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1.
J Virol ; 95(15): e0069221, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980596

RESUMO

Swine influenza virus (SIV) can cause respiratory illness in swine. Swine contribute to influenza virus reassortment, as avian, human, and/or swine influenza viruses can infect swine and reassort, and new viruses can emerge. Thus, it is important to determine the host antiviral responses that affect SIV replication. In this study, we examined the innate antiviral cytokine response to SIV by swine respiratory epithelial cells, focusing on the expression of interferon (IFN) and interferon-stimulated genes (ISGs). Both primary and transformed swine nasal and tracheal respiratory epithelial cells were examined following infection with field isolates. The results show that IFN and ISG expression is maximal at 12 h postinfection (hpi) and is dependent on cell type and virus genotype. IMPORTANCE Swine are considered intermediate hosts that have facilitated influenza virus reassortment events that have given rise pandemics or genetically related viruses have become established in swine. In this study, we examine the innate antiviral response to swine influenza virus in primary and immortalized swine nasal and tracheal epithelial cells, and show virus strain- and host cell type-dependent differential expression of key interferons and interferon-stimulated genes.


Assuntos
Citocinas/metabolismo , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N2/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Mucosa Respiratória/imunologia , Animais , Linhagem Celular , Citocinas/imunologia , Cães , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/imunologia , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Interferons/imunologia , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/imunologia , Mucosa Respiratória/citologia , Suínos , Replicação Viral/fisiologia
2.
PLoS Pathog ; 12(8): e1005804, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27505057

RESUMO

The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVß6 integrin, which is upregulated during injury. Once expressed, αVß6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of ß6 during influenza infection is involved in disease pathogenesis. ß6-deficient mice (ß6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the ß6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of ß6-activated transforming growth factor-ß (TGF-ß). Administration of exogenous TGF-ß to ß6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVß6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival.


Assuntos
Antígenos de Neoplasias/imunologia , Integrinas/imunologia , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Pulmão/imunologia , Infecções Respiratórias/imunologia , Transferência Adotiva , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Immunoblotting , Pulmão/microbiologia , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real
3.
J Virol ; 90(19): 8454-63, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27440882

RESUMO

UNLABELLED: The only licensed live attenuated influenza A virus vaccines (LAIVs) in the United States (FluMist) are created using internal protein-coding gene segments from the cold-adapted temperature-sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature-sensitive phenotype can revert and, if so, the types of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperatures in vitro generated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations, PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect the temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the roles of individual mutations were characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild-type 2009 H1N1 pandemic virus. IMPORTANCE: The live attenuated influenza vaccine FluMist has been proven safe and effective and is widely used in the United States. The phenotype and genotype of the vaccine virus are believed to be very stable, and mutants that cause disease in animals or humans have never been reported. By propagating the virus under well-controlled laboratory conditions, we found that the FluMist vaccine backbone could regain virulence to cause severe disease in mice. The identification of the responsible substitutions and elucidation of the underlying mechanisms provide unique insights into the attenuation of influenza virus, which is important to basic research on vaccines, attenuation reversion, and replication. In addition, this study suggests that the safety of LAIVs should be closely monitored after mass vaccination and that novel strategies to continue to improve LAIV vaccine safety should be investigated.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vacinas contra Influenza/genética , Animais , Modelos Animais de Doenças , Camundongos , Orthomyxoviridae , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , RNA Viral/genética , Genética Reversa , Análise de Sequência de DNA , Inoculações Seriadas , Supressão Genética , Temperatura , Vacinas Atenuadas/genética , Virulência , Replicação Viral
4.
PLoS Pathog ; 11(2): e1004642, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25668410

RESUMO

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8(+) cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K(b)PB(1703, D(b)PA(224), and D(b)NP(366) epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.


