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1.
Infez Med ; 28(4): 475-485, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33257621

RESUMO

While the world is focused on attending, controlling, and mitigating the current pandemic of COVID-19, caused by the SARS-CoV-2, other viral threats are possibly emerging and reemerging especially in Asia, posing a risk for the spread in that region and beyond. A predictable threat is the avian influenza virus, especially H5N6, which has recently led to significant outbreaks in China and the Philippines, deserving more attention and control. In the current review, we assess the history of this highly pathogenic reemerging virus, as well as the contemporary implications of poultry outbreaks occurring in some Asian countries. We also look at outbreaks due to other strains not only in Asia but also across Europe and Africa, according to recent reports from the World Organization of Animal Health (OIE).


Assuntos
/epidemiologia , Doenças Transmissíveis Emergentes/epidemiologia , Vírus da Influenza A/classificação , Influenza Humana/epidemiologia , Pandemias , Animais , Doenças Transmissíveis Emergentes/virologia , Humanos , Vírus da Influenza A/genética , Influenza Humana/virologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Aves Domésticas/virologia , /transmissão , /virologia
2.
Mol Biol (Mosk) ; 54(6): 980-989, 2020.
Artigo em Russo | MEDLINE | ID: mdl-33276361

RESUMO

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1 , Virulência/genética , Animais , Embrião de Galinha , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Virus da Influenza A Subtipo H5N1/fisiologia , Camundongos , Mutação , Infecções por Orthomyxoviridae/virologia , Replicação Viral
3.
Sci Adv ; 6(48)2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33239293

RESUMO

Acute respiratory distress syndrome is associated with a robust inflammatory response that damages the vascular endothelium, impairing gas exchange. While restoration of microcapillaries is critical to avoid mortality, therapeutic targeting of this process requires a greater understanding of endothelial repair mechanisms. Here, we demonstrate that lung endothelium possesses substantial regenerative capacity and lineage tracing reveals that native endothelium is the source of vascular repair after influenza injury. Ablation of chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TF2) (Nr2f2), a transcription factor implicated in developmental angiogenesis, reduced endothelial proliferation, exacerbating viral lung injury in vivo. In vitro, COUP-TF2 regulates proliferation and migration through activation of cyclin D1 and neuropilin 1. Upon influenza injury, nuclear factor κB suppresses COUP-TF2, but surviving endothelial cells ultimately reestablish vascular homeostasis dependent on restoration of COUP-TF2. Therefore, stabilization of COUP-TF2 may represent a therapeutic strategy to enhance recovery from pathogens, including H1N1 influenza and SARS-CoV-2.


Assuntos
Fator II de Transcrição COUP/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Vírus da Influenza A Subtipo H1N1 , Pulmão/citologia , Pulmão/fisiologia , Infecções por Orthomyxoviridae/metabolismo , Regeneração/genética , Animais , Fator II de Transcrição COUP/genética , Movimento Celular/genética , Proliferação de Células/genética , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Infecções por Orthomyxoviridae/virologia , Transfecção
4.
BMC Infect Dis ; 20(1): 823, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33176722

RESUMO

BACKGROUND: The highly pathogenic avian influenza A/H5N1 virus is one of the causative agents of acute lung injury (ALI) with high mortality rate. Studies on therapeutic administration of bone marrow-derived mesenchymal stem cells (MSCs) in ALI caused by the viral infection have been limited in number and have shown conflicting results. The aim of the present investigation is to evaluate the therapeutic potential of MSC administration in A/H5N1-caused ALI, using a mouse model. METHODS: MSCs were prepared from the bone marrow of 9 to 12 week-old BALB/c mice. An H5N1 virus of A/turkey/East Java/Av154/2013 was intranasally inoculated into BALB/c mice. On days 2, 4, and 6 after virus inoculation, MSCs were intravenously administered into the mice. To evaluate effects of the treatment, we examined for lung alveolar protein as an indicator for lung injury, PaO2/FiO2 ratio for lung functioning, and lung histopathology. Expressions of NF-κB, RAGE (transmembrane receptor for damage associated molecular patterns), TNFα, IL-1ß, Sftpc (alveolar cell type II marker), and Aqp5+ (alveolar cell type I marker) were examined by immunohistochemistry. In addition, body weight, virus growth in lung and brain, and duration of survival were measured. RESULTS: The administration of MSCs lowered the level of lung damage in the virus-infected mice, as shown by measuring lung alveolar protein, PaO2/FiO2 ratio, and histopathological score. In the MSC-treated group, the expressions of NF-κB, RAGE, TNFα, and IL-1ß were significantly suppressed in comparison with a mock-treated group, while those of Sftpc and Aqp5+ were enhanced. Body weight, virus growth, and survival period were not significantly different between the groups. CONCLUSION: The administration of MSCs prevented further lung injury and inflammation, and enhanced alveolar cell type II and I regeneration, while it did not significantly affect viral proliferation and mouse morbidity and mortality. The results suggested that MSC administration was a promissing strategy for treatment of acute lung injuries caused by the highly pathogenic avian influenza A/H5N1 virus, although further optimization and combination use of anti-viral drugs will be obviously required to achieve the goal of reducing mortality.


