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1.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199658

RESUMO

Influenza A virus (IAV) causes seasonal epidemics and sporadic pandemics, therefore is an important research subject for scientists around the world. Despite the high variability of its genome, the structure of viral RNA (vRNA) possesses features that remain constant between strains and are biologically important for virus replication. Therefore, conserved structural motifs of vRNA can represent a novel therapeutic target. Here, we focused on the presence of G-rich sequences within the influenza A/California/07/2009(H1N1) genome and their ability to form RNA G-quadruplex structures (G4s). We identified 12 potential quadruplex-forming sequences (PQS) and determined their conservation among the IAV strains using bioinformatics tools. Then we examined the propensity of PQS to fold into G4s by various biophysical methods. Our results revealed that six PQS oligomers could form RNA G-quadruplexes. However, three of them were confirmed to adopt G4 structures by all utilized methods. Moreover, we showed that these PQS motifs are present within segments encoding polymerase complex proteins indicating their possible role in the virus biology.


Assuntos
Quadruplex G , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Biologia Computacional , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/patologia , RNA Viral/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
2.
Nat Commun ; 12(1): 4427, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285233

RESUMO

The membrane-associated RING-CH (MARCH) proteins are E3 ligases that regulate the stability of various cellular membrane proteins. MARCH8 has been reported to inhibit the infection of HIV-1 and a few other viruses, thus plays an important role in host antiviral defense. However, the antiviral spectrum and the underlying mechanisms of MARCH8 are incompletely defined. Here, we demonstrate that MARCH8 profoundly inhibits influenza A virus (IAV) replication both in vitro and in mice. Mechanistically, MARCH8 suppresses IAV release through redirecting viral M2 protein from the plasma membrane to lysosomes for degradation. Specifically, MARCH8 catalyzes the K63-linked polyubiquitination of M2 at lysine residue 78 (K78). A recombinant A/Puerto Rico/8/34 virus carrying the K78R M2 protein shows greater replication and more severe pathogenicity in cells and mice. More importantly, we found that the M2 protein of the H1N1 IAV has evolved to acquire non-lysine amino acids at positions 78/79 to resist MARCH8-mediated ubiquitination and degradation. Together, our data support the important role of MARCH8 in host anti-IAV intrinsic immune defense by targeting M2, and suggest the inhibitory pressure of MARCH8 on H1N1 IAV transmission in the human population.


Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas da Matriz Viral/metabolismo , Células A549 , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Cães , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/virologia , Lisina/genética , Lisina/metabolismo , Lisossomos/metabolismo , Lisossomos/virologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Ubiquitinação/imunologia , Proteínas da Matriz Viral/genética , Replicação Viral
3.
Arch Virol ; 166(8): 2217-2224, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34091783

RESUMO

Swine influenza is an economically important respiratory disease in swine, but it also constantly poses a threat to human health. Therefore, developing rapid, sensitive, and efficient detection methods for swine influenza virus (SIV) is important. By aligning the haemagglutinin (HA) gene sequences of SIVs circulating in China over a 10-year period, an H1 primer-probe set targeting both Eurasian avian-like H1N1 (EA H1N1) and pandemic 2009 H1N1 ((H1N1)pdm09) lineages plus a H3 primer-probe set targeting the prevalent human-like H3N2 (HL H3N2) subtype were designed. Subsequently, a TaqMan-MGB-based duplex one-step real-time RT-PCR (RT-qPCR) assay was established and evaluated. The duplex RT-qPCR has a detection limit of 5 copies/µL of HA plasmid for EA H1N1, (H1N1)pdm09, and HL H3N2 subtype SIVs, and its overall detection sensitivity of 100% and specificity of 91.67% matches that of traditional virus isolation through chicken embryo inoculation using experimentally infected mouse lung samples. The method showed high repeatability both within run and between runs, and there was no cross-reactivity against several other porcine viruses that are commonly circulating in China. Furthermore, the duplex RT-qPCR method revealed a higher prevalence of subtype H1 than subtype H3 in 166 nasal swabs from pigs collected from one slaughterhouse between October and December 2019. This assay could be very helpful in the rapid differential detection and routine surveillance of EA H1N1, (H1N1)pdm09, and HL H3N2 SIVs in China.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Infecções por Orthomyxoviridae/diagnóstico , Animais , China , Modelos Animais de Doenças , Diagnóstico Precoce , Feminino , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/genética , Camundongos , Reação em Cadeia da Polimerase Multiplex , Nariz/virologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Suínos
4.
Emerg Microbes Infect ; 10(1): 1191-1199, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34049471