Assuntos
Epitopos de Linfócito T/imunologia , Imunidade Heteróloga/imunologia , Epitopos Imunodominantes/imunologia , Memória Imunológica/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
5.
J Biol Chem ; 289(51): 35246-63, 2014 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-25339175

RESUMO

Influenza infection exacerbates chronic pulmonary diseases, including idiopathic pulmonary fibrosis. A central pathway in the pathogenesis of idiopathic pulmonary fibrosis is epithelial injury leading to activation of transforming growth factor ß (TGFß). The mechanism and functional consequences of influenza-induced activation of epithelial TGFß are unclear. Influenza stimulates toll-like receptor 3 (TLR3), which can increase RhoA activity, a key event prior to activation of TGFß by the αvß6 integrin. We hypothesized that influenza would stimulate TLR3 leading to activation of latent TGFß via αvß6 integrin in epithelial cells. Using H1152 (IC50 6.1 µm) to inhibit Rho kinase and 6.3G9 to inhibit αvß6 integrins, we demonstrate their involvement in influenza (A/PR/8/34 H1N1) and poly(I:C)-induced TGFß activation. We confirm the involvement of TLR3 in this process using chloroquine (IC50 11.9 µm) and a dominant negative TLR3 construct (pZERO-hTLR3). Examination of lungs from influenza-infected mice revealed augmented levels of collagen deposition, phosphorylated Smad2/3, αvß6 integrin, and apoptotic cells. Finally, we demonstrate that αvß6 integrin-mediated TGFß activity following influenza infection promotes epithelial cell death in vitro and enhanced collagen deposition in vivo and that this response is diminished in Smad3 knock-out mice. These data show that H1N1 and poly(I:C) can induce αvß6 integrin-dependent TGFß activity in epithelial cells via stimulation of TLR3 and suggest a novel mechanism by which influenza infection may promote collagen deposition in fibrotic lung disease.


Assuntos
Antígenos de Neoplasias/metabolismo , Colágeno/metabolismo , Células Epiteliais/metabolismo , Integrinas/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Fator de Crescimento Transformador beta/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Antígenos de Neoplasias/genética , Antivirais/farmacologia , Apoptose , Linhagem Celular Transformada , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Humanos , Immunoblotting , Vírus da Influenza A Subtipo H1N1/fisiologia , Integrinas/genética , Pulmão/metabolismo , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , Fosforilação/efeitos dos fármacos , Poli I-C/farmacologia , Proteína Smad3/genética , Proteína Smad3/metabolismo , Receptor 3 Toll-Like/metabolismo , Fator de Crescimento Transformador beta/genética , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
6.
J Virol ; 88(22): 12982-91, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25210188

RESUMO

UNLABELLED: Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models. IMPORTANCE: The novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology, leading in some cases to the development of acute respiratory distress syndrome, is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo models, and found that H5N1 and H7N9 infections exhibit distinct pathological, immunological, and tissue tropism differences that could explain differences in clinical disease and viral transmission.


Assuntos
Citocinas/metabolismo , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/fisiologia , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Tropismo Viral , Animais , Linhagem Celular , Citocinas/toxicidade , Modelos Animais de Doenças , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/imunologia , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/patologia , Análise de Sobrevida
8.
Cell Rep ; 43(3): 113965, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38492217

RESUMO

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , DNA Helicases/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Virulência , RNA Guia de Sistemas CRISPR-Cas , Proteínas do Nucleocapsídeo , Replicação Viral , RNA Viral/genética
9.
FASEB J ; 26(4): 1372-86, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22247330

RESUMO

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Vírus da Influenza A/genética , Influenza Humana/terapia , Interferência de RNA , Proteínas Virais/genética , Animais , Humanos , Metanálise como Assunto , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia
10.
mBio ; 14(4): e0088723, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37341495

