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1.
Sci Transl Med ; 11(502)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31341062

RESUMO

The 2018-2019 period marks the centennial of the "Spanish" influenza pandemic, which caused at least 50 million deaths worldwide. The unprecedented nature of the pandemic's sudden appearance and high fatality rate serve as a stark reminder of the threat influenza poses. Unusual features of the 1918-1919 pandemic, including age-specific mortality and the high frequency of severe pneumonias, are still not fully understood. Sequencing and reconstruction of the 1918 virus has allowed scientists to answer many questions about its origin and pathogenicity, although many questions remain. This Review summarizes key findings and still-to-be answered questions about this deadliest of human events.

2.
Virology ; 534: 96-107, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31226666

RESUMO

Nasal wash samples from 15 human volunteers challenged with GMP manufactured influenza A/California/04/2009(H1N1) and from 5 naturally infected influenza patients of the 2009 pandemic were deep sequenced using viral targeted hybridization enrichment. Ten single nucleotide polymorphism (SNP) positions were found in the challenge virus. Some of the nonsynonymous changes in the inoculant virus were maintained in some challenge participants, but not in others, indicating that virus is evolving away from the Vero cell adapted inoculant, for example SNPs in the neuraminidase. Many SNP sites in challenge patients and naturally infected patients were found, many not identified previously. The SNPs identified, and phylogenetic analyses, showed that intrahost evolution of the virus are different in challenge participants and naturally infected patients. This study, using hybridization enrichment without PCR, provided an accurate and unbiased assessment of differential intrahost viral evolution from a uniform influenza inoculant in humans and comparison to naturally infected patients.

3.
MBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088926

RESUMO

In this study, we examined the relationships between anti-influenza virus serum antibody titers, clinical disease, and peripheral blood leukocyte (PBL) global gene expression during presymptomatic, acute, and convalescent illness in 83 participants infected with 2009 pandemic H1N1 virus in a human influenza challenge model. Using traditional statistical and logistic regression modeling approaches, profiles of differentially expressed genes that correlated with active viral shedding, predicted length of viral shedding, and predicted illness severity were identified. These analyses further demonstrated that challenge participants fell into three peripheral blood leukocyte gene expression phenotypes that significantly correlated with different clinical outcomes and prechallenge serum titers of antibodies specific for the viral neuraminidase, hemagglutinin head, and hemagglutinin stalk. Higher prechallenge serum antibody titers were inversely correlated with leukocyte responsiveness in participants with active disease and could mask expression of peripheral blood markers of clinical disease in some participants, including viral shedding and symptom severity. Consequently, preexisting anti-influenza antibodies may modulate PBL gene expression, and this must be taken into consideration in the development and interpretation of peripheral blood diagnostic and prognostic assays of influenza infection.IMPORTANCE Influenza A viruses are significant human pathogens that caused 83,000 deaths in the United States during 2017 to 2018, and there is need to understand the molecular correlates of illness and to identify prognostic markers of viral infection, symptom severity, and disease course. Preexisting antibodies against viral neuraminidase (NA) and hemagglutinin (HA) proteins play a critical role in lessening disease severity. We performed global gene expression profiling of peripheral blood leukocytes collected during acute and convalescent phases from a large cohort of people infected with A/H1N1pdm virus. Using statistical and machine-learning approaches, populations of genes were identified early in infection that correlated with active viral shedding, predicted length of shedding, or disease severity. Finally, these gene expression responses were differentially affected by increased levels of preexisting influenza antibodies, which could mask detection of these markers of contagiousness and disease severity in people with active clinical disease.


Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Influenza Humana/imunologia , Leucócitos/imunologia , Neuraminidase/imunologia , Doença Aguda , Adolescente , Adulto , Convalescença , Proteção Cruzada , Feminino , Perfilação da Expressão Gênica , Voluntários Saudáveis , Testes de Inibição da Hemaglutinação , Experimentação Humana , Humanos , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/sangue , Masculino , Pessoa de Meia-Idade , Eliminação de Partículas Virais , Adulto Jovem
4.
Clin Infect Dis ; 2019 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-30953061