RESUMO

The ongoing COVID-19 pandemic has led to more than 159 million confirmed cases with over 3.3 million deaths worldwide, but it remains mystery why most infected individuals (∼98%) were asymptomatic or only experienced mild illness. The same mystery applies to the deadly 1918 H1N1 influenza pandemic, which has puzzled the field for a century. Here we discuss dual potential properties of the 1918 H1N1 pandemic viruses that led to the high fatality rate in the small portion of severe cases, while about 98% infected persons in the United States were self-limited with mild symptoms, or even asymptomatic. These variations now have been postulated to be impacted by polymorphisms of the sialic acid receptors in the general population. Since coronaviruses (CoVs) also recognize sialic acid receptors and cause severe acute respiratory syndrome epidemics and pandemics, similar principles of influenza virus evolution and pandemicity may also apply to CoVs. A potential common principle of pathogen/host co-evolution of influenza and CoVs under selection of host sialic acids in parallel with different epidemic and pandemic influenza and coronaviruses is discussed.


Assuntos
COVID-19/patologia , Influenza Humana/patologia , Receptores de Superfície Celular/genética , Receptores Virais/genética , Ácidos Siálicos/metabolismo , Doenças Assintomáticas , Evolução Biológica , COVID-19/mortalidade , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Influenza Humana/mortalidade , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2/genética , Saliva/metabolismo , Saliva/virologia
5.
Anal Chim Acta ; 1165: 338542, 2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-33975694

RESUMO

Aerosol transmission is one of the three major transmission routes of respiratory viruses. However, the dynamics and significance of the aerosol transmission route are not well understood, partially due to the lack of rapid and efficient tools for on-the-spot detection of airborne viruses. We report a hand-held device that integrates a 3D-printed sample preparation unit with a laminated paper-based RNA amplification unit. The sample preparation unit features an innovative reagent delivery scheme based on a ball-based valve capable of storing and delivering reagents through the rotation of the unit without manual pipetting, while the paper-based unit enables RNA enrichment and reverse transcription loop-mediated isothermal amplification (RT-LAMP). We have determined the detection limit of the integrated sample-preparation/amplification device (SPAD) at 1 TCID50 H1N1 influenza viruses in 140 µL aqueous sample. Further, we integrated SPAD with a previously reported viable virus aerosol sampler (VIVAS), a water-vapor-based condensational growth system capable of collecting aerosolized virus particles (Pan et al., 2016) [1]. Using the combined VIVAS-SPAD platform, we have demonstrated the collection/detection of lab-generated, airborne H1N1 influenza viruses in 65 min, suggesting that the platform has a potential for detecting and monitoring airborne virus transmission during outbreaks. The effective sampling and rapid detection of airborne viruses by the sample-to-answer platform will also help us better understand the dynamics and significance of aerosol transmission of infectious disease.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A/genética , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA
6.
Sci Rep ; 11(1): 10793, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031464

RESUMO

Finding novel biomarkers for human pathologies and predicting clinical outcomes for patients is challenging. This stems from the heterogeneous response of individuals to disease and is reflected in the inter-individual variability of gene expression responses that obscures differential gene expression analysis. Here, we developed an alternative approach that could be applied to dissect the disease-associated molecular changes. We define gene ensemble noise as a measure that represents a variance for a collection of genes encoding for either members of known biological pathways or subunits of annotated protein complexes and calculated within an individual. The gene ensemble noise allows for the holistic identification and interpretation of gene expression disbalance on the level of gene networks and systems. By comparing gene expression data from COVID-19, H1N1, and sepsis patients we identified common disturbances in a number of pathways and protein complexes relevant to the sepsis pathology. Among others, these include the mitochondrial respiratory chain complex I and peroxisomes. This suggests a Warburg effect and oxidative stress as common hallmarks of the immune host-pathogen response. Finally, we showed that gene ensemble noise could successfully be applied for the prediction of clinical outcome namely, the mortality of patients. Thus, we conclude that gene ensemble noise represents a promising approach for the investigation of molecular mechanisms of pathology through a prism of alterations in the coherent expression of gene circuits.