RESUMO

Obesity has been epidemiologically and empirically linked with more severe diseases upon influenza infection. To ameliorate severe disease, treatment with antivirals, such as the neuraminidase inhibitor oseltamivir, is suggested to begin within days of infection especially in high-risk hosts. However, this treatment can be poorly effective and may generate resistance variants within the treated host. Here, we hypothesized that obesity would reduce oseltamivir treatment effectiveness in the genetically obese mouse model. We demonstrated that oseltamivir treatment does not improve viral clearance in obese mice. While no traditional variants associated with oseltamivir resistance emerged, we did note that drug treatment failed to quench the viral population and did lead to phenotypic drug resistance in vitro. Together, these studies suggest that the unique pathogenesis and immune responses in obese mice could have implications for pharmaceutical interventions and the within-host dynamics of the influenza virus population. IMPORTANCE Influenza virus infections, while typically resolving within days to weeks, can turn critical, especially in high-risk populations. Prompt antiviral administration is crucial to mitigating these severe sequalae, yet concerns remain if antiviral treatment is effective in hosts with obesity. Here, we show that oseltamivir does not improve viral clearance in genetically obese or type I interferon receptor-deficient mice. This suggests a blunted immune response may impair oseltamivir efficacy and render a host more susceptible to severe disease. This study furthers our understanding of oseltamivir treatment dynamics both systemically and in the lungs of obese mice, as well as the consequences of oseltamivir treatment for the within-host emergence of drug-resistant variants.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Camundongos , Animais , Humanos , Oseltamivir/uso terapêutico , Camundongos Obesos , Influenza Humana/tratamento farmacológico , Antivirais/uso terapêutico , Antivirais/farmacologia , Neuraminidase , Farmacorresistência Viral
11.
bioRxiv ; 2023 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-37425880

RESUMO

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

12.
Artigo em Inglês | MEDLINE | ID: mdl-31871227

RESUMO

Factoring significantly into the global burden of influenza disease are high-risk populations that suffer the bulk of infections. Classically, the very young, very old, and pregnant women have been identified as high-risk populations; however, recent research has uncovered several other conditions that contribute to severe infection. By using varied animal models, researchers have identified molecular mechanisms underpinning the increased likelihood for infection due to obesity and malnourishment, as well as insight into the role sex hormones play in antiviral immunity in males, in females, and across the life span. Additionally, novel comorbidity models have helped elucidate the role of chronic infectious and genetic diseases in influenza virus pathogenesis. Animal models play a vital role in understanding the contribution of host factors to influenza severity and immunity. An in-depth understanding of these host factors represents an important step in reducing the burden of influenza among the growing number of people living with one or more chronic medical conditions.


Assuntos
Modelos Animais de Doenças , Vírus da Influenza A/genética , Influenza Humana/genética , Animais , Feminino , Humanos , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Influenza Humana/complicações , Influenza Humana/prevenção & controle , Masculino , Camundongos , Gravidez , Medição de Risco , Fatores Sexuais
13.
mBio ; 11(2)2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127459

RESUMO

Obesity is associated with increased disease severity, elevated viral titers in exhaled breath, and significantly prolonged viral shed during influenza A virus infection. Due to the mutable nature of RNA viruses, we questioned whether obesity could also influence influenza virus population diversity. Here, we show that minor variants rapidly emerge in obese mice. The variants exhibit increased viral replication, resulting in enhanced virulence in wild-type mice. The increased diversity of the viral population correlated with decreased type I interferon responses, and treatment of obese mice with recombinant interferon reduced viral diversity, suggesting that the delayed antiviral response exhibited in obesity permits the emergence of a more virulent influenza virus population. This is not unique to obese mice. Obesity-derived normal human bronchial epithelial (NHBE) cells also showed decreased interferon responses and increased viral replication, suggesting that viral diversity also was impacted in this increasing population.IMPORTANCE Currently, 50% of the adult population worldwide is overweight or obese. In these studies, we demonstrate that obesity not only enhances the severity of influenza infection but also impacts viral diversity. The altered microenvironment associated with obesity supports a more diverse viral quasispecies and affords the emergence of potentially pathogenic variants capable of inducing greater disease severity in lean hosts. This is likely due to the impaired interferon response, which is seen in both obese mice and obesity-derived human bronchial epithelial cells, suggesting that obesity, aside from its impact on influenza virus pathogenesis, permits the stochastic accumulation of potentially pathogenic viral variants, raising concerns about its public health impact as the prevalence of obesity continues to rise.


