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1.
Nat Immunol ; 17(12): 1447-1458, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27798619

RESUMEN

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.


Asunto(s)
Centro Germinal/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Infecciones por Orthomyxoviridae/inmunología , Células TH1/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Células Cultivadas , Humanos , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
2.
Proc Natl Acad Sci U S A ; 115(28): E6622-E6629, 2018 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-29941581

RESUMEN

The pathophysiological changes that occur in lungs infected with influenza viruses are poorly understood. Here we established an in vivo imaging system that combines two-photon excitation microscopy and fluorescent influenza viruses of different pathogenicity. This approach allowed us to monitor and correlate several parameters and physiological changes including the spread of infection, pulmonary permeability, pulmonary perfusion speed, number of recruited neutrophils in infected lungs, and neutrophil motion in the lungs of live mice. Several physiological changes were larger and occurred earlier in mice infected with a highly pathogenic H5N1 influenza virus compared with those infected with a mouse-adapted human strain. These findings demonstrate the potential of our in vivo imaging system to provide novel information about the pathophysiological consequences of virus infections.


Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/metabolismo , Pulmón , Microscopía de Fluorescencia por Excitación Multifotónica , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/patología , Animales , Subtipo H5N1 del Virus de la Influenza A/genética , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Ratones , Infecciones por Orthomyxoviridae/genética
3.
J Infect Dis ; 222(7): 1155-1164, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32433769

RESUMEN

The avian influenza A(H7N9) virus has caused high mortality rates in humans, especially in the elderly; however, little is known about the mechanistic basis for this. In the current study, we used nonhuman primates to evaluate the effect of aging on the pathogenicity of A(H7N9) virus. We observed that A(H7N9) virus infection of aged animals (defined as age 20-26 years) caused more severe symptoms than infection of young animals (defined as age 2-3 years). In aged animals, lung inflammation was weak and virus infection was sustained. Although cytokine and chemokine expression in the lungs of most aged animals was lower than that in the lungs of young animals, 1 aged animal showed severe symptoms and dysregulated proinflammatory cytokine and chemokine production. These results suggest that attenuated or dysregulated immune responses in aged animals are responsible for the severe symptoms observed among elderly patients infected with A(H7N9) virus.


Asunto(s)
Envejecimiento , Subtipo H7N9 del Virus de la Influenza A , Pulmón/patología , Infecciones por Orthomyxoviridae/virología , Animales , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Pulmón/inmunología , Pulmón/virología , Macaca fascicularis , Infecciones por Orthomyxoviridae/inmunología , Replicación Viral
4.
Nature ; 501(7468): 551-5, 2013 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-23842494

RESUMEN

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.


Asunto(s)
Virus de la Influenza A , Gripe Humana/virología , Infecciones por Orthomyxoviridae/virología , Replicación Viral , Animales , Antivirales/farmacología , Células Cultivadas , Pollos/virología , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Perros , Inhibidores Enzimáticos/farmacología , Femenino , Hurones/virología , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Virus de la Influenza A/química , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/patogenicidad , Gripe Humana/tratamiento farmacológico , Macaca fascicularis/virología , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Enfermedades de los Monos/patología , Enfermedades de los Monos/virología , Neuraminidasa/antagonistas & inhibidores , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/transmisión , Codorniz/virología , Porcinos/virología , Porcinos Enanos/virología , Replicación Viral/efectos de los fármacos
5.
J Infect Dis ; 217(9): 1372-1382, 2018 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-29373693

RESUMEN

Exosomes regulate cell-cell communication by transferring functional proteins and RNAs between cells. Here, to clarify the function of exosomes during influenza virus infection, we characterized lung-derived exosomal microRNAs (miRNAs). Among the detected miRNAs, miR-483-3p was present at high levels in bronchoalveolar lavage fluid (BALF) exosomes during infection of mice with various strains of influenza virus, and miR-483-3p transfection potentiated gene expression of type I interferon and proinflammatory cytokine upon viral infection of MLE-12 cells. RNF5, a regulator of the RIG-I signaling pathway, was identified as a target gene of miR-483-3p. Moreover, we found that CD81, another miR-483-3p target, functions as a negative regulator of RIG-I signaling in MLE-12 cells. Taken together, this study indicates that BALF exosomal miRNAs may mediate the antiviral and inflammatory response to influenza virus infection.


