RESUMO
Amyloid deposition of human islet amyloid polypeptide (hIAPP) within the islets of Langerhans is a pathological feature of type 2 diabetes mellitus. Substantial evidence indicates that the membrane-mediated aggregation and subsequent deposition of hIAPP are linked to dysfunction and death of pancreatic ß-cells, but the molecular processes of hIAPP deposition are poorly understood. In this study, we examined the membrane-mediated aggregation and deposition of hIAPP at supported planar lipid bilayers with and without raft components (i.e. cholesterol and sphingomyelin) to provide insight into hIAPP-induced membrane dysfunction. The adsorption of hIAPP onto the bilayers was studied using a quartz crystal microbalance with dissipation monitoring, which showed enhanced accumulation of the peptide onto the bilayer containing raft components. Microscope observations demonstrated the growth of the aggregates formed from the membrane-adsorbed hIAPP. The examination of the membrane interfaces revealed that hIAPP aggregates retained the ability to associate with the membranes during the aggregation process, resulting in insertion of the aggregates into the bilayers. We also report the inhibitory effect of insulin on the hIAPP deposition. These findings demonstrate the aggregation of hIAPP at the membrane interfaces leading to amyloid deposits associated with the membrane and suggest a role for insulin in hIAPP deposition. A presumed mechanism regulating hIAPP deposition at the membrane interfaces is discussed.
Assuntos
Membrana Celular/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Amiloide/metabolismo , Humanos , Insulina/metabolismoRESUMO
Influenza A virus-associated encephalopathy triggered by influenza virus infection often occurs in children aged five and younger in Japan. However, the mechanisms behind Influenza A virus-associated encephalopathy are not yet well understood. This study developed an Influenza A virus-associated encephalopathy-like model using mice infected with Influenza A virus and given lipopolysaccharide treatment. The results showed that the mice used in the model suffered from brain edemas nearly three times more severe, as well as having higher cytokine levels in sera compared to those of the control groups. Using gene expression profiling, cytokine-related genes were found not to be up-regulated in the brain in situ, while protein coding genes, which are known to be involved in blood-brain barrier disruption, were up-regulated. Categorizing the functional groups using gene ontology revealed the terms "ion channels," "calcium oscillation," and "membrane transporter activities." The blood-brain barrier disruption found in this Influenza A virus-associated encephalopathy model can therefore be assumed to be due to a cellular electrolyte imbalance of the neuronal tissue, in addition to a cytokine storm.
Assuntos
Edema Encefálico/patologia , Perfilação da Expressão Gênica , Lipopolissacarídeos/toxicidade , Infecções por Orthomyxoviridae/patologia , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The human influenza A virus (H3N2) has been the predominant influenza strain since 1992, and one property of this virus is non-agglutination of chicken erythrocytes [Ch(-) virus]. The Ch(-) virus in our study was able to acquire chicken hemagglutination [Ch(+)] by trypsin passage but not by chymotrypsin passage. Moreover, the trypsin-passaged Ch(+) viruses reacquired the Ch(-) property after a further chymotrypsin passage. In particular, genetic analysis showed no evidence of mutations in the hemagglutinin (HA) gene during either trypsin or chymotrypsin passages: the only differences found were in the HA cleavage sites between the trypsin-passaged virus and the chymotrypsin-passaged virus as determined by the N-terminal amino acid sequence. These results suggested that protease-dependent differences at the viral HA cleavage site, rather than genetic mutations, are likely to have a significant effect on the viral ability to produce chicken hemagglutination.
