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1.
J Transl Med ; 18(1): 329, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867854

RESUMO

BACKGROUND: The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which was first detected in Wuhan (China) in December of 2019 is responsible for the current global pandemic. Phylogenetic analysis revealed that it is similar to other betacoronaviruses, such as SARS-CoV and Middle-Eastern Respiratory Syndrome, MERS-CoV. Its genome is ∼ 30 kb in length and contains two large overlapping polyproteins, ORF1a and ORF1ab that encode for several structural and non-structural proteins. The non-structural protein 1 (nsp1) is arguably the most important pathogenic determinant, and previous studies on SARS-CoV indicate that it is both involved in viral replication and hampering the innate immune system response. Detailed experiments of site-specific mutagenesis and in vitro reconstitution studies determined that the mechanisms of action are mediated by (a) the presence of specific amino acid residues of nsp1 and (b) the interaction between the protein and the host's small ribosomal unit. In fact, substitution of certain amino acids resulted in reduction of its negative effects. METHODS: A total of 17,928 genome sequences were obtained from the GISAID database (December 2019 to July 2020) from patients infected by SARS-CoV-2 from different areas around the world. Genomes alignment was performed using MAFFT (REFF) and the nsp1 genomic regions were identified using BioEdit and verified using BLAST. Nsp1 protein of SARS-CoV-2 with and without deletion have been subsequently modelled using I-TASSER. RESULTS: We identified SARS-CoV-2 genome sequences, from several Countries, carrying a previously unknown deletion of 9 nucleotides in position 686-694, corresponding to the AA position 241-243 (KSF). This deletion was found in different geographical areas. Structural prediction modelling suggests an effect on the C-terminal tail structure. CONCLUSIONS: Modelling analysis of a newly identified deletion of 3 amino acids (KSF) of SARS-CoV-2 nsp1 suggests that this deletion could affect the structure of the C-terminal region of the protein, important for regulation of viral replication and negative effect on host's gene expression. In addition, substitution of the two amino acids (KS) from nsp1 of SARS-CoV was previously reported to revert loss of interferon-alpha expression. The deletion that we describe indicates that SARS-CoV-2 is undergoing profound genomic changes. It is important to: (i) confirm the spreading of this particular viral strain, and potentially of strains with other deletions in the nsp1 protein, both in the population of asymptomatic and pauci-symptomatic subjects, and (ii) correlate these changes in nsp1 with potential decreased viral pathogenicity.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Deleção de Sequência , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Betacoronavirus/patogenicidade , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/epidemiologia , Frequência do Gene , Genoma Viral , Geografia , Humanos , Lisina/genética , Modelos Moleculares , Pandemias/estatística & dados numéricos , Fenilalanina/genética , Pneumonia Viral/epidemiologia , Domínios Proteicos/genética , Serina/genética , Proteínas não Estruturais Virais/química , Virulência/genética , Replicação Viral/genética
2.
J Transl Med ; 18(1): 362, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32967693

RESUMO

BACKGROUND: Since the first outbreak of SARS-CoV-2, the clinical characteristics of the Coronavirus Disease 2019 (COVID-19) have been progressively changed. Data reporting a viral intra-host and inter-host evolution favouring the appearance of mild SARS-CoV-2 strains are since being accumulating. To better understand the evolution of SARS-CoV-2 pathogenicity and its adaptation to the host, it is therefore crucial to investigate the genetic and phenotypic characteristics of SARS-CoV-2 strains circulating lately in the epidemic. METHODS: Nasopharyngeal swabs have been analyzed for viral load in the early (March 2020) and late (May 2020) phases of epidemic in Brescia, Italy. Isolation of SARS-CoV-2 from 2 high viral load specimens identified on March 9 (AP66) and on May 8 (GZ69) was performed on Vero E6 cells. Amount of virus released was assessed by quantitative PCR. Genotypic characterization of AP66 and GZ69 was performed by next generation sequencing followed by an in-depth in silico analysis of nucleotide mutations. RESULTS: The SARS-CoV-2 GZ69 strain, isolated in May from an asymptomatic healthcare worker, showed an unprecedented capability of replication in Vero E6 cells in the absence of any evident cytopathic effect. Vero E6 subculturing, up to passage 4, showed that SARS-CoV-2 GZ69 infection was as productive as the one sustained by the cytopathic strain AP66. Whole genome sequencing of the persistently replicating SARS-CoV-2 GZ69 has shown that this strain differs from the early AP66 variant in 9 nucleotide positions (C2939T; C3828T; G21784T; T21846C; T24631C; G28881A; G28882A; G28883C; G29810T) which lead to 6 non-synonymous substitutions spanning on ORF1ab (P892S; S1188L), S (K74N; I95T) and N (R203K, G204R) proteins. CONCLUSIONS: Identification of the peculiar SARS-CoV-2 GZ69 strain in the late Italian epidemic highlights the need to better characterize viral variants circulating among asymptomatic or paucisymptomatic individuals. The current approach could unravel the ways for future studies aimed at analyzing the selection process which favours viral mutations in the human host.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Variação Genética , Pneumonia Viral/virologia , Substituição de Aminoácidos , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/epidemiologia , Efeito Citopatogênico Viral/genética , Efeito Citopatogênico Viral/fisiologia , Genoma Viral , Humanos , Itália/epidemiologia , Mutação , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , Polimorfismo de Nucleotídeo Único , Pesquisa Médica Translacional , Células Vero , Proteínas Virais/genética , Proteínas Virais/fisiologia , Cultura de Vírus/métodos , Replicação Viral/genética , Replicação Viral/fisiologia , Sequenciamento Completo do Genoma
3.
PLoS Pathog ; 16(9): e1008853, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886726

RESUMO

HIV-1 transmission is associated with a severe bottleneck in which a limited number of variants from a pool of genetically diverse quasispecies establishes infection. The IAVI protocol C cohort of discordant couples, female sex workers, other heterosexuals and men who have sex with men (MSM) present varying risks of HIV infection, diverse HIV-1 subtypes and represent a unique opportunity to characterize transmitted/founder viruses (TF) where disease outcome is known. To identify the TF, the HIV-1 repertoire of 38 MSM participants' samples was sequenced close to transmission (median 21 days post infection, IQR 18-41) and assessment of multivariant infection done. Patient derived gag genes were cloned into an NL4.3 provirus to generate chimeric viruses which were characterized for replicative capacity (RC). Finally, an evaluation of how the TF virus predicted disease progression and modified the immune response at both acute and chronic HIV-1 infection was done. There was higher prevalence of multivariant infection compared with previously described heterosexual cohorts. A link was identified between multivariant infection and replicative capacity conferred by gag, whereby TF gag tended to be of lower replicative capacity in multivariant infection (p = 0.02) suggesting an overall lowering of fitness requirements during infection with multiple variants. Notwithstanding, multivariant infection was associated with rapid CD4+ T cell decline and perturbances in the CD4+ T cell and B cell compartments compared to single variant infection, which were reversible upon control of viremia. Strategies aimed at identifying and mitigating multivariant infection could contribute toward improving HIV-1 prognosis and this may involve strategies that tighten the stringency of the transmission bottleneck such as treatment of STI. Furthermore, the sequences and chimeric viruses help with TF based experimental vaccine immunogen design and can be used in functional assays to probe effective immune responses against TF.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Efeito Fundador , Infecções por HIV , HIV-1/fisiologia , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Doença Aguda , Adolescente , Adulto , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Feminino , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Viremia/genética , Viremia/imunologia , Viremia/patologia , Replicação Viral/genética , Replicação Viral/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
4.
PLoS Pathog ; 16(9): e1008773, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32881988

RESUMO

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and ß production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and ß induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and ß production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and ß induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and ß production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and ß production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Mutação , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Animais , Antivirais/farmacologia , Patos , Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/tratamento farmacológico , Encefalite Japonesa/virologia , Interações Hospedeiro-Patógeno , Camundongos , Ligação Proteica , Suínos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
5.
PLoS Pathog ; 16(8): e1008718, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32797103

RESUMO

APOBEC3 enzymes are innate immune effectors that introduce mutations into viral genomes. These enzymes are cytidine deaminases which transform cytosine into uracil. They preferentially mutate cytidine preceded by thymidine making the 5'TC motif their favored target. Viruses have evolved different strategies to evade APOBEC3 restriction. Certain viruses actively encode viral proteins antagonizing the APOBEC3s, others passively face the APOBEC3 selection pressure thanks to a depleted genome for APOBEC3-targeted motifs. Hence, the APOBEC3s left on the genome of certain viruses an evolutionary footprint. The aim of our study is the identification of these viruses having a genome shaped by the APOBEC3s. We analyzed the genome of 33,400 human viruses for the depletion of APOBEC3-favored motifs. We demonstrate that the APOBEC3 selection pressure impacts at least 22% of all currently annotated human viral species. The papillomaviridae and polyomaviridae are the most intensively footprinted families; evidencing a selection pressure acting genome-wide and on both strands. Members of the parvoviridae family are differentially targeted in term of both magnitude and localization of the footprint. Interestingly, a massive APOBEC3 footprint is present on both strands of the B19 erythroparvovirus; making this viral genome one of the most cleaned sequences for APOBEC3-favored motifs. We also identified the endemic coronaviridae as significantly footprinted. Interestingly, no such footprint has been detected on the zoonotic MERS-CoV, SARS-CoV-1 and SARS-CoV-2 coronaviruses. In addition to viruses that are footprinted genome-wide, certain viruses are footprinted only on very short sections of their genome. That is the case for the gamma-herpesviridae and adenoviridae where the footprint is localized on the lytic origins of replication. A mild footprint can also be detected on the negative strand of the reverse transcribing HIV-1, HIV-2, HTLV-1 and HBV viruses. Together, our data illustrate the extent of the APOBEC3 selection pressure on the human viruses and identify new putatively APOBEC3-targeted viruses.


Assuntos
Citidina Desaminase/metabolismo , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , Seleção Genética/genética , Replicação Viral/genética , Coronaviridae/genética , Humanos , Imunidade Inata/imunologia , Papillomaviridae/genética , Parvoviridae/genética , Polyomaviridae/genética , Proteínas Virais/genética
6.
Nat Commun ; 11(1): 4070, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792502

RESUMO

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.


Assuntos
Canais Iônicos/metabolismo , Mamastrovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Canais Iônicos/genética , Mamastrovirus/genética , Microscopia de Fluorescência , Plasmídeos/genética , Ribossomos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
7.
Nat Commun ; 11(1): 4089, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796830

RESUMO

Clonal expansions occur in the persistent HIV reservoir as shown by the duplication of proviral integration sites. However, the source of the proliferation of HIV-infected cells remains unclear. Here, we analyze the TCR repertoire of single HIV-infected cells harboring translation-competent proviruses in longitudinal samples from eight individuals on antiretroviral therapy (ART). When compared to uninfected cells, the TCR repertoire of reservoir cells is heavily biased: expanded clonotypes are present in all individuals, account for the majority of reservoir cells and are often maintained over time on ART. Infected T cell clones are detected at low frequencies in the long-lived central memory compartment and overrepresented in the most differentiated memory subsets. Our results indicate that clonal expansions highly contribute to the persistence of the HIV reservoir and suggest that reservoir cells displaying a differentiated phenotype are the progeny of infected central memory cells undergoing antigen-driven clonal expansion during ART.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Adulto , Células Cultivadas , Feminino , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/patogenicidade , Humanos , Masculino , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Carga Viral , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
8.
J Transl Med ; 18(1): 319, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811513

RESUMO

In less than 20 years, three deadly coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have emerged in human population causing hundreds to hundreds of thousands of deaths. Other coronaviruses are causing epizootic representing a significant threat for both domestic and wild animals. Members of this viral family have the longest genome of all RNA viruses, and express up to 29 proteins establishing complex interactions with the host proteome. Deciphering these interactions is essential to identify cellular pathways hijacked by these viruses to replicate and escape innate immunity. Virus-host interactions also provide key information to select targets for antiviral drug development. Here, we have manually curated the literature to assemble a unique dataset of 1311 coronavirus-host protein-protein interactions. Functional enrichment and network-based analyses showed coronavirus connections to RNA processing and translation, DNA damage and pathogen sensing, interferon production, and metabolic pathways. In particular, this global analysis pinpointed overlooked interactions with translation modulators (GIGYF2-EIF4E2), components of the nuclear pore, proteins involved in mitochondria homeostasis (PHB, PHB2, STOML2), and methylation pathways (MAT2A/B). Finally, interactome data provided a rational for the antiviral activity of some drugs inhibiting coronaviruses replication. Altogether, this work describing the current landscape of coronavirus-host interactions provides valuable hints for understanding the pathophysiology of coronavirus infections and developing effective antiviral therapies.


Assuntos
Infecções por Coronavirus/metabolismo , Coronavirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Mapas de Interação de Proteínas , Proteínas Virais/metabolismo , Animais , Betacoronavirus/fisiologia , Coronavirus/química , Infecções por Coronavirus/virologia , Bases de Dados de Proteínas , Humanos , Proteínas Mitocondriais/metabolismo , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Fatores de Transcrição/metabolismo , Replicação Viral/genética
9.
Viruses ; 12(8)2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32731335

RESUMO

Non-structural protein 1 (nsp1) is only characterized in alphacoronaviruses (α-CoVs) and betacoronaviruses (ß-CoVs). There have been extensive researches on how the ß-CoVs nsp1 regulates viral virulence by inhibiting host protein synthesis, but the regulatory mechanism of the α-CoVs nsp1 is still unclear. Here, we report the 2.1-Å full-length crystal structure of nsp1 in emerging porcine SADS-CoV and the 1.8-Å full-length crystal structure of nsp1 in the highly lethal cat FIPV. Although they belong to different subtypes of α-CoVs, these viruses all have a bucket-shaped fold composed of six ß-sheets, similar to the crystal structure of PEDV and TGEV nsp1. Comparing the above four structures, we found that the structure of α-CoVs nsp1 in the same subtype was more conserved. We then selected mammalian cells that were treated with SADS-CoV and FIPV nsp1 for RNA sequencing analysis and found that nsp1 had a specific inhibitory effect on interferon (IFN) and cell cycle genes. Using the Renilla luciferase (Rluc) assay and Western blotting, we confirmed that seven representative α-CoVs nsp1s could significantly inhibit the phosphorylation of STAT1-S727 and interfere with the effect of IFN-I. Moreover, the cell cycle experiment confirmed that α-CoVs nsp1 could encourage host cells to stay in the G0/G1 phase. Based on these findings, we not only greatly improved the crystal structure data on α-CoVs nsp1, but we also speculated that α-CoVs nsp1 regulated host proliferation and immune evasion-related biological functions by inhibiting the synthesis of host proteins, thus creating an environment conducive to the virus.


Assuntos
Alphacoronavirus/imunologia , Alphacoronavirus/fisiologia , Evasão da Resposta Imune/imunologia , Interferon Tipo I/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Alphacoronavirus/genética , Sequência de Aminoácidos , Animais , Gatos , Linhagem Celular , Cristalografia por Raios X , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Fosforilação , Estrutura Terciária de Proteína , Fator de Transcrição STAT1/metabolismo , Homologia de Sequência , Suínos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética
10.
JCI Insight ; 5(19)2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32841215

RESUMO

The emergence of SARS-CoV-2 has created an international health crisis, and small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection owing to low-affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here, we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promoter (K18). In contrast to nontransgenic mice, intranasal exposure of K18-hACE2 animals to 2 different doses of SARS-CoV-2 resulted in acute disease, including weight loss, lung injury, brain infection, and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals showed increases in transcripts involved in lung injury and inflammatory cytokines. In the low-dose challenge groups, there was a survival advantage in the female mice, with 60% surviving infection, whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared with female mice. To our knowledge, this is the first highly lethal murine infection model for SARS-CoV-2 and should be valuable for the study of SARS-CoV-2 pathogenesis and for the assessment of MCMs.


Assuntos
Causas de Morte , Infecções por Coronavirus/patologia , Progressão da Doença , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Síndrome Respiratória Aguda Grave/patologia , Animais , Infecções por Coronavirus/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pandemias , Pneumonia Viral/fisiopatologia , Síndrome Respiratória Aguda Grave/fisiopatologia , Índice de Gravidade de Doença , Taxa de Sobrevida , Replicação Viral/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-32738193

RESUMO

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Antivirais/farmacologia , Betacoronavirus/genética , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Avaliação Pré-Clínica de Medicamentos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Mutação , Testes de Neutralização , Pandemias/prevenção & controle , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , Receptores Virais/genética , Receptores Virais/fisiologia , Recombinação Genética , Serina Endopeptidases/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Internalização do Vírus , Replicação Viral/genética
12.
Indian J Med Res ; 152(1 & 2): 70-76, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32773420

