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2.
Harefuah ; 159(5): 320-325, 2020 May.
Artigo em Hebraico | MEDLINE | ID: mdl-32431119

RESUMO

INTRODUCTION: The spread of epidemics resulting in many deaths has been known since the dawn of civilization, for example, the typhus and smallpox epidemics and the plague. Early in the previous century there was an outbreak of the Spanish Flue and towards the end of the 60's, the AIDS epidemic (HIV). Since the start of the current century, several epidemics broke out and spread to various places around the world, for instance, SARS (Severe Acute Respiratory Syndrome), the Avian Influenza and the Swine Influenza. In 2014 there was an outbreak of Ebola (Ebola virus disease) and in 2015 the Zika virus emerged and there were more. Epidemics cause havoc and impact all areas of life. Each epidemic takes an unfathomable price in lives. It is estimated, for example, that the AIDS epidemic took the lives of some 30 million people. The Corona virus (Covid-19) broke out in China, towards the end of 2019 and spread to most parts of the world. The implications of the outbreak are similar in many countries, among others, due to the uncertainty regarding the way the virus spreads, the appropriate treatment, the lack of vaccination and the high rate of deaths. Naturally, at such times physical protection is a top priority. However, coping with the implications to people's mental health is no less important and these may result in long-term negative impacts.


Assuntos
Infecções por Coronavirus/psicologia , Saúde Mental , Pandemias , Pneumonia Viral/psicologia , Animais , Betacoronavirus , Infecções por Coronavirus/epidemiologia , Surtos de Doenças , HIV , Humanos , Infecções por Orthomyxoviridae , Pneumonia Viral/epidemiologia , Zika virus , Infecção por Zika virus
3.
Arch Virol ; 165(5): 1141-1150, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32222822

RESUMO

Pigs are capable of harbouring influenza A viruses of human and avian origin in their respiratory tracts and thus act as an important intermediary host to generate novel influenza viruses with pandemic potential by genetic reassortment between the two viruses. Here, we show that two distinct H1N2 swine influenza viruses contain avian-like or classical swine-like hemagglutinins with polymerase acidic (PA) and nucleoprotein (NP) genes from 2009 pandemic H1N1 influenza viruses that were found to be circulating in Korean pigs in 2018. Swine H1N2 influenza virus containing an avian-like hemagglutinin gene had enhanced pathogenicity, causing severe interstitial pneumonia in infected pigs and mice. The mortality rate of mice infected with swine H1N2 influenza virus containing an avian-like hemagglutinin gene was higher by 100% when compared to that of mice infected with swine H1N2 influenza virus harbouring classical swine-like hemagglutinin. Further, chemokines attracting inflammatory cells were strongly induced in lung tissues of pigs and mice infected by swine H1N2 influenza virus containing an avian-like hemagglutinin gene. In conclusion, it is necessary for the well-being of humans and pigs to closely monitor swine influenza viruses containing avian-like hemagglutinin with PA and NP genes from 2009 pandemic H1N1 influenza viruses.


Assuntos
Vírus da Influenza A Subtipo H1N2/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Fatores de Virulência/genética , Animais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Vírus da Influenza A Subtipo H1N2/patogenicidade , Camundongos , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Proteínas de Ligação a RNA/genética , Análise de Sobrevida , Suínos , Doenças dos Suínos/patologia , Proteínas do Core Viral/genética , Virulência
4.
Emerg Microbes Infect ; 9(1): 664-675, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32193996

RESUMO

The H7N9 viruses have been circulating for six years. The insertion of a polybasic cleavage site in the haemagglutinin (HA) protein of H7N9 has resulted in the emergence of a highly pathogenic (HP) avian influenza virus. Currently, there are limited studies on neutralizing monoclonal antibodies(mAbs) against HP H7N9 AIVs. In this study, mice were immunized with inactivated H7N9 vaccine of A/ZJU01/PR8/2013 to produce murine mAbs. Finally, two murine mAbs against the HA of low pathogenic (LP) virus were produced and characterized. Characterization included determining mAbs binding breadth and affinity, in vitro neutralization capacity, and potential in vivo protection. Two of these mAbs, 1H10 and 2D1, have been identified to have therapeutic and prophylactic efficacy against the HP strain in mouse passive transfer-viral challenge experiments. The mAb 1H10 was most efficacious, even if the treatment-time was as late as 72 h post-infection, or the therapeutic dose was as low as 1 mg/kg; and it was confirmed to have haemagglutination inhibition and neutralizing activity on both LP-and HP-H7N9 strains. Further study indicated that the protection provided by 2D1 was mediated by antibody-dependent cellular cytotoxicity. The mAbs described here provide promising results and merit further development into potential antiviral therapeutics for H7N9 infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Amplamente Neutralizantes/imunologia , Linhagem Celular , Mapeamento de Epitopos , Feminino , Testes de Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Imunização Passiva , Subtipo H7N9 do Vírus da Influenza A/metabolismo , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Testes de Neutralização , Filogenia
5.
Emerg Microbes Infect ; 9(1): 691-706, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32208814

