RESUMO
The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.
Assuntos
Alelos , Influenza Humana , Macrófagos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , Transativadores , Humanos , Transativadores/genética , Transativadores/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Macrófagos/metabolismo , Células U937 , Influenza Humana/genética , Sítios de Ligação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Regulação da Expressão GênicaRESUMO
The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-ß in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.
Assuntos
Motivos de Aminoácidos , Vírus da Influenza A Subtipo H1N1 , Infecções por Orthomyxoviridae , Proteínas não Estruturais Virais , Replicação Viral , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/química , Animais , Cães , Virulência , Camundongos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/fisiologia , Infecções por Orthomyxoviridae/virologia , Humanos , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Doenças do Cão/virologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H3N8/patogenicidade , FemininoRESUMO
Accurate airborne virus monitoring is important for preventing the spread of infectious diseases. Although standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) can efficiently detect viral ribonucleic acid (RNA), it cannot determine whether the RNA is associated with active (infectious) or inactive (non-infectious) viruses. Plaque assay is the gold standard for determining viral infectivity but is laborious and time-consuming. This study explored the viral infectivity of H1N1 influenza virus and human coronavirus (HCoV-229E) using capsid integrity RT-qPCR, where virus samples were pretreated with reagents penetrating viruses with damaged capsids, impeding amplification by binding to their RNA. Therefore, the amplified signals corresponded solely to active viruses with undamaged capsids. Propidium monoazide (PMA) and platinum (IV) chloride (PtCl4) were used to investigate the effects of reagent concentration. Feasibility tests revealed that PtCl4 was more efficient than PMA, with optimal concentrations of 125-250 µM and 250-500 µM for H1N1 influenza virus and HCoV-229E, respectively. The results of percentage of active virus showed that capsid integrity RT-qPCR provided a trend similar to that of plaque assay, indicating an accurate measure of viral infectivity. Virus sampling in the laboratory and field highlighted the precision of this methodology for determining viral infectivity. Therefore, this methodology enables rapid and accurate detection of infectious airborne H1N1 influenza virus and HCoV-229E, allowing swift response to outbreaks.
Assuntos
Azidas , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Azidas/química , Humanos , RNA Viral/genética , Microbiologia do Ar , Capsídeo/metabolismo , Coronavirus Humano 229E/genética , Propídio/análogos & derivados , Propídio/química , Animais , Células Madin Darby de Rim Canino , Cães , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Swine influenza viruses (SIVs) have been circulating in swine globally and are potential threats to human health. During the surveillance of SIVs in Shandong Province, China, from 2019 to 2022, 21 reassortant G4 genotype Eurasian avian-like (EA) H1N1 subtypes containing genes from the EA H1N1 (HA and NA), 2009 pandemic (pdm/09) H1N1 virus (PB2, PB1, PA, NP, and M), and classical swine (CS) H1N1 (NS) lineages were isolated. The analysis of the key functional amino acid sites in the isolated viruses showed that two mutation sites (190D and 225E) that preferentially bind to the human α2-6 sialic acid receptor were found in HA. In PB2, three mutation sites (271A, 590S, and 591R) that may increase mammalian fitness and a mutation site (431M) that increases pathogenicity in mice were found. A typical human signature marker that may promote infection in humans, 357K, was found in NP. The viruses could replicate efficiently in mouse lungs and turbinates, and one of the H1N1 isolates could replicate in mouse kidneys and brains without prior adaption, which indicates that the viruses potentially pose a threat to human health. Histopathological results showed that the isolated viruses caused typical bronchopneumonia and encephalitis in mice. The results indicate that G4 genotype H1N1 has potential transmissibility to humans, and surveillance should be enhanced, which could provide important information for assessing the pandemic potential of the viruses.
