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1.
Diagn Microbiol Infect Dis ; 99(1): 115206, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33045499

RESUMO

The diagnosis of coronavirus disease-19 (COVID-19) relies on the detection of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA by real-time reverse-transcription polymerase chain reaction in respiratory samples. Rapid increase in the COVID-19 cases across the world requires fast and efficient testing as testing capacity is a bottleneck in diagnosis. In this context, pooling strategy can be opted for rapid testing in a cost-effective manner. In this study, the authors have optimized and compared the effect of pooling (5 and 10 samples) before and after nucleic acid extraction. It was concluded that there was no significant difference in the SARS CoV-2 RNA detection in the pools prepared at sample or RNA level. Even after pooling, 10-fold dilution was detectable with 3-cycle threshold value change in both type of pools when compared with individual samples. Hence, sample pool size of 10 can be used in low-prevalent areas, and testing capacity can be substantially increased.


Assuntos
/métodos , /isolamento & purificação , Manejo de Espécimes/métodos , /normas , Genes Virais/genética , Humanos , Índia/epidemiologia , Nasofaringe/virologia , Faringe/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Manejo de Espécimes/normas , Centros de Atenção Terciária
2.
J Virol Methods ; 287: 114008, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33160015

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.


Assuntos
/métodos , RNA Viral/isolamento & purificação , /isolamento & purificação , Testes Diagnósticos de Rotina , Genes Virais/genética , Humanos , Limite de Detecção , Faringe/virologia , RNA Viral/análise , RNA Viral/genética , Reprodutibilidade dos Testes , /genética
3.
PLoS Genet ; 16(12): e1009272, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33332358

RESUMO

The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.


Assuntos
Betacoronavirus/classificação , Betacoronavirus/genética , Recombinação Genética/genética , Glicoproteína da Espícula de Coronavírus/genética , Troca Genética/genética , Genes Virais/genética , Genoma Viral/genética , Especificidade de Hospedeiro/genética , Modelos Genéticos , Polimorfismo Genético , /genética , Tropismo Viral/genética
4.
Sci Rep ; 10(1): 22214, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33335187

RESUMO

Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test's reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.


Assuntos
/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , /genética , Austrália , Genes Virais/genética , Humanos , Mutação/genética , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
5.
Nat Commun ; 11(1): 6009, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33243984

RESUMO

Leviviruses are bacteriophages with small single-stranded RNA genomes consisting of 3-4 genes, one of which (sgl) encodes a protein that induces the host to undergo autolysis and liberate progeny virions. Recent meta-transcriptomic studies have uncovered thousands of leviviral genomes, but most of these lack an annotated sgl, mainly due to the small size, lack of sequence similarity, and embedded nature of these genes. Here, we identify sgl genes in 244 leviviral genomes and functionally characterize them in Escherichia coli. We show that leviviruses readily evolve sgl genes and sometimes have more than one per genome. Moreover, these genes share little to no similarity with each other or to previously known sgl genes, thus representing a rich source for potential protein antibiotics.


Assuntos
Bacteriólise/genética , Evolução Molecular , Genes Virais/genética , Levivirus/genética , Proteínas Virais/metabolismo , Escherichia coli/virologia , Levivirus/patogenicidade , Mutagênese Sítio-Dirigida , Mutação , RNA Viral/genética , Proteínas Virais/genética
6.
Proc Natl Acad Sci U S A ; 117(49): 31519-31526, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33203681

RESUMO

Genome-wide epistasis analysis is a powerful tool to infer gene interactions, which can guide drug and vaccine development and lead to deeper understanding of microbial pathogenesis. We have considered all complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes deposited in the Global Initiative on Sharing All Influenza Data (GISAID) repository until four different cutoff dates, and used direct coupling analysis together with an assumption of quasi-linkage equilibrium to infer epistatic contributions to fitness from polymorphic loci. We find eight interactions, of which three are between pairs where one locus lies in gene ORF3a, both loci holding nonsynonymous mutations. We also find interactions between two loci in gene nsp13, both holding nonsynonymous mutations, and four interactions involving one locus holding a synonymous mutation. Altogether, we infer interactions between loci in viral genes ORF3a and nsp2, nsp12, and nsp6, between ORF8 and nsp4, and between loci in genes nsp2, nsp13, and nsp14. The paper opens the prospect to use prominent epistatically linked pairs as a starting point to search for combinatorial weaknesses of recombinant viral pathogens.