RESUMO

BACKGROUND: Identification of correlates of protection against human influenza A virus infection is important in development of broadly protective ("universal") influenza vaccines. Certain assumptions underlie current vaccine developmental strategies including that infection with a particular influenza A virus should offer long term or lifelong protection against that strain, preventing reinfection. In this study we report observations made when 7 volunteers participated in sequential influenza challenge studies where they were challenged intranasally using the identical influenza A(H1N1)pdm09 virus approximately 1 year apart. We evaluate and describe the outcomes of these seven re-challenge participants and discuss what these results may suggest about correlates of protection and development of more broadly protective influenza vaccines. METHODS: Seven participants were enrolled in two viral challenge studies at 7.5 to 18.5 month intervals. Both challenge studies used the identical lot of influenza A (H1N1)pdm09 virus administered intranasally. We evaluated pre- and post-challenge HAI, NAI, and stalk antibody titers, peripheral blood leukocyte (PBL) host gene expression response profiles, daily viral detection via nasal wash, and clinical signs and symptoms. RESULTS: At least 3 of 7 participants demonstrated confirmed laboratory evidence of sequential infection with 5 of 7 demonstrating clinical evidence. CONCLUSION: The data presented in this report demonstrate that sequential infection with the identical influenza A virus can occur and suggest it may not be rare. These data raise questions about immune memory responses in an acute superficial respiratory mucosal infection and their implications in development of broadly protective influenza vaccines. Further investigation of these observations is warranted.

5.
mSphere ; 3(5)2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30232169

RESUMO

Influenza A virus (IAV) infections are a major public health concern, including annual epidemics, epizootic outbreaks, and pandemics. A significant IAV epizootic outbreak was the H7N9 avian influenza A outbreak in China, which was first detected in 2013 and which has spread over 5 waves from 2013 to 2017, causing human infections in many different Chinese provinces. Here, RNA from primary clinical throat swab samples from 20 H7N9-infected local patients with different clinical outcomes, who were admitted and treated at one hospital in Shanghai, China, from April 2013 to April 2015, was analyzed. Whole-transcriptome amplification, with positive enrichment of IAV RNA, was performed, all 20 samples were subjected to deep sequencing, and data from 16 samples were analyzed in detail. Many single-nucleotide polymorphisms, including ones not previously reported, and many nonsynonymous changes that could affect hemagglutinin head and stalk antibody binding epitopes were observed. Minor populations representing viral quasispecies, including nonsynonymous hemagglutinin changes shared by antigenically variant H7N9 clades identified in the most recent wave of H7N9 infections in 2016 to 2017, were also identified.IMPORTANCE H7N9 subtype avian influenza viruses caused infections in over 1,400 humans from 2013 to 2017 and resulted in almost 600 deaths. It is important to understand how avian influenza viruses infect and cause disease in humans and to assess their potential for efficient person-to-person transmission. In this study, we used deep sequencing of primary clinical material to assess the evolution and potential for human adaptation of H7N9 influenza viruses.

6.
mSphere ; 3(1)2018 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29299535

RESUMO

Neutrophils are essential cells of host innate immunity. Although the role of neutrophils in defense against bacterial and fungal infections is well characterized, there is a relative paucity of information about their role against viral infections. Influenza A virus (IAV) infection can be associated with secondary bacterial coinfection, and it has long been posited that the ability of IAV to alter normal neutrophil function predisposes individuals to secondary bacterial infections. To better understand this phenomenon, we evaluated the interaction of pandemic or seasonal H1N1 IAV with human neutrophils isolated from healthy persons. These viruses were ingested by human neutrophils and elicited changes in neutrophil gene expression that are consistent with an interferon-mediated immune response. The viability of neutrophils following coculture with either pandemic or seasonal H1N1 IAV was similar for up to 18 h of culture. Notably, neutrophil exposure to seasonal (but not pandemic) IAV primed these leukocytes for enhanced functions, including production of reactive oxygen species and bactericidal activity. Taken together, our results are at variance with the universal idea that IAV impairs neutrophil function directly to predispose individuals to secondary bacterial infections. Rather, we suggest that some strains of IAV prime neutrophils for enhanced bacterial clearance. IMPORTANCE A long-standing notion is that IAV inhibits normal neutrophil function and thereby predisposes individuals to secondary bacterial infections. Here we report that seasonal H1N1 IAV primes human neutrophils for enhanced killing of Staphylococcus aureus. Moreover, we provide a comprehensive view of the changes in neutrophil gene expression during interaction with seasonal or pandemic IAV and report how these changes relate to functions such as bactericidal activity. This study expands our knowledge of IAV interactions with human neutrophils.