Assuntos
COVID-19/patologia , Expressão Gênica , Influenza Humana/patologia , Sepse/patologia , Área Sob a Curva , COVID-19/complicações , COVID-19/virologia , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Redes Reguladoras de Genes/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/complicações , Influenza Humana/virologia , Estresse Oxidativo/genética , Peroxissomos/genética , Peroxissomos/metabolismo , Modelos de Riscos Proporcionais , Curva ROC , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sepse/complicações , Sepse/genética , Sepse/mortalidade , Índice de Gravidade de Doença , Taxa de Sobrevida , Interface Usuário-Computador
7.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33789991

RESUMO

Recombinant influenza A viral (IAV) vectors are potential to stimulate systemic and mucosal immunity, but the packaging capacity is limited and only one or a few epitopes can be carried. Here, we report the generation of a replication-competent IAV vector that carries a full-length HIV-1 p24 gene linked to the 5'-terminal coding region of the neuraminidase segment via a protease cleavage sequence (IAV-p24). IAV-p24 was successfully rescued and stably propagated, and P24 protein was efficiently expressed in infected mammalian cells. In BALB/c mice, IAV-p24 showed attenuated pathogenicity compared to that of the parental A/PR/8/34 (H1N1) virus. An intranasal inoculation with IAV-p24 elicited moderate HIV-specific cell-mediated immune (CMI) responses in the airway and vaginal tracts and in the spleen, and an intranasal boost with a replication-incompetent adenovirus type 2 vector expressing the HIV-1 gag gene (Ad2-gag) greatly improved these responses. Importantly, compared to an Ad2-gag prime plus IAV-p24 boost regimen, the IAV-p24 prime plus Ad2-gag boost regimen had a greater efficacy in eliciting HIV-specific CMI responses. P24-specific CD8+ T cells and antibodies were robustly provoked both systemically and in mucosal sites and showed long-term durability, revealing that IAV-p24 may be used as a mucosa-targeted priming vaccine. Our results illustrate that IAV-p24 is able to prime systemic and mucosal immunity against HIV-1 and warrants further evaluation in nonhuman primates.IMPORTANCE An effective HIV-1 vaccine remains elusive despite nearly 40 years of research. CD8+ T cells and protective antibodies may both be desirable for preventing HIV-1 infection in susceptible mucosal sites. Recombinant influenza A virus (IAV) vector has the potential to stimulate these immune responses, but the packaging capacity is extremely limited. Here, we describe a replication-competent IAV vector expressing the HIV-1 p24 gene (IAV-p24). Unlike most other IAV vectors that carried one or several antigenic epitopes, IAV-p24 stably expressed the full-length P24 protein, which contains multiple epitopes and is highly conserved among all known HIV-1 sequences. Compared to the parental A/PR/8/34 (H1N1) virus, IAV-p24 showed an attenuated pathogenicity in BALB/c mice. When combined with an adenovirus vector expressing the HIV-1 gag gene, IAV-p24 was able to prime P24-specific systemic and mucosal immune responses. IAV-p24 as an alternative priming vaccine against HIV-1 warrants further evaluation in nonhuman primates.


Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD8-Positivos/imunologia , Anticorpos Anti-HIV/análise , Proteína do Núcleo p24 do HIV/imunologia , HIV-1/imunologia , Imunidade nas Mucosas , Adenoviridae/genética , Animais , Anticorpos Antivirais/sangue , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Genes gag , Anticorpos Anti-HIV/sangue , Proteína do Núcleo p24 do HIV/genética , Infecções por HIV/prevenção & controle , Imunidade Celular , Imunização Secundária , Imunogenicidade da Vacina , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/imunologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas Sintéticas/imunologia
8.
FEBS J ; 288(10): 3164-3185, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33830641

RESUMO

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.


Assuntos
Apresentação do Antígeno/genética , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A Subtipo H1N1/genética , Macroautofagia/genética , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/imunologia , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/virologia , Brefeldina A/farmacologia , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Feminino , Expressão Gênica , Células HEK293 , Antígenos de Histocompatibilidade Classe II/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Macroautofagia/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Plasmídeos/química , Plasmídeos/metabolismo , Transfecção
9.
Front Immunol ; 12: 656350, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868301

