Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.111
Filtrar
1.
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 , RNA Replicase/genética , 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
2.
PLoS Pathog ; 16(9): e1008825, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886709

RESUMO

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other's templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses.


Assuntos
Alphavirus , Genoma Viral , Conformação de Ácido Nucleico , RNA Replicase , RNA Viral , Proteínas Virais , Alphavirus/química , Alphavirus/genética , Alphavirus/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , RNA Replicase/química , RNA Replicase/genética , RNA Replicase/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
3.
Int J Mol Sci ; 21(15)2020 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-32759818

RESUMO

The current COronaVIrus Disease 2019 (COVID-19) pandemic started in December 2019. COVID-19 cases are confirmed by the detection of SARS-CoV-2 RNA in biological samples by RT-qPCR. However, limited numbers of SARS-CoV-2 genomes were available when the first RT-qPCR methods were developed in January 2020 for initial in silico specificity evaluation and to verify whether the targeted loci are highly conserved. Now that more whole genome data have become available, we used the bioinformatics tool SCREENED and a total of 4755 publicly available SARS-CoV-2 genomes, downloaded at two different time points, to evaluate the specificity of 12 RT-qPCR tests (consisting of a total of 30 primers and probe sets) used for SARS-CoV-2 detection and the impact of the virus' genetic evolution on four of them. The exclusivity of these methods was also assessed using the human reference genome and 2624 closely related other respiratory viral genomes. The specificity of the assays was generally good and stable over time. An exception is the first method developed by the China Center for Disease Control and prevention (CDC), which exhibits three primer mismatches present in 358 SARS-CoV-2 genomes sequenced mainly in Europe from February 2020 onwards. The best results were obtained for the assay of Chan et al. (2020) targeting the gene coding for the spiking protein (S). This demonstrates that our user-friendly strategy can be used for a first in silico specificity evaluation of future RT-qPCR tests, as well as verifying that the former methods are still capable of detecting circulating SARS-CoV-2 variants.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Genoma Viral , Pneumonia Viral/diagnóstico , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Bases de Dados Genéticas , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , RNA Replicase/genética , RNA Viral/análise , Sensibilidade e Especificidade , Sequenciamento Completo do Genoma
4.
Euro Surveill ; 25(30)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32734855

RESUMO

We analysed consecutive RT-qPCR results of 537 symptomatic coronavirus disease (COVID-19) patients in home quarantine. Respectively 2, 3, and 4 weeks after symptom onset, 50%, 25% and 10% of patients had detectable RNA from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In patients with mild COVID-19, RNA detection is likely to outlast currently known periods of infectiousness by far and fixed time periods seem more appropriate in determining the length of home isolation than laboratory-based approaches.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Coronavirus/genética , Pandemias , Pneumonia Viral , RNA Replicase/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas não Estruturais Virais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Coronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Alemanha/epidemiologia , Humanos , Pessoa de Meia-Idade , Isolamento de Pacientes , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Quarentena , Análise de Sobrevida , Fatores de Tempo
5.
Arch Virol ; 165(10): 2401-2404, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757057

RESUMO

A novel mycovirus, named "Corynespora cassiicola bipartite mycovirus 1" (CcBV1), was isolated from a phytopathogenic fungus, Corynespora cassiicola, the causal agent of rubber leaf fall disease. The nucleotide sequence of the complete genome of CcBV1, which consists of two double-stranded RNA (dsRNA) segments, was determined. The first dsRNA is 2,002 bp in length and contains a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) (69 kDa), while the second is 1,738 bp in length and contains a single ORF encoding a hypothetical protein of unknown function, with an approximately molecular weight of 36 kDa. The amino acid sequences of the both deduced proteins are most similar (58.9% and 45.1% identity, respectively) to those of Cryphonectria parasitica bipartite mycovirus 1 (CpBV1). Phylogenetic analysis indicated that CcBV1 clusters together with CpBV1 and other unassigned dsRNA mycoviruses. To the best of our knowledge, this represents the first report of a mycovirus infecting C. cassiicola.