Asunto(s)
Inmunidad Innata/fisiología , MicroARNs/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Animales , Líquido del Lavado Bronquioalveolar , Línea Celular , Femenino , Regulación de la Expresión Génica/inmunología , Pulmón/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , FN-kappa B , Infecciones por Orthomyxoviridae/virología , Tetraspanina 28/genética , Tetraspanina 28/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
6.
J Virol ; 91(7)2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-28100622

RESUMEN

The highly pathogenic avian influenza (HPAI) H5N1 viruses continue to circulate in nature and threaten public health. Although several viral determinants and host factors that influence the virulence of HPAI H5N1 viruses in mammals have been identified, the detailed molecular mechanism remains poorly defined and requires further clarification. In our previous studies, we characterized two naturally isolated HPAI H5N1 viruses that had similar viral genomes but differed substantially in their lethality in mice. In this study, we explored the molecular determinants and potential mechanism for this difference in virulence. By using reverse genetics, we found that a single amino acid at position 158 of the hemagglutinin (HA) protein substantially affected the systemic replication and pathogenicity of these H5N1 influenza viruses in mice. We further found that the G158N mutation introduced an N-linked glycosylation at positions 158 to 160 of the HA protein and that this N-linked glycosylation enhanced viral productivity in infected mammalian cells and induced stronger host immune and inflammatory responses to viral infection. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.IMPORTANCE Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to evolve in nature and threaten human health. Key mutations in the virus hemagglutinin (HA) protein or reassortment with other pandemic viruses endow HPAI H5N1 viruses with the potential for aerosol transmissibility in mammals. A thorough understanding of the pathogenic mechanisms of these viruses will help us to develop more effective control strategies; however, such mechanisms and virulent determinants for H5N1 influenza viruses have not been fully elucidated. In this study, we identified glycosylation at positions 158 to 160 of the HA protein of two naturally occurring H5N1 viruses as an important virulence determinant. This glycosylation event enhanced viral productivity, exacerbated the host response, and thereby contributed to the high pathogenicity of H5N1 virus in mice.


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Inmunidad Innata , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/virología , Procesamiento Proteico-Postraduccional , Secuencias de Aminoácidos , Animales , Proliferación Celular , Perros , Femenino , Glicosilación , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Células de Riñón Canino Madin Darby , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Virulencia , Replicación Viral
7.
J Infect Dis ; 216(5): 582-593, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28931216

RESUMEN

Antiviral compounds (eg, the neuraminidase inhibitor oseltamivir) are invaluable for the treatment of individuals infected with influenza A viruses of the H7N9 subtype (A[H7N9]), which have infected and killed hundreds of persons. However, oseltamivir treatment often leads to the emergence of resistant viruses in immunocompromised individuals. To better understand the emergence and properties of oseltamivir-resistant A(H7N9) viruses in immunosuppressed individuals, we infected immunosuppressed cynomolgus macaques with an A(H7N9) virus and treated them with oseltamivir. Disease severity and mortality were higher in immunosuppressed than in immunocompetent animals. Oseltamivir treatment at 2 different doses reduced A(H7N9) viral titers in infected animals, but even high-dose oseltamivir did not block viral replication sufficiently to suppress the emergence of resistant variants. Some resistant variants were not appreciably attenuated in cultured cells, but an oseltamivir-resistant A(H7N9) virus did not transmit among ferrets. These findings are useful for the control of A(H7N9) virus infections in clinical settings.


Asunto(s)
Farmacorresistencia Viral Múltiple , Huésped Inmunocomprometido , Subtipo H7N9 del Virus de la Influenza A/efectos de los fármacos , Macaca fascicularis/virología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oseltamivir/uso terapéutico , Animales , Antivirales/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Subtipo H7N9 del Virus de la Influenza A/fisiología , Masculino , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/veterinaria , Infecciones por Orthomyxoviridae/virología , Replicación Viral
8.
PLoS Pathog ; 11(6): e1004856, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26046528

RESUMEN

Influenza viruses present major challenges to public health, evident by the 2009 influenza pandemic. Highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. However, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. Here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. We found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. Cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin 6 and monocyte chemotactic protein 1, exhibit ultrasensitive behavior. A systematic exploration of the pathways regulating the inflammatory-associated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the Toll-like receptor pathway that regulates STAT1 phosphorylation. This study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. The approach developed here should facilitate the construction of gene regulatory models of other infectious diseases.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Infecciones por Orthomyxoviridae/inmunología , Animales , Western Blotting , Femenino , Citometría de Flujo , Inflamación/genética , Inflamación/inmunología , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Orthomyxoviridae/genética , Transcriptoma , Virulencia
9.
J Virol ; 89(22): 11337-46, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26339046