Assuntos
Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/cirurgia , Virus da Influenza A Subtipo H5N1 , Transplante de Células-Tronco Mesenquimais , Infecções por Orthomyxoviridae/complicações , Pneumonia/etiologia , Pneumonia/cirurgia , Lesão Pulmonar Aguda/prevenção & controle , Lesão Pulmonar Aguda/virologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Pulmão/metabolismo , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologia , Pneumonia/prevenção & controle , Pneumonia/virologia , Resultado do Tratamento
5.
PLoS One ; 15(11): e0241266, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33180828

RESUMO

Respiratory mucosal infection by airborne microbes is a common event that occurs every day. We report here that intranasal administration of non-replicating adenovirus (Ad) particles to mice could either confer rapid protection against influenza virus (IFV) challenge independent of adaptive immunity, or exacerbate influenza by triggering rapid death. The life-or-death outcome hinges on the time interval between Ad administration and IFV challenge in conjunction with specific mouse/IFV strains. Intranasal instillation of Ad particles 1-47 days prior to IFV challenge conferred rapid protection against influenza in Balb/c mice whereas exposure to Ad 39 days prior to challenge with a specific IFV strain or 1 day post-challenge with that IFV strain induced rapid death in C57BL/6 mice. Notably, consecutive administrations of Ad prior to IFV challenge conferred a synergy in triggering a potent anti-influenza state; even a detrimental Ad exposure 39 days before challenge with the deadly IFV strain was reversed to a beneficial one by subsequent Ad boosts. Results revealed an intricate relationship between infection and innate immunity that is a linchpin around which effects revolve from protective immunity to collateral damage. It is urgent to repeat the experiments with an expanded scope for characterizing the status that defines susceptibility or resistance to IFV infection and subsequently reveal the underlying mechanisms. Whether broad heterologous protective effects induced by AdE and adaptive immunity elicited by vaccination could confer synergy during mitigation of a pandemic remains to be seen.


Assuntos
Adenoviridae/fisiologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Vírion/fisiologia , Replicação Viral , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Progressão da Doença , Cães , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Camundongos Endogâmicos C57BL , Fatores de Tempo
6.
Emerg Top Life Sci ; 4(4): 389-398, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33210707

RESUMO

Influenza virus causes an acute febrile respiratory disease in humans that is commonly known as 'flu'. Influenza virus has been around for centuries and is one of the most successful, and consequently most studied human viruses. This has generated tremendous amount of data and information, thus it is pertinent to summarise these for, particularly interdisciplinary readers. Viruses are acellular organisms and exist at the interface of living and non-living. Due to this unique characteristic, viruses require another organism, i.e. host to survive. Viruses multiply inside the host cell and are obligate intracellular pathogens, because their relationship with the host is almost always harmful to host. In mammalian cells, the life cycle of a virus, including influenza is divided into five main steps: attachment, entry, synthesis, assembly and release. To complete these steps, some viruses, e.g. influenza utilise all three parts - plasma membrane, cytoplasm and nucleus, of the cell; whereas others, e.g. SARS-CoV-2 utilise only plasma membrane and cytoplasm. Hence, viruses interact with numerous host factors to complete their life cycle, and these interactions are either exploitative or antagonistic in nature. The host factors involved in the life cycle of a virus could be divided in two broad categories - proviral and antiviral. This perspective has endeavoured to assimilate the information about the host factors which promote and suppress influenza virus infection. Furthermore, an insight into host factors that play a dual role during infection or contribute to influenza virus-host adaptation and disease severity has also been provided.


