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1.
Comp Immunol Microbiol Infect Dis ; 65: 213-218, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31300116

RESUMO

Human respiratory syncytial virus (hRSV) is the most important respiratory pathogen in young children worldwide. Experimental modelling of hRSV disease by bovine RSV (bRSV) infection in calves provides an important tool for developing new strategies for prevention and treatment. Depending on the scientific hypothesis under investigation, this cognate host-virus model might have the disadvantage of using a highly related but not genetically identical virus. In this study, we aim to describe viral kinetics and (clinical) disease characteristics in calves inoculated with hRSV. Our results show that hRSV infects the upper and, to a lesser extent, the lower respiratory tract of calves. Infection causes upper airway clinical disease symptoms and neutrophilic infiltration of the lower airways. We conclude that a hRSV model in calves may aid future research involving distinct scientific questions related to hRSV disease in children.


Assuntos
Modelos Animais de Doenças , Interações entre Hospedeiro e Microrganismos , Infecções por Vírus Respiratório Sincicial/veterinária , Vírus Sincicial Respiratório Bovino/fisiologia , Vírus Sincicial Respiratório Humano/fisiologia , Infecções Respiratórias/veterinária , Fatores Etários , Animais , Bovinos , Feminino , Humanos , Cinética , Pulmão/imunologia , Pulmão/virologia , Masculino , Projetos Piloto , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia
2.
Immunol Allergy Clin North Am ; 39(3): 321-334, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31284923

RESUMO

Severe lower respiratory tract infection in infants and young children is most frequently caused by respiratory syncytial virus (RSV). RSV infects the smallest airways, making breathing difficult and in some infants requiring medical support. Severity is affected by viral dose, infant age, virus genotype, and effectiveness of the innate/adaptive immune responses. Severe disease correlates with later wheezing and asthma in some children. The adaptive immune response is protective but wanes after each infection, likely due to the ability of the RSV NS1/NS2 proteins to inhibit the innate immune response. Several vaccine approaches and candidates are currently in clinical trials.


Assuntos
Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Antígenos Virais/química , Antígenos Virais/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/imunologia , Índice de Gravidade de Doença , Proteínas Virais/química , Proteínas Virais/imunologia , Vacinas Virais , Montagem de Vírus
3.
Virology ; 534: 1-13, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31163351

RESUMO

Alum adjuvanted formalin-inactivated respiratory syncytial virus (RSV) vaccination resulted in enhanced respiratory disease in young children upon natural infection. Here, we investigated the adjuvant effects of monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG (CpG) on vaccine-enhanced respiratory disease after fusion (F) protein prime vaccination and RSV challenge in infant and adult mouse models. Combination CpG + MPL adjuvant in RSV F protein single dose priming of infant and adult age mice was found to promote the induction of IgG2a isotype antibodies and neutralizing activity, and lung viral clearance after challenge. CpG + MPL adjuvanted F protein (Fp) priming of infant and adult age mice was effective in avoiding lung histopathology, in reducing interleukin-4+ CD4 T cells and cellular infiltration of monocytes and neutrophils after RSV challenge. This study suggests that combination CpG and MPL adjuvant in RSV subunit vaccination might contribute to priming protective immune responses and preventing inflammatory RSV disease after infection.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/administração & dosagem , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/fisiologia , Vacinação , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia
4.
Arch Virol ; 164(9): 2231-2241, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31177351

