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1.
PLoS Pathog ; 16(8): e1008845, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866210

RESUMO

Modified vaccinia virus Ankara (MVA) is an approved smallpox vaccine and a promising vaccine vector for other pathogens as well as for cancer therapeutics with more than 200 current or completed clinical trials. MVA was derived by passaging the parental Ankara vaccine virus hundreds of times in chick embryo fibroblasts during which it lost the ability to replicate in human and most other mammalian cells. Although this replication deficiency is an important safety feature, the genetic basis of the host restriction is not understood. Here, an unbiased human genome-wide RNAi screen in human A549 cells revealed that the zinc-finger antiviral protein (ZAP), previously shown to inhibit certain RNA viruses, is a host restriction factor for MVA, a DNA virus. Additional studies demonstrated enhanced MVA replication in several human cell lines following knockdown of ZAP. Furthermore, CRISPR-Cas9 knockout of ZAP in human A549 cells increased MVA replication and spread by more than one log but had no effect on a non-attenuated strain of vaccinia virus. The intact viral C16 protein, which had been disrupted in MVA, antagonized ZAP by binding and sequestering the protein in cytoplasmic punctate structures. Studies aimed at exploring the mechanism by which ZAP restricts MVA replication in the absence of C16 showed that knockout of ZAP had no discernible effect on viral DNA or individual mRNA or protein species as determined by droplet digital polymerase chain reaction, deep RNA sequencing and mass spectrometry, respectively. Instead, inactivation of ZAP reduced the number of aberrant, dense, spherical particles that typically form in MVA-infected human cells, suggesting that ZAP has a novel role in interfering with a late step in the assembly of infectious MVA virions in the absence of the C16 protein.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Vírus Vaccinia/fisiologia , Replicação Viral/fisiologia , Células A549 , Animais , Galinhas , Citoplasma/metabolismo , Citoplasma/virologia , DNA Viral/genética , DNA Viral/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Proteínas Repressoras/genética
2.
PLoS One ; 15(9): e0238614, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32936826

RESUMO

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions.


Assuntos
Betacoronavirus/isolamento & purificação , Pandemias , Cultura de Vírus/métodos , Animais , Austrália , Betacoronavirus/genética , Betacoronavirus/fisiologia , Canadá , Linhagem Celular , Chlorocebus aethiops , Busca de Comunicante , Infecções por Coronavirus , Efeito Citopatogênico Viral , DNA Complementar/genética , DNA Viral/genética , Inglaterra , Genoma Viral , Células HeLa , Humanos , Irã (Geográfico) , Kuweit , Macaca mulatta , Nasofaringe/virologia , Filogenia , Pneumonia Viral , Análise de Sequência de DNA , Viagem , Turquia/epidemiologia , Células Vero , Replicação Viral
3.
Nat Commun ; 11(1): 4506, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908149

RESUMO

Bacteriophages play critical roles in the biosphere, but their vast genomic diversity has obscured their evolutionary origins, and phylogenetic analyses have traditionally been hindered by their lack of universal phylogenetic marker genes. In this study we mine metagenomic data and identify a clade of Caudovirales that encodes the ß and ß' subunits of multi-subunit RNA polymerase (RNAP), a high-resolution phylogenetic marker which enables detailed evolutionary analyses. Our RNAP phylogeny revealed that the Caudovirales RNAP forms a clade distinct from cellular homologs, suggesting an ancient acquisition of this enzyme. Within these multimeric RNAP-encoding Caudovirales (mReC), we find that the similarity of major capsid proteins and terminase large subunits further suggests they form a distinct clade with common evolutionary origin. Our study characterizes a clade of RNAP-encoding Caudovirales and suggests the ancient origin of this enzyme in this group, underscoring the important role of viruses in the early evolution of life on Earth.