RESUMO

Infection of influenza A virus (IAV) can trigger exaggerated pulmonary inflammation and induce acute lung injury (ALI). Limiting IAV replication and alleviation of pulmonary inflammation are two important therapeutic strategies for influenza virus infection. Recent studies have shown that hypoxia inducible factor-1α (HIF-1α) is an essential factor for the development and repair of ALI; however, the role and the underlying mechanisms of HIF-1α in IAV-induced ALI remain elusive. Here, we demonstrated that lung epithelial cell-specific Hif1α knockout mice infected with IAV developed more lung IAV replication and severe lung inflammation, which led to increased mortality compared to IAV-infected control mice. Moreover, knockdown of HIF1A in A549 cells (human alveolar type II epithelial cell line) promoted IAV replication in vitro. Mechanistically, knockdown of HIF1A reduced glycolysis by regulating transcription of glycolysis-related enzymes, which subsequently activated the AMPKα-ULK1 signalling pathway. Interestingly, AMPKα-ULK1 signalling promoted autophagy and augmented IAV replication. Taken together, deficiency of HIF-1α in lung epithelial cells reduces glycolysis and enhances AMPKα-ULK1-mediated autophagy, which finally facilitates IAV replication. These findings have deepened our understanding of the role of HIF-1α in regulating IAV replication and provided us novel therapeutic targets for combating influenza infection.


Assuntos
Células Epiteliais Alveolares/fisiologia , Células Epiteliais Alveolares/virologia , Autofagia , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Vírus da Influenza A/fisiologia , Pulmão/virologia , Infecções por Orthomyxoviridae/virologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Citocinas/biossíntese , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/fisiopatologia , Pneumonia Viral/fisiopatologia , Pneumonia Viral/virologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Regulação para Cima , Replicação Viral
6.
PLoS One ; 15(2): e0229326, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32078666

RESUMO

As high-throughput sequencing technologies are becoming more widely adopted for analysing pathogens in disease outbreaks there needs to be assurance that the different sequencing technologies and approaches to data analysis will yield reliable and comparable results. Conversely, understanding where agreement cannot be achieved provides insight into the limitations of these approaches and also allows efforts to be focused on areas of the process that need improvement. This manuscript describes the next-generation sequencing of three closely related viruses, each analysed using different sequencing strategies, sequencing instruments and data processing pipelines. In order to determine the comparability of consensus sequences and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed alignment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated that variation in the final result could be attributed to all stages in the process, but the most critical were the well-known homopolymer errors introduced by 454 sequencing, and the alignment processes in the different data processing pipelines which affected the consistency of mSNV detection. However, homopolymer errors aside, there was generally a good agreement between consensus sequences that were obtained for all combinations of sequencing platforms and data processing pipelines. Nevertheless, minority variant analysis will need a different level of careful standardization and awareness about the possible limitations, as shown in this study.