Assuntos
Genótipo , Vírus da Influenza A Subtipo H1N1 , Infecções por Orthomyxoviridae , Animais , Suínos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , China/epidemiologia , Camundongos , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/patologia , Humanos , Doenças dos Suínos/virologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/patologia , Filogenia , Influenza Humana/virologia , Influenza Humana/epidemiologia , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Vírus Reordenados/isolamento & purificação , Células Madin Darby de Rim Canino , Mutação , Replicação Viral/genética , Proteínas Virais/genéticaRESUMO
Influenza A virus can infect respiratory tracts and may cause severe illness in humans. Proteins encoded by influenza A virus can interact with cellular factors and dysregulate host biological processes to support viral replication and cause pathogenicity. The influenza viral PA protein is not only a subunit of influenza viral polymerase but also a virulence factor involved in pathogenicity during infection. To explore the role of the influenza virus PA protein in regulating host biological processes, we performed immunoprecipitation and LCâMS/MS to globally identify cellular factors that interact with the PA proteins of the influenza A H1N1, 2009 pandemic H1N1, and H3N2 viruses. The results demonstrated that proteins located in the mitochondrion, proteasome, and nucleus are associated with the PA protein. We further discovered that the PA protein is partly located in mitochondria by immunofluorescence and mitochondrial fractionation and that overexpression of the PA protein reduces mitochondrial respiration. In addition, our results revealed the interaction between PA and the mitochondrial matrix protein PYCR2 and the antiviral role of PYCR2 during influenza A virus replication. Moreover, we found that the PA protein could also trigger autophagy and disrupt mitochondrial homeostasis. Overall, our research revealed the impacts of the influenza A virus PA protein on mitochondrial function and autophagy.
Assuntos
Mitocôndrias , Proteínas Virais , Replicação Viral , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , Vírus da Influenza A/fisiologia , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus da Influenza A Subtipo H3N2/metabolismo , Autofagia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Células HEK293 , Influenza Humana/virologia , Influenza Humana/metabolismo , Células A549 , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Espectrometria de Massas em TandemRESUMO
Patients with pulmonary fibrosis (PF) often experience exacerbations of their disease, characterised by a rapid, severe deterioration in lung function that is associated with high mortality. Whilst the pathobiology of such exacerbations is poorly understood, virus infection is a trigger. The present study investigated virus-induced injury responses of alveolar and bronchial epithelial cells (AECs and BECs, respectively) from patients with PF and age-matched controls (Ctrls). Air-liquid interface (ALI) cultures of AECs, comprising type I and II pneumocytes or BECs were inoculated with influenza A virus (H1N1) at 0.1 multiplicity of infection (MOI). Levels of interleukin-6 (IL-6), IL-36γ and IL-1ß were elevated in cultures of AECs from PF patients (PF-AECs, n = 8-11), being markedly higher than Ctrl-AECs (n = 5-6), 48 h post inoculation (pi) (P<0.05); despite no difference in H1N1 RNA copy numbers 24 h pi. Furthermore, the virus-induced inflammatory responses of PF-AECs were greater than BECs (from either PF patients or controls), even though viral loads in the BECs were overall 2- to 3-fold higher than AECs. Baseline levels of the senescence and DNA damage markers, nuclear p21, p16 and H2AXγ were also significantly higher in PF-AECs than Ctrl-AECs and further elevated post-infection. Senescence induction using etoposide augmented virus-induced injuries in AECs (but not viral load), whereas selected senotherapeutics (rapamycin and mitoTEMPO) were protective. The present study provides evidence that senescence increases the susceptibility of AECs from PF patients to severe virus-induced injury and suggests targeting senescence may provide an alternative option to prevent or treat the exacerbations that worsen the underlying disease.
Assuntos
Células Epiteliais Alveolares , Vírus da Influenza A Subtipo H1N1 , Fibrose Pulmonar , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Células Epiteliais Alveolares/virologia , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/metabolismo , Fibrose Pulmonar/virologia , Fibrose Pulmonar/patologia , Masculino , Influenza Humana/virologia , Influenza Humana/complicações , Influenza Humana/patologia , Pessoa de Meia-Idade , Feminino , Células Cultivadas , Idoso , Senescência Celular , Estudos de Casos e Controles , Citocinas/metabolismoRESUMO
Swine are regarded as "intermediate hosts" or "mixing vessels" of influenza viruses, capable of generating strains with pandemic potential. From 2020 to 2021, we conducted surveillance on swine H1N2 influenza (swH1N2) viruses in swine farms located in Guangdong, Yunnan, and Guizhou provinces in southern China, as well as Henan and Shandong provinces in northern China. We systematically analyzed the evolution and pathogenicity of swH1N2 isolates, and characterized their replication and transmission abilities. The isolated viruses are quadruple reassortant H1N2 viruses containing genes from pdm/09 H1N1 (PB2, PB1, PA and NP genes), triple-reassortant swine (NS gene), Eurasian Avian-like (HA and M genes), and recent human H3N2 (NA gene) lineages. The NA, PB2, and NP of SW/188/20 and SW/198/20 show high gene similarities to A/Guangdong/Yue Fang277/2017 (H3N2). The HA gene of swH1N2 exhibits a high evolutionary rate. The five swH1N2 isolates replicate efficiently in human, canine, and swine cells, as well as in the turbinate, trachea, and lungs of mice. A/swine/Shandong/198/2020 strain efficiently replicates in the respiratory tract of pigs and effectively transmitted among them. Collectively, these current swH1N2 viruses possess zoonotic potential, highlighting the need for strengthened surveillance of swH1N2 viruses.
Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H1N2 , Infecções por Orthomyxoviridae , Vírus Reordenados , Doenças dos Suínos , Animais , Suínos , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Vírus Reordenados/isolamento & purificação , China/epidemiologia , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Doenças dos Suínos/transmissão , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/patogenicidade , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Humanos , Camundongos , Cães , Filogenia , Replicação Viral , Saúde Pública , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , Influenza Humana/transmissão , Camundongos Endogâmicos BALB C , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Virulência , FemininoRESUMO
Previous studies have shown that the Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses circulated widely in pigs around the world and formed multiple genotypes by acquiring non-hemagglutinin and neuraminidase segments derived from other swine influenza viruses. Swine influenza control is not a priority for the pig industry in many countries, and it is worrisome that some strains may become more pathogenic and/or transmissible during their circulation in nature. Our routine surveillance indicated that the EA H1N1 viruses obtained different internal genes from different swine influenza viruses and formed various new genotypes. In this study, we found that a naturally isolated swine influenza reassortant, A/swine/Liaoning/265/2017 (LN265), a representative strain of one of the predominant genotypes in recent years, is lethal in mice and transmissible in ferrets. LN265 contains the hemagglutinin, neuraminidase, and matrix of the EA H1N1 virus; the basic polymerase 2, basic polymerase 1, acidic polymerase (PA), and nucleoprotein of the 2009 H1N1 pandemic virus; and the nonstructural protein of the North American triple-reassortment H1N2 virus. By generating and testing a series of reassortants and mutants, we found that four gradually accumulated mutations in PA are responsible for the increased pathogenicity and transmissibility of LN265. We further revealed that these mutations increase the messenger RNA transcription of viral proteins by enhancing the endonuclease cleavage activity and viral RNA-binding ability of the PA protein. Our study demonstrates that EA H1N1 swine influenza virus became pathogenic and transmissible in ferrets by acquiring key mutations in PA and provides important insights for monitoring field strains with pandemic potential.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Infecções por Orthomyxoviridae , RNA Polimerase Dependente de RNA , Doenças dos Suínos , Animais , Furões , Genótipo , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Camundongos , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Filogenia , RNA Polimerase Dependente de RNA/genética , Vírus Reordenados/genética , Suínos , Doenças dos Suínos/virologia , Proteínas Virais/genéticaRESUMO
OBJECTIVES: Because of the spread of the Omicron variant, many countries have experienced COVID-19 case numbers unseen since the start of the pandemic. We aimed to compare the epidemiological characteristics of Omicron with previous variants and different strains of influenza to provide context for public health responses. METHODS: We developed transmission models for SARS-CoV-2 variants and influenza, in which transmission, death, and vaccination rates were taken to be time-varying. We fit our model based on publicly available data in South Africa, the United States, and Canada. We used this model to evaluate the relative transmissibility and mortality of Omicron compared with previous variants and influenza. RESULTS: We found that Omicron is more transmissible and less fatal than both seasonal and 2009 H1N1 influenza and the Delta variant; these characteristics make Omicron epidemiologically more similar to influenza than it is to Delta. We estimate that as of February 7, 2022, booster doses have prevented 4.29×107 and 1.14×106 Omicron infections in the United States and Canada, respectively. CONCLUSION: Our findings indicate that the high infectivity of Omicron will keep COVID-19 endemic, similar to influenza. However, because of Omicron's lower fatality rate, our work suggests that human populations living with SARS-CoV-2 are most likely.
Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Mutação , SARS-CoV-2 , COVID-19/epidemiologia , COVID-19/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/prevenção & controle , Influenza Humana/virologia , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Estados Unidos/epidemiologiaRESUMO
H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene MAN2A1 were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the MAN2A1 gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.
Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/patogenicidade , Macrófagos Alveolares/virologia , MicroRNAs/genética , RNA Circular/genética , alfa-Manosidase/genética , Animais , Linhagem Celular , Biologia Computacional , Cães , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Macrófagos Alveolares/química , Macrófagos Alveolares/citologia , Células Madin Darby de Rim Canino , Modelos Biológicos , SuínosRESUMO
Influenza A virus infection causes a series of diseases, but the factors associated with disease severity are not fully understood. Disruption of the endothelial glycocalyx contributes to acute lung injury in sepsis, but has not been well studied in H1N1 influenza. We aim to determine whether the plasma glycocalyx components levels are predictive of disease severity in H1N1 influenza. This prospective observational study included 53 patients with influenza A (H1N1) during the influenza season, and 30 healthy controls in our hospital. Patients were grouped by severity and survival. We collected clinical data and blood samples at admission. Inflammatory factors (tumor necrosis factor-α, interleukin-6, interleukin-10) and endothelial glycocalyx components (syndecan-1, hyaluronan, heparan sulfate) were measured. The plasma levels of syndecan-1, hyaluronan, and heparan sulfate were significantly higher in patients with severe influenza A (H1N1) than in mild cases. Syndecan-1 and hyaluronan were positively correlated with disease severity, which was indicated by the APACHE II and SOFA scores and lactate levels, and negatively correlated with albumin levels. At a cutoff point ≥ 173.9 ng/mL, syndecan-1 had a 81.3% sensitivity and 70.3% specificity for predicting of 28-day mortality. Kaplan-Meier analysis demonstrated a strong association between syndecan-1 levels and 28-day mortality (log-rank 11.04, P = 0.001). Elevated plasma levels of syndecan-1 has a potential role in systemic organ dysfunction and may be indicative of disease severity in patients with influenza A (H1N1).
Assuntos
Células Endoteliais/metabolismo , Glicocálix/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Sindecana-1/sangue , Adulto , Idoso , Biomarcadores/sangue , Células Endoteliais/virologia , Feminino , Glicocálix/virologia , Heparitina Sulfato/sangue , Humanos , Ácido Hialurônico/sangue , Influenza Humana/sangue , Influenza Humana/diagnóstico , Influenza Humana/mortalidade , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Here we describe a protocol for identifying metabolites in respiratory specimens of patients that are SARS-CoV-2 positive, SARS-CoV-2 negative, or H1N1 positive. This protocol provides step-by-step instructions on sample collection from patients, followed by metabolite extraction. We use ultra-high-pressure liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) for data acquisition and describe the steps for data analysis. The protocol was standardized with specific customization for SARS-CoV-2-containing respiratory specimens. For complete details on the use and execution of this protocol, please refer to Maras et al. (2021).
Assuntos
COVID-19/diagnóstico , Cromatografia Líquida de Alta Pressão/métodos , Metabolômica/métodos , COVID-19/metabolismo , Biologia Computacional , Testes Diagnósticos de Rotina , Perfilação da Expressão Gênica , Técnicas Genéticas , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Espectrometria de Massas/métodos , Metaboloma , Padrões de Referência , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Manejo de Espécimes/métodosRESUMO
In this protocol, we describe global proteome profiling for the respiratory specimen of COVID-19 patients, patients suspected with COVID-19, and H1N1 patients. In this protocol, details for identifying host, viral, or bacterial proteome (Meta-proteome) are provided. Major steps of the protocol include virus inactivation, protein quantification and digestion, desalting of peptides, high-resolution mass spectrometry (HRMS)-based analysis, and downstream bioinformatics analysis. For complete details on the use and execution of this profile, please refer to Maras et al. (2021).
Assuntos
COVID-19/diagnóstico , Genômica/métodos , Proteômica/métodos , COVID-19/metabolismo , Cromatografia Líquida/métodos , Biologia Computacional , Testes Diagnósticos de Rotina , Perfilação da Expressão Gênica , Técnicas Genéticas , Genoma Viral/genética , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Peptídeos , Proteoma , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Manejo de Espécimes/métodos , Espectrometria de Massas em Tandem/métodos , Viroma/genética , Viroma/fisiologiaRESUMO
Virus-like particles (VLPs) modified through different molecular technologies are employed as delivery vehicles or platforms for heterologous antigen display. We have recently created a norovirus (NoV) VLP platform, where two influenza antigens, the extracellular domain of matrix protein M2 (M2e) or the stem domain of the major envelope glycoprotein hemagglutinin (HA2) are displayed on the surface of the NoV VLPs by SpyTag/SpyCatcher conjugation. To demonstrate the feasibility of the platform to deliver foreign antigens, this study examined potential interference of the conjugation with induction of antibodies against conjugated M2e peptide, HA2, and NoV VLP carrier. High antibody response was induced by HA2 but not M2e decorated VLPs. Furthermore, HA2-elicited antibodies did not neutralize the homologous influenza virus in vitro. Conjugated NoV VLPs retained intact receptor binding capacity and self-immunogenicity. The results demonstrate that NoV VLPs could be simultaneously used as a platform to deliver foreign antigens and a NoV vaccine.