Assuntos
Evolução Biológica , Caudovirales/genética , RNA Polimerases Dirigidas por DNA/genética , Subunidades Proteicas/genética , Proteínas Virais/genética , DNA Viral/genética , Conjuntos de Dados como Assunto , Transferência Genética Horizontal , Metagenômica , Filogenia , Análise de Sequência de DNA
4.
Nat Commun ; 11(1): 4884, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985507

RESUMO

Gene drives are genetic modifications designed to propagate in a population with high efficiency. Current gene drive strategies rely on sexual reproduction and are thought to be restricted to sexual organisms. Here, we report on a gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently target and replace wildtype populations in cell culture experiments. Moreover, by targeting sequences necessary for viral replication, our results indicate that a viral gene drive can be used as a strategy to suppress a viral infection. Taken together, this work offers a proof of principle for the design of a gene drive in viruses.


Assuntos
Tecnologia de Impulso Genético/métodos , Herpesviridae/genética , Linhagem Celular , Citomegalovirus/genética , Citomegalovirus/fisiologia , DNA Viral/genética , Edição de Genes , Herpesviridae/fisiologia , Humanos , Replicação Viral
5.
PLoS One ; 15(9): e0234532, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32991587

RESUMO

This article describes the isolation, molecular characterization, and genotyping of two fowl adenovirus (FAdVs) strains with GenBank Accession numbers (MT478054, JSN-G033-18-L and MT478055, JSN-G033-18-B) obtained from the internal organs of black grouse (Lyrurus tetrix). This study also reveals the first confirmation of fowl adenovirus in Poland, supporting one of the hypotheses about the probability of fowl adenovirus interspecies transmission. The adenovirus strain sequences were investigated via phylogenetic analysis and were found to have an overall mean pairwise distance of 2.189. The heterogeneity, Relative Synonymous Codon Usage (RSCU), codon composition, and nucleotide frequencies were examined. Statistical analyses and Tajima's test for the examined sequences were carried out. The Maximum Likelihood for the examined sequences substitutions was performed. The results of the sequence analysis identified MT478054, JSN-G033-18-L and MT478055, JSN-G033-18-B as strains of fowl adenovirus 2/11/D, with the Fowl adenovirus D complete sequence showing a 93% match. Wild birds may act as a natural reservoir for FAdVs and likely play an important role in the spreading of these viruses in the environment. The findings reported here suggest horizontal transmission within and between avian species.


Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/isolamento & purificação , Galliformes/virologia , Doenças das Aves Domésticas/virologia , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/classificação , Aviadenovirus/genética , Uso do Códon , DNA Viral/genética , Filogenia , Polônia
6.
Biosens Bioelectron ; 166: 112436, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32750677

RESUMO

Our recent experience of the COVID-19 pandemic has highlighted the importance of easy-to-use, quick, cheap, sensitive and selective detection of virus pathogens for the efficient monitoring and treatment of virus diseases. Early detection of viruses provides essential information about possible efficient and targeted treatments, prolongs the therapeutic window and hence reduces morbidity. Graphene is a lightweight, chemically stable and conductive material that can be successfully utilized for the detection of various virus strains. The sensitivity and selectivity of graphene can be enhanced by its functionalization or combination with other materials. Introducing suitable functional groups and/or counterparts in the hybrid structure enables tuning of the optical and electrical properties, which is particularly attractive for rapid and easy-to-use virus detection. In this review, we cover all the different types of graphene-based sensors available for virus detection, including, e.g., photoluminescence and colorimetric sensors, and surface plasmon resonance biosensors. Various strategies of electrochemical detection of viruses based on, e.g., DNA hybridization or antigen-antibody interactions, are also discussed. We summarize the current state-of-the-art applications of graphene-based systems for sensing a variety of viruses, e.g., SARS-CoV-2, influenza, dengue fever, hepatitis C virus, HIV, rotavirus and Zika virus. General principles, mechanisms of action, advantages and drawbacks are presented to provide useful information for the further development and construction of advanced virus biosensors. We highlight that the unique and tunable physicochemical properties of graphene-based nanomaterials make them ideal candidates for engineering and miniaturization of biosensors.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Grafite , Pneumonia Viral/diagnóstico , Vírus/isolamento & purificação , Reações Antígeno-Anticorpo , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/tendências , Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/estatística & dados numéricos , Colorimetria , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , DNA Viral/análise , DNA Viral/genética , Técnicas Eletroquímicas , Desenho de Equipamento , Grafite/química , Humanos , Luminescência , Nanoestruturas/química , Hibridização de Ácido Nucleico , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Pontos Quânticos/química , Análise Espectral Raman , Ressonância de Plasmônio de Superfície , Virologia/métodos , Vírus/genética , Vírus/patogenicidade
7.
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 , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase/economia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
8.
Nat Commun ; 11(1): 3813, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732913