Assuntos
Ascomicetos/virologia , Micovírus/genética , Genoma Viral , Filogenia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Ascomicetos/patogenicidade , Sequência de Bases , China , Micovírus/classificação , Micovírus/isolamento & purificação , Fases de Leitura Aberta , Doenças das Plantas/microbiologia , Plantas/microbiologia , RNA Replicase/genética , Alinhamento de Sequência , Proteínas Virais/genética , Sequenciamento Completo do Genoma
6.
Arch Virol ; 165(10): 2405-2408, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32766954

RESUMO

The genome sequence of a mitovirus found in an isolate of Diaporthe rudis, one of the causal agents of Phomopsis dieback on grapevines, was determined by two high-throughput sequencing approaches, small RNA and total RNA sequencing. The genome of this mitovirus is 2,455 nt in length and includes a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). A BLASTx comparison of the full-length genome sequence showed the highest similarity (54.15%) with that of Colletotrichum falcatum mitovirus 1 (CfMV1). Our results reveal a new member of the genus Mitovirus first detected in D. rudis (Fr.) Nitschke, with the proposed name "Diaporthe rudis mitovirus 1" (DrMV1).


Assuntos
Micovírus/genética , Genoma Viral , Filogenia , RNA Replicase/genética , Saccharomycetales/virologia , Proteínas Virais/genética , Micovírus/classificação , Micovírus/isolamento & purificação , Expressão Gênica , Tamanho do Genoma , Fases de Leitura Aberta , Doenças das Plantas/microbiologia , Vitis/microbiologia , Sequenciamento Completo do Genoma
7.
Methods Mol Biol ; 2203: 41-53, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833202

RESUMO

Wild birds are natural hosts of multiple microbial agents, including a wide diversity of coronaviruses. Here we describe a pan-Coronavirus detection RT-PCR method to identify those viruses regardless of the coronavirus genus or nature of the specimen. We also describe a protocol using high-throughput sequencing technologies to obtain their entire genome, which overcomes the inherent difficulties of wild bird coronavirus sequencing, that is, their genetic diversity and the lack of virus isolation methods.


Assuntos
Doenças das Aves/virologia , Infecções por Coronavirus/veterinária , Coronavirus/genética , Coronavirus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Animais Selvagens , Infecções por Coronavirus/genética , RNA Replicase/genética , Manejo de Espécimes/métodos
8.
Euro Surveill ; 25(32)2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32794443

RESUMO

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Coronavirus/genética , Genoma Viral/genética , Pandemias , Pneumonia Viral/epidemiologia , RNA Replicase/genética , RNA Viral/análise , Sequência de Bases , Betacoronavirus/patogenicidade , Coronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Europa (Continente)/epidemiologia , Humanos , Filogeografia , Pneumonia Viral/virologia , RNA Viral/genética , Síndrome Respiratória Aguda Grave , Análise Espaço-Temporal , Organização Mundial da Saúde
9.
PLoS One ; 15(8): e0238253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857812

RESUMO

The origins of life on Earth have been the subject of inquiry since the early days of philosophical thought and are still intensively investigated by the researchers around the world. One of the theories explaining the life emergence, that gained the most attention recently is the RNA World hypothesis, which assumes that life on Earth was sparked by replicating RNA chains. Since wet lab analysis is time-consuming, many mathematical and computational approaches have been proposed that try to explain the origins of life. Recently proposed one, based on the work by Takeuchi and Hogeweg, addresses the problem of interplay between RNA replicases and RNA parasitic species, which is crucial for understanding the first steps of prebiotic evolution. In this paper, the aforementioned model has been extended and modified by introducing RNA sequence (structure) information and mutation rate close to real one. It allowed to observe the simple evolution mechanisms, which could have led to the more complicated systems and eventually, to the formation of the first cells. The main goal of this study was to determine the conditions that allowed the spontaneous emergence and evolution of the prebiotic replicases equipped with simple functional domains within a large population. Here we show that polymerase ribozymes could have appeared randomly and then quickly started to copy themselves in order for the system to reach equilibrium. It has been shown that evolutionary selection works even in the simplest systems.


Assuntos
Sequência de Bases , Simulação por Computador , Modelos Teóricos , Conformação de Ácido Nucleico , Origem da Vida , RNA , Algoritmos , Difusão , Hidrólise , Mutação , RNA/química , RNA Replicase/química , RNA Replicase/genética
10.
PLoS One ; 15(8): e0237162, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750098