RESUMO

Viral persistence alters cellular antiviral activities. Nitric oxide (NO), a highly reactive free radical and a potent antiviral molecule, can inhibit replication of RNA and DNA viruses, but its production and effect during viral persistence are largely unknown. NO synthesis is stimulated in epithelial cells during acute infection with respiratory syncytial virus (RSV) and interferes with viral replication. In this study, we compared the levels of production of NO and expression of its regulatory enzymes, inducible nitric oxide synthase (NOS II) and arginase 1 (Arg-1), during acute and persistent RSV infection in a macrophage cell line to investigate their role in the control and maintenance of viral infection. We observed that NO and NOS II mRNA were induced at higher levels in acutely infected macrophages than in persistently infected macrophages, while the kinetics of NOS II protein expression were similar in both types of infected cultures, except that its disappearance was delayed during acute infection. Thus, NOS II was inducible and expressed at high levels during persistent infection, but production of NO was low relative to acute infection. This was not associated with a lack of enzymatic activity but instead was due to constitutive expression of the Arg-1 enzyme at the mRNA and protein levels, suggesting that arginase restricts availability of L-arginine as a substrate for NOS II to synthesize NO. This hypothesis was supported by showing that arginase enzymatic activity was inhibited in persistently RSV-infected cells by Nω-hydroxy-nor-L-arginine, increasing L-arginine availability in conditioned medium and producing increased levels of nitrites, concurrently with a significant reduction in virus genome replication, implying that Arg-1 overexpression contributes to the maintenance of the RSV genome in the host in persistent infection.


Assuntos
Arginase/metabolismo , Óxido Nítrico/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Arginase/genética , Arginina/metabolismo , Regulação para Baixo , Humanos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Infecções por Vírus Respiratório Sincicial/enzimologia , Infecções por Vírus Respiratório Sincicial/genética , Vírus Sincicial Respiratório Humano/genética , Replicação Viral
5.
Nat Commun ; 10(1): 2105, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068578

RESUMO

The respiratory syncytial virus (RSV) F glycoprotein is a class I fusion protein that mediates viral entry and is a major target of neutralizing antibodies. Structures of prefusion forms of RSV F, as well as other class I fusion proteins, have revealed compact trimeric arrangements, yet whether these trimeric forms can transiently open remains unknown. Here, we perform structural and biochemical studies on a recently isolated antibody, CR9501, and demonstrate that it enhances the opening of prefusion-stabilized RSV F trimers. The 3.3 Å crystal structure of monomeric RSV F bound to CR9501, combined with analysis of over 25 previously determined RSV F structures, reveals a breathing motion of the prefusion conformation. We also demonstrate that full-length RSV F trimers transiently open and dissociate on the cell surface. Collectively, these findings have implications for the function of class I fusion proteins, as well as antibody prophylaxis and vaccine development for RSV.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Virais de Fusão/metabolismo , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Linfócitos B/virologia , Simulação por Computador , Cristalografia por Raios X , Desenvolvimento de Medicamentos , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Multimerização Proteica/fisiologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia
6.
Biomolecules ; 9(5)2019 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035368

RESUMO

Human respiratory syncytial virus (HRSV) infection is a common cause of severe lower respiratory tract diseases such as bronchiolitis and pneumonia. Both virus replication and the associated inflammatory immune response are believed to be behind these pathologies. So far, no vaccine or effective treatment is available for this viral infection. With the aim of finding new strategies to counteract HRSV replication and modulate the immune response, specific small interfering RNAs (siRNAs) were generated targeting the mRNA coding for the viral fusion (F) protein or nucleoprotein (N), or for two proteins involved in intracellular immune signaling, which are named tripartite motif-containing protein 25 (TRIM25) and retinoic acid-inducible gene-I (RIG-I). Furthermore, two additional bispecific siRNAs were designed that silenced F and TRIM25 (TRIM25/HRSV-F) or N and RIG-I (RIG-I/HRSV-N) simultaneously. All siRNAs targeting N or F, but not those silencing TRIM25 or RIG-I alone, significantly reduced viral titers. However, while siRNAs targeting F inhibited only the expression of the F mRNA and protein, the siRNAs targeting N led to a general inhibition of viral mRNA and protein expression. The N-targeting siRNAs also induced a drastic decrease in the expression of genes of the innate immune response. These results show that both virus replication and the early innate immune response can be regulated by targeting distinct viral products with siRNAs, which may be related to the different role of each protein in the life cycle of the virus.