Assuntos
Surtos de Doenças/veterinária , Patos/virologia , Vírus da Influenza A Subtipo H5N8/classificação , Vírus da Influenza A Subtipo H5N8/genética , Infecções por Orthomyxoviridae/veterinária , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma/métodos , Animais , Genoma Viral , Infecções por Orthomyxoviridae/virologia , RNA Viral/análise , RNA Viral/genética , Análise de Sequência de DNA
7.
Nat Commun ; 11(1): 791, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034141

RESUMO

The conserved hemagglutinin (HA) stem has been a focus of universal influenza vaccine efforts. Influenza A group 1 HA stem-nanoparticles have been demonstrated to confer heterosubtypic protection in animals; however, the protection does not extend to group 2 viruses, due in part to differences in glycosylation between group 1 and 2 stems. Here, we show that introducing the group 2 glycan at Asn38HA1 to a group 1 stem-nanoparticle (gN38 variant) based on A/New Caledonia/20/99 (H1N1) broadens antibody responses to cross-react with group 2 HAs. Immunoglobulins elicited by the gN38 variant provide complete protection against group 2 H7N9 virus infection, while the variant loses protection against a group 1 H5N1 virus. The N38HA1 glycan thus is pivotal in directing antibody responses by controlling access to group-determining stem epitopes. Precise targeting of stem-directed antibody responses to the site of vulnerability by glycan repositioning may be a step towards achieving cross-group influenza protection.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Nanopartículas/química , Polissacarídeos/química , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Asparagina/química , Asparagina/metabolismo , Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas , Epitopos/imunologia , Feminino , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Imunoglobulinas/imunologia , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle
8.
Nat Commun ; 11(1): 766, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034144

RESUMO

Human influenza A viruses are known to be transmitted via the air from person to person. It is unknown from which anatomical site of the respiratory tract influenza A virus transmission occurs. Here, pairs of genetically tagged and untagged influenza A/H1N1, A/H3N2 and A/H5N1 viruses that are transmissible via the air are used to co-infect donor ferrets via the intranasal and intratracheal routes to cause an upper and lower respiratory tract infection, respectively. In all transmission cases, we observe that the viruses in the recipient ferrets are of the same genotype as the viruses inoculated intranasally, demonstrating that they are expelled from the upper respiratory tract of ferrets rather than from trachea or the lower airways. Moreover, influenza A viruses that are transmissible via the air preferentially infect ferret and human nasal respiratory epithelium. These results indicate that virus replication in the upper respiratory tract, the nasal respiratory epithelium in particular, of donors is a driver for transmission of influenza A viruses via the air.


Assuntos
Furões/virologia , Vírus da Influenza A/fisiologia , Mucosa Nasal/virologia , Infecções por Orthomyxoviridae/transmissão , Ar , Animais , Cães , Feminino , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/veterinária , Tropismo Viral
9.
Science ; 367(6480)2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32079747

RESUMO

Current influenza vaccines only confer protection against homologous viruses. We synthesized pulmonary surfactant (PS)-biomimetic liposomes encapsulating 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an agonist of the interferon gene inducer STING (stimulator of interferon genes). The adjuvant (PS-GAMP) vigorously augmented influenza vaccine-induced humoral and CD8+ T cell immune responses in mice by simulating the early phase of viral infection without concomitant excess inflammation. Two days after intranasal immunization with PS-GAMP-adjuvanted H1N1 vaccine, strong cross-protection was elicited against distant H1N1 and heterosubtypic H3N2, H5N1, and H7N9 viruses for at least 6 months while maintaining lung-resident memory CD8+ T cells. Adjuvanticity was then validated in ferrets. When alveolar epithelial cells (AECs) lacked Sting or gap junctions were blocked, PS-GAMP-mediated adjuvanticity was substantially abrogated in vivo. Thus, AECs play a pivotal role in configuring heterosubtypic immunity.


Assuntos
Materiais Biomiméticos , Vacinas contra Influenza/imunologia , Nanopartículas , Nucleotídeos Cíclicos/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Surfactantes Pulmonares/imunologia , Vacinação/métodos , Adjuvantes Imunológicos/administração & dosagem , Administração Intranasal , Animais , Materiais Biomiméticos/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Furões , Memória Imunológica , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Lipossomos , Proteínas de Membrana/agonistas , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Nanopartículas/administração & dosagem , Nucleotídeos Cíclicos/farmacologia , Surfactantes Pulmonares/administração & dosagem
10.
Emerg Microbes Infect ; 9(1): 78-87, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31894728

RESUMO

The H7N9 influenza virus has been circulating in China for more than six years. The neuraminidase (NA) has gained great concern for the development of antiviral drugs, therapeutic antibodies, and new vaccines. In this study, we screened seven mouse monoclonal antibodies (mAbs) and compared their protective effects against H7N9 influenza virus. The epitope mapping from escape mutants showed that all the seven mAbs could bind to the head region of the N9 NA close to the enzyme activity sites, and four key sites of N9 NA were reported for the first time. The mAbs D3 and 7H2 could simultaneously inhibit the cleavage of the sialic acid of fetuin protein with large molecular weight and NA-XTD with small molecule weight in the NA inhibition experiment, prevent the formation of virus plaque at a low concentration, and effectively protect the mice from the challenge of the lethal dose of H7N9 virus.