RESUMO

Viral diversity is an important feature of hepatitis C virus (HCV) infection and an important predictor of disease progression and treatment response. HIV/HCV co-infection is associated with enhanced HCV replication, increased fibrosis, and the development of liver disease. HIV also increases quasispecies diversity of HCV structural genes, although limited data are available regarding the impact of HIV on non-structural genes of HCV, particularly in the absence of direct-acting therapies. The genetic diversity and presence of drug resistance mutations within the RNA-dependent RNA polymerase (NS5B) gene were examined in 3 groups of women with HCV genotype 1a infection, including those with HCV mono-infection, antiretroviral (ART)-naïve women with HIV/HCV co-infection and CD4 cell count <350 cells/mm3, and ART-naïve women with HIV/HCV co-infection and CD4 cell count ≥350 cells/mm3. None had ever been treated for HCV infection. There was evidence of significant diversity across the entire NS5B gene in all women. There were several nucleotides and amino acids with distinct distributions across the three study groups, although no obvious clustering of NS5B sequences was observed based on HIV co-infection or CD4 cell count. Polymorphisms at amino acid positions associated with resistance to dasabuvir and sofosbuvir were limited, although the Q309R variant associated with ribavirin resistance was present in 12 individuals with HCV mono-infection, 8 HIV/HCV co-infected individuals with CD4 <350 cells/mm3, and 12 HIV/HCV co-infected individuals with CD4 ≥350 cells/mm3. Previously reported fitness altering mutations were rare. CD8+ T cell responses against the human leukocyte antigen (HLA) B57-restricted epitopes NS5B2629-2637 and NS5B2936-2944 are critical for HCV control and were completely conserved in 44 (51.8%) and 70 (82.4%) study participants. These data demonstrate extensive variation across the NS5B gene. Genotypic variation may have a profound impact on HCV replication and pathogenesis and deserves careful evaluation.


Assuntos
Infecções Oportunistas Relacionadas com a AIDS/genética , Coinfecção/genética , Variação Genética , HIV , Hepacivirus/genética , Hepatite C/genética , Proteínas não Estruturais Virais/genética , Infecções Oportunistas Relacionadas com a AIDS/tratamento farmacológico , Infecções Oportunistas Relacionadas com a AIDS/virologia , Adulto , Sequência de Aminoácidos , Antivirais/uso terapêutico , Contagem de Linfócito CD4 , Estudos de Coortes , Coinfecção/tratamento farmacológico , Coinfecção/virologia , Farmacorresistência Viral/genética , Feminino , Genótipo , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Filogenia , RNA Replicase/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Sofosbuvir/uso terapêutico , Sulfonamidas/uso terapêutico , Uracila/análogos & derivados , Uracila/uso terapêutico
11.
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 , Pneumonia Viral/diagnóstico , RNA Replicase/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Mucosa Respiratória/virologia , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genética
12.
Viruses ; 12(6)2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32630601

RESUMO

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has highlighted the importance of reliable and rapid diagnostic testing to prevent and control virus circulation. Dozens of monoplex in-house RT-qPCR assays are already available; however, the development of dual-target assays is suited to avoid false-negative results caused by polymorphisms or point mutations, that can compromise the accuracy of diagnostic and screening tests. In this study, two mono-target assays recommended by WHO (E-Sarbeco (enveloppe gene, Charite University, Berlin, Germany) and RdRp-IP4 (RdRp, Institut Pasteur, Paris, France)) were selected and combined in a unique robust test; the resulting duo SARS-CoV-2 RT-qPCR assay was compared to the two parental monoplex tests. The duo SARS-CoV-2 assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. We demonstrated that combining two single systems into a dual-target assay (with or without an MS2-based internal control) did not impair performances, providing a potent tool adapted for routine molecular diagnosis in clinical microbiology laboratories.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Replicase/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , RNA Viral/análise , Sensibilidade e Especificidade , Organização Mundial da Saúde
13.
Interdiscip Sci ; 12(3): 335-348, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32617855

RESUMO

Most recently, an outbreak of severe pneumonia caused by the infection of SARS-CoV-2, a novel coronavirus first identified in Wuhan, China, imposes serious threats to public health. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Therefore, the role and inhibition of nsp12 are indispensable. A cryo-EM structure of RdRp from SARs-CoV-2 was used to identify novel drugs from Northern South African medicinal compounds database (NANPDB) by using computational virtual screening and molecular docking approaches. Considering Remdesivir as the control, 42 compounds were shortlisted to have docking score better than Remdesivir. The top 5 hits were validated by using molecular dynamics simulation approach and free energy calculations possess strong inhibitory properties than the Remdesivir. Thus, this study paved a way for designing novel drugs by decoding the architecture of an important enzyme and its inhibition with compounds from natural resources. This disclosing of necessary knowledge regarding the screening and the identification of top hits could help to design effective therapeutic candidates against the coronaviruses and design robust preventive measurements.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Produtos Biológicos/farmacologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Replicase/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Antivirais/química , Betacoronavirus/genética , Produtos Biológicos/química , Domínio Catalítico/genética , Simulação por Computador , Infecções por Coronavirus/epidemiologia , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos , Genoma Viral , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Humanos , Ligantes , Simulação de Acoplamento Molecular , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , RNA Replicase/química , RNA Replicase/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
14.
Nat Commun ; 11(1): 3656, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694517

RESUMO

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.