RESUMEN

UNLABELLED: We previously reported that an H5N1 virus carrying the Venus reporter gene, which was inserted into the NS gene segment from the A/Puerto Rico/8/1934(H1N1) virus (Venus-H5N1 virus), became more lethal to mice, and the reporter gene was stably maintained after mouse adaptation compared with the wild-type Venus-H5N1 (WT-Venus-H5N1) virus. However, the basis for this difference in virulence and Venus stability was unclear. Here, we investigated the molecular determinants behind this virulence and reporter stability by comparing WT-Venus-H5N1 virus with a mouse-adapted Venus-H5N1 (MA-Venus-H5N1) virus. To determine the genetic basis for these differences, we used reverse genetics to generate a series of reassortants of these two viruses. We found that reassortants with PB2 from MA-Venus-H5N1 (MA-PB2), MA-PA, or MA-NS expressed Venus more stably than did WT-Venus-H5N1 virus. We also found that a single mutation in PB2 (V25A) or in PA (R443K) increased the virulence of the WT-Venus-H5N1 virus in mice and that the presence of both of these mutations substantially enhanced the pathogenicity of the virus. Our results suggest roles for PB2 and PA in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus. IMPORTANCE: The ability to visualize influenza viruses has far-reaching benefits in influenza virus research. Previously, we reported that an H5N1 virus bearing the Venus reporter gene became more pathogenic to mice and that its reporter gene was more highly expressed and more stably maintained after mouse adaptation. Here, we investigated the molecular determinants behind this enhanced virulence and reporter stability. We found that mutations in PB2 (V25A) and PA (R443K) play crucial roles in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus and in the virulence of influenza virus in mice. Our findings further our knowledge of the pathogenicity of influenza virus in mammals and will help advance influenza virus-related live-imaging studies in vitro and in vivo.


Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Virus Reordenados/patogenicidad , Proteínas no Estructurales Virales/genética , Replicación Viral/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Perros , Femenino , Genes Reporteros/genética , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Virus Reordenados/genética , Análisis de Secuencia de ARN , Virulencia/genética
10.
Antimicrob Agents Chemother ; 60(3): 1902-6, 2015 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-26711748

RESUMEN

New strategies to develop novel broad-spectrum antiviral drugs against influenza virus infections are needed due to the emergence of antigenic variants and drug-resistant viruses. Here, we evaluated C646, a novel p300/CREB-binding protein-specific inhibitor of histone acetyltransferase (HAT), as an anti-influenza virus agent in vitro and in vivo and explored how C646 affects the viral life cycle and host response. Our studies highlight the value of targeting HAT activity for anti-influenza drug development.


Asunto(s)
Antivirales/farmacología , Benzoatos/farmacología , Proteína de Unión a CREB/antagonistas & inhibidores , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Histona Acetiltransferasas/antagonistas & inhibidores , Virus de la Influenza A/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Pirazoles/farmacología , Animales , Proteína de Unión a CREB/metabolismo , Línea Celular , Perros , Proteína p300 Asociada a E1A/metabolismo , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Nitrobencenos , Pirazolonas
11.
J Virol ; 88(6): 3127-34, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24371069

RESUMEN

UNLABELLED: Novel avian-origin influenza A(H7N9) viruses were first reported to infect humans in March 2013. To date, 143 human cases, including 45 deaths, have been recorded. By using sequence comparisons and phylogenetic and ancestral inference analyses, we identified several distinct amino acids in the A(H7N9) polymerase PA protein, some of which may be mammalian adapting. Mutant viruses possessing some of these amino acid changes, singly or in combination, were assessed for their polymerase activities and growth kinetics in mammalian and avian cells and for their virulence in mice. We identified several mutants that were slightly more virulent in mice than the wild-type A(H7N9) virus, A/Anhui/1/2013. These mutants also exhibited increased polymerase activity in human cells but not in avian cells. Our findings indicate that the PA protein of A(H7N9) viruses has several amino acid substitutions that are attenuating in mammals. IMPORTANCE: Novel avian-origin influenza A(H7N9) viruses emerged in the spring of 2013. By using computational analyses of A(H7N9) viral sequences, we identified several amino acid changes in the polymerase PA protein, which we then assessed for their effects on viral replication in cultured cells and mice. We found that the PA proteins of A(H7N9) viruses possess several amino acid substitutions that cause attenuation in mammals.