RESUMO

Spatial organization of biological processes allows for variability in molecular outcomes and coordinated development. Here, we investigate how organization underpins phage lambda development and decision-making by characterizing viral components and processes in subcellular space. We use live-cell and in situ fluorescence imaging at the single-molecule level to examine lambda DNA replication, transcription, virion assembly, and resource recruitment in single-cell infections, uniting key processes of the infection cycle into a coherent model of phage development encompassing space and time. We find that different viral DNAs establish separate subcellular compartments within cells, which sustains heterogeneous viral development in single cells. These individual phage compartments are physically separated by the E. coli nucleoid. Our results provide mechanistic details describing how separate viruses develop heterogeneously to resemble single-cell phenotypes.


Assuntos
Bacteriófago lambda/genética , Replicação do DNA/genética , Escherichia coli/virologia , Montagem de Vírus/genética , Bacteriófago lambda/crescimento & desenvolvimento , DNA Viral/biossíntese , DNA Viral/genética , Escherichia coli/genética , Lisogenia/genética , Transcrição Genética/genética
9.
Arch Virol ; 165(10): 2355-2359, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32748178

RESUMO

Two Staphylococcus aureus bacteriophages, KSAP7 and KSAP11, were isolated from sewage and characterized. Based on morphology and DNA sequences, they were assigned to the genus Silviavirus, subfamily Twortvirinae, family Herelleviridae, whose members are hypothesized to be suitable for bacteriophage therapy. The KSAP7 and KSAP11 genomes were 137,950 and 138,307 bp in size, respectively. Although their DNA sequences were almost identical, evidence of site-specific DNA rearrangements was found in two regions. Changes in the number of PIEPEK amino acid sequence repeats encoded by orf10 and the insertion/deletion of a 541-bp sequence that includes a possible tail-related gene were identified.


Assuntos
Caudovirales/genética , DNA Viral/genética , Genoma Viral , Filogenia , Fagos de Staphylococcus/genética , Staphylococcus aureus/virologia , Sequência de Aminoácidos , Caudovirales/classificação , Caudovirales/isolamento & purificação , Rearranjo Gênico , Tamanho do Genoma , Mutação INDEL , Japão , Fases de Leitura Aberta , Terapia por Fagos , Alinhamento de Sequência , Fagos de Staphylococcus/classificação , Fagos de Staphylococcus/isolamento & purificação
10.
Arch Virol ; 165(10): 2397-2400, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32748177

RESUMO

Enterobacter aerogenes is a member of the ESKAPE group of bacteria, and multi-drug-resistant strains are increasingly being found. In this study, a novel bacteriophage, ATCEA85, which infects E. aerogenes, has been isolated and characterized. ATCEA85 is seen to have a circularly permuted linear double-stranded DNA genome of 47,484 base pairs in length. The closest related phage found in the databases is the Klebsiella phage Kp3, which exhibits 77% identity over a 34% query coverage. The G+C content of ATCEA85 is 56.2%, and 15 putative open reading frames are functionally annotated.