7.
Sci Transl Med ; 9(385)2017 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-28404864

RESUMO

The 2013-2015 outbreak of Ebola virus disease in Guinea, Liberia, and Sierra Leone was unprecedented in the number of documented cases, but there have been few published reports on immune responses in clinical cases and their relationships with the course of illness and severity of Ebola virus disease. Symptoms of Ebola virus disease can include severe headache, myalgia, asthenia, fever, fatigue, diarrhea, vomiting, abdominal pain, and hemorrhage. Although experimental treatments are in development, there are no current U.S. Food and Drug Administration-approved vaccines or therapies. We report a detailed study of host gene expression as measured by microarray in daily peripheral blood samples collected from a patient with severe Ebola virus disease. This individual was provided with supportive care without experimental therapies at the National Institutes of Health Clinical Center from before onset of critical illness to recovery. Pearson analysis of daily gene expression signatures revealed marked gene expression changes in peripheral blood leukocytes that correlated with changes in serum and peripheral blood leukocytes, viral load, antibody responses, coagulopathy, multiple organ dysfunction, and then recovery. This study revealed marked shifts in immune and antiviral responses that preceded changes in medical condition, indicating that clearance of replicating Ebola virus from peripheral blood leukocytes is likely important for systemic viral clearance.


Assuntos
Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/virologia , Leucócitos/metabolismo , Surtos de Doenças , Doença pelo Vírus Ebola/sangue , Humanos , Estudos Longitudinais , RNA Viral/sangue , RNA Viral/genética , Replicação Viral/fisiologia
8.
Antiviral Res ; 129: 120-129, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26923881

RESUMO

BACKGROUND: Influenza results in up to 500,000 deaths annually. Seasonal influenza vaccines have an estimated 60% effectiveness, but provide little or no protection against novel subtypes, and may be less protective in high-risk groups. Neuraminidase inhibitors are recommended for the treatment of severe influenza infection, but are not proven to reduce mortality in severe disease. Preclinical models of severe influenza infection that closely correlate to human disease are needed to assess efficacy of new vaccines and therapeutics. METHODS: We developed a nonhuman primate model of influenza and bacterial co-infection that recapitulates severe pneumonia in humans. Animals were infected with influenza A virus via intra-bronchial or small-particle aerosol inoculation, methicillin-resistant Staphylococcus aureus, or co-infected with influenza and methicillin-resistant S. aureus combined. We assessed the severity of disease in animals over the course of our study using tools available to evaluate critically ill human patients including high-resolution computed tomography imaging of the lungs, arterial blood gas analyses, and bronchoalveolar lavage. RESULTS: Using an intra-bronchial route of inoculation we successfully induced severe pneumonia following influenza infection alone and following influenza and bacterial co-infection. Peak illness was observed at day 6 post-influenza infection, manifested by bilateral pulmonary infiltrates and hypoxemia. The timing of radiographic and physiologic manifestations of disease in our model closely match those observed in severe human influenza infection. DISCUSSION: This was the first nonhuman primate study of influenza and bacterial co-infection where high-resolution computed tomography scanning of the lungs was used to quantitatively assess pneumonia over the course of illness and where hypoxemia was correlated with pneumonia severity. With additional validation this model may serve as a pathway for regulatory approval of vaccines and therapeutics for the prevention and treatment of severe influenza pneumonia.


Assuntos
Coinfecção , Vírus da Influenza A , Modelos Animais , Infecções por Orthomyxoviridae/complicações , Pneumonia Estafilocócica/complicações , Pneumonia Viral/complicações , Animais , Humanos , Vírus da Influenza A/patogenicidade , Vacinas contra Influenza , Influenza Humana/complicações , Influenza Humana/microbiologia , Pulmão/microbiologia , Pulmão/patologia , Pulmão/virologia , Macaca mulatta , Masculino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons
9.
J Pathol ; 238(1): 85-97, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26383585