Asunto(s)
Sustitución de Aminoácidos , Subtipo H7N9 del Virus de la Influenza A/enzimología , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Gripe Aviar/virología , Gripe Humana/virología , Enfermedades de las Aves de Corral/virología , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Animales , Pollos , Patos , Femenino , Humanos , Subtipo H7N9 del Virus de la Influenza A/clasificación , Subtipo H7N9 del Virus de la Influenza A/genética , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Ratones , Ratones Endogámicos BALB C , Filogenia , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Virulencia
12.
J Virol ; 88(16): 8981-97, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24899188

RESUMEN

UNLABELLED: Occasional transmission of highly pathogenic avian H5N1 influenza viruses to humans causes severe pneumonia with high mortality. To better understand the mechanisms via which H5N1 viruses induce severe disease in humans, we infected cynomolgus macaques with six different H5N1 strains isolated from human patients and compared their pathogenicity and the global host responses to the virus infection. Although all H5N1 viruses replicated in the respiratory tract, there was substantial heterogeneity in their replicative ability and in the disease severity induced, which ranged from asymptomatic to fatal. A comparison of global gene expression between severe and mild disease cases indicated that interferon-induced upregulation of genes related to innate immunity, apoptosis, and antigen processing/presentation in the early phase of infection was limited in severe disease cases, although interferon expression was upregulated in both severe and mild cases. Furthermore, coexpression analysis of microarray data, which reveals the dynamics of host responses during the infection, demonstrated that the limited expression of these genes early in infection led to a failure to suppress virus replication and to the hyperinduction of genes related to immunity, inflammation, coagulation, and homeostasis in the late phase of infection, resulting in a more severe disease. Our data suggest that the attenuated interferon-induced activation of innate immunity, apoptosis, and antigen presentation in the early phase of H5N1 virus infection leads to subsequent severe disease outcome. IMPORTANCE: Highly pathogenic avian H5N1 influenza viruses sometimes transmit to humans and cause severe pneumonia with ca. 60% lethality. The continued circulation of these viruses poses a pandemic threat; however, their pathogenesis in mammals is not fully understood. We, therefore, investigated the pathogenicity of six H5N1 viruses and compared the host responses of cynomolgus macaques to the virus infection. We identified differences in the viral replicative ability of and in disease severity caused by these H5N1 viruses. A comparison of global host responses between severe and mild disease cases identified the limited upregulation of interferon-stimulated genes early in infection in severe cases. The dynamics of the host responses indicated that the limited response early in infection failed to suppress virus replication and led to hyperinduction of pathological condition-related genes late in infection. These findings provide insight into the pathogenesis of H5N1 viruses in mammals.


Asunto(s)
Regulación Viral de la Expresión Génica/genética , Expresión Génica/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/virología , Primates/virología , Animales , Presentación de Antígeno/inmunología , Apoptosis/inmunología , Células Cultivadas , Perros , Expresión Génica/inmunología , Regulación Viral de la Expresión Génica/inmunología , Humanos , Inmunidad Innata/inmunología , Inflamación/inmunología , Inflamación/virología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Macaca/inmunología , Macaca/virología , Macaca fascicularis/inmunología , Macaca fascicularis/virología , Células de Riñón Canino Madin Darby , Infecciones por Orthomyxoviridae/inmunología , Primates/inmunología , Sistema Respiratorio/inmunología , Sistema Respiratorio/virología , Índice de Severidad de la Enfermedad , Replicación Viral/genética , Replicación Viral/inmunología
13.
Nat Rev Immunol ; 4(9): 699-710, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15343369

RESUMEN

Recent studies indicate that the mechanism of nasopharynx-associated lymphoid tissue (NALT) organogenesis is different from that of other lymphoid tissues. NALT has an important role in the induction of mucosal immune responses, including the generation of T helper 1 and T helper 2 cells, and IgA-committed B cells. Moreover, intranasal immunization can lead to the induction of antigen-specific protective immunity in both the mucosal and systemic immune compartments. Therefore, a greater understanding of the differences between NALT and other organized lymphoid tissues, such as Peyer's patches, should facilitate the development of nasal vaccines.