Assuntos
DNA Viral/genética , Enterobacter aerogenes/virologia , Genoma Viral , Fases de Leitura Aberta , Filogenia , Siphoviridae/genética , Composição de Bases , DNA/genética , Ontologia Genética , Anotação de Sequência Molecular , Siphoviridae/classificação , Siphoviridae/isolamento & purificação , Sequenciamento Completo do Genoma
11.
Arch Virol ; 165(10): 2177-2191, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32748179

RESUMO

The canonical frameworks of viral evolution describe viruses as cellular predecessors, reduced forms of cells, or entities that escaped cellular control. The discovery of giant viruses has changed these standard paradigms. Their genetic, proteomic and structural complexities resemble those of cells, prompting a redefinition and reclassification of viruses. In a previous genome-wide analysis of the evolution of structural domains in proteomes, with domains defined at the fold superfamily level, we found the origins of viruses intertwined with those of ancient cells. Here, we extend these data-driven analyses to the study of fold families confirming the co-evolution of viruses and ancient cells and the genetic ability of viruses to foster molecular innovation. The results support our suggestion that viruses arose by genomic reduction from ancient cells and validate a co-evolutionary 'symbiogenic' model of viral origins.


Assuntos
Evolução Biológica , DNA Viral/genética , Genoma Viral , Vírus Gigantes/genética , Filogenia , Proteínas Virais/genética , Archaea/genética , Archaea/virologia , Bactérias/genética , Bactérias/virologia , DNA Viral/química , Eucariotos/genética , Eucariotos/virologia , Tamanho do Genoma , Vírus Gigantes/classificação , Proteogenômica/métodos , Proteoma/genética , Proteínas Virais/química
12.
PLoS Pathog ; 16(8): e1008752, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760121

RESUMO

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Núcleo Celular/metabolismo , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Macrófagos/imunologia , Proteínas Nucleares/metabolismo , Fator de Transcrição Sp1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Núcleo Celular/genética , DNA Viral/genética , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Células Hep G2 , Humanos , Imunidade Inata/imunologia , Inflamassomos/genética , Inflamassomos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas Nucleares/genética , Fator de Transcrição Sp1/genética , Replicação Viral
13.
Proc Natl Acad Sci U S A ; 117(33): 19643-19652, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759221

RESUMO

Living organisms expend metabolic energy to repair and maintain their genomes, while viruses protect their genetic material by completely passive means. We have used cryo-electron microscopy (cryo-EM) to solve the atomic structures of two filamentous double-stranded DNA viruses that infect archaeal hosts living in nearly boiling acid: Saccharolobus solfataricus rod-shaped virus 1 (SSRV1), at 2.8-Å resolution, and Sulfolobus islandicus filamentous virus (SIFV), at 4.0-Å resolution. The SIFV nucleocapsid is formed by a heterodimer of two homologous proteins and is membrane enveloped, while SSRV1 has a nucleocapsid formed by a homodimer and is not enveloped. In both, the capsid proteins wrap around the DNA and maintain it in an A-form. We suggest that the A-form is due to both a nonspecific desolvation of the DNA by the protein, and a specific coordination of the DNA phosphate groups by positively charged residues. We extend these observations by comparisons with four other archaeal filamentous viruses whose structures we have previously determined, and show that all 10 capsid proteins (from four heterodimers and two homodimers) have obvious structural homology while sequence similarity can be nonexistent. This arises from most capsid residues not being under any strong selective pressure. The inability to detect homology at the sequence level arises from the sampling of viruses in this part of the biosphere being extremely sparse. Comparative structural and genomic analyses suggest that nonenveloped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution.


Assuntos
Vírus de Archaea/química , Vírus de DNA/química , DNA Viral/química , Sulfolobales/virologia , Sulfolobus/virologia , Vírus de Archaea/classificação , Vírus de Archaea/genética , Vírus de Archaea/ultraestrutura , Evolução Biológica , Capsídeo/química , Capsídeo/ultraestrutura , Vírus de DNA/classificação , Vírus de DNA/genética , Vírus de DNA/ultraestrutura , DNA Viral/genética , Ambientes Extremos , Genoma Viral , Filogenia
14.
PLoS One ; 15(7): e0235012, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32663205

RESUMO

Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription ("uracilation"). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that the kinetics for MC infection is compatible with their lifetime in vivo and their near absence of hUNG2 activity is consistent with the retention of viral dUMP at high levels at least until differentiation into macrophages, where UBER becomes possible. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.