Assuntos
Anticorpos Monoclonais/química , Subtipo H7N9 do Vírus da Influenza A/imunologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/química , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos Virais , Domínio Catalítico , Linhagem Celular , Cães , Mapeamento de Epitopos , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/tratamento farmacológico
11.
Emerg Microbes Infect ; 9(1): 88-94, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31900060

RESUMO

Infection with a novel H10N8 influenza virus in humans was first described in China in December 2013, which raised concerns related to public health. This novel virus was subsequently confirmed to have originated from a live poultry market. However, whether this virus can infect other mammals remains unclear. In the present study, antibody specific for H10N8 influenza virus was detected in swine herds in southern China during serological monitoring for swine influenza virus. The pathogenicity and transmissibility of this H10N8 influenza virus to swine was examined. The results showed that swine are susceptible to infection with human-origin H10N8 influenza virus, which causes viral shedding, severe tissue lesions, and seroconversion, while infection with avian-origin H10N8 influenza virus causes only seroconversion and no viral shedding. Importantly, human-origin H10N8 influenza virus can inefficiently be transmitted between swine and cause seroconversion through direct contact. This study provides a new perspective regarding the ecology of H10N8 influenza virus and highlights the importance of epidemiological monitoring of the H10N8 influenza virus in different animal species, which will be helpful for preventing and controlling future infections by this virus.


Assuntos
Vírus da Influenza A Subtipo H10N8/fisiologia , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/imunologia , Doenças dos Suínos/transmissão , Animais , Anticorpos Antivirais/imunologia , China , Humanos , Vírus da Influenza A Subtipo H10N8/patogenicidade , Pulmão/patologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/transmissão , Sus scrofa , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Zoonoses
12.
Nat Commun ; 11(1): 162, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919357

RESUMO

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.


Assuntos
Antivirais/farmacologia , Hemaglutininas Virais/metabolismo , Vírus da Influenza A/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/prevenção & controle , Peptídeos/farmacologia , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Autofagossomos/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Endossomos/metabolismo , Feminino , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Biblioteca de Peptídeos , Células Sf9 , Spodoptera
13.
Phytomedicine ; 67: 153150, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31958713