Assuntos
Anticorpos Antivirais/biossíntese , Hemaglutininas Virais/genética , Imunoglobulina G/biossíntese , Vacinas contra Influenza/genética , Influenza Humana/prevenção & controle , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/genética , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Feminino , Hemaglutininas Virais/imunologia , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/biossíntese , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Norovirus/genética , Norovirus/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinação/métodos , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
Annual repeat influenza vaccination raises concerns about protective efficacy against mismatched viruses. We investigated the impact of heterologous prime-boost vaccination on inducing cross protection by designing recombinant influenza viruses with chimeric hemagglutinin (HA) carrying M2 extracellular domains (M2e-HA). Heterologous prime-boost vaccination of C57BL/6 mice with M2e-HA chimeric virus more effectively induced M2e and HA stalk specific IgG antibodies correlating with cross protection than homologous prime-boost vaccination. Induction of M2e and HA stalk specific IgG antibodies was compromised in 1-year old mice, indicating significant aging effects on priming subdominant M2e and HA stalk IgG antibody responses. This study demonstrates that a heterologous prime-boost strategy with recombinant influenza virus expressing extra M2e epitopes provides more effective cross protection than homologous vaccination.
Assuntos
Envelhecimento/imunologia , Anticorpos Antivirais/biossíntese , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Imunoglobulina G/biossíntese , Vacinas contra Influenza/genética , Influenza Humana/prevenção & controle , Infecções por Orthomyxoviridae/prevenção & controle , Fatores Etários , Envelhecimento/genética , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteção Cruzada , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização Secundária/métodos , Imunogenicidade da Vacina , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/biossíntese , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinação/métodos , Vacinas Sintéticas , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologiaRESUMO
Influenza is a respiratory virus that alone or in combination with secondary bacterial pathogens can contribute to the development of acute pneumonia in persons >65 years of age. Host innate immune antiviral signaling early in response to influenza is essential to inhibit early viral replication and guide the initiation of adaptive immune responses. Using young adult (3 months) and aged adult mice infected with mouse adapted H1N1 or H3N2, the results of our study illustrate dysregulated and/or diminished activation of key signaling pathways in aged lung contribute to increased lung inflammation and morbidity. Specifically, within the first seven days of infection, there were significant changes in genes associated with TLR and RIG-I signaling detected in aged murine lung in response to H1N1 or H3N2. Taken together, the results of our study expand our current understanding of age-associated changes in antiviral signaling in the lung.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/genética , Pneumonia/genética , Células A549 , Animais , Proteína DEAD-box 58/genética , Modelos Animais de Doenças , Regulação Viral da Expressão Gênica/genética , Humanos , Imunidade Inata/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/microbiologia , Influenza Humana/virologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/microbiologia , Infecções por Orthomyxoviridae/virologia , Pneumonia/microbiologia , Pneumonia/virologia , Receptores Toll-Like/genética , Replicação Viral/genéticaRESUMO
The spatiotemporal patterns of spread of influenza A(H1N1)pdm09 viruses on a countrywide scale are unclear in many tropical/subtropical regions mainly because spatiotemporally representative sequence data are lacking. We isolated, sequenced, and analyzed 383 A(H1N1)pdm09 viral genomes from hospitalized patients between 2009 and 2018 from seven locations across Kenya. Using these genomes and contemporaneously sampled global sequences, we characterized the spread of the virus in Kenya over several seasons using phylodynamic methods. The transmission dynamics of A(H1N1)pdm09 virus in Kenya were characterized by (i) multiple virus introductions into Kenya over the study period, although only a few of those introductions instigated local seasonal epidemics that then established local transmission clusters, (ii) persistence of transmission clusters over several epidemic seasons across the country, (iii) seasonal fluctuations in effective reproduction number (Re) associated with lower number of infections and seasonal fluctuations in relative genetic diversity after an initial rapid increase during the early pandemic phase, which broadly corresponded to epidemic peaks in the northern and southern hemispheres, (iv) high virus genetic diversity with greater frequency of seasonal fluctuations in 2009-2011 and 2018 and low virus genetic diversity with relatively weaker seasonal fluctuations in 2012-2017, and (v) virus spread across Kenya. Considerable influenza virus diversity circulated within Kenya, including persistent viral lineages that were unique to the country, which may have been capable of dissemination to other continents through a globally migrating virus population. Further knowledge of the viral lineages that circulate within understudied low-to-middle-income tropical and subtropical regions is required to understand the full diversity and global ecology of influenza viruses in humans and to inform vaccination strategies within these regions.