Assuntos
Suscetibilidade a Doenças , Vírus da Influenza A/fisiologia , Influenza Humana/etiologia , Obesidade/complicações , Animais , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/metabolismo , Camundongos , Mutação , Fenótipo , RNA Viral , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Índice de Gravidade de Doença , Virulência , Replicação Viral
14.
mSystems ; 5(5)2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32873612

RESUMO

Influenza A virus (IAV) is a major pathogen of the human respiratory tract, where the virus coexists and interacts with bacterial populations comprising the respiratory tract microbiome. Synergies between IAV and respiratory bacterial pathogens promote enhanced inflammation and disease burden that exacerbate morbidity and mortality. We demonstrate that direct interactions between IAV and encapsulated bacteria commonly found in the respiratory tract promote environmental stability and infectivity of IAV. Antibiotic-mediated depletion of the respiratory bacterial flora abrogated IAV transmission in ferret models, indicating that these virus-bacterium interactions are operative for airborne transmission of IAV. Restoring IAV airborne transmission in antibiotic-treated ferrets by coinfection with Streptococcus pneumoniae confirmed a role for specific members of the bacterial respiratory community in promoting IAV transmission. These results implicate a role for the bacterial respiratory flora in promoting airborne transmission of IAV.IMPORTANCE Infection with influenza A virus (IAV), especially when complicated with a secondary bacterial infection, is a leading cause of global mortality and morbidity. Gaining a greater understanding of the transmission dynamics of IAV is important during seasonal IAV epidemics and in the event of a pandemic. Direct bacterium-virus interactions are a recently appreciated aspect of infectious disease biology. Direct interactions between IAV and specific bacterial species of the human upper respiratory tract were found to promote the stability and infectivity of IAV during desiccation stress. Viral environmental stability is an important aspect during transmission, suggesting a potential role for bacterial respiratory communities in IAV transmission. Airborne transmission of IAV was abrogated upon depletion of nasal bacterial flora with topical antibiotics. This defect could be functionally complemented by S. pneumoniae coinfection. These data suggest that bacterial coinfection may be an underappreciated aspect of IAV transmission dynamics.

15.
Nat Microbiol ; 4(8): 1328-1336, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31110359

RESUMO

Epidemiological observations and animal models have long shown synergy between influenza virus infections and bacterial infections. Influenza virus infection leads to an increase in both the susceptibility to secondary bacterial infections and the severity of the bacterial infections, primarily pneumonias caused by Streptococcus pneumoniae or Staphylococcus aureus. We show that, in addition to the widely described immune modulation and tissue-remodelling mechanisms of bacterial-viral synergy, the virus interacts directly with the bacterial surface. Similar to the recent observation of direct interactions between enteric bacteria and enteric viruses, we observed a direct interaction between influenza virus on the surface of Gram-positive, S. pneumoniae and S. aureus, and Gram-negative, Moraxella catarrhalis and non-typeable Haemophilus influenzae, bacterial colonizers and pathogens in the respiratory tract. Pre-incubation of influenza virus with bacteria, followed by the removal of unbound virus, increased bacterial adherence to respiratory epithelial cells in culture. This result was recapitulated in vivo, with higher bacterial burdens in murine tissues when infected with pneumococci pre-incubated with influenza virus versus control bacteria without virus. These observations support an additional mechanism of bacteria-influenza virus synergy at the earliest steps of pathogenesis.