RESUMO

The new SARS-CoV-2 virus differs from the pandemic Influenza A virus H1N1 subtype (H1N1pmd09) how it induces a pro-inflammatory response in infected patients. This study aims to evaluate the involvement of SNPs and tissue expression of IL-17A and the neutrophils recruitment in post-mortem lung samples from patients who died of severe forms of COVID-19 comparing to those who died by H1N1pdm09. Twenty lung samples from patients SARS-CoV-2 infected (COVID-19 group) and 10 lung samples from adults who died from a severe respiratory H1N1pdm09 infection (H1N1 group) were tested. The tissue expression of IL-8/IL-17A was identified by immunohistochemistry, and hematoxylin and eosin (H&E) stain slides were used for neutrophil scoring. DNA was extracted from paraffin blocks, and genotyping was done in real time-PCR for two IL17A target polymorphisms. Tissue expression increasing of IL-8/IL-17A and a higher number of neutrophils were identified in samples from the H1N1 group compared to the COVID-19 group. The distribution of genotype frequencies in the IL17A gene was not statistically significant between groups. However, the G allele (GG and GA) of rs3819025 was correlated with higher tissue expression of IL-17A in the COVID-19 group. SARS-CoV-2 virus evokes an exacerbated response of the host's immune system but differs from that observed in the H1N1pdm09 infection since the IL-8/IL-17A tissue expression, and lung neutrophilic recruitment may be decreased. In SNP rs3819025 (G/A), the G allele may be considered a risk allele in the patients who died for COVID-19.


Assuntos
COVID-19 , Regulação da Expressão Gênica/imunologia , Interleucina-17 , Interleucina-8 , Pulmão/imunologia , Neutrófilos/imunologia , Polimorfismo de Nucleotídeo Único , SARS-CoV-2 , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/genética , COVID-19/imunologia , COVID-19/patologia , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/genética , Influenza Humana/imunologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Neutrófilos/virologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia
10.
Life Sci ; 277: 119484, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33862119

RESUMO

As a type of non-coding RNA, microRNAs are considered to be a new regulator in viral infections. Influenza A (H1N1) virus infection is a serious threat to human health. There is growing evidence supporting that microRNAs play important roles in various cellular infection stages and host antiviral response during H1N1 infection. Some microRNAs defend against H1N1 invasion, while others may promote viral replication. MicroRNAs are implicated in the host-viral interactions and serve versatile functions in it. In this review, we focus on the innate immune response and virus replication regulated by microRNAs during H1N1 infection. MicroRNAs can influence H1N1 virus replication by directly binding to viral compositions and through host cellular pathways. Moreover, microRNAs are involved in multiple antiviral response, including production of interferons (IFNs), retinoic acid-inducible gene I (RIG-I) signaling pathway, immune cells development and secretion, activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). Furthermore, these regulatory effects of microRNAs suggest its potential clinical significance. In addition, another non-coding RNA, lncRNA, are also mentioned in the review, which can regulate innate immune response and influence virus replication during H1N1 infection as well.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , MicroRNAs/genética , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Imunidade Inata/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/metabolismo , Interferons/metabolismo , MicroRNAs/fisiologia , Transdução de Sinais/genética , Replicação Viral/genética
11.
J Gen Virol ; 102(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33616517

RESUMO

Since the influenza pandemic in 2009, the causative agent 'A(H1N1)pdm09 virus', has been circulating in both human and swine populations. Although phylogenetic analyses of the haemagglutinin (HA) gene segment have revealed broader genetic diversity of A(H1N1)pdm09-related swine influenza A viruses (swIAVs) compared with human A(H1N1)pdm09 viruses, it remains unclear whether the genetic diversity reflects the antigenic differences in HA. To assess the impact of the diversity of the HA gene of A(H1N1)pdm09-related swIAVs on HA antigenicity, we characterized 12 swIAVs isolated in Japan from 2013 to 2018. We used a ferret antiserum and a panel of anti-HA mouse monoclonal antibodies (mAbs) raised against an early A(H1N1)pdm09 isolate. The neutralization assay with the ferret antiserum revealed that five of the 12 swIAVs were significantly different in their HA antigenicity from the early A(H1N1)pdm09 isolate. The mAbs also showed differential neutralization patterns depending on the swIAV strains. In addition, the single amino acid substitution at position 190 of HA, which was found in one of the five antigenically different swIAVs but not in human isolates, was shown to be one of the critical determinants for the antigenic difference of swIAV HAs. Two potential N-glycosylation sites at amino acid positions 185 and 276 of the HA molecule were identified in two antigenically different swIAVs. These results indicated that the genetic diversity of HA in the A(H1N1)pdm09-related swIAVs is associated with their HA antigenic variation. Our findings highlighted the need for surveillance to monitor the emergence of swIAV antigenic variants with public health importance.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Substituição de Aminoácidos , Animais , Variação Antigênica , Cães , Feminino , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , Japão , Camundongos , Infecções por Orthomyxoviridae/virologia , Filogenia , Suínos/virologia
12.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33408177