Assuntos
Imunidade Inata , RNA Interferente Pequeno/metabolismo , Vírus Sincicial Respiratório Humano/imunologia , Vírus Sincicial Respiratório Humano/fisiologia , Replicação Viral/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica , Inativação Gênica , Genoma Viral , Humanos , Imunidade Inata/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Proteínas Virais/genética , Proteínas Virais/metabolismo
7.
PLoS One ; 14(4): e0214708, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30986239

RESUMO

Many aspects of the respiratory syncytial virus (RSV) are still poorly understood. Yet these knowledge gaps have had and could continue to have adverse, unintended consequences for the efficacy and safety of antivirals and vaccines developed against RSV. Mathematical modelling was used to test and evaluate hypotheses about the rate of loss of RSV infectivity and the mechanisms and kinetics of RSV infection spread in SIAT cells in vitro. While the rate of loss of RSV integrity, as measured via qRT-PCR, is well-described by an exponential decay, the latter mechanism failed to describe the rate at which RSV A Long loses infectivity over time in vitro based on the data presented herein. This is unusual given that other viruses (HIV, HCV, influenza) have been shown to lose their infectivity exponentially in vitro, and indeed an exponential rate of loss of infectivity is always assumed in mathematical modelling and experimental analyses. The infectivity profile of RSV in HEp-2 and SIAT cells remained consistent over the course of an RSV infection, over time and a large range of infectivity. However, SIAT cells were found to be ∼ 100× less sensitive to RSV infection than HEp-2 cells. In particular, we found that RSV spreads inefficiently in SIAT cells, in a manner we show is consistent with the establishment of infection resistance in uninfected cells. SIAT cells are a good in vitro model in which to study RSV in vivo dissemination, yielding similar infection timescales. However, the higher sensitivity of HEp-2 cells to RSV together with its RSV infectivity profile being similar to that of SIAT cells, makes HEp-2 cells more suitable for quantifying RSV infectivity over the course of in vitro RSV infections in SIAT cells. Our findings highlight the importance and urgency of resolving the mechanisms at play in the dissemination of RSV infections in vitro, and the processes by which this infectivity is lost.


Assuntos
Modelos Teóricos , Vírus Sincicial Respiratório Humano/fisiologia , Linhagem Celular , Humanos , Vírus da Influenza A/fisiologia , Cinética , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Vírus Sincicial Respiratório Humano/genética
8.
Virus Res ; 266: 58-68, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31004621

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F N-glycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F N-glycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Vírus Sincicial Respiratório Humano/patogenicidade , Proteínas Virais de Fusão/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular Tumoral , Feminino , Células Gigantes/virologia , Glicosilação , Humanos , Imunização , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB C , Mutação , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/imunologia , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Replicação Viral
9.
PLoS One ; 14(3): e0214517, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30921408

RESUMO

INTRODUCTION: The epidemiology, clinical features and outcomes of hospitalized adult patients with Influenza A (FluA), Influenza B (FluB) and Respiratory Syncytial Virus (RSV) have not been thoroughly compared. The aim of this study was to describe the differences between these viruses during 3 winter seasons. METHODS: A retrospective observational study was conducted consisting of all the polymerase chain reaction (PCR)-based diagnoses of FluA, FluB and RSV among adults during 2015-2018, in one regional hospital. Epidemiology, clinical symptoms and outcome-related data were comparatively analyzed. RESULTS: Between November 2015 and April 2018, 759 patients were diagnosed with FluA, FluB or RSV. Study cohort included 539 adult patients (306 FluA, 148 FluB and 85 RSV). FluB was predominant during the winter of 2017-18. RSV caused 15.7% of hospitalizations with diagnosed viral infection and in comparison to influenza, had distinct epidemiological, clinical features and outcomes, including older age (74.2 vs 66.2, p = 0.001) and higher rates of co-morbidities; complications including bacterial pneumonia (31 vs 18%, p = 0.02), mechanical ventilation (20 vs 7%, p = 0.001), and viral-related death (13 vs 6.6%, p = 0.04). FluA and FluB had similar epidemiology, clinical symptoms and outcomes, but vaccinated patients were less prone to be hospitalized with FluB as compared with FluA (3 vs 14%, p = 0.001). Paroxysmal atrial fibrillation and falls were common (8.7 and 8.5% respectively). CONCLUSIONS: FluA and FluB had similar epidemiological, clinical features and contributed equally to hospitalization burden and complications. RSV had a major impact on hospitalizations, occurring among the more elderly and sick populations and causing significantly worse outcomes, when compared to influenza patients. Vaccination appeared as a protective factor against hospitalizations with FluB as compared with FluA.