Assuntos
Reparo do DNA , Infecções por HIV/genética , Macrófagos/virologia , Monócitos/virologia , Provírus/genética , Uracila/metabolismo , DNA Viral/genética , Nucleotídeos de Desoxiuracil/deficiência , Nucleotídeos de Desoxiuracil/metabolismo , HIV-1/genética , Humanos , Uracila-DNA Glicosidase/metabolismo
15.
PLoS One ; 15(7): e0235832, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706778

RESUMO

Porcine circovirus type 3 (PCV3) is a newly emerging virus in the swine industry, first reported recently in 2016. PCV3 assembles into a 2000 bp circular genome; slightly larger than PCV1 (1758-1760 bp), PCV2 (1766-1769 bp) and PCV4 (1770 bp). Apart from being associated with porcine dermatitis and nephropathy syndrome (PDNS), PCV3 has been isolated from pigs with clinical signs of reproductive failures, myocarditis, porcine respiratory disease complex (PRDC) and neurologic disease. Given that PCV3 is increasingly reported in countries including Thailand and U.S. with whom Malaysia shares trade and geographical relationship; and that PCV3 is associated with several clinical presentations that affect productivity, there is a need to study the presence and molecular characteristics of PCV3 in Malaysian swine farms. Twenty-four commercial swine farms, three abattoirs and retail shops in Peninsular Malaysia were sampled using convenience sampling method. A total of 281 samples from 141 pigs, including 49 lung archive samples were tested for PCV3 by conventional PCR. Twenty-eight lung samples from wild boar population in Peninsular Malaysia were also included. Nucleotide sequences were analyzed for maximum likelihood phylogeny relationship and pairwise distances. Results revealed that PCV3 is present in Peninsular Malaysia at a molecular prevalence of 17.02%, with inguinal lymph nodes and lungs showing the highest molecular detection rates of 81.82% and 71.43% respectively. Despite wide reports of PCV3 in healthy animals and wild boars, no positive samples were detected in clinically healthy finishers and wild boar population of this study. PCV3 strain A1 and A2 were present in Malaysia, and Malaysian PCV3 strains were found to be phylogenetically related to Spanish, U.S. and Mexico strains.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/genética , Genoma Viral , Doenças dos Suínos/virologia , Suínos/virologia , Animais , Sequência de Bases , Infecções por Circoviridae/virologia , Circovirus/isolamento & purificação , DNA Viral/genética , Dermatite/veterinária , Dermatite/virologia , Nefropatias/veterinária , Nefropatias/virologia
16.
Nat Commun ; 11(1): 3279, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32606306

RESUMO

Recombinant adeno-associated viruses (rAAVs) are currently considered the safest and most reliable gene delivery vehicles for human gene therapy. Three serotype capsids, AAV1, AAV2, and AAV9, have been approved for commercial use in patients, but they may not be suitable for all therapeutic contexts. Here, we describe a novel capsid identified in a human clinical sample by high-throughput, long-read sequencing. The capsid, which we have named AAVv66, shares high sequence similarity with AAV2. We demonstrate that compared to AAV2, AAVv66 exhibits enhanced production yields, virion stability, and CNS transduction. Unique structural properties of AAVv66 visualized by cryo-EM at 2.5-Å resolution, suggest that critical residues at the three-fold protrusion and at the interface of the five-fold axis of symmetry likely contribute to the beneficial characteristics of AAVv66. Our findings underscore the potential of AAVv66 as a gene therapy vector.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Animais , Capsídeo/ultraestrutura , Proteínas do Capsídeo/classificação , Sistema Nervoso Central/virologia , Microscopia Crioeletrônica , DNA Viral/análise , DNA Viral/genética , Dependovirus/classificação , Dependovirus/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Filogenia , Sorogrupo , Transdução Genética , Montagem de Vírus/genética
17.
PLoS Negl Trop Dis ; 14(7): e0008361, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667912