RESUMO

Influenza A virus (IAV) nonstructural protein 1 (NS1) is a protein with multiple functions that are regulated by phosphorylation. Phosphoproteomic screening of H1N1 virus-infected cells revealed that NS1 was phosphorylated at serine 205 in intermediate stages of the viral life cycle. Interestingly, S205 is one of six amino acid changes in NS1 of post-pandemic H1N1 viruses currently circulating in humans compared to the original swine-origin 2009 pandemic (H1N1pdm09) virus, suggesting a role in host adaptation. To identify NS1 functions regulated by S205 phosphorylation, we generated recombinant PR8 H1N1 NS1 mutants with S205G (nonphosphorylatable) or S205N (H1N1pdm09 signature), as well as H1N1pdm09 viruses harboring the reverse mutation NS1 N205S or N205D (phosphomimetic). Replication of PR8 NS1 mutants was attenuated relative to wild-type (WT) virus replication in a porcine cell line. However, PR8 NS1 S205N showed remarkably higher attenuation than PR8 NS1 S205G in a human cell line, highlighting a potential host-independent advantage of phosphorylatable S205, while an asparagine at this position led to a potential host-specific attenuation. Interestingly, PR8 NS1 S205G did not show polymerase activity-enhancing functions, in contrast to the WT, which can be attributed to diminished interaction with cellular restriction factor DDX21. Analysis of the respective kinase mediating S205 phosphorylation indicated an involvement of casein kinase 2 (CK2). CK2 inhibition significantly reduced the replication of WT viruses and decreased NS1-DDX21 interaction, as observed for NS1 S205G. In summary, NS1 S205 is required for efficient NS1-DDX21 binding, resulting in enhanced viral polymerase activity, which is likely to be regulated by transient phosphorylation.IMPORTANCE Influenza A viruses (IAVs) still pose a major threat to human health worldwide. As a zoonotic virus, IAV can spontaneously overcome species barriers and even reside in new hosts after efficient adaptation. Investigation of the functions of specific adaptational mutations can lead to a deeper understanding of viral replication in specific hosts and can probably help to find new targets for antiviral intervention. In the present study, we analyzed the role of NS1 S205, a phosphorylation site that was reacquired during the circulation of pandemic H1N1pdm09 "swine flu" in the human host. We found that phosphorylation of human H1N1 virus NS1 S205 is mediated by the cellular kinase CK2 and is needed for efficient interaction with human host restriction factor DDX21, mediating NS1-induced enhancement of viral polymerase activity. Therefore, targeting CK2 activity might be an efficient strategy for limiting the replication of IAVs circulating in the human population.


Assuntos
Vírus da Influenza A/fisiologia , RNA Polimerase Dependente de RNA/metabolismo , Serina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Adaptação Fisiológica/genética , Animais , Caseína Quinase II/metabolismo , Linhagem Celular , RNA Helicases DEAD-box/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Mutação , Fosforilação , Ligação Proteica , Suínos , Proteínas não Estruturais Virais/genética , Replicação Viral
13.
PLoS One ; 16(1): e0244669, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33471840

RESUMO

The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.


Assuntos
Vírus da Influenza A/isolamento & purificação , Influenza Aviária/diagnóstico , Influenza Humana/diagnóstico , Infecções por Orthomyxoviridae/diagnóstico , Doenças dos Suínos/diagnóstico , Animais , Aves/virologia , 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 A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Saúde Única , Infecções por Orthomyxoviridae/virologia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Suínos , Doenças dos Suínos/virologia
14.
J Int Med Res ; 49(1): 300060520982832, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33472481