Assuntos
Hospitalização , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Influenza Humana/epidemiologia , Laboratórios , Vírus Sincicial Respiratório Humano/fisiologia , Infecções Respiratórias/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Influenza Humana/terapia , Pessoa de Meia-Idade , Infecções Respiratórias/terapia , Estudos Retrospectivos , Estações do Ano , Análise de Sobrevida , Resultado do Tratamento , Adulto Jovem
11.
Nat Rev Microbiol ; 17(4): 233-245, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30723301

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease in young children and elderly people. Although the virus was isolated in 1955, an effective RSV vaccine has not been developed, and the only licensed intervention is passive immunoprophylaxis of high-risk infants with a humanized monoclonal antibody. During the past 5 years, however, there has been substantial progress in our understanding of the structure and function of the RSV glycoproteins and their interactions with host cell factors that mediate entry. This period has coincided with renewed interest in developing effective interventions, including the isolation of potent monoclonal antibodies and small molecules and the design of novel vaccine candidates. In this Review, we summarize the recent findings that have begun to elucidate RSV entry mechanisms, describe progress on the development of new interventions and conclude with a perspective on gaps in our knowledge that require further investigation.


Assuntos
Antivirais/farmacologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/fisiologia , Internalização do Vírus/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Ensaios Clínicos como Assunto , Humanos , Infecções por Vírus Respiratório Sincicial/terapia , Bibliotecas de Moléculas Pequenas/farmacologia , Vacinas Virais
12.
Antiviral Res ; 161: 125-133, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30503888

RESUMO

Human respiratory syncytial virus (HRSV) infection is a significant cause of morbidity and mortality, particularly among the children and the elderly. Despite extensive efforts, there is currently no formally approved vaccine and effective antiviral options against HRSV infection are limited. The development of vaccines and antiviral strategies for HRSV was partly hampered by the lack of an efficient lethal mouse model to evaluate the efficacy of the candidate vaccines or antivirals. In this study, we established a lethal HRSV mouse model by consecutively passaging a clinical HRSV isolate, GZ08-0. GZ08-18 was isolated from mouse bronchioalveolar lavage fluids at the 50th passage of GZ08-0. Importantly, all GZ08-18-inoculated mice succumbed to the infection by day 7 post infection, whereas all GZ08-0-inoculated mice recovered from the infection. Subsequent investigations demonstrated that GZ08-18 replicated to a higher titer in mouse lungs, induced more prominent lung pathology, and resulted in higher expression levels of a number of key pro-inflammatory cytokines including IFN-γ, MIP-1α, and TNF-α in comparison to that of GZ08-0. The cyclophosphamide pretreatment rendered the mice more susceptible to a lethal outcome with less rounds of virus inoculation. Full genome sequencing revealed 17 mutations in GZ08-18, some of which might account for the dramatically increased pathogenicity over GZ08-0. In addition, by using ribavirin as a positive control, we demonstrated the potential application of this lethal mouse model as a tool in HRSV investigations. Overall, we have successfully established a practical lethal mouse model for HRSV with a mouse-adapted virus, which may facilitate future in vivo studies on the evaluation of candidate vaccines and drugs against HRSV.


Assuntos
Envelhecimento , Modelos Animais de Doenças , Infecções por Vírus Respiratório Sincicial/mortalidade , Vírus Sincicial Respiratório Humano/patogenicidade , Animais , Antivirais/uso terapêutico , Linhagem Celular , Ciclofosfamida/farmacologia , Genoma Viral , Humanos , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/fisiologia , Ribavirina/uso terapêutico , Replicação Viral
13.
Infection ; 47(2): 201-207, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30132249