RESUMO

To study bacterial co-infection following 1918 H1N1 influenza virus infection, mice were inoculated with the 1918 influenza virus, followed by Streptococcus pneumoniae (SP) 72 h later. Co-infected mice exhibited markedly more severe disease, shortened survival time and more severe lung pathology, including widespread thrombi. Transcriptional profiling revealed activation of coagulation only in co-infected mice, consistent with the extensive thrombogenesis observed. Immunohistochemistry showed extensive expression of tissue factor (F3) and prominent deposition of neutrophil elastase on endothelial and epithelial cells in co-infected mice. Lung sections of SP-positive 1918 autopsy cases showed extensive thrombi and prominent staining for F3 in alveolar macrophages, monocytes, neutrophils, endothelial and epithelial cells, in contrast to co-infection-positive 2009 pandemic H1N1 autopsy cases. This study reveals that a distinctive feature of 1918 influenza virus and SP co-infection in mice and humans is extensive expression of tissue factor and activation of the extrinsic coagulation pathway leading to widespread pulmonary thrombosis.


Assuntos
Coinfecção/complicações , Influenza Humana/microbiologia , Infecções por Orthomyxoviridae/microbiologia , Infecções Pneumocócicas/microbiologia , Embolia Pulmonar/microbiologia , Animais , Coagulação Sanguínea , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Vírus da Influenza A Subtipo H1N1 , Influenza Pandêmica, 1918-1919 , Influenza Humana/complicações , Influenza Humana/patologia , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/patologia , Infecções Pneumocócicas/complicações , Infecções Pneumocócicas/patologia , Embolia Pulmonar/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Streptococcus pneumoniae
10.
MBio ; 6(4): e01044, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26199334

RESUMO

UNLABELLED: Influenza virus infections are a global public health problem, with a significant impact of morbidity and mortality from both annual epidemics and pandemics. The current strategy for preventing annual influenza is to develop a new vaccine each year against specific circulating virus strains. Because these vaccines are unlikely to protect against an antigenically divergent strain or a new pandemic virus with a novel hemagglutinin (HA) subtype, there is a critical need for vaccines that protect against all influenza A viruses, a so-called "universal" vaccine. Here we show that mice were broadly protected against challenge with a wide variety of lethal influenza A virus infections (94% aggregate survival following vaccination) with a virus-like particle (VLP) vaccine cocktail. The vaccine consisted of a mixture of VLPs individually displaying H1, H3, H5, or H7 HAs, and vaccinated mice showed significant protection following challenge with influenza viruses expressing 1918 H1, 1957 H2, and avian H5, H6, H7, H10, and H11 hemagglutinin subtypes. These experiments suggest a promising and practical strategy for developing a broadly protective "universal" influenza vaccine. IMPORTANCE: The rapid and unpredictable nature of influenza A virus evolution requires new vaccines to be produced annually to match circulating strains. Human infections with influenza viruses derived from animals can cause outbreaks that may be associated with high mortality, and such strains may also adapt to humans to cause a future pandemic. Thus, there is a large public health need to create broadly protective, or "universal," influenza vaccines that could prevent disease from a wide variety of human and animal influenza A viruses. In this study, a noninfectious virus-like particle (VLP) vaccine was shown to offer significant protection against a variety of influenza A viruses in mice, suggesting a practical strategy to develop a universal influenza vaccine.


Assuntos
Proteção Cruzada , Imunidade Heteróloga , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Administração Intranasal , Animais , Modelos Animais de Doenças , Camundongos , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem
11.
Am J Pathol ; 185(6): 1528-36, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25747532

RESUMO

Influenza A virus infections in humans generally cause self-limited infections, but can result in severe disease, secondary bacterial pneumonias, and death. Influenza viruses can replicate in epithelial cells throughout the respiratory tree and can cause tracheitis, bronchitis, bronchiolitis, diffuse alveolar damage with pulmonary edema and hemorrhage, and interstitial and airspace inflammation. The mechanisms by which influenza infections result in enhanced disease, including development of pneumonia and acute respiratory distress, are multifactorial, involving host, viral, and bacterial factors. Host factors that enhance risk of severe influenza disease include underlying comorbidities, such as cardiac and respiratory disease, immunosuppression, and pregnancy. Viral parameters enhancing disease risk include polymerase mutations associated with host switch and adaptation, viral proteins that modulate immune and antiviral responses, and virulence factors that increase disease severity, which can be especially prominent in pandemic viruses and some zoonotic influenza viruses causing human infections. Influenza viral infections result in damage to the respiratory epithelium that facilitates secondary infection with common bacterial pneumopathogens and can lead to secondary bacterial pneumonias that greatly contribute to respiratory distress, enhanced morbidity, and death. Understanding the molecular mechanisms by which influenza and secondary bacterial infections, coupled with the role of host risk factors, contribute to enhanced morbidity and mortality is essential to develop better therapeutic strategies to treat severe influenza.