Assuntos
Quimotripsina/metabolismo , Hemaglutinação/fisiologia , Hemaglutininas/química , Hemaglutininas/metabolismo , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/fisiologia , Tripsina/metabolismo , Animais , Galinhas , Quimotripsina/farmacologia , Cães , Eritrócitos/química , Eritrócitos/efeitos dos fármacos , Eritrócitos/virologia , Cobaias , Hemaglutinação/efeitos dos fármacos , Células Madin Darby de Rim Canino , Inoculações Seriadas , Tripsina/farmacologiaRESUMO
Influenza A(H1N1)pdm virus caused the first human pandemic of the 21st century. Although various probiotic Lactobacillus species have been shown to have anti-microbial effects against pneumonia-inducing pathogens, the prophylactic efficacy and mechanisms behind their protection remain largely unknown. Here, we evaluated the prophylactic efficacy of heat-killed Lactobacillus pentosus b240 against lethal influenza A(H1N1)pdm virus infection in a mouse model. To further define the protective responses induced by b240, we performed virologic, histopathologic, and transcriptomic analyses on the mouse lungs. Although we did not observe an appreciable effect of b240 on virus growth, cytokine production, or histopathology, gene expressional analysis revealed that oral administration of b240 differentially regulates antiviral gene expression in mouse lungs. Our results unveil the possible mechanisms behind the protection mediated by b240 against influenza virus infection and provide new insights into probiotic therapy.
Assuntos
Antivirais/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Lactobacillus , Infecções por Orthomyxoviridae/terapia , Probióticos/uso terapêutico , Administração Oral , Animais , Antivirais/administração & dosagem , Linhagem Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Cães , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Feminino , Imunidade Inata/imunologia , Vírus da Influenza A Subtipo H1N1 , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Probióticos/administração & dosagemRESUMO
Alzheimer's disease (AD) is the most prevalent age-dependent form of dementia, characterized by extracellular amyloid deposits comprising amyloid ß-peptide (Aß) in the cerebral cortex. Increasing evidence has indicated that ganglioside GM1 (GM1) in lipid rafts plays a pivotal role in amyloid deposition of Aß and the related cytotoxicity in AD. Despite recent efforts to characterize Aß-lipid interactions, the effect of Aß aggregation on dynamic properties and organization of lipid membranes is poorly understood. In this study, we examined the aggregation of Aß on supported lipid bilayers containing raft components (i.e., cholesterol, sphingomyelin, and GM1) and its effects on the membrane properties. We showed that the lateral fluidity of membranes was significantly affected by membrane binding and subsequent aggregation of Aß. Microscopic observations of the membrane surfaces demonstrated an enhancement in phase separation of lipids as a result of interactions between Aß and GM1 during induced aggregation of Aß. The uptake of GM1 into Aß aggregates and the attendant membrane damage were also observed under a microscope when the membrane-anchored aggregates were formed. On the basis of these observations, we propose that Aß aggregates formed in the presence of lipid membranes have a latent ability to trigger the uptake of raft components accompanied by phase separation of lipids.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Colesterol/química , Colesterol/metabolismo , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/metabolismo , Humanos , Bicamadas Lipídicas/química , Lipossomos/química , Lipossomos/metabolismo , Microdomínios da Membrana/química , Modelos Moleculares , Transição de Fase , Ligação Proteica , Esfingomielinas/química , Esfingomielinas/metabolismoRESUMO
A novel swine-origin H1N1 influenza virus [A(H1N1)pdm09 virus] caused the 2009 influenza pandemic. Most patients exhibited mild symptoms similar to seasonal influenza, but some experienced severe clinical signs and, in the worst cases, died. Such differences in symptoms are generally associated with preexisting medical conditions, but recent reports indicate the possible involvement of viral factors in clinical severity. To better understand the mechanism of pathogenicity of the A(H1N1)pdm09 virus, here, we compared five viruses that are genetically similar but were isolated from patients with either severe or mild symptoms. In a mouse model, A/Norway/3487/2009 (Norway3487) virus exhibited greater pathogenicity than did A/Osaka/164/2009 (Osaka164) virus. By exploiting reassortant viruses between these two viruses, we found that viruses possessing the hemagglutinin (HA) gene of Norway3487 in the genetic background of Osaka164 were more pathogenic in mice than other reassortant viruses, indicating a role for HA in the high virulence of Norway3487 virus. Intriguingly, a virus possessing HA, NA, and NS derived from Norway3487 exhibited greater pathogenicity in mice in concert with PB2 and PB1 derived from Osaka164 than did the parental Norway3487 virus. These findings demonstrate that reassortment between A(H1N1)pdm09 viruses can lead to increased pathogenicity and highlight the need for continued surveillance of A(H1N1)pdm09 viruses.
Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Vírus Reordenados/patogenicidade , Proteínas Virais/genética , Virulência , Fatores de Virulência/genéticaRESUMO
During the influenza pandemic of 2009, the number of viral pneumonia cases showed a marked increase in comparison with seasonal influenza viruses. Mutations at amino acid 222 (D222G mutations) in the virus hemagglutinin (HA) molecule, known to alter the receptor-recognition properties of the virus, were detected in a number of the more severely-affected patients in the early phases of the pandemic. To understand the background for the emergence of the mutant amino acid D222G in human lungs, we conducted histological examinations on lung specimens of patients from Mexico who had succumbed in the pandemic. Prominent regenerative and hyperplastic changes in the alveolar type II pneumocytes, which express avian-type sialoglycan receptors in the respiratory tract of severely affected individuals, were observed in the Mexican patients. An infection model utilizing guinea pigs, which was chosen in order to best simulate the sialic acid distribution of severe pneumonia in human patients, demonstrated an increase of D222G mutants and a delay in the diminution of mutants in the lower respiratory tract in comparison to the upper respiratory tract. Our data suggests that the predominance of avian-type sialoglycan receptors in the pneumonic lungs may contribute to the emergence of viral HA mutants. This data comprehensively illustrates the mechanisms for the emergence of mutants in the clinical samples.
Assuntos
Modelos Animais de Doenças , Hemaglutininas Virais/genética , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Viral/virologia , Células Epiteliais Alveolares/virologia , Animais , Surtos de Doenças , Feminino , Genes Virais , Cobaias , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/patologia , Células Madin Darby de Rim Canino , Mutação , Pneumonia Viral/patologia , RNA Viral/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Although the viral factors of host adaptation from domestic poultry to humans have been studied several times since the first cases of direct transmission of highly pathogenic avian influenza viruses from domestic poultry to humans were confirmed in 1997, the host-specific adaptation mechanisms from waterfowl to domestic poultry remain unknown. To study the mechanisms involved, a waterfowl-derived virus was passaged in a chicken fibroblast cell line. This passaged virus was found to have much higher growth titer than that of the original virus and several mutations were discovered in its genome. One of the most characteristics was an increase of the polymorphism of the internal genes. In addition, the general applicability of this property to the field isolates of influenza A viruses by database sequences analysis was confirmed, with the smallest amount of amino acid polymorphism in viral internal proteins observed in waterfowl-derived viruses, more in domestic poultry and the most in human-derived viruses. Although specific amino acid changes conserved in human-derived viruses were found, such amino acid changes were not observed in poultry-derived viruses.
Assuntos
Genoma Viral , Vírus da Influenza A Subtipo H1N1/genética , Animais , Embrião de Galinha , Cães , Patos/virologia , Humanos , Células Madin Darby de Rim Canino , Mutação , Análise de Sequência de RNARESUMO
Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.
Assuntos
Adaptação Fisiológica/genética , Furões/virologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Vírus Reordenados/patogenicidade , Sistema Respiratório/virologia , Animais , Bioterrorismo/prevenção & controle , Aves/virologia , Líquidos Corporais/virologia , Linhagem Celular , Cães , Evolução Molecular , Feminino , Células HEK293 , Células HeLa , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Temperatura Alta , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/fisiologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/transmissão , Influenza Aviária/virologia , Influenza Humana/prevenção & controle , Influenza Humana/transmissão , Influenza Humana/virologia , Epidemiologia Molecular/métodos , Pandemias , Vigilância da População/métodos , Estabilidade Proteica , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Vírus Reordenados/fisiologia , Receptores Virais/química , Receptores Virais/metabolismo , Sistema Respiratório/anatomia & histologia , Medidas de Segurança , Zoonoses/transmissão , Zoonoses/virologiaRESUMO
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).