Assuntos
Proteínas Aviárias/ultraestrutura , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas Nucleares/ultraestrutura , Domínios Proteicos/genética , RNA Replicase/ultraestrutura , Proteínas de Ligação a RNA/ultraestrutura , Proteínas Virais/ultraestrutura , Animais , Proteínas Aviárias/metabolismo , Aves/virologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Influenza Aviária/virologia , Influenza Humana/virologia , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , RNA Replicase/genética , RNA Replicase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
15.
Arch Virol ; 165(10): 2389-2392, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32699979

RESUMO

A novel tobamovirus, brugmansia latent virus (BrLV), was discovered during a study of brugmansia (Brugmansia spp.) in the living collections held at the Royal Botanic Gardens, Kew. Here, we report the complete genome sequence of BrLV, which is 6,397 nucleotides long and contains the four open reading frames (RNA-dependent RNA polymerase, methyltransferase/helicase, movement, and coat proteins) typical of tobamoviruses. The complete genome sequence of BrLV shares 69.7% nucleotide sequence identity with brugmansia mild mottle virus (BrMMV) and 66.7 to 68.7% identity with other tobamoviruses naturally infecting members of the Solanaceae plant family. Phylogenetic analysis of the complete genome nucleotide sequence and the deduced amino acid sequences of the four tobamovirus proteins place BrLV in a subcluster with BrMMV within the Solanaceae-infecting tobamovirus subgroup as a new species.


Assuntos
Brugmansia/virologia , Proteínas do Capsídeo/genética , Genoma Viral , RNA Viral/genética , Tobamovirus/genética , Sequência de Bases , Sequência Conservada , Metiltransferases/genética , Fases de Leitura Aberta , Filogenia , Doenças das Plantas/virologia , RNA Replicase/genética , Alinhamento de Sequência , Tobamovirus/classificação , Tobamovirus/isolamento & purificação , Reino Unido , Sequenciamento Completo do Genoma
16.
Nat Commun ; 11(1): 3590, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681014

RESUMO

Bunyavirales is an order of segmented negative-strand RNA viruses comprising several life-threatening pathogens against which no effective treatment is currently available. Replication and transcription of the RNA genome constitute essential processes performed by the virally encoded multi-domain RNA-dependent RNA polymerase. Here, we describe the complete high-resolution cryo-EM structure of La Crosse virus polymerase. It reveals the presence of key protruding C-terminal domains, notably the cap-binding domain, which undergoes large movements related to its role in transcription initiation, and a zinc-binding domain that displays a fold not previously observed. We capture the polymerase structure at pre-initiation and elongation states, uncovering the coordinated movement of the priming loop, mid-thumb ring linker and lid domain required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. These structural details and the observed dynamics of key functional elements will be instrumental for structure-based development of polymerase inhibitors.


Assuntos
Vírus La Crosse/enzimologia , RNA Replicase/química , RNA Replicase/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Cristalografia por Raios X , Vírus La Crosse/química , Vírus La Crosse/genética , Conformação Proteica , Domínios Proteicos , RNA Replicase/genética , Transcrição Genética , Proteínas Virais/genética
17.
Nature ; 584(7821): 425-429, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32604404

RESUMO

On 21 February 2020, a resident of the municipality of Vo', a small town near Padua (Italy), died of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection1. This was the first coronavirus disease 19 (COVID-19)-related death detected in Italy since the detection of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. Here we collected information on the demography, clinical presentation, hospitalization, contact network and the presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo' at two consecutive time points. From the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI): 2.1-3.3%). From the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI: 0.8-1.8%). Notably, 42.5% (95% CI: 31.5-54.6%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic (that is, did not have symptoms at the time of swab testing and did not develop symptoms afterwards). The mean serial interval was 7.2 days (95% CI: 5.9-9.6). We found no statistically significant difference in the viral load of symptomatic versus asymptomatic infections (P = 0.62 and 0.74 for E and RdRp genes, respectively, exact Wilcoxon-Mann-Whitney test). This study sheds light on the frequency of asymptomatic SARS-CoV-2 infection, their infectivity (as measured by the viral load) and provides insights into its transmission dynamics and the efficacy of the implemented control measures.