Assuntos
Coinfecção/patologia , Influenza Humana/patologia , Pulmão/patologia , Pneumonia Bacteriana/patologia , Mucosa Respiratória/patologia , Coinfecção/microbiologia , Coinfecção/virologia , Progressão da Doença , Humanos , Influenza Humana/microbiologia , Influenza Humana/virologia , Pulmão/microbiologia , Pulmão/virologia , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/virologia , Mucosa Respiratória/microbiologia , Mucosa Respiratória/virologia
12.
MBio ; 5(6): e02116, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25406382

RESUMO

UNLABELLED: Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. IMPORTANCE: Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections with these subtypes may be important in controlling future outbreaks.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/crescimento & desenvolvimento , Influenza Aviária/virologia , Fatores de Virulência/metabolismo , Animais , Aves , Efeito Citopatogênico Viral , Modelos Animais de Doenças , Células Epiteliais/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Mamíferos , Camundongos , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Vírus Reordenados/genética , Vírus Reordenados/crescimento & desenvolvimento , Vírus Reordenados/patogenicidade , Genética Reversa , Fatores de Virulência/genética
13.
Free Radic Biol Med ; 67: 235-47, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24140866

RESUMO

The 1918 influenza pandemic caused over 40 million deaths worldwide, with 675,000 deaths in the United States alone. Studies in several experimental animal models showed that 1918 influenza virus infection resulted in severe lung pathology associated with dysregulated immune and cell death responses. To determine if reactive oxygen species produced by host inflammatory responses play a central role in promoting severity of lung pathology, we treated 1918 influenza virus-infected mice with the catalytic catalase/superoxide dismutase mimetic, salen-manganese complex EUK-207 beginning 3 days postinfection. Postexposure treatment of mice infected with a lethal dose of the 1918 influenza virus with EUK-207 resulted in significantly increased survival and reduced lung pathology without a reduction in viral titers. In vitro studies also showed that EUK-207 treatment did not affect 1918 influenza viral replication. Immunohistochemical analysis showed a reduction in the detection of the apoptosis marker cleaved caspase-3 and the oxidative stress marker 8-oxo-2'-deoxyguanosine in lungs of EUK-207-treated animals compared to vehicle controls. High-throughput sequencing and RNA expression microarray analysis revealed that treatment resulted in decreased expression of inflammatory response genes and increased lung metabolic and repair responses. These results directly demonstrate that 1918 influenza virus infection leads to an immunopathogenic immune response with excessive inflammatory and cell death responses that can be limited by treatment with the catalytic antioxidant EUK-207.


Assuntos
Depuradores de Radicais Livres/farmacologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Pandêmica, 1918-1919 , Compostos Organometálicos/farmacologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Biomarcadores/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Reparo do DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Cães , Feminino , Expressão Gênica , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/mortalidade , Inflamação/virologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sobrevida , Carga Viral , Replicação Viral
14.
Virulence ; 4(8): 707-15, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24104465

RESUMO

Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.


Assuntos
Coinfecção/microbiologia , Coinfecção/virologia , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Interações Microbianas , Infecções por Orthomyxoviridae/complicações , Pneumonia Estafilocócica/complicações , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Coinfecção/patologia , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/patologia , Macaca fascicularis , Masculino , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Estafilocócica/microbiologia , Pneumonia Estafilocócica/patologia
15.
J Pathol ; 229(4): 535-45, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23180419

RESUMO

Most biopsy and autopsy tissues are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. The RNA genome of the 1918 pandemic influenza virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Here, the full genome of the 1918 virus at 3000× coverage was determined in one high-throughput sequencing run of a library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. Bacterial sequences associated with secondary bacterial pneumonias were also detected. Host transcripts were well represented in the library. Compared to a 2009 pandemic influenza virus FFPE post-mortem library, the 1918 sample showed significant enrichment for host defence and cell death response genes, concordant with prior animal studies. This methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of diseases.