RESUMO

Human T-cell leukemia virus type 1 (HTLV-1) causes incurable adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Patients with HAM/TSP have increased levels of HTLV-1-infected cells compared with asymptomatic HTLV-1 carriers. However, the roles of cellular genes in HTLV-1-infected CD4+ T cells await discovery. We performed microarray analysis of CD4+ T cells from HAM/TSP patients and found that the ABL1 is an important gene in HAM/TSP. ABL1 is a known survival factor for T- and B-lymphocytes and is part of the fused gene (BCR-ABL) known to be responsible for chronic myelogenous leukemia (CML). ABL1 tyrosine kinase inhibitors (TKIs), including imatinib, nilotinib, and dasatinib, are used clinically for treating CML. To evaluate whether ABL1 is indeed important for HAM/TSP, we investigated the effect of TKIs on HTLV-1-infected cells. We developed a propidium monoazide-HTLV-1 viability quantitative PCR assay, which distinguishes DNA from live cells and dead cells. Using this method, we were able to measure the HTLV-1 proviral load (PVL) in live cells alone when peripheral blood mononuclear cells (PBMCs) from HAM/TSP cases were treated with TKIs. Treating the PBMCs with nilotinib or dasatinib induced significant reductions in PVL (21.0% and 17.5%, respectively) in live cells. Furthermore, ABL1 siRNA transfection reduced cell viability in HTLV-1-infected cell lines, but not in uninfected cell lines. A retrospective survey based on our clinical records found a rare case of HAM/TSP who also suffered from CML. The patient showed an 84.2% PVL reduction after CML treatment with imatinib. We conclude that inhibiting the ABL1 tyrosine kinase specifically reduced the PVL in PBMCs from patients with HAM/TSP, suggesting that ABL1 is an important gene for the survival of HTLV-1-infected cells and that TKIs may be potential therapeutic agents for HAM/TSP.


Assuntos
Infecções por HTLV-I/complicações , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Leucócitos Mononucleares/virologia , Paraparesia Espástica Tropical/enzimologia , Doenças da Medula Espinal/enzimologia , Adulto , Idoso , DNA Viral/genética , Feminino , Infecções por HTLV-I/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Paraparesia Espástica Tropical/tratamento farmacológico , Paraparesia Espástica Tropical/etiologia , Paraparesia Espástica Tropical/genética , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Provírus/genética , Provírus/fisiologia , Estudos Retrospectivos , Doenças da Medula Espinal/tratamento farmacológico , Doenças da Medula Espinal/etiologia , Doenças da Medula Espinal/genética , Carga Viral
18.
BMC Infect Dis ; 20(1): 552, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727389

RESUMO

BACKGROUND: Hepatitis B virus (HBV) infections are a severe health concern worldwide. HBV is a DNA virus with a rapid rate of mutation. Based on heterogeneity of the nucleotide sequence, the HBV strains are divided into nine genotypes, each with a characteristic geographical distribution. Identifying and tracking alterations of HBV genotypes is important in epidemiological and transmission studies, and contributes to predicting the risk for development of severe liver disease and response to antiviral treatment. The present study was undertaken to detect HBV genotypes and sub-genotypes in the general population of different states and regions in Myanmar. METHODS: In 2015, a total of 5547 adults of the general population, residing in seven states, seven regions and the Nay Pyi Taw Union Territory, were screened for Hepatitis B Surface antigen (HBsAg) by the immunochromatographic test (ICT). Of the 353 HBsAg positive samples, the HBVDNA was identified using polymerase chain reactions (PCR) targeting the DNA sequences encoding the Pre-S region. A total of 153 PCR positive samples were subsequently subjected to genotyping by partial genome sequencing in both directions. The resulting sequences were then edited, aligned, and compared with reference sequences using the National Centre for Biotechnology Information (NCBI) web-based genotyping tool. RESULTS: Three HBV genotypes (HBV genotype B, genotype C and genotype D) were detected in Myanmar, of which genotype HBV genotype C (66.7%) was the most prevalent, followed by HBV genotype D (32%) and HBV genotype B (1.3%). Sub-genotyping revealed a total of 7 variants within the B, C and D genotypes: 2 (B4 and B5) in HBV genotype B, 3 (C1, C5 and C7) in HBV genotype C, and 2 (D3 and D6) in HBV genotype D. CONCLUSION: HBV genotype C, sub-genotype C1 was predominantly distributed in all states and regions of Myanmar. This study is the first report on the nationwide distribution of HBV genotypes and sub-genotypes in Myanmar. We believe our findings will enable huge support for the hepatitis disease surveillance program, since HBV infection is one of the National Priority Diseases in Myanmar.