RESUMO

BACKGROUND: Influenza virus is one of the most important human pathogens, causing substantial seasonal and pandemic morbidity and mortality. Houttuynia cordata is a traditionally used medicinal plant for the treatment of pneumonia. Flavonoids are one of the major bioactive constituents of Houttuynia cordata. PURPOSE: This study was designed to investigate the therapeutic effect and mechanism of flavonoid glycosides from H. cordata on influenza A virus (IAV)-induced acute lung injury (ALI) in mice. METHODS: Flavonoids from H. cordata (HCF) were extracted from H. cordata and identified by high-performance liquid chromatography. Mice were infected intranasally with influenza virus H1N1 (A/FM/1/47). HCF (50, 100, or 200 mg/kg) or Ribavirin (100 mg/kg, the positive control) were administered intragastrically. Survival rates, life spans, weight losses, lung indexes, histological changes, inflammatory infiltration, and inflammatory markers in the lungs were measured. Lung virus titers and neuraminidase (NA) activities were detected. The expression of Toll-like receptors (TLRs) and levels of NF-κB p65 phosphorylation (NF-κB p65(p)) in the lungs were analysed. The effects of HCF on viral replication and TLR signalling were further evaluated in cells. RESULTS: HCF contained 78.5% flavonoid glycosides. The contents of rutin, hyperin, isoquercitrin, and quercitrin in HCF were 8.8%, 26.7%, 9.9% and 31.7%. HCF (50, 100 and 200 mg/kg) increased the survival rate and life span of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with HCF (50, 100 and 200 mg/kg) showed lesser weight loss and lower lung index than the model group. The lungs of HCF-treated ALI mice presented more intact lung microstructural morphology, milder inflammatory infiltration, and lower levels of monocyte chemotactic protein 1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) than in the model group. Further investigation revealed that HCF exerted antiviral and TLR-inhibitory effects in vivo and in vitro. HCF (50, 100 and 200 mg/kg) reduced lung H1N1 virus titers and inhibited viral NA activity in mice. HCF (100 and 200 mg/kg) elevated the levels of interferon-ß in lungs. HCF also decreased the expression of TLR3/4/7 and level of NF-κB p65(p) in lung tissues. In vitro experiments showed that HCF (50, 100 and 200 µg/ml) significantly inhibited viral proliferation and suppressed NA activity. In RAW 264.7 cells, TLR3, TLR4, and TLR7 agonist-stimulated cytokine secretion, NF-κB p65 phosphorylation, and nuclear translocation were constrained by HCF treatment. Furthermore, among the four major flavonoid glycosides in HCF, hyperin and quercitrin inhibited both viral replication and TLR signalling in cells. CONCLUSION: HCF significantly alleviated H1N1-induced ALI in mice, which were associated with its dual antiviral and anti-inflammatory effects via inhibiting influenzal NA activity and TLR signalling. among the four major flavonoid glycosides in HCF, hyperin and quercitrin played key roles in the therapeutic effect of HCF.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/virologia , Antivirais/farmacologia , Flavonoides/farmacologia , Houttuynia/química , Vírus da Influenza A Subtipo H1N1/patogenicidade , Lesão Pulmonar Aguda/metabolismo , Animais , Antivirais/química , Cães , Flavonoides/química , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/fisiologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/metabolismo , Receptores Toll-Like/metabolismo , Fator de Transcrição RelA/metabolismo , Replicação Viral/efeitos dos fármacos
14.
Nat Protoc ; 15(3): 1041-1065, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31996843

RESUMO

In vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. In this protocol, we describe a methodology for analyzing influenza virus-infected lung in vivo by two-photon imaging microscopy. We describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. Further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (Color-flu) for multicolor analysis. Setup of this model typically takes ~30 min and enables the observation of influenza virus-infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. This imaging system, which we termed two-photon IMPRESS (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo.


Assuntos
Vírus da Influenza A/genética , Proteínas Luminescentes/metabolismo , Infecções por Orthomyxoviridae/fisiopatologia , Infecções por Orthomyxoviridae/virologia , Animais , Cães , Regulação Viral da Expressão Gênica , Genes Reporter , Proteínas Luminescentes/química , Pulmão/fisiopatologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos
15.
Nat Immunol ; 21(3): 309-320, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31953534

RESUMO

Tissue-resident memory T cells (TRM cells) are critical for cellular immunity to respiratory pathogens and reside in both the airways and the interstitium. In the present study, we found that the airway environment drove transcriptional and epigenetic changes that specifically regulated the cytolytic functions of airway TRM cells and promoted apoptosis due to amino acid starvation and activation of the integrated stress response. Comparison of airway TRM cells and splenic effector-memory T cells transferred into the airways indicated that the environment was necessary to activate these pathways, but did not induce TRM cell lineage reprogramming. Importantly, activation of the integrated stress response was reversed in airway TRM cells placed in a nutrient-rich environment. Our data defined the genetic programs of distinct lung TRM cell populations and show that local environmental cues altered airway TRM cells to limit cytolytic function and promote cell death, which ultimately leads to fewer TRM cells in the lung.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Epigênese Genética/imunologia , Memória Imunológica/genética , Pulmão/imunologia , Animais , Apoptose/imunologia , Linfócitos T CD8-Positivos/citologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Microambiente Celular/genética , Microambiente Celular/imunologia , Feminino , Pulmão/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia
16.
Nat Immunol ; 21(2): 145-157, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932810

RESUMO

Despite the prevalence and clinical importance of influenza, its long-term effect on lung immunity is unclear. Here we describe that following viral clearance and clinical recovery, at 1 month after infection with influenza, mice are better protected from Streptococcus pneumoniae infection due to a population of monocyte-derived alveolar macrophages (AMs) that produce increased interleukin-6. Influenza-induced monocyte-derived AMs have a surface phenotype similar to resident AMs but display a unique functional, transcriptional and epigenetic profile that is distinct from resident AMs. In contrast, influenza-experienced resident AMs remain largely similar to naive AMs. Thus, influenza changes the composition of the AM population to provide prolonged antibacterial protection. Monocyte-derived AMs persist over time but lose their protective profile. Our results help to understand how transient respiratory infections, a common occurrence in human life, can constantly alter lung immunity by contributing monocyte-derived, recruited cells to the AM population.


Assuntos
Imunidade Inata/imunologia , Macrófagos Alveolares/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções Pneumocócicas/imunologia , Animais , Camundongos
17.
Cell Prolif ; 53(1): e12721, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31782850

RESUMO

OBJECTIVES: Secondary bacterial pneumonia is common following influenza infection. However, it remains unclear about the underlying molecular mechanisms. MATERIALS AND METHODS: We established a mouse model of post-influenza S aureus pneumonia using conditional Shp2 knockout mice (LysMCre/+ :Shp2flox/flox ). The survival, bacterial clearance, pulmonary histology, phenotype of macrophages, and expression of type I interferons and chemokines were assessed between SHP2 deletion and control mice (Shp2flox/flox ). We infused additional KC and MIP-2 to examine the reconstitution of antibacterial immune response in LysMCre/+ :Shp2flox/flox mice. The effect of SHP2 on signal molecules including MAPKs (JNK, p38 and Erk1/2), NF-κB p65 and IRF3 was further detected. RESULTS: LysMCre/+ :Shp2flox/flox mice displayed impaired antibacterial immunity and high mortality compared with control mice in post-influenza S aureus pneumonia. The attenuated antibacterial ability was associated with the induction of type I interferon and suppression of chemo-attractants KC and MIP-2, which reduced the infiltration of neutrophils into the lung upon secondary bacterial invasion. In additional, Shp2 knockout mice displayed enhanced polarization to alternatively activated macrophages (M2 phenotype). Further in vitro analyses consistently demonstrated that SHP2-deficient macrophages were skewed towards an M2 phenotype and had a decreased antibacterial capacity. Moreover, SHP2 modulated the inflammatory response to secondary bacterial infection via interfering with NF-κB and IRF3 signalling in macrophages. CONCLUSIONS: Our findings reveal that the SHP2 expression enhances the host immune response and prompts bacterial clearance in post-influenza S aureus pneumonia.


Assuntos
Vírus da Influenza A/imunologia , Macrófagos/imunologia , Infecções por Orthomyxoviridae/imunologia , Pneumonia Estafilocócica/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Staphylococcus aureus/imunologia , Animais , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Pneumonia Estafilocócica/etiologia , Pneumonia Estafilocócica/genética , Pneumonia Estafilocócica/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia
18.
Arch Virol ; 165(1): 55-67, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31696308

RESUMO

A swine influenza survey was conducted between 2003 and 2015 in Germany. During this period, 8122 snout swabs or other respiratory specimens from pigs of 5178 herds, mainly from Germany, were investigated for the presence of swine influenza A virus (S-IAV). In total, 1310 S-IAV isolates were collected. Of this collection, the complete genome of 267 H1N2 S-IAV isolates was sequenced and phylogenetically analyzed. The data demonstrate the incursion of human-like swine H1N2 viruses (Gent/1999-like) in 2000 and prevalent circulation until 2010. From 2008 onward, a sustained and broad change of the genetic constellation of the swine H1N2 subtype commenced. The Gent/1999-like swine H1N2 viruses ceased and several new swine H1N2 reassortants emerged and became prevalent in Germany. Of these, the upsurge of the Diepholz/2008-like, Emmelsbuell/2009-like and Papenburg/2010-like viruses is notable. The data reveal the importance of reassortment events in S-IAV evolution. The strong circulation of S-IAV of different lineages in the swine population throughout the year underlines that pigs are important reservoir hosts.