RESUMO

OBJECTIVE: Influenza season occurs every year in China, but its presentation was unusual in the period from December 2017 to early 2018. During this period, influenza activity was increasing across the country and was much greater than during the same period in previous years, with great harm to people's health. METHODS: In this study, we isolated two human influenza virus strains-A/Hebei/F076/2018(H1N1) and B/Hebei/16275B/2018-from patients with severe influenza in Hebei, China, during the flu season in January 2018, and explored their genetic characteristics, pathogenicity, and transmissibility. RESULTS: A/Hebei/F076/2018(H1N1) belongs to the human-like H1N1 influenza virus lineage, whereas B/Hebei/16275B/2018 belongs to the Victoria lineage and is closely related to the World Health Organization reference strain B/Brisbane/60/2008. Pathogenicity tests revealed that A/Hebei/F076/2018(H1N1) replicated much more strongly in mice, with mice exhibiting 40% mortality, whereas B/Hebei/16275B/2018 was not lethal. Both viruses could be transmitted through direct contact and by the aerosol route between guinea pigs, but the H1N1 strain exhibited higher airborne transmissibility. CONCLUSIONS: These results may contribute to the monitoring of influenza mutation and the prevention of an influenza outbreak.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A , Influenza Humana , Animais , China , Cobaias , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/epidemiologia , Camundongos , Virulência
15.
BMC Genomics ; 22(1): 77, 2021 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-33485319

RESUMO

BACKGROUND: Influenza viruses are dangerous pathogens. Seventy-Seven genomes of recently emerged genotype 4 reassortant Eurasian avian-like H1N1 virus (G4-EA-H1N1) are currently available. We investigated the presence and variation of potential G-quadruplex forming sequences (PQS), which can serve as targets for antiviral treatment. RESULTS: PQS were identified in all 77 genomes. The total number of PQS in G4-EA-H1N1 genomes was 571. Interestingly, the number of PQS per genome in individual close relative viruses varied from 4 to 12. PQS were not randomly distributed in the 8 segments of the G4-EA-H1N1 genome, the highest frequency of PQS being found in the NP segment (1.39 per 1000 nt), which is considered a potential target for antiviral therapy. In contrast, no PQS was found in the NS segment. Analyses of variability pointed the importance of some PQS; even if genome variation of influenza virus is extreme, the PQS with the highest G4Hunter score is the most conserved in all tested genomes. G-quadruplex formation in vitro was experimentally confirmed using spectroscopic methods. CONCLUSIONS: The results presented here hint several G-quadruplex-forming sequences in G4-EA-H1N1 genomes, that could provide good therapeutic targets.


Assuntos
Quadruplex G , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Genoma Viral , Genótipo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus Reordenados/genética
16.
J Hazard Mater ; 412: 125219, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33516114

RESUMO

Capturing virus aerosols in a small volume of liquid is essential when monitoring airborne viruses. As such, aerosol-to-hydrosol enrichment is required to produce a detectable viral sample for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays. To meet this requirement, the efficient and non-destructive collection of airborne virus particles is needed, while the incoming air flow rate should be sufficiently high to quickly collect a large number of virus particles. To achieve this, we introduced a high air flow-rate electrostatic sampler (HAFES) that collected virus aerosols (human coronavirus 229E, influenza A virus subtypes H1N1 and H3N2, and bacteriophage MS2) in a continuously flowing liquid. Viral collection efficiency was evaluated using aerosol particle counts, while viral recovery rates were assessed using real-time qRT-PCR and plaque assays. An air sampling period of 20 min was sufficient to produce a sample suitable for use in real-time qRT-PCR in a viral epidemic scenario.


Assuntos
Coronavirus , Vírus da Influenza A Subtipo H1N1 , Aerossóis , Microbiologia do Ar , Coronavirus/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2 , Eletricidade Estática
17.
Eur J Clin Microbiol Infect Dis ; 40(1): 141-149, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32814996

RESUMO

Emerging evidence highlights the role of non-coding small RNAs in host-influenza interaction. We have identified a Y RNA-derived small RNA, miR-1975, which is upregulated upon influenza A virus infection in A549 cells. The aim of this study is to investigate whether miR-1975 serves as an indicator of clinical severity upon influenza infection. We investigate the abundance of miR-1975 in sera from clinical patients and its correlation with hypoxemia status. We quantified its amounts in sera from influenza virus-infected patients and healthy volunteers by means of stem-loop RT-PCR. Median values of miR-1975 were significantly higher in influenza virus-infected patients, especially in hypoxemic patients. miR-1975 levels at the acute stage of the disease were highly correlated with the fraction of inspired oxygen used by the patients and total ventilator days. Receiver operator characteristic curve analysis revealed that miR-1975 levels in combination with days of fever before presenting to hospital had significant predictive value for hypoxemia and respiratory failure for patients infected with influenza virus. Our results reveal that circulating miR-1975 has great potential to serve as a biomarker for predicting prognosis in patients infected with influenza virus.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Adulto , Feminino , Humanos , Influenza Humana/sangue , Masculino , MicroRNAs/análise , Pessoa de Meia-Idade , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Adulto Jovem
18.
Int J Infect Dis ; 103: 352-357, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33249287