RESUMO

BACKGROUND: Respiratory infections are the main causes for hospitalization in children and a common reason for the initiation of antibiotic treatment. Rapid antigen detection tests and point-of-care mPCR-based assays provide a fast detection of viral pathogens. Nonetheless, the prescription rate of antibiotics for respiratory infections is exceedingly high. In particular, human metapneumovirus (hMPV) infections frequently cause antibiotic treatment. METHODS: Children hospitalized in our clinic with an acute respiratory infection between January 2008 and January 2013 were included in the present study. Data of 3799 children were analyzed retrospectively for clinical symptoms, laboratory findings, and antibiotic and inhalation treatment. We performed an in-house m-RT-PCR-ELISA method for pathogen detection. RESULTS: Pathogen detection was possible in 2464 patients. In 6.3%, hMPV and, in 24.0%, RSV were detected. Patients positively tested for hMPV received inhalation therapy in 62.9%; patients positive for RSV in 73.8%. Patients positive for hMPV were treated with antibiotics in 62.3%. Patients with RSV infection received antibiotic treatment in 44.4%; all others in 43.5%. Notably, a positive result in RSV-RADT was associated with reduced number of antibiotic treatment. CONCLUSION: hMPV infections inherit a two times higher probability of antibiotic treatment. There was no significant difference in laboratory findings or body temperature between hMPV infection and infections caused by other pathogens. Clinical symptoms seem not to differ from those in RSV illness. Nonetheless, RSV infections triggered significantly lower antibiotic prescription rates. A considerate application of a POC-mPCR for patients with RSV-like symptoms and age of 1 year and older with a negative RSV-RADT might lead to higher detection rates of hMPV and a reduction in prescription of antibiotics.


Assuntos
Antibacterianos/uso terapêutico , Infecções por Paramyxoviridae/tratamento farmacológico , Sistemas Automatizados de Assistência Junto ao Leito/estatística & dados numéricos , Prescrições/estatística & dados numéricos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Pré-Escolar , Feminino , Alemanha , Hospitalização , Humanos , Lactente , Recém-Nascido , Masculino , Metapneumovirus/fisiologia , Infecções por Paramyxoviridae/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Estudos Retrospectivos
14.
Cell Microbiol ; 21(1): e12955, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30223301

RESUMO

Activation of the Raf/MEK/ERK cascade is required for efficient propagation of several RNA and DNA viruses, including human respiratory syncytial virus (RSV). In RSV infection, activation of the Raf/MEK/ERK cascade is biphasic. An early induction within minutes after infection is associated with viral attachment. Subsequently, a second activation occurs with, so far, unknown function in the viral life cycle. In this study, we aimed to characterise the role of Raf/MEK/ERK-mediated signalling during ongoing RSV infection. Our data show that inhibition of the kinase MEK after the virus has been internalised results in a reduction of viral titers. Further functional investigations revealed that the late-stage activation of ERK is required for a specific step in RSV replication, namely, the secretory transport of the RSV fusion protein F. Thus, MEK inhibition resulted in impaired surface accumulation of the F protein. F protein surface expression is essential for efficient replication as it is involved in viral filament formation, cell fusion, and viral transmission. In summary, we provide detailed insights of how host cell signalling interferes with RSV replication and identified the Raf/MEK/ERK kinase cascade as potential target for novel anti-RSV strategies.


Assuntos
Membrana Celular/metabolismo , Interações Hospedeiro-Patógeno , Sistema de Sinalização das MAP Quinases , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Virais de Fusão/metabolismo , Replicação Viral , Animais , Células Cultivadas , Humanos , Transporte Proteico
15.
Viruses ; 10(12)2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30558248

RESUMO

Respiratory syncytial virus (RSV) causes severe acute lower respiratory tract disease. Retinoic acid-inducible gene-I (RIG-I) serves as an innate immune sensor and triggers antiviral responses upon recognizing viral infections including RSV. Since tripartite motif-containing protein 25 (TRIM25)-mediated K63-polyubiquitination is crucial for RIG-I activation, several viruses target initial RIG-I activation through ubiquitination. RSV NS1 and NS2 have been shown to interfere with RIG-I-mediated antiviral signaling. In this study, we explored the possibility that NS1 suppresses RIG-I-mediated antiviral signaling by targeting TRIM25. Ubiquitination of ectopically expressed RIG-I-2Cards domain was decreased by RSV infection, indicating that RSV possesses ability to inhibit TRIM25-mediated RIG-I ubiquitination. Similarly, ectopic expression of NS1 sufficiently suppressed TRIM25-mediated RIG-I ubiquitination. Furthermore, interaction between NS1 and TRIM25 was detected by a co-immunoprecipitation assay. Further biochemical assays showed that the SPRY domain of TRIM25, which is responsible for interaction with RIG-I, interacted sufficiently with NS1. Suppression of RIG-I ubiquitination by NS1 resulted in decreased interaction between RIG-I and its downstream molecule, MAVS. The suppressive effect of NS1 on RIG-I signaling could be abrogated by overexpression of TRIM25. Collectively, this study suggests that RSV NS1 interacts with TRIM25 and interferes with RIG-I ubiquitination to suppress type-I interferon signaling.