Assuntos
Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Surtos de Doenças/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções Assintomáticas/epidemiologia , Betacoronavirus/enzimologia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Criança , Pré-Escolar , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Surtos de Doenças/estatística & dados numéricos , Feminino , Humanos , Lactente , Recém-Nascido , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , Prevalência , RNA Replicase/genética , Proteínas do Envelope Viral/genética , Carga Viral , Proteínas não Estruturais Virais/genética , Adulto Jovem
18.
Sci Rep ; 10(1): 9294, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: covidwho-592060

RESUMO

As of today, there is no antiviral for the treatment of the SARS-CoV-2 infection, and the development of a vaccine might take several months or even years. The structural superposition of the hepatitis C virus polymerase bound to sofosbuvir, a nucleoside analog antiviral approved for hepatitis C virus infections, with the SARS-CoV polymerase shows that the residues that bind to the drug are present in the latter. Moreover, a multiple alignment of several SARS-CoV-2, SARS and MERS-related coronaviruses polymerases shows that these residues are conserved in all these viruses, opening the possibility to use sofosbuvir against these highly infectious pathogens.


Assuntos
Antivirais/química , Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Pandemias/prevenção & controle , Pneumonia Viral/virologia , RNA Replicase/química , Sofosbuvir/química , Proteínas não Estruturais Virais/química , Antivirais/uso terapêutico , Sequência de Bases , Domínio Catalítico , Simulação por Computador , Infecções por Coronavirus/tratamento farmacológico , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Pneumonia Viral/tratamento farmacológico , Ligação Proteica , Estrutura Terciária de Proteína , RNA Replicase/genética , Vírus da SARS/enzimologia , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/virologia , Sofosbuvir/uso terapêutico , Proteínas não Estruturais Virais/genética
19.
Sci Rep ; 10(1): 9294, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518317

RESUMO

As of today, there is no antiviral for the treatment of the SARS-CoV-2 infection, and the development of a vaccine might take several months or even years. The structural superposition of the hepatitis C virus polymerase bound to sofosbuvir, a nucleoside analog antiviral approved for hepatitis C virus infections, with the SARS-CoV polymerase shows that the residues that bind to the drug are present in the latter. Moreover, a multiple alignment of several SARS-CoV-2, SARS and MERS-related coronaviruses polymerases shows that these residues are conserved in all these viruses, opening the possibility to use sofosbuvir against these highly infectious pathogens.


Assuntos
Antivirais/química , Betacoronavirus/enzimologia , Infecções por Coronavirus/virologia , Pandemias/prevenção & controle , Pneumonia Viral/virologia , RNA Replicase/química , Sofosbuvir/química , Proteínas não Estruturais Virais/química , Antivirais/uso terapêutico , Sequência de Bases , Domínio Catalítico , Simulação por Computador , Infecções por Coronavirus/tratamento farmacológico , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Pneumonia Viral/tratamento farmacológico , Ligação Proteica , Estrutura Terciária de Proteína , RNA Replicase/genética , Vírus da SARS/enzimologia , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/virologia , Sofosbuvir/uso terapêutico , Proteínas não Estruturais Virais/genética
20.
Arch Virol ; 165(8): 1911-1914, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32488617

RESUMO

To our knowledge, there have been no reports of mycoviruses infecting Rhodosporidiobolus odoratus. Here, we describe the sequence of a novel mycovirus isolated from R. odoratus, which was designated "Rhodosporidiobolus odoratus RNA virus 1" (RoRV1). Sequence analysis revealed that RoRV1 has two discontinuous open reading frames (ORFs), ORF1 and ORF2, potentially encoding a hypothetical protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on RdRp sequences clearly placed RoRV1 in the genus Totivirus, family Totiviridae. The fungus also contains two additional, smaller dsRNAs, which might represent RoRV1 satellite RNAs.


Assuntos
Fungos/virologia , Vírus de RNA/genética , Totivirus/genética , Totivirus/isolamento & purificação , Proteínas do Capsídeo/genética , Genoma Viral/genética , Fases de Leitura Aberta/genética , Filogenia , RNA Replicase/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , Análise de Sequência de DNA/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA