RESUMO
Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications.IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets.
Assuntos
Replicação do DNA , Células Epiteliais/virologia , Genoma Viral , Corpos de Inclusão Viral/fisiologia , Metapneumovirus/genética , Metapneumovirus/fisiologia , Brônquios/citologia , Brônquios/virologia , Linhagem Celular , Citoplasma/virologia , Células Epiteliais/citologia , Humanos , Hibridização in Situ Fluorescente , RNA Viral , Análise Espaço-Temporal , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites.IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly.
Assuntos
Vírus Hendra/genética , Multimerização Proteica , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Virossomos/metabolismo , Linhagem Celular , Endossomos/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Domínios Proteicos , Transporte Proteico , Proteínas da Matriz Viral/metabolismo , Virossomos/genéticaRESUMO
Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses.
RESUMO
How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.
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Orthohantavírus/metabolismo , Virus Puumala/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Western Blotting , Reações Cruzadas/imunologia , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Orthohantavírus/genética , Humanos , Virus Puumala/genética , Vírion/genéticaRESUMO
IMPORTANCE: Human metapneumovirus is an important respiratory pathogen that causes significant morbidity and mortality, particularly in the very young, the elderly, and the immunosuppressed. However, the molecular details of how this virus spreads to new target cells are unclear. This work provides important new information on the formation of filamentous structures that are consistent with virus particles and adds critical new insight into the structure of extensions between cells that form during infection. In addition, it demonstrates for the first time the movement of viral replication centers through these intercellular extensions, representing a new mode of direct cell-to-cell spread that may be applicable to other viral systems.
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Metapneumovirus , Humanos , Idoso , Linhagem Celular , Citoesqueleto , Corpos de Inclusão , VírionRESUMO
Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.
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Bacteriófagos , Salmonella enterica , Animais , Galinhas/microbiologia , Antibacterianos , BrasilRESUMO
Deformed wing virus (DWV) is one of the most common viruses affecting honey bee specimens. Although the presence of DWV has been reported in many countries, there is no data of the current situation in Chile. In this report, we detected the presence of DWV in apiaries from two different locations in central Chile. Furthermore, the genome of a Chilean DWV isolate was completely sequenced. This is the first report of the presence of a honey bee virus in Chile.
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Abelhas/virologia , Genoma Viral , Vírus de Insetos/genética , Picornaviridae/genética , Animais , Sequência de Bases , Chile , Vírus de Insetos/classificação , Vírus de Insetos/isolamento & purificação , Vírus de Insetos/patogenicidade , Filogenia , Picornaviridae/classificação , Picornaviridae/isolamento & purificação , Picornaviridae/patogenicidade , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Asas de Animais/patologia , Asas de Animais/virologiaRESUMO
Control of the COVID-19 pandemic largely depends on the effectiveness of the vaccination process. An understanding of the factors that underlie the willingness to accept vaccination contributes pivotal information to controlling the pandemic. We analyzed the association between the willingness to accept the available COVID-19 vaccines and vaccine determinants amidst the Chilean vaccination process. Individual-level survey data was collected from 744 nationally representative respondents and multivariate regression models were used to estimate the association between outcome and explanatory variables. We found that trust in COVID-19 vaccines, scientists, and medical professionals significantly increased the willingness to: accept the vaccines and booster doses, as well as annual vaccinations and the vaccination of children. Our results are critical to understanding the acceptance of COVID-19 vaccines in the context of a country with one of the world's highest vaccination rates. We provide useful information for decision-making and policy design, in addition to establishing guidelines regarding how to effectively explain vaccination programs to citizens.
RESUMO
Background: Little is known about the interaction between the nasopharyngeal bacterial profile and the nutritional status in children. In this study, our main goal was to evaluate the associations between overnutrition and the presence of four potentially pathogenic bacteria in the nasopharynx of infants with viral lower respiratory tract infections (LRTI). In addition, we determined whether changes in the nasopharyngeal bacterial profile were associated with mucosal and serum proinflammatory cytokines and with clinical disease severity. Methods: We enrolled 116 children less than 2 years old hospitalized for viral LRTI during two consecutive respiratory seasons (May 2016 to August 2017); their nutritional status was assessed, and nasopharyngeal and blood samples were obtained. S. aureus, S. pneumoniae, H. influenzae, M. catarrhalis, and respiratory viruses were identified in nasopharyngeal samples by qPCR. Cytokine concentrations were measured in nasopharyngeal and blood samples. Disease severity was assessed by the length of hospitalization and oxygen therapy. Results: Nasopharyngeal pathogenic bacteria were identified in 96.6% of the enrolled children, and 80% of them tested positive for two or more bacteria. The presence and loads of M. catarrhalis was higher (p = 0.001 and p = 0.022, respectively) in children with overnutrition (n = 47) compared with those with normal weights (n = 69). In addition, the detection of >2 bacteria was more frequent in children with overnutrition compared to those with normal weight (p = 0.02). Multivariate regression models showed that the presence and loads of S. pneumoniae and M. catarrhalis were associated with higher concentrations of IL-6 in plasma and TNF-α in mucosal samples in children with overnutrition. Conclusions: The nasopharyngeal profile of young children with overnutrition was characterized by an over representation of pathogenic bacteria and proinflammatory cytokines.
Assuntos
Hipernutrição , Infecções Respiratórias , Bactérias , Criança , Pré-Escolar , Citocinas , Haemophilus influenzae , Humanos , Lactente , Moraxella catarrhalis , Nasofaringe , Infecções Respiratórias/microbiologia , Staphylococcus aureus , Streptococcus pneumoniaeRESUMO
Human metapneumovirus (HMPV) inclusion bodies (IBs) are dynamic structures required for efficient viral replication and transcription. The minimum components needed to form IB-like structures in cells are the nucleoprotein (N) and the tetrameric phosphoprotein (P). HMPV P binds to the following two versions of the N protein in infected cells: N-terminal P residues interact with monomeric N (N0) to maintain a pool of protein to encapsidate new RNA and C-terminal P residues interact with oligomeric, RNA-bound N (N-RNA). Recent work on other negative-strand viruses has suggested that IBs are, at least in part, liquid-like phase-separated membraneless organelles. Here, HMPV IBs in infected or transfected cells were shown to possess liquid organelle properties, such as fusion and fission. Recombinant versions of HMPV N and P proteins were purified to analyze the interactions required to drive phase separation in vitro. Purified HMPV P was shown to form liquid droplets in isolation. This observation is distinct from other viral systems that also form IBs. Partial removal of nucleic acid from purified P altered phase-separation dynamics, suggesting that nucleic acid interactions play a role in IB formation. HMPV P also recruits monomeric N (N0-P) and N-RNA to droplets in vitro. These findings suggest that HMPV P may also act as a scaffold protein to mediate multivalent interactions with monomeric and oligomeric N, as well as RNA, to promote phase separation of IBs. Together, these findings highlight an additional layer of regulation in HMPV replication by the viral P and N proteins. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of respiratory disease among children, immunocompromised individuals, and the elderly. Currently, no vaccines or antivirals are available for the treatment of HMPV infections. Cytoplasmic inclusion bodies (IBs), where HMPV replication and transcription occur, represent a promising target for the development of novel antivirals. The HMPV nucleoprotein (N) and phosphoprotein (P) are the minimal components needed for IB formation in eukaryotic cells. However, interactions that regulate the formation of these dynamic structures are poorly understood. Here, we showed that HMPV IBs possess the properties of liquid organelles and that purified HMPV P phase separates independently in vitro. Our work suggests that HMPV P phase-separation dynamics are altered by nucleic acid. We provide strong evidence that, unlike results reported from other viral systems, HMPV P alone can serve as a scaffold for multivalent interactions with monomeric (N0) and oligomeric (N-RNA) HMPV N for IB formation.
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Corpos de Inclusão Viral , Metapneumovirus , Ácidos Nucleicos , Humanos , Antivirais , Metapneumovirus/genética , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA , Replicação ViralRESUMO
Hantaviruses infect human cells through cell attachment and subsequent fusion of viral and cellular membranes at low pH. This largely unknown entry process is mediated by the Gn and Gc glycoproteins, anchored at the viral envelope membrane. Performing bioinformatic analysis and peptide-liposome-binding assays we suggested in a former report that Gc of Andes virus (ANDV) and other hantaviruses corresponds to the viral fusion protein sharing characteristics with class II fusion proteins. To gain insights into the fusion protein of hantaviruses, residues within the previously predicted fusion peptide of ANDV Gc were substituted and mutant proteins tested in fusion and infection assays. To ensure proper folding of mutant proteins, they were first characterized for trafficking to the plasma membrane and incorporation on to ANDV Gn/Gc-pseudotyped lentiviral particles. Cell attachment of these particles was assessed using a newly developed binding assay and their subsequent entry properties determined by FACS analysis of transduced cells expressing the GFP reporter gene. Furthermore, a three-colour-based cell-cell fusion assay of ANDV Gn/Gc expressing cells was performed. The results indicate an essential role of conserved Gc residues W115 and N118 in membrane fusion. Conversely, substitutions of the non-conserved Gc residue G116 did not considerably affect fusion and infection. Altogether, the findings are fully consistent with our earlier prediction suggesting Gc residues 115-121 as an internal fusion peptide and further emphasize the importance of aromatic and polar residues in hantavirus-cell membrane fusion.
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Aminoácidos/genética , Fusão de Membrana , Orthohantavírus/patogenicidade , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Substituição de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Fusão Celular , Linhagem Celular , Chlorocebus aethiops , Citometria de Fluxo/métodos , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Coloração e Rotulagem/métodosRESUMO
A critical step in the life cycle of a virus is spread to a new target cell, which generally involves the release of new viral particles from the infected cell which can then initiate infection in the next target cell. While cell-free viral particles released into the extracellular environment are necessary for long distance spread, there are disadvantages to this mechanism. These include the presence of immune system components, the low success rate of infection by single particles, and the relative fragility of viral particles in the environment. Several mechanisms of direct cell-to-cell spread have been reported for animal viruses which would avoid the issues associated with cell-free particles. A number of viruses can utilize several different mechanisms of direct cell-to-cell spread, but our understanding of the differential usage by these pathogens is modest. Although the mechanisms of cell-to-cell spread differ among viruses, there is a common exploitation of key pathways and components of the cellular cytoskeleton. Remarkably, some of the viral mechanisms of cell-to-cell spread are surprisingly similar to those used by bacteria. Here we summarize the current knowledge of the conventional and non-conventional mechanisms of viral spread, the common methods used to detect viral spread, and the impact that these mechanisms can have on viral pathogenesis.
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Células/virologia , Vírion/fisiologia , Fenômenos Fisiológicos Virais , Replicação Viral/fisiologia , Animais , Livros , Humanos , Vírus/patogenicidadeRESUMO
Pneumoviruses represent a major public health burden across the world. Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV), two of the most recognizable pediatric infectious agents, belong to this family. These viruses are enveloped with a non-segmented negative-sense RNA genome, and their replication occurs in specialized cytosolic organelles named inclusion bodies (IB). The critical role of IBs in replication of pneumoviruses has begun to be elucidated, and our current understanding suggests they are highly dynamic structures. From IBs, newly synthesized nucleocapsids are transported to assembly sites, potentially via the actin cytoskeleton, to be incorporated into nascent virions. Released virions, which generally contain one genome, can then diffuse in the extracellular environment to target new cells and reinitiate the process of infection. This is a challenging business for virions, which must face several risks including the extracellular immune responses. In addition, several recent studies suggest that successful infection may be achieved more rapidly by multiple, rather than single, genomic copies being deposited into a target cell. Interestingly, recent data indicate that pneumoviruses have several mechanisms that permit their transmission en bloc, i.e. transmission of multiple genomes at the same time. These mechanisms include the well-studied syncytia formation as well as the newly described formation of long actin-based intercellular extensions. These not only permit en bloc viral transmission, but also bypass assembly of complete virions. In this review we describe several aspects of en bloc viral transmission and how these mechanisms are reshaping our understanding of pneumovirus replication, assembly and spread.
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Infecções por Paramyxoviridae/transmissão , Pneumovirus/fisiologia , Montagem de Vírus , Animais , Linhagem Celular , Humanos , Metapneumovirus/genética , Metapneumovirus/fisiologia , Camundongos , Pneumovirus/genética , RNA Viral , Vírion/genética , Vírion/fisiologia , Replicação ViralRESUMO
In recent years, ultrastructural studies of viral surface spikes from three different genera within the Bunyaviridae family have revealed a remarkable diversity in their spike organization. Despite this structural heterogeneity, in every case the spikes seem to be composed of heterodimers formed by Gn and Gc envelope glycoproteins. In this review, current knowledge of the Gn and Gc structures and their functions in virus cell entry and exit is summarized. During virus cell entry, the role of Gn and Gc in receptor binding has not yet been determined. Nevertheless, biochemical studies suggest that the subsequent virus-membrane fusion activity is accomplished by Gc. Further, a class II fusion protein conformation has been predicted for Gc of hantaviruses, and novel crystallographic data confirmed such a fold for the Rift Valley fever virus (RVFV) Gc protein. During virus cell exit, the assembly of different viral components seems to be established by interaction of Gn and Gc cytoplasmic tails (CT) with internal viral ribonucleocapsids. Moreover, recent findings show that hantavirus glycoproteins accomplish important roles during virus budding since they self-assemble into virus-like particles. Collectively, these novel insights provide essential information for gaining a more detailed understanding of Gn and Gc functions in the early and late steps of the hantavirus infection cycle.
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Glicoproteínas/metabolismo , Orthohantavírus/fisiologia , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Internalização do VírusRESUMO
To infect target cells, enveloped viruses use their virion surface proteins to direct cell attachment and subsequent entry via virus-cell membrane fusion. How hantaviruses enter cells has been largely unexplored. To study early steps of Andes virus (ANDV) cell infection, a lentiviral vector system was developed based on a Simian immunodeficiency virus (SIV) vector pseudotyped with the ANDV-Gn/Gc envelope glycoproteins. The incorporation of Gn and Gc onto SIV-derived vector particles was assessed using newly generated monoclonal antibodies against ANDV glycoproteins. In addition, sera of ANDV infected humans were able to block cell entry of the SIV vector pseudotyped with ANDV glycoproteins, suggesting that their antigenic conformation is similar to that in the native virus. The use of such SIV vector pseudotyped with ANDV-Gn/Gc glycoproteins should facilitate studies on ANDV cell entry. Along this line, it was found that depletion of cholesterol from target cells strongly diminished cell infection, indicating a possible role of lipid rafts in ANDV cell entry. The Gn/Gc pseudotyped SIV vector has several advantages, notably high titer vector production and easy quantification of cell infection by monitoring GFP reporter gene expression by flow cytometry. Such pseudotyped SIV vectors can be used to identify functional domains in the Gn/Gc glycoproteins and to screen for potential hantavirus cell entry inhibitors.