RESUMO

Background & objectives: The genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonging to the family Coronaviridae, encodes for structural, non-structural, and accessory proteins, which are required for replication of the virus. These proteins are encoded by different genes present on the SARS-CoV-2 genome. The expression pattern of these genes in the host cells needs to be assessed. This study was undertaken to understand the transcription pattern of the SARS-CoV-2 genes in the Vero CCL-81 cells during the course of infection. Methods: Vero CCL-81 cells were infected with the SARS-CoV-2 virus inoculum having a 0.1 multiplicity of infection. The supernatants and cell pellets were harvested after centrifugation at different time points, post-infection. The 50% tissue culture infective dose (TCID50)and cycle threshold (Ct) values of the E and the RdRp-2 genes were calculated. Next-generation sequencing of the harvested sample was carried out to observe the expression pattern of the virus by mapping to the SARS-CoV-2 Wuhan HU-1 reference sequence. The expressions were in terms of the reads per kilobase million (RPKM) values. Results: In the inital six hours post-infection, the copy numbers of E and RdRp-2 genes were approximately constant, which raised 10 log-fold and continued to increase till the 12 h post-infection (hpi). The TCID50 was observed in the supernatant after 7 hpi, indicating the release of the viral progeny. ORF8 and ORF7a, along with the nucleocapsid transcript, were found to express at higher levels. Interpretation & conclusions: This study was a step towards understanding the growth kinetics of the SARS-CoV-2 replication cycle. The findings indicated that ORF8 and ORF7b gene transcripts were expressed in higher amounts indicating their essential role in viral replication. Future studies need to be conducted to explore their role in the SARS-CoV-2 replication.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/genética , Pneumonia Viral/genética , Transcriptoma/genética , Animais , Betacoronavirus/patogenicidade , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Células Vero/virologia , Replicação Viral/genética
13.
J Exp Med ; 217(12)2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-32750141

RESUMO

Severe acute respiratory syndrome-coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus's inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Modelos Animais de Doenças , Interferon Tipo I/metabolismo , Camundongos/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Animais , Linhagem Celular Tumoral , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Dependovirus/genética , Feminino , Humanos , Inflamação/metabolismo , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pandemias , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/virologia , Peptidil Dipeptidase A/genética , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Transdução de Sinais/genética , Replicação Viral/genética
14.
Nat Commun ; 11(1): 3505, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665593

RESUMO

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.


Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Linfócitos T CD4-Positivos/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Viral/genética , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Microscopia de Fluorescência , Virologia , Integração Viral/genética , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
15.
BMC Infect Dis ; 20(1): 546, 2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32711474

RESUMO

BACKGROUND: Human immunodeficiency virus (HIV-1) infection is characterized by high viral replication and a decrease in CD4+ T cells (CD4+TC), resulting in AIDS, which can lead to death. In elite controllers and viremia controllers, viral replication is naturally controlled, with maintenance of CD4+TC levels without the use of antiretroviral therapy (ART). METHODS: The aim of the present study was to describe virological and immunological risk factors among HIV-1-infected individuals according to characteristics of progression to AIDS. The sample included 30 treatment-naive patients classified into three groups based on infection duration (> 6 years), CD4+TC count and viral load: (i) 2 elite controllers (ECs), (ii) 7 viremia controllers (VCs) and (iii) 21 nonviremia controllers (NVCs). Nested PCR was employed to amplify the virus genome, which was later sequenced using the Ion PGM platform for subtyping and analysis of immune escape mutations. RESULTS: Viral samples were classified as HIV-1 subtypes B and F. Greater selection pressure on mutations was observed in the group of viremia controllers, with a higher frequency of immunological escape mutations in the genes investigated, including two new mutations in gag. The viral sequences of viremia controllers and nonviremia controllers did not differ significantly regarding the presence of immune escape mutations. CONCLUSION: The results suggest that progression to AIDS is not dependent on a single variable but rather on a set of characteristics and pressures exerted by virus biology and interactions with immunogenetic host factors.