Asunto(s)
Inmunidad Mucosa/inmunología , Nasofaringe/inmunología , Ganglios Linfáticos Agregados/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Ganglios Linfáticos/embriología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos Agregados/embriología , Ganglios Linfáticos Agregados/metabolismo
14.
J Virol ; 87(14): 7874-81, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23658445

RESUMEN

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/genética , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Western Blotting , Líquido del Lavado Bronquioalveolar/inmunología , Perros , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/genética , Cinética , Células de Riñón Canino Madin Darby , Ratones , Infecciones por Orthomyxoviridae/inmunología , Plásmidos/genética , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética
15.
J Virol ; 86(11): 6055-66, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22491448

RESUMEN

Viral pneumonia has been frequently reported during early stages of influenza virus pandemics and in many human cases of highly pathogenic avian influenza (HPAI) H5N1 virus infection. To better understand the pathogenesis of this disease, we produced nonlethal viral pneumonia in rhesus macaques by using an HPAI H5N1 virus (A/Anhui/2/2005; referred to as Anhui/2). Infected macaques were monitored for 14 days, and tissue samples were collected at 6 time points for virologic, histopathologic, and transcriptomic analyses. Anhui/2 efficiently replicated in the lung from 12 h to 3 days postinfection (p.i.) and caused temporal but severe pneumonia that began to resolve by day 14. Lung transcriptional changes were first observed at 6 h, and increased expression of vascular permeability regulators and neutrophil chemoattractants correlated with increased serum leakage and neutrophil infiltration in situ. Additional inflammatory, antiviral, and apoptotic genes were upregulated from 12 h, concurrent with viral antigen detection and increasing immune cell populations. A shift toward upregulation of acquired immunity was apparent after day 6. Expression levels of established immune cell molecular markers revealed remarkable similarity with pathological findings, indicating early and robust neutrophil infiltration, a slight delay in macrophage accumulation, and abundant late populations of T lymphocytes. We also characterized the putative mechanisms regulating a unique, pneumonia-associated biphasic fever pattern. Thus, this study is the first to use a comprehensive and integrative approach to delineate specific molecular mechanisms regulating influenza virus-induced pneumonia in nonhuman primates, an important first step toward better management of human influenza virus disease.


Asunto(s)
Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Neumonía Viral/patología , Neumonía Viral/virología , Transcriptoma , Animales , Modelos Animales de Enfermedad , Femenino , Histocitoquímica , Pulmón/patología , Pulmón/virología , Macaca mulatta , Macrófagos/inmunología , Masculino , Neutrófilos/inmunología , Linfocitos T/inmunología , Factores de Tiempo
16.
J Virol ; 86(17): 9361-8, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22718834

RESUMEN

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. Molecular evolutionary analyses of the 2009 pandemic influenza A H1N1 [A(H1N1)pdm09] virus revealed two major clusters, cluster I and cluster II. Although the pathogenicity of viruses belonging to cluster I, which became extinct by the end of 2009, has been examined in a nonhuman primate model, the pathogenic potential of viruses belonging to cluster II, which has spread more widely in the world, has not been studied in this animal model. Here, we characterized two Norwegian isolates belonging to cluster II, namely, A/Norway/3568/2009 (Norway3568) and A/Norway/3487-2/2009 (Norway3487), which caused distinct clinical symptoms, despite their genetic similarity. We observed more efficient replication in cultured cells and delayed virus clearance from ferret respiratory organs for Norway3487 virus, which was isolated from a severe case, compared with the efficiency of replication and time of clearance of Norway3568 virus, which was isolated from a mild case. Moreover, Norway3487 virus to some extent caused more severe lung damage in nonhuman primates than did Norway3568 virus. Our data suggest that the distinct replicative and pathogenic potentials of these two viruses may result from differences in their biological properties (e.g., the receptor-binding specificity of hemagglutinin and viral polymerase activity).


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Secuencia de Aminoácidos , Animales , Línea Celular , Femenino , Hurones , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H1N1 del Virus de la Influenza A/fisiología , Gripe Humana/epidemiología , Macaca , Datos de Secuencia Molecular , Noruega/epidemiología , Pandemias , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virulencia , Replicación Viral
17.
J Immunol ; 186(12): 6972-80, 2011 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-21555530

