA element that is important for efficient encapsidation of antigenome RNA.


Assuntos
Regulação Viral da Expressão Gênica , Vírus Nipah/genética , Regiões Promotoras Genéticas , RNA Viral , Montagem de Vírus , Regiões 5' não Traduzidas , Linhagem Celular , Mutagênese , Proteínas do Nucleocapsídeo/genética , RNA Mensageiro , RNA Viral/fisiologia , Proteínas Recombinantes/genética , Transcrição Genética
18.
Arch Virol ; 165(10): 2373-2377, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761270
19.
J Infect Dis ; 222(8): 1270-1279, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32726441

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China as the cause of coronavirus disease 2019 in December 2019 and reached Europe by late January 2020, when community-acquired respiratory viruses (CARVs) are at their annual peak. We validated the World Health Organization (WHO)-recommended SARS-CoV-2 assay and analyzed the epidemiology of SARS-CoV-2 and CARVs. METHODS: Nasopharyngeal/oropharyngeal swabs (NOPS) from 7663 patients were prospectively tested by the Basel S-gene and WHO-based E-gene (Roche) assays in parallel using the Basel N-gene assay for confirmation. CARVs were prospectively tested in 2394 NOPS by multiplex nucleic acid testing, including 1816 (75%) simultaneously for SARS-CoV-2. RESULTS: The Basel S-gene and Roche E-gene assays were concordant in 7475 cases (97.5%) including 825 (11%) SARS-CoV-2 positives. In 188 (2.5%) discordant cases, SARS-CoV-2 loads were significantly lower than in concordant positive ones and confirmed in 105 (1.4%). Adults were more frequently SARS-CoV-2 positive, whereas children tested more frequently CARV positive. CARV coinfections with SARS-CoV-2 occurred in 1.8%. SARS-CoV-2 replaced CARVs within 3 weeks, reaching 48% of all detected respiratory viruses followed by rhinovirus/enterovirus (13%), influenza virus (12%), coronavirus (9%), respiratory syncytial virus (6%), and metapneumovirus (6%). CONCLUSIONS: Winter CARVs were dominant during the early SARS-CoV-2 pandemic, impacting infection control and treatment decisions, but were rapidly replaced, suggesting competitive infection. We hypothesize that preexisting immune memory and innate immune interference contribute to the different SARS-CoV-2 epidemiology among adults and children.


Assuntos
Coinfecção/epidemiologia , Doenças Transmissíveis Emergentes/epidemiologia , Infecções por Coronavirus/epidemiologia , Pneumonia Viral/epidemiologia , Infecções Respiratórias/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Criança , Pré-Escolar , Técnicas de Laboratório Clínico , Coinfecção/imunologia , Coinfecção/virologia , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Infecções Respiratórias/virologia , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral , Organização Mundial da Saúde , Adulto Jovem
20.
Sci Transl Med ; 12(556)2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32719001

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Colorimetria/métodos , Colorimetria/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Humanos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , RNA-Seq , Sensibilidade e Especificidade , Pesquisa Médica Translacional
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