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Feminino , Furões , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/epidemiologia , Macaca , Dados de Sequência Molecular , Noruega/epidemiologia , Pandemias , Proteínas Virais/genética , Proteínas Virais/metabolismo , Virulência , Replicação ViralRESUMO
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.
Assuntos
Virus da Influenza A Subtipo H5N1/patogenicidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Transcriptoma , Animais , Modelos Animais de Doenças , Feminino , Histocitoquímica , Pulmão/patologia , Pulmão/virologia , Macaca mulatta , Macrófagos/imunologia , Masculino , Neutrófilos/imunologia , Linfócitos T/imunologia , Fatores de TempoRESUMO
Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Transdução de Sinais , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Linhagem Celular , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Humana/genética , Influenza Humana/virologia , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 4 Toll-Like/genéticaRESUMO
Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)--and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.
Assuntos
Adaptação Fisiológica , Patos/virologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Aviária/virologia , Codorniz/virologia , Animais , Linhagem Celular , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H3N2/genética , Mucosa Intestinal/metabolismo , Modelos Moleculares , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácidos Siálicos/metabolismoRESUMO
Oseltamivir-resistant H1N1 influenza viruses emerged in 2007 to 2008 and have subsequently circulated widely. However, prior to 2007 to 2008, viruses possessing the neuraminidase (NA) H274Y mutation, which confers oseltamivir resistance, generally had low growth capability. NA mutations that compensate for the deleterious effect of the NA H274Y mutation have since been identified. Given the importance of the functional balance between hemagglutinin (HA) and NA, we focused on amino acid changes in HA. Reverse genetic analysis showed that a mutation at residue 82, 141, or 189 of the HA protein promotes virus replication in the presence of the NA H274Y mutation. Our findings thus identify HA mutations that contributed to the replacement of the oseltamivir-sensitive viruses of 2007 to 2008.
Assuntos
Substituição de Aminoácidos , Antivirais/farmacologia , Farmacorresistência Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Oseltamivir/farmacologia , Replicação Viral , Sequência de Aminoácidos , Substituição de Aminoácidos/efeitos dos fármacos , Animais , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Neuraminidase/genética , Neuraminidase/metabolismo , Filogenia , Alinhamento de Sequência , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The matrix protein VP40 of Marburg virus promotes the formation and release of virus-like particles (VLPs). Marburg virus VP40 interacts with cellular Tsg101 via its L domain motif; however, mutation of this motif does not affect VLP budding or the accumulation of VP40 in multivesicular bodies (MVBs), which are platforms for virus particle formation. To identify regions of Marburg virus VP40 that are important for VLP budding, we examined deletion mutants and alanine-scanning mutants at the N- and C-terminus of VP40 for their involvement in VLP budding. VLPs were not detected in the presence of alanine-replacement mutants at Ile39 and Thr40, and the level of VLP budding for the alanine mutant at Asn297 was decreased. Moreover, these mutants did not accumulate in MVBs. Our results suggest the involvement of a novel host factor(s) in VLP budding and VP40 transport to MVBs.