Assuntos
Interações entre Hospedeiro e Microrganismos , Orthomyxoviridae/fisiologia , Animais , Humanos , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia
7.
Nat Commun ; 11(1): 5597, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154358

RESUMO

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells. We show that this leads to simultaneous inhibition of virus release, and immune-mediated elimination of both free virus and virus-infected cells. Intranasal or intraperitoneal administration of a single dose of drug to mice infected with 100x MLD50 virus is shown to eradicate advanced infections from representative strains of both influenza A and B viruses. Since treatments of severe infections remain effective up to three days post lethal inoculation, our approach may successfully treat infections refractory to current therapies.


Assuntos
Antivirais/administração & dosagem , Antivirais/farmacologia , Imunoterapia/métodos , Infecções por Orthomyxoviridae/tratamento farmacológico , 2,4-Dinitrofenol/administração & dosagem , 2,4-Dinitrofenol/química , 2,4-Dinitrofenol/imunologia , Administração Intranasal , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Antivirais/química , Linhagem Celular , Citotoxicidade Imunológica/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/fisiologia , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/enzimologia , Vírus da Influenza B/fisiologia , Infusões Parenterais , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Ligação Proteica , Resultado do Tratamento , Liberação de Vírus/efeitos dos fármacos , Zanamivir/administração & dosagem , Zanamivir/química , Zanamivir/farmacologia
8.
Sci Immunol ; 5(53)2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33158975

RESUMO

Lower respiratory viral infections, such as influenza virus and severe acute respiratory syndrome coronavirus 2 infections, often cause severe viral pneumonia in aged individuals. Here, we report that influenza viral pneumonia leads to chronic nonresolving lung pathology and exacerbated accumulation of CD8+ tissue-resident memory T cells (TRM) in the respiratory tract of aged hosts. TRM cell accumulation relies on elevated TGF-ß present in aged tissues. Further, we show that TRM cells isolated from aged lungs lack a subpopulation characterized by expression of molecules involved in TCR signaling and effector function. Consequently, TRM cells from aged lungs were insufficient to provide heterologous protective immunity. The depletion of CD8+ TRM cells dampens persistent chronic lung inflammation and ameliorates tissue fibrosis in aged, but not young, animals. Collectively, our data demonstrate that age-associated TRM cell malfunction supports chronic lung inflammatory and fibrotic sequelae after viral pneumonia.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Pulmão/imunologia , Pneumonia Viral/imunologia , /imunologia , Fatores Etários , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , /virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Influenza Humana/imunologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Pulmão/metabolismo , Pulmão/virologia , Camundongos Endogâmicos C57BL , Orthomyxoviridae/imunologia , Orthomyxoviridae/fisiologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo
9.
PLoS Pathog ; 16(10): e1008974, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33064776

RESUMO

During viral infection, the numbers of virions infecting individual cells can vary significantly over time and space. The functional consequences of this variation in cellular multiplicity of infection (MOI) remain poorly understood. Here, we rigorously quantify the phenotypic consequences of cellular MOI during influenza A virus (IAV) infection over a single round of replication in terms of cell death rates, viral output kinetics, interferon and antiviral effector gene transcription, and superinfection potential. By statistically fitting mathematical models to our data, we precisely define specific functional forms that quantitatively describe the modulation of these phenotypes by MOI at the single cell level. To determine the generality of these functional forms, we compare two distinct cell lines (MDCK cells and A549 cells), both infected with the H1N1 strain A/Puerto Rico/8/1934 (PR8). We find that a model assuming that infected cell death rates are independent of cellular MOI best fits the experimental data in both cell lines. We further observe that a model in which the rate and efficiency of virus production increase with cellular co-infection best fits our observations in MDCK cells, but not in A549 cells. In A549 cells, we also find that induction of type III interferon, but not type I interferon, is highly dependent on cellular MOI, especially at early timepoints. This finding identifies a role for cellular co-infection in shaping the innate immune response to IAV infection. Finally, we show that higher cellular MOI is associated with more potent superinfection exclusion, thus limiting the total number of virions capable of infecting a cell. Overall, this study suggests that the extent of cellular co-infection by influenza viruses may be a critical determinant of both viral production kinetics and cellular infection outcomes in a host cell type-dependent manner.