Assuntos
Síndrome de Imunodeficiência Adquirida/imunologia , HIV-1/genética , Evasão da Resposta Imune/genética , Mutação/imunologia , Síndrome de Imunodeficiência Adquirida/virologia , Adulto , Brasil , Linfócitos T CD4-Positivos/imunologia , Estudos Transversais , Feminino , Genes gag/genética , Humanos , Masculino , Filogenia , Conformação Proteica , Estudos Retrospectivos , Carga Viral , Viremia/genética , Replicação Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
16.
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
17.
Viruses ; 12(7)2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32674326

RESUMO

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious economically important respiratory pathogen of domestic fowl. Reverse genetics allows for the molecular study of pathogenic determinants to enable rational vaccine design. The recombinant IBV (rIBV) Beau-R, a molecular clone of the apathogenic Beaudette strain, has previously been investigated as a vaccine platform. To determine tissues in which Beau-R could effectively deliver antigenic genes, an in vivo study in chickens, the natural host, was used to compare the pattern of viral dissemination of Beau-R to the pathogenic strain M41-CK. Replication of Beau-R was found to be restricted to soft tissue within the beak, whereas M41-CK was detected in beak tissue, trachea and eyelid up to seven days post infection. In vitro assays further identified that, unlike M41-CK, Beau-R could not replicate at 41 °C, the core body temperature of a chicken, but is able to replicate a 37 °C, a temperature relatable to the very upper respiratory tract. Using a panel of rIBVs with defined mutations in the structural and accessory genes, viral replication at permissive and non-permissive temperatures was investigated, identifying that the Beau-R replicase gene was a determinant of temperature sensitivity and that sub-genomic mRNA synthesis had been affected. The identification of temperature sensitive allelic lesions within the Beau-R replicase gene opens up the possibility of using this method of attenuation in other IBV strains for future vaccine development as well as a method to investigate the functions of the IBV replicase proteins.


Assuntos
Infecções por Coronavirus/prevenção & controle , Vírus da Bronquite Infecciosa/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinação/veterinária , Vacinas Virais/imunologia , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , RNA Viral/genética , Temperatura , Vacinas Atenuadas/imunologia , Replicação Viral/genética , Replicação Viral/fisiologia
19.
PLoS Biol ; 18(7): e3000562, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32730280

RESUMO

Virus proliferation involves gene replication inside infected cells and transmission to new target cells. Once positive-strand RNA virus has infected a cell, the viral genome serves as a template for copying ("stay-strategy") or is packaged into a progeny virion that will be released extracellularly ("leave-strategy"). The balance between genome replication and virion release determines virus production and transmission efficacy. The ensuing trade-off has not yet been well characterized. In this study, we use hepatitis C virus (HCV) as a model system to study the balance of the two strategies. Combining viral infection cell culture assays with mathematical modeling, we characterize the dynamics of two different HCV strains (JFH-1, a clinical isolate, and Jc1-n, a laboratory strain), which have different viral release characteristics. We found that 0.63% and 1.70% of JFH-1 and Jc1-n intracellular viral RNAs, respectively, are used for producing and releasing progeny virions. Analysis of the Malthusian parameter of the HCV genome (i.e., initial proliferation rate) and the number of de novo infections (i.e., initial transmissibility) suggests that the leave-strategy provides a higher level of initial transmission for Jc1-n, whereas, in contrast, the stay-strategy provides a higher initial proliferation rate for JFH-1. Thus, theoretical-experimental analysis of viral dynamics enables us to better understand the proliferation strategies of viruses, which contributes to the efficient control of virus transmission. Ours is the first study to analyze the stay-leave trade-off during the viral life cycle and the significance of the replication-release switching mechanism for viral proliferation.


Assuntos
Genoma Viral , Hepacivirus/genética , Interações Hospedeiro-Patógeno/genética , Envelhecimento/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Hepatite C , Humanos , Modelos Biológicos , Replicação Viral/genética
20.
Emerg Infect Dis ; 26(9): 2054-2063, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32558639

RESUMO

Since its emergence in Wuhan, China, in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected ≈6 million persons worldwide. As SARS-CoV-2 spreads across the planet, we explored the range of human cells that can be infected by this virus. We isolated SARS-CoV-2 from 2 infected patients in Toronto, Canada; determined the genomic sequences; and identified single-nucleotide changes in representative populations of our virus stocks. We also tested a wide range of human immune cells for productive infection with SARS-CoV-2. We confirm that human primary peripheral blood mononuclear cells are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single-nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype by using in vitro and in vivo infection models.


Assuntos
Betacoronavirus , Infecções por Coronavirus/virologia , Leucócitos Mononucleares/virologia , Pneumonia Viral/virologia , Replicação Viral/genética , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , DNA Viral/genética , DNA Viral/isolamento & purificação , Genótipo , Humanos , Cinética , Pandemias , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma
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