Assuntos
Epistasia Genética/genética , Genes Virais/genética , /genética , /patologia , /genética , Exorribonucleases/genética , Genoma Viral/genética , Humanos , Metiltransferases/genética , RNA Helicases/genética , Seleção Genética/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , /genética
7.
ACS Chem Neurosci ; 11(20): 3180-3184, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-32997488

RESUMO

After several months of rapid pandemic expansion, it is now apparent that the SARS-CoV-2 coronavirus interferes with smell and taste sensation in a substantial proportion of COVID-19 patients. Recent epidemiological data documented intriguing differences in prevalence of chemosensory dysfunctions between different world regions. Viral genetic factors as well as host genetic factors appear to be relevant; however, it is not yet known which mutations or polymorphisms actually contribute to such phenotypic differences between populations. Here, we discuss recent genetic and epidemiological data on the D614G spike protein variant and assess whether current evidence is consistent with the notion that this single nucleotide polymorphism augments chemosensory impairments in COVID-19 patients. We hypothesize that this spike variant is an important viral genetic factor that facilitates infection of chemosensory epithelia, possibly acting together with yet to be identified host factors, and thereby increases smell and taste impairment. We suggest that the prevalence of chemosensory deficits may reflect the pandemic potential for transmissibility and spread which differs between populations.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/genética , Transtornos do Olfato/virologia , Pneumonia Viral/genética , Glicoproteína da Espícula de Coronavírus/genética , Distúrbios do Paladar/virologia , Infecções por Coronavirus/complicações , Genes Virais/genética , Humanos , Transtornos do Olfato/genética , Pandemias , Pneumonia Viral/complicações , Polimorfismo de Nucleotídeo Único , Distúrbios do Paladar/genética
8.
J Virol ; 94(22)2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-32878889

RESUMO

Oryctes rhinoceros nudivirus (OrNV) is a double-stranded DNA (dsDNA) virus which has been used as a biocontrol agent to suppress the coconut rhinoceros beetle (Oryctes rhinoceros) in Southeast Asia and the Pacific Islands. A new wave of O. rhinoceros incursions in Oceania is thought to be related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles. In this study, chronically infected beetles were collected from Philippines, Fiji, Papua New Guinea (PNG), and the Solomon Islands (SI). RNA sequencing (RNA-seq) was performed to investigate the global viral gene expression profiles and for comparative genomic analysis of structural variations. Maximum likelihood phylogenic analysis indicated that OrNV strains from the SI and Philippines are closely related, while OrNV strains from PNG and Fiji formed a distinct adjacent clade. We detected several polymorphic sites with a frequency higher than 35% in 892 positions of the viral genome. Nonsynonymous mutations were detected in several hypothetical proteins and 15 nudivirus core genes, such as gp034, lef-8, lef-4, and vp91 We found limited evidence of variation in viral gene expression among geographic populations. Only a few genes, such as gp01, gp022, and gp107, were differentially expressed among different strains. Additionally, small RNA sequencing from the SI population suggested that OrNV is targeted by the host RNA interference (RNAi) response with abundant 21-nucleotide small RNAs. Some of these genomic changes are specific to the geographic population and could be related to particular phenotypic characteristics of the strain, such as viral pathogenicity or transmissibility, and this requires further investigation.IMPORTANCE Oryctes rhinoceros nudivirus has been an effective biocontrol agent against the coconut rhinoceros beetle in Southeast Asia and the Pacific Islands for decades. The recent outbreak of these beetles in many South Pacific islands has had a significant impact on livelihoods in the region. It has been suggested that the resurgence and spread of the pest are related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles. We examined viral genomic and transcriptional variations in chronically infected beetles from different geographical populations. A high number of polymorphic sites among several geographical strains of OrNV were identified, but potentially only a few of these variations in the genome are involved in functional changes and can potentially alter the typical function. These findings provide valuable resources for future studies to improve our understanding of the OrNV genetic variations in different geographic regions and their potential link to virus pathogenicity.