Assuntos
Genótipo , Vírus da Hepatite B/genética , Hepatite B/epidemiologia , Adulto , Sequência de Bases , Cromatografia de Afinidade , Estudos Transversais , DNA Viral/genética , Feminino , Hepatite B/sangue , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Mianmar/epidemiologia , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Adulto Jovem
19.
Nat Commun ; 11(1): 3748, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719311

RESUMO

Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a ß-hairpin loop, and interconnected along the tail by the splayed ß-hairpins. By contrast, we show that the ß-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.


Assuntos
Bacteriófagos/fisiologia , Flagelos/fisiologia , Motivos de Aminoácidos , Bacteriófagos/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , DNA/genética , DNA Viral/genética , Flagelos/ultraestrutura , Herpesviridae/ultraestrutura , Multimerização Proteica , Estrutura Secundária de Proteína , Vírion/ultraestrutura , Vitrificação
20.
Life Sci ; 257: 118089, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659369

RESUMO

AIM: Hepatitis B virus (HBV) is a major cause of a variety of liver diseases. Existing antiviral drugs cannot eradicate HBV from our body, and the main reason is unclear on the molecular mechanism of HBV replication. Flap endonuclease 1 (FEN1) can repair relaxed circular DNA (HBV rcDNA) to covalently closed circular DNA (HBV cccDNA) that promotes HBV DNA replication, while its specific regulatory detail remains unclear. In addition, miR-146a is close related to regulation in HBV replication. This study aims to explore whether miR-146a regulates HBV cccDNA formation through FEN1. MAIN METHODS: We investigated the expression of miR-146a, FEN1 and HBV copies in HBV stable replication cell line HepG2.2.15 and its parent cell line HepG2 transfected miR-146a and FEN1 plasmid by qRT-PCR and western blot, to identify the cooperation of Argonaute-2 (Ago2) and miR-146a by Ago2 siRNA and Ago2 RNA Binding Protein Immunoprecipitation (RIP). KEY FINDINGS: Compared with the control group, we found that the expression of miR-146a was significantly up-regulated in HepG2.2.15, and the expression of FEN1 and HBV copies were also significantly up-regulated. On contrary, the expression of target gene of miR-146a, interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor-6 (TRAF6), was significantly decreased in HepG2.2.15. With the use of Ago2 siRNA and then Ago2 RIP, we found that Ago2 performed as a carrier for miR-146a to promote HBV replication. SIGNIFICANCE: The results suggest a novel miR-146a â†’ FEN1 â†’ HBV DNA regulatory axis in HBV replication life. Ago2 cooperates with miR-146a to regulate the transcription and expression level of FEN1 protein through the downstream target gene IRAK1/TRAF6, and to promote HBV replication.


Assuntos
Proteínas Argonauta/genética , Vírus da Hepatite B/fisiologia , MicroRNAs/genética , Replicação Viral/genética , DNA Circular/genética , DNA Viral/genética , Endonucleases Flap/genética , Células Hep G2 , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética
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