Assuntos
Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Vírus da Influenza A/genética , Influenza Humana/virologia , Pulmão/virologia , Células Epiteliais/virologia , Feminino , Formaldeído , Biblioteca Gênica , Humanos , Vírus da Influenza A/isolamento & purificação , Pulmão/microbiologia , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Pandemias , Inclusão em Parafina , RNA/genética , Estabilidade de RNA , Análise de Sequência de RNA , Adulto Jovem
16.
PLoS Pathog ; 8(11): e1002998, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23133386

RESUMO

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.


Assuntos
Processamento Alternativo , Vírus da Influenza A Subtipo H5N2/metabolismo , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Proteínas da Matriz Viral/biossíntese , Animais , Aves , Linhagem Celular Tumoral , Surtos de Doenças , Cães , Humanos , Vírus da Influenza A Subtipo H5N2/genética , Influenza Aviária/epidemiologia , Influenza Aviária/genética , Influenza Aviária/metabolismo , Influenza Humana/epidemiologia , Influenza Humana/genética , Influenza Humana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , América do Norte/epidemiologia , RNA Mensageiro/genética , RNA Viral/genética , Proteínas da Matriz Viral/genética
17.
J Virol ; 86(17): 9211-20, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22718825

RESUMO

The 1918-1919 "Spanish" influenza pandemic is estimated to have caused 50 million deaths worldwide. Understanding the origin, virulence, and pathogenic properties of past pandemic influenza viruses, including the 1918 virus, is crucial for current public health preparedness and future pandemic planning. The origin of the 1918 pandemic virus has not been resolved, but its coding sequences are very like those of avian influenza virus. The proteins encoded by the 1918 virus differ from typical low-pathogenicity avian influenza viruses at only a small number of amino acids in each open reading frame. In this study, a series of chimeric 1918 influenza viruses were created in which each of the eight 1918 pandemic virus gene segments was replaced individually with the corresponding gene segment of a prototypical low-pathogenicity avian influenza (LPAI) H1N1 virus in order to investigate functional compatibility of the 1918 virus genome with gene segments from an LPAI virus and to identify gene segments and mutations important for mammalian adaptation. This set of eight "7:1" chimeric viruses was compared to the parental 1918 and LPAI H1N1 viruses in intranasally infected mice. Seven of the 1918 LPAI 7:1 chimeric viruses replicated and caused disease equivalent to the fully reconstructed 1918 virus. Only the chimeric 1918 virus containing the avian influenza PB2 gene segment was attenuated in mice. This attenuation could be corrected by the single E627K amino acid change, further confirming the importance of this change in mammalian adaptation and mouse pathogenicity. While the mechanisms of influenza virus host switch, and particularly mammalian host adaptation are still only partly understood, these data suggest that the 1918 virus, whatever its origin, is very similar to avian influenza virus.


Assuntos
Influenza Aviária/virologia , Influenza Humana/virologia , Vírus Reordenados/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Aves , Linhagem Celular , Embrião de Galinha , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Influenza Aviária/patologia , Influenza Humana/epidemiologia , Influenza Humana/patologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Pandemias , Vírus Reordenados/química , Vírus Reordenados/metabolismo , Vírus Reordenados/patogenicidade , Recombinação Genética , Alinhamento de Sequência , Espanha/epidemiologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Virulência
18.
Virology ; 432(1): 39-44, 2012 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-22727831

RESUMO

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Animais , Proteção Cruzada , Modelos Animais de Doenças , Feminino , Vacinas contra Influenza/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae , Análise de Sobrevida , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/imunologia
19.
Proc Natl Acad Sci U S A ; 108(39): 16416-21, 2011 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-21930918