Assuntos
Besouros/virologia , Genoma Viral , Genômica , Nudiviridae/genética , Animais , Cocos , DNA , Vírus de DNA/genética , Feminino , Regulação Viral da Expressão Gênica , Genes Virais/genética , Haplótipos , MicroRNAs , Oceania , Perissodáctilos , Filogenia , Interferência de RNA , Análise de Sequência de RNA
9.
Nat Commun ; 11(1): 4894, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994400

RESUMO

Identification of the complete set of translated genes of viruses is important to understand viral replication and pathogenesis as well as for therapeutic approaches to control viral infection. Here, we use chemical proteomics, integrating bio-orthogonal non-canonical amino acid tagging and high-resolution mass spectrometry, to characterize the newly synthesized herpes simplex virus 1 (HSV-1) proteome in infected cells. In these infected cells, host cellular protein synthesis is shut-off, increasing the chance to preferentially detect viral proteomes. We identify nine previously cryptic orphan protein coding sequences whose translated products are expressed in HSV-1-infected cells. Functional characterization of one identified protein, designated piUL49, shows that it is critical for HSV-1 neurovirulence in vivo by regulating the activity of virally encoded dUTPase, a key enzyme that maintains accurate DNA replication. Our results demonstrate that cryptic orphan protein coding genes of HSV-1, and probably other large DNA viruses, remain to be identified.


Assuntos
Encefalite por Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Pirofosfatases/metabolismo , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Encéfalo/patologia , Encéfalo/virologia , Chlorocebus aethiops , Replicação do DNA , Modelos Animais de Doenças , Encefalite por Herpes Simples/patologia , Feminino , Genes Virais/genética , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Camundongos , Biossíntese de Proteínas , Proteômica/métodos , Células Vero , Proteínas Virais/genética , Fatores de Virulência/genética , Replicação Viral
10.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
11.
PLoS Negl Trop Dis ; 14(8): e0008574, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853274