Assuntos
Marburgvirus/metabolismo , Proteínas da Matriz Viral/metabolismo , Liberação de Vírus , Motivos de Aminoácidos , Sequência de Aminoácidos , Regulação Viral da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Marburgvirus/genética , Dados de Sequência Molecular , Corpos Multivesiculares/metabolismo , Mutação , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
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.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Receptores Virais/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Pulmão/patologia , Pulmão/virologia , Macaca , Receptores Virais/genéticaRESUMO
Human pandemic H1N1 2009 influenza virus rapidly infected millions worldwide and was associated with significant mortality. Antiviral drugs that inhibit influenza virus replication are the primary therapy used to diminish disease; however, there are two significant limitations to their effective use: (i) antiviral drugs exert selective pressure on the virus, resulting in the generation of more fit viral progeny that are resistant to treatment; and (ii) antiviral drugs do not directly inhibit immune-mediated pulmonary injury that is a significant component of disease. Here we show that dampening the host's immune response against influenza virus using an immunomodulatory drug, AAL-R, provides significant protection from mortality (82%) over that of the neuraminidase inhibitor oseltamivir alone (50%). AAL-R combined with oseltamivir provided maximum protection against a lethal challenge of influenza virus (96%). Mechanistically, AAL-R inhibits cellular and cytokine/chemokine responses to limit immunopathologic damage, while maintaining host control of virus replication. With cytokine storm playing a role in the pathogenesis of a wide assortment of viral, bacterial, and immunologic diseases, a therapeutic approach using sphingosine analogs is of particular interest.
Assuntos
Citocinas/imunologia , Imunomodulação/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/farmacologia , Esfingosina/farmacologia , Alternaria/química , Animais , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Citocinas/metabolismo , Cães , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Infecções por Orthomyxoviridae/imunologia , Oseltamivir/metabolismo , Oseltamivir/uso terapêutico , Esfingosina/metabolismo , Esfingosina/uso terapêuticoRESUMO
The isolation of an H5N1 influenza A virus from a tree sparrow (Passer montanus) captured in East Java, Indonesia in 2010 is reported here. Its hemagglutinin and neuraminidase were genetically similar to those of human isolates from 2006-2007 in Indonesia. The finding of a tree sparrow H5N1 virus that possesses genetically similar surface molecules to those of human viruses highlights the importance of monitoring resident wild birds, as well as migratory birds, for pandemic preparedness.
Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Pardais/virologia , Animais , Análise por Conglomerados , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Indonésia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Dados de Sequência Molecular , Neuraminidase/genética , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas Virais/genéticaRESUMO
Despite the high prevalence of highly pathogenic H5N1 influenza A viruses in Indonesia, epidemiology information on seasonal human influenza is lacking. The present authors, therefore, conducted virologic surveillance in Surabaya, East Java from October 2008 to March 2010. Influenza viruses, including pandemic (H1N1) 2009 viruses, were isolated from 71 of 635 individuals tested. Seasonal influenza peaked in the rainy season. Compared with seasonal influenza viruses, pandemic 2009 viruses were isolated from younger patients with milder symptoms. Given the high prevalence of H5N1 infections in humans, continued influenza surveillance is essential for pandemic preparedness.
Assuntos
Influenza Humana/epidemiologia , Influenza Humana/virologia , Adolescente , Adulto , Distribuição por Idade , Criança , Pré-Escolar , Humanos , Indonésia/epidemiologia , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Dados de Sequência Molecular , Orthomyxoviridae/classificação , Orthomyxoviridae/genética , Orthomyxoviridae/isolamento & purificação , Prevalência , RNA Viral/genética , Estações do Ano , Análise de Sequência de DNA , Adulto JovemRESUMO
Although oral exposure to H5N1 highly pathogenic avian influenza viruses is a risk factor for infection in humans, it is unclear how oral exposure to these virus results in lethal respiratory infections. To address this issue, we inoculated ferrets and hamsters with two highly pathogenic H5N1 strains. These viruses, inoculated directly into the stomach, were isolated from the large intestine and the mesenteric lymph nodes within 1 day of inoculation and subsequently spread to multiple tissues, including lung, liver, and brain. Histopathologic analysis of ferrets infected with virus via direct intragastric inoculation revealed lymph folliculitis in the digestive tract and mesenteric lymph nodes and focal interstitial pneumonia. Comparable results were obtained with the hamster model. We conclude that, in mammals, ingested H5N1 influenza viruses can disseminate to nondigestive organs, possibly through the lymphatic system of the gastrointestinal tract.