Assuntos
Aderência Bacteriana/fisiologia , Coinfecção , Interações Microbianas/fisiologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/virologia , Células A549 , Animais , Bactérias , Infecções Bacterianas/complicações , Infecções Bacterianas/microbiologia , Infecções Bacterianas/virologia , Modelos Animais de Doenças , Feminino , Humanos , Influenza Humana , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/complicações , Infecções Respiratórias/complicações , Infecções Estafilocócicas/complicações , Staphylococcus aureus , Streptococcus pneumoniae
16.
Methods Mol Biol ; 1836: 431-459, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30151586

RESUMO

To streamline standard virological assays, we developed bioluminescent replication-competent influenza reporter viruses that mimic their parental counterparts. These reporter viruses provide a rapid and quantitative readout of viral infection and replication. Moreover, they permit real-time in vivo measures of viral load, tissue distribution, and transmission in the same cohort of animals over the entire course of infection-measurements that were not previously possible. Here we provide detailed protocols using bioluminescent reporter viruses for in vivo imaging in mice and ferrets. We also describe cell culture-based techniques using reporter viruses for quantification of viral titers and performing microneutralization assays. The ease, speed, and adaptability of these approaches have the potential to accelerate multiple areas of influenza virus research.


Assuntos
Genes Reporter , Medições Luminescentes , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/fisiologia , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Furões , Humanos , Medições Luminescentes/métodos , Masculino , Camundongos , Testes de Neutralização , Carga Viral
17.
Viruses ; 9(1)2017 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-28117758

RESUMO

Astroviruses are a major cause of diarrhea in the young, elderly, and the immunocompromised. Since the discovery of human astrovirus type 1 (HAstV-1) in 1975, the family Astroviridae has expanded to include two more human clades and numerous mammalian and avian-specific genotypes. Despite this, there is still little known about pathogenesis. The following review highlights the current knowledge of astrovirus pathogenesis, and outlines the critical steps needed to further astrovirus research, including the development of animal models of cell culture systems.


Assuntos
Infecções por Astroviridae/virologia , Astroviridae/fisiologia , Interações Hospedeiro-Patógeno , Animais , Humanos
18.
Annu Rev Virol ; 4(1): 327-348, 2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28715976

RESUMO

Astroviruses are nonenveloped, positive-sense single-stranded RNA viruses that cause gastrointestinal illness. Although a leading cause of pediatric diarrhea, human astroviruses are among the least characterized enteric RNA viruses. However, by using in vitro methods and animal models to characterize virus-host interactions, researchers have discovered several important properties of astroviruses, including the ability of the astrovirus capsid to act as an enterotoxin, disrupting the gut epithelial barrier. Improved animal models are needed to study this phenomenon, along with the pathogenesis of astroviruses, particularly in those strains that can cause extraintestinal disease. Much like for other enteric viruses, the current dogma states that astroviruses infect in a species-specific manner; however, this assumption is being challenged by growing evidence that these viruses have potential to cross species barriers. This review summarizes these remarkable facets of astrovirus biology, highlighting critical steps toward increasing our understanding of this unique enteric pathogen.


Assuntos
Infecções por Astroviridae/virologia , Gastroenterite/virologia , Mamastrovirus/fisiologia , Mamastrovirus/patogenicidade , Animais , Infecções por Astroviridae/fisiopatologia , Infecções por Astroviridae/veterinária , Capsídeo/metabolismo , Galinhas/virologia , Diarreia/virologia , Modelos Animais de Doenças , Gastroenterite/fisiopatologia , Humanos , Mucosa Intestinal/fisiopatologia , Mucosa Intestinal/virologia , Mamastrovirus/genética , Camundongos , Permeabilidade , Filogenia , Especificidade da Espécie , Suínos/virologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-28638805