RESUMO

BACKGROUND: Shifts have occurred in the epidemiological characteristics of Japanese encephalitis (JE), extending from the molecular level to the population level. The aim of this study was to investigate the seroprevalence of JE neutralizing antibodies in healthy populations from different age groups in Zhejiang Province, and to conduct mosquito monitoring to evaluate the infection rate of Japanese encephalitis virus (JEV) among vectors, as well as the molecular characteristics of the E gene of isolated JEV strains. METHODOLOGY/PRINCIPAL FINDINGS: A total of 1190 sera samples were screened by a microseroneutralization test, including 429 infants (28d-11m) and 761 participants (2y-82y). For those under 1 year old, the geometric mean titers (GMTs) of the JE neutralizing antibody was 9.49 at birth and significantly declined as the age of month increased (r = -0.225, P<0.001). For those above 1-year old, seropositive proportions were higher in subjects aged 1-3 years old as well as ≥25 years old (65%-75%), and relatively lower in subjects aged between 4-25 years old (22%-55%). Four or more years after the 2nd dose of JEV-L (first dose administered at 8 months and the second at 2 years of age), the seropositive proportion decreased to 32.5%, and GMTs decreased to 8.08. A total of 87,201 mosquitoes were collected from livestock sheds in 6 surveillance sites during 2015-2018, from which 139 E gene sequences were successfully amplified. The annual infection rate according to bias-corrected maximum likelihood estimation of JEV in Culex tritaeniorhynchus was 1.56, 2.36, 5.65 and 1.77 per 1000, respectively. JEV strains isolated during 2015-2018 all belonged to Genotype I. The E gene of amplified 139 samples differed from the JEV-L vaccine strain at fourteen amino acid residues, including the eight key residues related to virulence and virus attenuation. No divergence was observed at the sites related to antigenicity. CONCLUSIONS/SIGNIFICANCE: Zhejiang Province was at a high risk of JE exposure due to relatively lower neutralizing antibody levels among the younger-aged population and higher infection rates of JEV in mosquitoes. Continuous, timely and full coverage of JE vaccination are essential, as well as the separation of human living areas and livestock shed areas. In addition, annual mosquito surveillance and periodic antibody level monitoring are important for providing evidence for improvement in JE vaccines and immunization schedules.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/epidemiologia , Encefalite Japonesa/virologia , Epidemiologia Molecular , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Neutralizantes , Criança , Pré-Escolar , China/epidemiologia , Estudos Transversais , Culex/virologia , Culicidae/virologia , Vírus da Encefalite Japonesa (Espécie)/classificação , Vírus da Encefalite Japonesa (Espécie)/imunologia , Vírus da Encefalite Japonesa (Espécie)/isolamento & purificação , Genes Virais/genética , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Mosquitos Vetores/virologia , Estudos Soroepidemiológicos , Adulto Jovem
12.
PLoS Pathog ; 16(8): e1008779, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853289

RESUMO

The pandemic spread of African swine fever virus (ASFV) genotype II (GTII) has led to a global crisis. Since the circulating strains are almost identical, time and money have been mis-invested in whole-genome sequencing the last years. New methods, harmonised protocols for sample selection, sequencing, and bioinformatics are therefore urgently needed.


Assuntos
Vírus da Febre Suína Africana/classificação , Vírus da Febre Suína Africana/genética , Febre Suína Africana/diagnóstico , Genes Virais/genética , Variação Genética , Genoma Viral , Sequenciamento Completo do Genoma/métodos , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/isolamento & purificação , Animais , Biologia Computacional/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Filogenia , Controle de Qualidade , Suínos
13.
PLoS Pathog ; 16(8): e1008705, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853291

RESUMO

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.


Assuntos
Betacoronavirus/genética , Sistemas CRISPR-Cas/genética , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/genética , Pneumonia Viral/diagnóstico , Pneumonia Viral/genética , Bactérias/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genes Virais/genética , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade
14.
Virol J ; 17(1): 86, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32605577

RESUMO

The need for timely establishment of a complete diagnostic protocol of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is demanded worldwide. We selected 15 positive novel coronavirus disease 19 (COVID-19) patients with mild or no symptom. Initially, fecal samples were negative in the 67% (10/15) of the cases, while 33% (5/10) of the cases were positive. After serial virus RNA testing, 73% (11/15) of the cases resulted positive to fecal specimens. In particular, 15 days after the first positive respiratory specimens test, 6 fecal specimens became positive for SARS-CoV-2 RNA, while 13 respiratory test returned negative result. In conclusion, qRT-PCR assays of fecal specimens, is an important step to control infection, suggesting that samples remained positive for SARS-CoV-2 RNA longer time then respiratory tract samples. Our results enhance the recent knowledge on this emerging infectious disease and offer suggestions for a more complete diagnostic strategy.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Fezes/virologia , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Feminino , Genes Virais/genética , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Sistema Respiratório/virologia , Fatores de Tempo , Eliminação de Partículas Virais
15.
Drug Dev Ind Pharm ; 46(8): 1345-1353, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32643448