RESUMO

The 1918 to 1919 "Spanish" influenza pandemic virus killed up to 50 million people. We report here clinical, pathological, bacteriological, and virological findings in 68 fatal American influenza/pneumonia military patients dying between May and October of 1918, a period that includes ~4 mo before the 1918 pandemic was recognized, and 2 mo (September-October 1918) during which it appeared and peaked. The lung tissues of 37 of these cases were positive for influenza viral antigens or viral RNA, including four from the prepandemic period (May-August). The prepandemic and pandemic peak cases were indistinguishable clinically and pathologically. All 68 cases had histological evidence of bacterial pneumonia, and 94% showed abundant bacteria on Gram stain. Sequence analysis of the viral hemagglutinin receptor-binding domain performed on RNA from 13 cases suggested a trend from a more "avian-like" viral receptor specificity with G222 in prepandemic cases to a more "human-like" specificity associated with D222 in pandemic peak cases. Viral antigen distribution in the respiratory tree, however, was not apparently different between prepandemic and pandemic peak cases, or between infections with viruses bearing different receptor-binding polymorphisms. The 1918 pandemic virus was circulating for at least 4 mo in the United States before it was recognized epidemiologically in September 1918. The causes of the unusually high mortality in the 1918 pandemic were not explained by the pathological and virological parameters examined. These findings have important implications for understanding the origins and evolution of pandemic influenza viruses.


Assuntos
Autopsia , Influenza Humana/mortalidade , Antígenos Virais/análise , História do Século XX , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Influenza Humana/epidemiologia , Influenza Humana/história , Dados de Sequência Molecular , RNA Viral/análise
20.
MBio ; 2(5)2011.
Artigo em Inglês | MEDLINE | ID: mdl-21933918

RESUMO

UNLABELLED: Secondary bacterial infections increase disease severity of influenza virus infections and contribute greatly to increased morbidity and mortality during pandemics. To study secondary bacterial infection following influenza virus infection, mice were inoculated with sublethal doses of 2009 seasonal H1N1 virus (NIH50) or pandemic H1N1 virus (Mex09) followed by inoculation with Streptococcus pneumoniae 48 h later. Disease was characterized by assessment of weight loss and survival, titration of virus and bacteria by quantitative reverse transcription-PCR (qRT-PCR), histopathology, expression microarray, and immunohistochemistry. Mice inoculated with virus alone showed 100% survival for all groups. Mice inoculated with Mex09 plus S. pneumoniae showed severe weight loss and 100% mortality with severe alveolitis, denuded bronchiolar epithelium, and widespread expression of apoptosis marker cleaved caspase 3. In contrast, mice inoculated with NIH50 plus S. pneumoniae showed increased weight loss, 100% survival, and slightly enhanced lung pathology. Mex09-S. pneumoniae coinfection also resulted in increased S. pneumoniae replication in lung and bacteremia late in infection. Global gene expression profiling revealed that Mex09-S. pneumoniae coinfection did not induce significantly more severe inflammatory responses but featured significant loss of epithelial cell reproliferation and repair responses. Histopathological examination for cell proliferation marker MCM7 showed significant staining of airway epithelial cells in all groups except Mex09-S. pneumoniae-infected mice. This study demonstrates that secondary bacterial infection during 2009 H1N1 pandemic virus infection resulted in more severe disease and loss of lung repair responses than did seasonal influenza viral and bacterial coinfection. Moreover, this study provides novel insights into influenza virus and bacterial coinfection by showing correlation of lethal outcome with loss of airway basal epithelial cells and associated lung repair responses. IMPORTANCE: Secondary bacterial pneumonias lead to increased disease severity and have resulted in a significant percentage of deaths during influenza pandemics. To understand the biological basis for the interaction of bacterial and viral infections, mice were infected with sublethal doses of 2009 seasonal H1N1 and pandemic H1N1 viruses followed by infection with Streptococcus pneumoniae 48 h later. Only infection with 2009 pandemic H1N1 virus and S. pneumoniae resulted in severe disease with a 100% fatality rate. Analysis of the host response to infection during lethal coinfection showed a significant loss of responses associated with lung repair that was not observed in any of the other experimental groups. This group of mice also showed enhanced bacterial replication in the lung. This study reveals that the extent of lung damage during viral infection influences the severity of secondary bacterial infections and may help explain some differences in mortality during influenza pandemics.


Assuntos
Coinfecção/mortalidade , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/mortalidade , Pulmão/fisiopatologia , Infecções Pneumocócicas/mortalidade , Streptococcus pneumoniae/fisiologia , Animais , Coinfecção/epidemiologia , Feminino , Regulação da Expressão Gênica , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/microbiologia , Influenza Humana/fisiopatologia , Influenza Humana/virologia , Pulmão/microbiologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Pandemias , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/fisiopatologia , Infecções Pneumocócicas/virologia , Streptococcus pneumoniae/patogenicidade , Virulência , Perda de Peso
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