RESUMEN

TGF-ß can induce Foxp3(+) inducible regulatory T cells (Treg) and also synergize with IL-6 and IL-4 to induce Th17 and Th9 cells. We now report that NO modulates TGF-ß activity away from Treg but toward the Th1 lineage. NO potentiated Th1 differentiation in the presence of TGF-ß in both IL-12-independent and -dependent fashions by augmenting IFN-γ-activated STAT-1 and T-bet. Differentiation into Treg, Th1, and Th17 lineages could be modulated by NO competing with other cofactors, such as IL-6 and retinoic acid. NO antagonized IL-6 to block TGF-ß-directed Th17 differentiation, and together with IL-6, NO suppressed Treg development induced by TGF-ß and retinoic acid. Furthermore, we show that physiologically produced NO from TNF and inducible NO synthase-producing dendritic cells can contribute to Th1 development predominating over Treg development through a synergistic activity induced when these cells cocluster with conventional dendritic cells presenting Ag to naive Th cells. This illustrates that NO is another cofactor allowing TGF-ß to participate in development of multiple Th lineages and suggests a new mechanism by which NO, which is associated with protection against intracellular pathogens, might maintain effective Th1 immunity.


Asunto(s)
Diferenciación Celular/inmunología , Óxido Nítrico/farmacología , Linfocitos T Reguladores/citología , Células TH1/citología , Factor de Crecimiento Transformador beta/inmunología , Animales , Presentación de Antígeno , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/inmunología , Ratones , Óxido Nítrico/inmunología , Transducción de Señal/inmunología , Células TH1/efectos de los fármacos
18.
J Immunol ; 186(7): 4253-62, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-21357262

RESUMEN

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.


Asunto(s)
Antígenos Bacterianos/administración & dosificación , Tejido Linfoide/inmunología , Mucosa Nasal/inmunología , Nasofaringe/inmunología , Lectinas de Plantas/administración & dosificación , Cornetes Nasales/inmunología , Administración por Inhalación , Animales , Antígenos Bacterianos/inmunología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Recuento de Linfocitos , Tejido Linfoide/microbiología , Tejido Linfoide/ultraestructura , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Noqueados , Cavidad Nasal/inmunología , Cavidad Nasal/microbiología , Cavidad Nasal/ultraestructura , Mucosa Nasal/microbiología , Mucosa Nasal/ultraestructura , Nasofaringe/microbiología , Nasofaringe/ultraestructura , Lectinas de Plantas/biosíntesis , Lectinas de Plantas/inmunología , Salmonella typhimurium/inmunología , Streptococcus pyogenes/inmunología , Cornetes Nasales/microbiología , Cornetes Nasales/ultraestructura , Ulex/inmunología , Aglutininas del Germen de Trigo/inmunología
19.
J Virol ; 85(24): 13195-203, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21937653

RESUMEN

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.


Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Receptores Virales/metabolismo , Internalización del Virus , Animales , Línea Celular , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Pulmón/patología , Pulmón/virología , Macaca , Receptores Virales/genética
20.
Proc Natl Acad Sci U S A ; 106(15): 6244-9, 2009 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-19332782

RESUMEN

The herpesvirus entry mediator (HVEM; TNFRSF14) activates NF-kappaB through the canonical TNF-related cytokine LIGHT, serving as a costimulatory pathway during activation of T cells. HVEM also functions as a ligand for the Ig superfamily members B and T lymphocyte attenuator (BTLA) and CD160, both of which limit inflammatory responses initiated by T cells. Emerging evidence indicates BTLA also promotes T cell survival, but its structural differences from LIGHT intimate BTLA is unlikely to function as an activator of HVEM. We demonstrate here that BTLA, CD160, and herpes simplex virus envelope glycoprotein D (gD) function as activating ligands for HVEM, promoting NF-kappaB activation and cell survival. Membrane-expressed BTLA and CD160, as well as soluble dimeric receptor surrogates BTLA-Fc and gD-Fc specifically activated HVEM-dependent NF-kappaB. BTLA and CD160 engagement induced recruitment of TNF receptor-associated factor 2 (TRAF2), but not TRAF3, to HVEM that specifically activated the RelA but not the RelB form of NF-kappaB in a mucosal epithelial tumor cell line. Moreover, Btla(-/-) T cells survived poorly following activation but were rescued with BTLA-Fc, indicating HVEM-BTLA bidirectional signaling may serve as a critical cell-survival system for lymphoid and epithelial cells.


Asunto(s)
Miembro 14 de Receptores del Factor de Necrosis Tumoral/inmunología , Transducción de Señal/inmunología , Animales , Antígenos CD/inmunología , Línea Celular , Supervivencia Celular/inmunología , Proteínas Ligadas a GPI , Humanos , Inmunoglobulinas/inmunología , Ligandos , Activación de Linfocitos/inmunología , Ratones , Receptores Inmunológicos/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteínas del Envoltorio Viral/inmunología
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