Assuntos
Vírus da Influenza A Subtipo H1N2/classificação , Infecções por Orthomyxoviridae/epidemiologia , Vírus Reordenados/classificação , Análise de Sequência de RNA/métodos , Animais , Alemanha/epidemiologia , Humanos , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Infecções por Orthomyxoviridae/virologia , Filogenia , Prevalência , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Suínos
19.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31740528

RESUMO

Influenza A virus (H1N1) is an acute, highly contagious respiratory virus. The use of lactic acid bacteria (LAB) to deliver mucosal vaccines against influenza virus infection is a research hot spot. In this study, two recombinant Lactobacillus plantarum strains expressing hemagglutinin (HA) alone or coexpressing aCD11c-HA to target HA protein to dendritic cells (DCs) by fusion to an anti-CD11c single-chain antibody (aCD11c) were constructed. The activation of bone marrow dendritic cells (BMDCs) by recombinant strains and the interaction of activated BMDCs and sorted CD4+ or CD8+ T cells were evaluated through flow cytometry in vitro, and cellular supernatants were assessed by using an enzyme-linked immunosorbent assay kit. The results demonstrated that, compared to the HA strain, the aCD11c-HA strain significantly increased the activation of BMDCs and increased the production of CD4+ gamma interferon-positive (IFN-γ+) T cells, CD8+ IFN-γ+ T cells, and IFN-γ in the cell culture supernatant in vitro Consistent with these results, the aCD11c-HA strain clearly increased the activation and maturation of DCs, the HA-specific responses of CD4+ IFN-γ+ T cells, CD8+ IFN-γ+ T cells, and CD8+ CD107a+ T cells, and the proliferation of T cells in the spleen, finally increasing the levels of specific antibodies and neutralizing antibodies in mice. In addition, the protection of immunized mice was observed after viral infection, as evidenced by improved weight loss, survival, and lung pathology. The adoptive transfer of CD8+ T cells from the aCD11c-HA mice to NOD/Lt-SCID mice resulted in a certain level of protection after influenza virus infection, highlighting the efficacy of the aCD11c targeting strategy.


Assuntos
Antígeno CD11c/imunologia , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Lactobacillus plantarum/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/virologia , Feminino , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia
20.
Int J Nanomedicine ; 14: 9337-9349, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31819435

RESUMO

Background: Enzyme-linked immunosorbent assay (ELISA) is a common method for diagnosing swine influenza. However, the production of classical antibodies is both costly and time-consuming. As a promising alternative diagnostic tool, single-domain antibodies (sdAbs) offer the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. Methods: Phage display technology was used to isolate sdAbs against the SIV-NP protein from a camel VHH library. The sdAb5 was fused to the biotin acceptor peptide (BAP) and a His-Tag for its expression as monomeric and site-specific biotinylation in E.coli to develop an sdAb-based blocking ELISA (sdAb-ELISA). In the sdAb-ELISA, the anti-SIV antibodies from swine samples were used to block the binding between the biotinylated sdAb5 and SIV-NP protein coated on the ELISA plate. The specificity, sensitivity, and reproducibility of sdAb-ELISA were determined. In addition, consistency among sdAb-ELISA, commercial ELISA kit, and Western blot was evaluated. Results: Six SIV-NP-specific sdAbs were isolated, among which sdAb5 was identified as a dominant sdAb with higher reactivity. The cut-off value of biotinylated sdAb5-based bELISA was determined to be 29.8%. Compared with the positive reference serum against five different types of swine viruses, the developed sdAb-ELISA showed 100% specificity. The detection limit of sdAb-ELISA was 1:160 in an anti-SIV positive reference serum, which is lower than that of the commercial ELISA kit (1:20). In 78 diluted anti-SIV positive serum (1:80), 21 and 42 samples were confirmed as positive by the commercial ELISA kit and sdAb-ELISA, respectively. The coefficients of variation of intra- and inter-assay were 1.79-4.57% and 5.54-9.98%, respectively. The sdAb-ELISA and commercial ELISA kit showed a consistency of 94.17% in clinical swine serum samples. Furthermore, the coincidence rate was 96.67% between the results detected by sdAb-ELISA and Western blot. Conclusion: A specific, sensitive, and reproducible sdAb-ELISA was successfully developed, which offers a new, promising method to detect anti-SIV antibodies in swine serum.


Assuntos
Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/imunologia , Anticorpos de Domínio Único/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Biotina/química , Biotinilação , Camelus , Nucleoproteínas/isolamento & purificação , Nucleoproteínas/metabolismo , Infecções por Orthomyxoviridae/sangue , Infecções por Orthomyxoviridae/virologia , Biblioteca de Peptídeos , Reprodutibilidade dos Testes , Anticorpos de Domínio Único/química , Suínos
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