Assuntos
Coinfecção/virologia , Imunidade Inata/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Interferon Tipo I/farmacologia , Interferons/farmacologia , Infecções por Orthomyxoviridae/virologia , Células A549 , Animais , Coinfecção/imunologia , Coinfecção/patologia , Cães , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/imunologia , Influenza Humana/patologia , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Proteínas não Estruturais Virais , Replicação Viral
10.
PLoS Pathog ; 16(10): e1008957, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33104753

RESUMO

Infection with the influenza virus triggers an innate immune response that initiates the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Although influenza increases parasitized cell metabolism, it does not productively replicate in dendritic cells. To dissect these mechanisms, we compared the metabolism of dendritic cells to that of those infected with active and inactive influenza A virus and those treated with toll-like receptor agonists. Using quantitative mass spectrometry, pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes in dendritic cells 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as mitochondrial respiration. Influenza infection of dendritic cells led to a metabolic phenotype distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified c-Myc as one transcription factor modulating this response. Restriction of c-Myc activity or mitochondrial substrates significantly changed the immune functions of dendritic cells, such as reducing motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process, dendritic cells respond with global metabolic restructuring, that is present in inflammatory lung dendritic cells after infection, and this is important for effector function. These findings suggest metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Ativação Linfocitária/imunologia , Infecções por Orthomyxoviridae/imunologia , Replicação Viral , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/virologia , Feminino , Glicólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteoma/análise , Proteoma/metabolismo , Receptores Toll-Like/metabolismo
11.
Virology ; 551: 10-15, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33010670

RESUMO

Bovine respiratory disease (BRD) is the costliest disease affecting the cattle industry globally. Orthomyxoviruses, influenza C virus (ICV) and influenza D virus (IDV) have recently been implicated to play a role in BRD. However, there are contradicting reports about the association of IDV and ICV to BRD. Using the largest cohort study (cattle, n = 599) to date we investigated the association of influenza viruses in cattle with BRD. Cattle were scored for respiratory symptoms and pooled nasal and pharyngeal swabs were tested for bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus, bovine coronavirus, ICV and IDV by real-time PCR. Cattle that have higher viral loads of IDV and ICV also have greater numbers of co-infecting viruses than controls. More strikingly, 2 logs higher IDV viral RNA in BRD-symptomatic cattle that are co-infected animals than those infected with IDV alone. Our results strongly suggest that ICV and IDV may be significant contributors to BRD.


Assuntos
Complexo Respiratório Bovino/virologia , Influenzavirus C/patogenicidade , Infecções por Orthomyxoviridae/veterinária , Thogotovirus/patogenicidade , Carga Viral/veterinária , Animais , Complexo Respiratório Bovino/epidemiologia , Bovinos , Coinfecção/epidemiologia , Coinfecção/veterinária , Coinfecção/virologia , Feminino , Influenzavirus C/isolamento & purificação , Gado , Masculino , Razão de Chances , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Prevalência , RNA Viral/análise , Thogotovirus/isolamento & purificação
12.
Euro Surveill ; 25(42)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33094718

RESUMO

A zoonotic A/sw/H1avN1 1C.2.2 influenza virus infection was detected in a German child that presented with influenza-like illness, including high fever. There was a history of close contact with pigs 3 days before symptom onset. The child recovered within 3 days. No other transmissions were observed. Serological investigations of the virus isolate revealed cross-reactions with ferret antisera against influenza A(H1N1)pdm09 virus, indicating a closer antigenic relationship with A(H1N1)pdm09 than with the former seasonal H1N1 viruses.


Assuntos
Variação Antigênica/genética , Furões/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/diagnóstico , Infecções por Orthomyxoviridae/diagnóstico , Doenças dos Suínos/transmissão , Zoonoses/virologia , Animais , Anticorpos Antivirais/sangue , Variação Antigênica/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/transmissão , Influenza Humana/virologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Reação em Cadeia da Polimerase , Análise de Sequência , Suínos , Doenças dos Suínos/virologia , Zoonoses/transmissão
13.
Sci Rep ; 10(1): 17090, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33051497

RESUMO

The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.