Assuntos
Proteína DEAD-box 58/genética , Vírus Sincicial Respiratório Humano/fisiologia , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas não Estruturais Virais/genética , Células A549 , Linhagem Celular , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Reação em Cadeia da Polimerase , Ligação Proteica , Vírus Sincicial Respiratório Humano/genética , Transdução de Sinais , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/imunologia , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/metabolismo
16.
PLoS One ; 13(11): e0206474, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30439987

RESUMO

OBJECTIVES: Understanding respiratory syncytial virus (RSV) morbidity may help to plan health care and future vaccine recommendations. We aim to describe the characteristics and temporal distribution of children diagnosed with RSV admitted in a Spanish hospital. METHODS: Descriptive study for which the hospital discharges of children < 5 years of age with RSV infection were analyzed. The information was extracted from the hospital discharge database of a reference pediatric hospital in northern Spain for the 2010-2011 to 2014-2015 RSV seasons. RESULTS: Six hundred and forty-seven hospitalizations of children with RSV infection were analyzed, 94% of which occurred between the second week of November and the last week of March. Most children (72%) were under one year of age and 95% were previously healthy infants. Infants born from October to December had the highest risk of hospitalization in the first year of life. The median length of hospital stay of children with and without comorbidities was six and three days, respectively. 6.5% of the hospitalized cases were admitted to the pediatric intensive care unit; this percentage was higher among children < 2 months (adjusted odds ratio 4.15; 95% confidence interval: 1.37-12.61) or with comorbidities (adjusted odds ratio 4.15; 95% confidence interval: 1.53-11.28). The case lethality was 0.3%. CONCLUSIONS: The risk of hospitalizations for RSV is high during the first year of life and increases among infants born in the fall. Being under two months of age and presenting comorbidities are the main risk factors associated to pediatric intensive care unit admission.


Assuntos
Hospitalização/estatística & dados numéricos , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/fisiologia , Pré-Escolar , Comorbidade , Feminino , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Pediátrica , Tempo de Internação , Masculino , Infecções por Vírus Respiratório Sincicial/terapia , Fatores de Risco , Estações do Ano , Espanha/epidemiologia
17.
Virol J ; 15(1): 182, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30477508

RESUMO

Cellular protein eukaryotic translation elongation factor 1A (eEF1A) is an actin binding protein that plays a role in the formation of filamentous actin (F-actin) bundles. F-Actin regulates multiple stages of respiratory syncytial virus (RSV) replication including assembly and budding. Our previous study demonstrated that eEF1A knock-down significantly reduced RSV replication. Here we investigated if the eEF1A function in actin bundle formation was important for RSV replication and release. To investigate this, eEF1A function was impaired in HEp-2 cells by either knock-down of eEF1A with siRNA, or treatment with an eEF1A inhibitor, didemnin B (Did B). Cell staining and confocal microscopy analysis showed that both eEF1A knock-down and treatment with Did B resulted in disruption of cellular stress fiber formation and elevated accumulation of F-actin near the plasma membrane. When treated cells were then infected with RSV, there was also reduced formation of virus-induced cellular filopodia. Did B treatment, similarly to eEF1A knock-down, reduced the release of infectious RSV, but unlike eEF1A knock-down, did not significantly affect RSV genome replication. The lower infectious virus production in Did B treated cells also reduced RSV-induced cell death. In conclusion, the cellular factor eEF1A plays an important role in the regulation of F-actin stress fiber formation required for RSV assembly and release.