RESUMO

Bacteria growing within biofilms are protected from antibiotics and the immune system. Within these structures, horizontal transfer of genes encoding virulence factors, and promoting antibiotic resistance occurs, making biofilms an extremely important aspect of pneumococcal colonization and persistence. Identifying environmental cues that contribute to the formation of biofilms is critical to understanding pneumococcal colonization and infection. Iron has been shown to be essential for the formation of pneumococcal biofilms; however, the role of other physiologically important metals such as copper, zinc, and manganese has been largely neglected. In this study, we investigated the effect of metals on pneumococcal aggregation and early biofilm formation. Our results show that biofilms increase as zinc concentrations increase. The effect was found to be zinc-specific, as altering copper and manganese concentrations did not affect biofilm formation. Scanning electron microscopy analysis revealed structural differences between biofilms grown in varying concentrations of zinc. Analysis of biofilm formation in a mutant strain lacking the peroxide-generating enzyme pyruvate oxidase, SpxB, revealed that zinc does not protect against pneumococcal H2O2. Further, analysis of a mutant strain lacking the major autolysin, LytA, indicated the role of zinc as a negative regulator of LytA-dependent autolysis, which could affect biofilm formation. Additionally, analysis of cell-cell aggregation via plating and microscopy revealed that high concentrations of zinc contribute to intercellular interaction of pneumococci. The findings from this study demonstrate that metal availability contributes to the ability of pneumococci to form aggregates and subsequently, biofilms.


Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/metabolismo , Zinco/farmacologia , Animais , Autólise/microbiologia , Linhagem Celular , Cobre/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Humanos , Peróxido de Hidrogênio , Manganês/metabolismo , Camundongos Endogâmicos C57BL , Viabilidade Microbiana , Microscopia Eletrônica de Varredura , Mutação , N-Acetil-Muramil-L-Alanina Amidase/genética , Piruvato Oxidase/metabolismo , Streptococcus pneumoniae/patogenicidade , Fatores de Virulência
20.
mBio ; 8(5)2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28928207

RESUMO

Obesity is a risk factor for developing severe disease following influenza virus infection; however, the comorbidity of obesity and secondary bacterial infection, a serious complication of influenza virus infections, is unknown. To fill this gap in knowledge, lean and obese C57BL/6 mice were infected with a nonlethal dose of influenza virus followed by a nonlethal dose of Streptococcus pneumoniae Strikingly, not only did significantly enhanced death occur in obese coinfected mice compared to lean controls, but also high mortality was seen irrespective of influenza virus strain, bacterial strain, or timing of coinfection. This result was unexpected, given that most influenza virus strains, especially seasonal human A and B viruses, are nonlethal in this model. Both viral and bacterial titers were increased in the upper respiratory tract and lungs of obese animals as early as days 1 and 2 post-bacterial infection, leading to a significant decrease in lung function. This increased bacterial load correlated with extensive cellular damage and upregulation of platelet-activating factor receptor, a host receptor central to pneumococcal invasion. Importantly, while vaccination of obese mice against either influenza virus or pneumococcus failed to confer protection, antibiotic treatment was able to resolve secondary bacterial infection-associated mortality. Overall, secondary bacterial pneumonia could be a widespread, unaddressed public health problem in an increasingly obese population.IMPORTANCE Worldwide obesity rates have continued to increase. Obesity is associated with increased severity of influenza virus infection; however, very little is known about respiratory coinfections in this expanding, high-risk population. Our studies utilized a coinfection model to show that obesity increases mortality from secondary bacterial infection following influenza virus challenge through a "perfect storm" of host factors that lead to excessive viral and bacterial outgrowth. In addition, we found that vaccination of obese mice against either virus or bacteria failed to confer protection against coinfection, but antibiotic treatment did alleviate mortality. Combined, these results represent an understudied and imminent public health concern in a weighty portion of the global population.


Assuntos
Coinfecção/etiologia , Vírus da Influenza A/isolamento & purificação , Vacinas contra Influenza/administração & dosagem , Obesidade/complicações , Infecções por Orthomyxoviridae/complicações , Vacinas Pneumocócicas/administração & dosagem , Animais , Coinfecção/microbiologia , Coinfecção/virologia , Comorbidade , Vírus da Influenza A/crescimento & desenvolvimento , Pulmão/microbiologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/microbiologia , Obesidade/virologia , Infecções por Orthomyxoviridae/microbiologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/prevenção & controle , Infecções Pneumocócicas/virologia , Falha de Tratamento , Vacinação
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