Assuntos
Adjuvantes Imunológicos/química , Vacinas contra Influenza/imunologia , Triterpenos/química , Animais , Anticorpos Antivirais/sangue , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Citocinas/sangue , Imunoglobulina G/sangue , Vacinas contra Influenza/química , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos
14.
Nat Methods ; 17(10): 1025-1032, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32929269

RESUMO

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/virologia , Papillomaviridae/imunologia , Tomografia por Emissão de Pósitrons/métodos , Animais , Antígenos , Clonagem Molecular , Epitopos/genética , Epitopos/metabolismo , Feminino , Regulação da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/classificação , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoglobulina G/classificação , Imunoglobulina G/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia
15.
PLoS One ; 15(9): e0238615, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32915821

RESUMO

Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher/fluorescence oligonucleotides connected by a 5' backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A/H1 and A/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 103 copies and 102 copies of RNA for influenza A and influenza B, respectively. The sensitivities of the multiplex influenza A/B/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A/B/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A/B/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses.


Assuntos
Influenza Humana/diagnóstico , Técnicas de Diagnóstico Molecular , Infecções por Orthomyxoviridae/diagnóstico , Orthomyxoviridae/isolamento & purificação , Animais , Epidemias , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Influenza Humana/genética , Influenza Humana/virologia , Influenzavirus C/genética , Influenzavirus C/isolamento & purificação , Orthomyxoviridae/genética , Orthomyxoviridae/patogenicidade , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Pandemias
16.
Toxicol Appl Pharmacol ; 404: 115167, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32771490

RESUMO

Growing use of carbon nanotubes (CNTs) have garnered concerns regarding their association with adverse health effects. Few studies have probed how CNTs affect a host's susceptibility to pathogens, particularly respiratory viruses. We reported that exposure of lung cells and mice to pristine single-walled CNTs (SWCNTs) leads to significantly increased influenza virus H1N1 strain A/Mexico/4108/2009 (IAV) titers in concert with repressed antiviral immune responses. In the present study, we investigated if hydroxylated multi-walled CNTs (MWCNTs), would result in similar outcomes. C57BL/6 mice were exposed to 20 µg MWCNTs on day 0 and IAV on day 3 and samples were collected on day 7. We investigated pathological changes, viral titers, immune-related gene expression in lung tissue, and quantified differential cell counts and cytokine and chemokine levels in bronchoalveolar lavage fluid. MWCNTs alone caused mild inflammation with no apparent changes in immune markers whereas IAV alone presented typical infection-associated inflammation, pathology, and titers. The co-exposure (MWCNTs + IAV) did not alter titers or immune cell profiles compared to the IAV only but increased concentrations of IL-1ß, TNFα, GM-CSF, KC, MIPs, and RANTES and inhibited mRNA expression of Tlr3, Rig-i, Mda5, and Ifit2. Our findings suggest MWCNTs modulate immune responses to IAV with no effect on the viral titer and modest pulmonary injury, a result different from those reported for SWCNT exposures. This is the first study to show that MWCNTs modify cytokine and chemokine responses that control aspects of host defenses which may play a greater role in mitigating IAV infections.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Lesão Pulmonar/induzido quimicamente , Nanotubos de Carbono , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
17.
Nat Commun ; 11(1): 4062, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811826

RESUMO

Influenza viruses are presumed, but not conclusively known, to spread among humans by several possible routes. We provide evidence of a mode of transmission seldom considered for influenza: airborne virus transport on microscopic particles called "aerosolized fomites." In the guinea pig model of influenza virus transmission, we show that the airborne particulates produced by infected animals are mainly non-respiratory in origin. Surprisingly, we find that an uninfected, virus-immune guinea pig whose body is contaminated with influenza virus can transmit the virus through the air to a susceptible partner in a separate cage. We further demonstrate that aerosolized fomites can be generated from inanimate objects, such as by manually rubbing a paper tissue contaminated with influenza virus. Our data suggest that aerosolized fomites may contribute to influenza virus transmission in animal models of human influenza, if not among humans themselves, with important but understudied implications for public health.