RESUMO

PURPOSE: Huashi Baidu formula (HSBDF) was developed to treat the patients with severe COVID-19 in China. The purpose of this study was to explore its active compounds and demonstrate its mechanisms against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through network pharmacology and molecular docking. METHODS: All the components of HSBDF were retrieved from the pharmacology database of TCM system. The genes corresponding to the targets were retrieved using UniProt and GeneCards database. The herb-compound-target network was constructed by Cytoscape. The target protein-protein interaction network was built using STRING database. The core targets of HSBDF were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The main active compounds of HSBDF were docked with SARS-CoV-2 and angiotensin converting enzyme II (ACE2). RESULTS: Compound-target network mainly contained 178 compounds and 272 corresponding targets. Key targets contained MAPK3, MAPK8, TP53, CASP3, IL6, TNF, MAPK1, CCL2, PTGS2, etc. There were 522 GO items in GO enrichment analysis (p < .05) and 168 signaling pathways (p < .05) in KEGG, mainly including TNF signaling pathway, PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, and HIF-1 signaling pathway. The results of molecular docking showed that baicalein and quercetin were the top two compounds of HSBDF, which had high affinity with ACE2. CONCLUSION: Baicalein and quercetin in HSBDF may regulate multiple signaling pathways through ACE2, which might play a therapeutic role on COVID-19.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Simulação de Acoplamento Molecular/métodos , Farmacologia Clínica/métodos , Pneumonia Viral/tratamento farmacológico , Betacoronavirus/química , Betacoronavirus/genética , China , Bases de Dados Factuais , Ontologia Genética , Marcação de Genes , Genes Virais/efeitos dos fármacos , Genes Virais/genética , Humanos , Medicina Tradicional Chinesa , Pandemias , Peptidil Dipeptidase A/efeitos dos fármacos , Peptidil Dipeptidase A/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
16.
Arch Virol ; 165(8): 1827-1835, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32507978

RESUMO

Human cytomegalovirus (HCMV) infection causes high morbidity and mortality among immunocompromised patients and can remain in a latent state in host cells. Expression of the immediate-early (IE) genes sustains HCMV replication and reactivation. As a novel genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been extensively utilized to modify and edit genomic DNA. In the present study, the CRISPR/Cas9 system was used to target the IE region of the HCMV genome via specific single-guide RNAs (sgRNAs). Infection with CRISPR/Cas9/sgRNA lentiviral constructs significantly reduced viral gene expression and virion production in HFF primary fibroblasts and inhibited viral DNA production and reactivation in the THP-1 monocytic cell line. Thus, the CRISPR/Cas9/sgRNA system can accurately and efficiently target HCMV replication and reactivation and represents a novel therapeutic strategy against latent HCMV infection.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citomegalovirus/genética , Endonucleases/genética , Genes Virais/genética , Replicação Viral/genética , Linhagem Celular , Infecções por Citomegalovirus/virologia , DNA Viral/genética , Fibroblastos/virologia , Edição de Genes/métodos , Expressão Gênica/genética , Células HEK293 , Humanos , RNA Guia/genética , Células THP-1
17.
Environ Microbiol ; 22(6): 2001-2006, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32367648

RESUMO

The origin of the SARS-CoV-2 virus remains enigmatic. It is likely to be a continuum resulting from inevitable mutations and recombination events. These genetic changes keep developing in the present epidemic. Mutations tending to deplete the genome in its cytosine content will progressively lead to attenuation as a consequence of Muller's ratchet, but this is counteracted by recombination when different mutants co-infect the same host, in particular, in clusters of infection. Monitoring as a function of time the genome sequences in closely related cases is critical to anticipate the future of SARS-CoV-2 and hence of COVID-19.


Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Sequência de Bases , Betacoronavirus/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Evolução Molecular , Genes Virais/genética , Humanos , Mutação , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Recombinação Genética , Vacinas Virais/imunologia
18.
Sci Rep ; 10(1): 5928, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32246084

RESUMO

Vermamoeba vermiformis is a predominant free-living amoeba in human environments and amongst the most common amoebae that can cause severe infections in humans. It is a niche for numerous amoeba-resisting microorganisms such as bacteria and giant viruses. Differences in the susceptibility to these giant viruses have been observed. V. vermiformis and amoeba-resisting microorganisms share a sympatric lifestyle that can promote exchanges of genetic material. This work analyzed the first draft genome sequence of a V. vermiformis strain (CDC-19) through comparative genomic, transcriptomic and phylogenetic analyses. The genome of V. vermiformis is 59.5 megabase pairs in size, and 22,483 genes were predicted. A high proportion (10% (n = 2,295)) of putative genes encoded proteins showed the highest sequence homology with a bacterial sequence. The expression of these genes was demonstrated for some bacterial homologous genes. In addition, for 30 genes, we detected best BLAST hits with members of the Candidate Phyla Radiation. Moreover, 185 genes (0.8%) best matched with giant viruses, mostly those related to the subfamily Klosneuvirinae (101 genes), in particular Bodo saltans virus (69 genes). Lateral sequence transfers between V. vermiformis and amoeba-resisting microorganisms were strengthened by Sanger sequencing, transcriptomic and phylogenetic analyses. This work provides important insights and genetic data for further studies about this amoeba and its interactions with microorganisms.


Assuntos
Amoeba/genética , Bactérias/genética , Transferência Genética Horizontal , Genoma de Protozoário/genética , Vírus Gigantes/genética , Amoeba/classificação , Amoeba/microbiologia , Sequência de Bases/genética , Código de Barras de DNA Taxonômico , Resistência à Doença/genética , Genes Bacterianos/genética , Genes Virais/genética , Humanos , Filogenia , Homologia de Sequência
20.
Proc Natl Acad Sci U S A ; 117(11): 5987-5996, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123111

RESUMO

Endogenous retroviruses and long terminal repeat (LTR) retrotransposons are mobile genetic elements that are closely related to retroviruses. Desilenced endogenous retroviruses are associated with human autoimmune disorders and neurodegenerative diseases. Caenorhabditis elegans and related Caenorhabditis spp. contain LTR retrotransposons and, as described here, numerous integrated viral genes including viral envelope genes that are part of LTR retrotransposons. We found that both LTR retrotransposons and endogenous viral elements are silenced by ADARs [adenosine deaminases acting on double-stranded RNA (dsRNA)] together with the endogenous RNA interference (RNAi) factor ERI-6/7, a homolog of MOV10 helicase, a retrotransposon and retrovirus restriction factor in human. siRNAs corresponding to integrated viral genes and LTR retrotransposons, but not to DNA transposons, are dependent on the ADARs and ERI-6/7. siRNAs corresponding to palindromic repeats are independent of the ADARs and ERI-6/7, and are in fact increased in adar- and eri-6/7-defective mutants because of an antiviral RNAi response to dsRNA. Silencing of LTR retrotransposons is dependent on downstream RNAi factors and P granule components but is independent of the viral sensor DRH-1/RIG-I and the nuclear Argonaute NRDE-3. The activation of retrotransposons in the ADAR- and ERI-6/7/MOV10-defective mutant is associated with the induction of the unfolded protein response (UPR), a common response to viral infection. The overlap between genes induced upon viral infection and infection with intracellular pathogens and genes coexpressed with retrotransposons suggests that there is a common response to different types of foreign elements that includes a response to proteotoxicity presumably caused by the burden of replicating pathogens and expressed retrotransposons.


Assuntos
Caenorhabditis elegans/genética , Retrovirus Endógenos/genética , Interações entre Hospedeiro e Microrganismos/genética , Interferência de RNA , Retroelementos/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Caenorhabditis elegans/virologia , Proteínas de Caenorhabditis elegans/metabolismo , DNA Helicases/metabolismo , DNA Viral/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação Viral da Expressão Gênica , Genes Virais/genética , Humanos , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Homologia de Sequência de Aminoácidos , Sequências Repetidas Terminais/genética , Resposta a Proteínas não Dobradas/genética
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