Assuntos
Epidemias , Genoma Viral , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/epidemiologia , Variação Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Quênia/epidemiologia , Filogenia , RNA Viral/genética , Estações do AnoRESUMO
Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/epidemiologia , Animais , Surtos de Doenças/veterinária , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H5N2/patogenicidade , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/imunologia , Filogenia , Suínos/virologia , Doenças dos Suínos/virologiaRESUMO
Seasonal Influenza A virus (IAV) infections can promote dissemination of upper respiratory tract commensals such as Streptococcus pneumoniae to the lower respiratory tract resulting in severe life-threatening pneumonia. Here, we aimed to compare innate immune responses in the lungs of healthy colonized and non-colonized mice after IAV challenge at the initial asymptomatic stage of infection. Responses during a severe bacterial pneumonia were profiled for comparison. Cytokine and innate immune cell imprints of the lungs were analyzed. Irrespective of the colonization status, mild H1N1 IAV infection was characterized by a bi-phasic disease progression resulting in full recovery of the animals. Already at the asymptomatic stage of viral infection, the pro-inflammatory cytokine response was as high as in pneumococcal pneumonia. Flow cytometry analyses revealed an early influx of inflammatory monocytes into the lungs. Neutrophil influx was mostly limited to bacterial infections. The majority of cells, except monocytes, displayed an activated phenotype characterized by elevated CCR2 and MHCII expression. In conclusion, we show that IAV challenge of colonized healthy mice does not automatically result in severe co-infection. However, a general local inflammatory response was noted at the asymptomatic stage of infection irrespective of the infection type.
Assuntos
Imunidade Inata/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções Pneumocócicas/imunologia , Animais , Portador Sadio/imunologia , Coinfecção/virologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Pulmão/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Infecções por Orthomyxoviridae/virologia , Infecções Pneumocócicas/complicações , Pneumonia Bacteriana , Pneumonia Pneumocócica/imunologia , Cultura Primária de Células , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Streptococcus pneumoniae/patogenicidadeRESUMO
Seasonal influenza outbreaks represent a large burden for the health care system as well as the economy. While the role of the microbiome has been elucidated in the context of various diseases, the impact of respiratory viral infections on the human microbiome is largely unknown. In this study, swine was used as an animal model to characterize the temporal dynamics of the respiratory and gastrointestinal microbiome in response to an influenza A virus (IAV) infection. A multi-omics approach was applied on fecal samples to identify alterations in microbiome composition and function during IAV infection. We observed significantly altered microbial richness and diversity in the gastrointestinal microbiome after IAV infection. In particular, increased abundances of Prevotellaceae were detected, while Clostridiaceae and Lachnospiraceae decreased. Moreover, our metaproteomics data indicated that the functional composition of the microbiome was heavily affected by the influenza infection. For instance, we identified decreased amounts of flagellin, correlating with reduced abundances of Lachnospiraceae and Clostridiaceae, possibly indicating involvement of a direct immune response toward flagellated Clostridia during IAV infection. Furthermore, enzymes involved in short-chain fatty acid (SCFA) synthesis were identified in higher abundances, while metabolome analyses revealed rather stable concentrations of SCFAs. In addition, 16S rRNA gene sequencing was used to characterize effects on the composition and natural development of the upper respiratory tract microbiome. Our results showed that IAV infection resulted in significant changes in the abundance of Moraxellaceae and Pasteurellaceae in the upper respiratory tract. Surprisingly, temporal development of the respiratory microbiome structure was not affected. IMPORTANCE Here, we used swine as a biomedical model to elucidate the impact of influenza A H1N1 infection on structure and function of the respiratory and gastrointestinal tract microbiome by employing a multi-omics analytical approach. To our knowledge, this is the first study to investigate the temporal development of the porcine microbiome and to provide insights into the functional capacity of the gastrointestinal microbiome during influenza A virus infection.