Assuntos
Fômites , Vírus da Influenza A , Influenza Humana/transmissão , Influenza Humana/virologia , Material Particulado , Aerossóis , Animais , Modelos Animais de Doenças , Feminino , Cobaias , Humanos , Vírus da Influenza A Subtipo H3N2 , Infecções por Orthomyxoviridae/virologia , Tamanho da Partícula
18.
BMC Infect Dis ; 20(1): 585, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32762666

RESUMO

BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. METHODS: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays. RESULTS: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3' termini of each genomic segment and subsequent qPCR of the 5' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R2 = 0.931, P = 0.000066). CONCLUSIONS: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virulência/genética , Animais , Cães , Genoma Viral/genética , Genoma Viral/efeitos da radiação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/efeitos da radiação , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/virologia , Estabilidade de RNA/efeitos da radiação , RNA Viral/genética , RNA Viral/efeitos da radiação , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação
19.
PLoS Pathog ; 16(8): e1008816, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32853241

RESUMO

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics. Most pandemics occurred upon adaptation of avian IAVs to humans. This adaptation includes a hallmark receptor-binding specificity switch of hemagglutinin (HA) from avian-type α2,3- to human-type α2,6-linked sialic acids. Complementary changes of the receptor-destroying neuraminidase (NA) are considered to restore the precarious, but poorly described, HA-NA-receptor balance required for virus fitness. In comparison to the detailed functional description of adaptive mutations in HA, little is known about the functional consequences of mutations in NA in relation to their effect on the HA-NA balance and host tropism. An understudied feature of NA is the presence of a second sialic acid-binding site (2SBS) in avian IAVs and absence of a 2SBS in human IAVs, which affects NA catalytic activity. Here we demonstrate that mutation of the 2SBS of avian IAV H5N1 disturbs the HA-NA balance. Passaging of a 2SBS-negative H5N1 virus on MDCK cells selected for progeny with a restored HA-NA balance. These viruses obtained mutations in NA that restored a functional 2SBS and/or in HA that reduced binding of avian-type receptors. Importantly, a particular HA mutation also resulted in increased binding of human-type receptors. Phylogenetic analyses of avian IAVs show that also in the field, mutations in the 2SBS precede mutations in HA that reduce binding of avian-type receptors and increase binding of human-type receptors. Thus, 2SBS mutations in NA can drive acquisition of mutations in HA that not only restore the HA-NA balance, but may also confer increased zoonotic potential.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/virologia , Ácidos Siálicos/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/isolamento & purificação , Células Madin Darby de Rim Canino , Neuraminidase/química , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Ligação Proteica
20.
PLoS Pathog ; 16(8): e1008760, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32790753

RESUMO

Influenza A viruses (IAVs) remain a significant global health burden. Activation of the innate immune response is important for controlling early virus replication and spread. It is unclear how early IAV replication events contribute to immune detection. Additionally, while many cell types in the lung can be infected, it is not known if all cell types contribute equally to establish the antiviral state in the host. Here, we use single-cycle influenza A viruses (scIAVs) to characterize the early immune response to IAV in vitro and in vivo. We found that the magnitude of virus replication contributes to antiviral gene expression within infected cells prior to the induction of a global response. We also developed a scIAV that is only capable of undergoing primary transcription, the earliest stage of virus replication. Using this tool, we uncovered replication stage-specific responses in vitro and in vivo. Using several innate immune receptor knockout cell lines, we identify RIG-I as the predominant antiviral detector of primary virus transcription and amplified replication in vitro. Through a Cre-inducible reporter mouse, we used scIAVs expressing Cre-recombinase to characterize cell type-specific responses in vivo. Individual cell types upregulate unique sets of antiviral genes in response to both primary virus transcription and amplified replication. We also identified antiviral genes that are only upregulated in response to direct infection. Altogether, these data offer insight into the early mechanisms of antiviral gene activation during influenza A infection.


Assuntos
Células Epiteliais/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Replicação Viral , Células A549 , Animais , Antivirais/farmacologia , Proteína DEAD-box 58/metabolismo , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Células HEK293 , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia
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