Assuntos
Actinas/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Vírus Sincicial Respiratório Humano/fisiologia , Fibras de Estresse/fisiologia , Replicação Viral , Actinas/genética , Linhagem Celular Tumoral , Depsipeptídeos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Técnicas de Silenciamento de Genes , Humanos , Pseudópodes/fisiologia , Pseudópodes/virologia , Vírus Sincicial Respiratório Humano/genética
18.
Viruses ; 10(10)2018 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-30274351

RESUMO

The virus⁻host protein interactions that underlie respiratory syncytial virus (RSV) assembly are still not completely defined, despite almost 60 years of research. RSV buds from the apical surface of infected cells, once virion components have been transported to the budding sites. Association of RSV matrix (M) protein with the actin cytoskeleton may play a role in facilitating this transport. We have investigated the interaction of M with actin in vitro and cell culture. Purified wildtype RSV M protein was found to bind directly to polymerized actin in vitro. Vero cells were transfected to express full-length M (1⁻256) as a green fluorescent protein-(GFP) tagged protein, followed by treatment with the microfilament destabilizer, cytochalasin D. Destabilization of the microfilament network resulted in mislocalization of full-length M, from mostly cytoplasmic to diffused across both cytoplasm and nucleus, suggesting that M interacts with microfilaments in this system. Importantly, treatment of RSV-infected cells with cytochalasin D results in lower infectious virus titers, as well as mislocalization of M to the nucleus. Finally, using deletion mutants of M in a transfected cell system, we show that both the N- and C-terminus of the protein are required for the interaction. Together, our data suggest a possible role for M⁻actin interaction in transporting virion components in the infected cell.


Assuntos
Actinas/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas da Matriz Viral/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Citocalasina D/farmacologia , Citoplasma/metabolismo , Deleção de Genes , Humanos , Ligação Proteica/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Vírion/metabolismo , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
19.
Acta Virol ; 62(3): 310-325, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30160147

RESUMO

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infection in infants. Winter outbreaks in Chile result in 5% of infected children hospitalized, with 0.01% mortality. Increased evidence indicates that viral and host factors modulate the severity of infection. Using DNA microarrays, we characterized the genome-wide transcriptional response of lung mucoepidermoid cells (NCI-H292) at 0, 24, 48, 72 and 96 hours post-infection (hpi) with a single dose of RSV/A. During the whole studied period, a bi-phasic gene expression profile was observed by a total of 330 differentially expressed genes. About 60% of them were up-regulated between 24-72 hpi and then turned-off at 96 hpi. This transient, early gene expression pattern was significantly enriched in biological processes like interferon signaling, antigen processing and presentation, double-stranded RNA binding and chemokine activity. We detected 27 common genes up-regulated between 24-72 hpi, from which IFIT1, IFI44, MX1, CXCL11 and OAS1 had the highest expression. The second pattern comprised over 120 genes, which remained silenced until 72 hpi, but were steeply up-regulated by 96 hpi. Biological processes of this late-response profile included cell cycle division and microtubule cytoskeleton organization. Conversely, the genes belonging to virus response pathway showed a decreased expression at 96 hpi. We conclude that RSV induces an early innate immune activation profile response until 72 hpi. Thereafter, the viral response is inhibited, leading to host cell recovery. The presented cellular model allows to study the specific pathways involved in elimination of infection at prolonged time intervals and their subsequent analysis in severe RSV disease of infants and/or older adults.


Assuntos
Células Epiteliais/metabolismo , Infecções por Vírus Respiratório Sincicial/genética , Vírus Sincicial Respiratório Humano/fisiologia , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Pulmão/metabolismo , Pulmão/virologia , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Fatores de Tempo , Transcriptoma
20.
Viruses ; 10(8)2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30127286

RESUMO

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 µm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.


Assuntos
Vírus Sincicial Respiratório Humano/ultraestrutura , Vírion/ultraestrutura , Montagem de Vírus/fisiologia , Células A549 , Animais , Brônquios/virologia , Linhagem Celular , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Células HeLa , Humanos , Microtomia , Vírus Sincicial Respiratório Humano/fisiologia , Células Vero , Vírion/fisiologia
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