RESUMO

BACKGROUND: Global influenza virus circulation decreased during the COVID-19 pandemic, possibly due to widespread community mitigation measures. Cambodia eased some COVID-19 mitigation measures in June and July 2020. On 20 August a cluster of respiratory illnesses occurred among residents of a pagoda, including people who tested positive for influenza A but none who were positive for SARS-CoV-2. METHODS: A response team was deployed on 25 August 2020. People with influenza-like illness (ILI) were asked questions regarding demographics, illness, personal prevention measures, and residential arrangements. Respiratory swabs were tested for influenza and SARS-Cov-2 by real-time reverse transcription PCR, and viruses were sequenced. Sentinel surveillance data were analyzed to assess recent trends in influenza circulation in the community. RESULTS: Influenza A (H3N2) viruses were identified during sentinel surveillance in Cambodia in July 2020 prior to the reported pagoda outbreak. Among the 362 pagoda residents, 73 (20.2%) ILI cases were identified and 40 were tested, where 33/40 (82.5%) confirmed positive for influenza A (H3N2). All 40 were negative for SARS-CoV-2. Among the 73 residents with ILI, none were vaccinated against influenza, 47 (64%) clustered in 3/8 sleeping quarters, 20 (27%) reported often wearing a mask, 27 (36%) reported often washing hands, and 11 (15%) reported practicing social distancing. All viruses clustered within clade 3c2.A1 close to strains circulating in Australia in 2020. CONCLUSIONS: Circulation of influenza viruses began in the community following the relaxation of national COVID-19 mitigation measures, and prior to the outbreak in a pagoda with limited social distancing. Continued surveillance and influenza vaccination are required to limit the impact of influenza globally.


Assuntos
COVID-19/epidemiologia , Vírus da Influenza A Subtipo H3N2 , Influenza Humana/epidemiologia , Adolescente , Adulto , Camboja/epidemiologia , Criança , Surtos de Doenças , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vacinas contra Influenza/administração & dosagem , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Pandemias , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Vigilância de Evento Sentinela , Adulto Jovem
19.
Arch Virol ; 166(1): 179-189, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33145635

RESUMO

We investigated and analysed the molecular evolution of hemagglutinin (HA) and neuraminidase (NA) of influenza A(H1N1)pdm09 virus during the 2017-2018 and 2018-2019 influenza seasons in Beijing, China. We collected and extracted RNA from influenza A(H1N1)pdm09 strains from Peking University People's Hospital and analyzed their HA and NA genes by RT-PCR and sequencing. Phylogenetic analysis of HA and NA sequences was used to compare the amino acid sequences of 51 strains with those of reference strains. All strains belonged to subclade 6B.1, with S162N and I216T substitutions (H1 numbering). Our strains differed from strain A/Michigan/45/2015, with the substitutions S91R, S181T and I312V in the HA antigenic epitope. An E189G mutation was detected in the 190 helix of the receptor binding region of HA. A new potential glycosylation site, 179 (NQT), which was not detected before the 2015 influenza season, was identified. Two strains were mutated at I223, the NA inhibitor resistance site. During 2012-2019, amino acids of HA and NA mutated over time. Co-occurrence mutations N146D, S200P, S202I and A273T in HA appeared along with Q51K, F74S and D416N in NA in six strains during two influenza seasons. Our work reveals the molecular changes and phylogenetic characteristics of influenza A(H1N1)pdm09 virus and suggests that a vaccine probably provides suboptimal protection. The biological characteristics of the new glycosylation and drug-resistance sites detected in this work need to be studied further. The co-occurrence of mutations in HA and NA might affect the characteristics of the virus and need to be given more attention.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Neuraminidase/genética , Substituição de Aminoácidos/genética , Antígenos Virais/genética , Pequim , Evolução Molecular , Variação Genética/genética , Humanos , Filogenia , Estações do Ano , Análise de Sequência de DNA/métodos
20.
Adv Exp Med Biol ; 1324: 29-34, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33346902

RESUMO

This paper presents a case of coinfection of influenza A virus (H1N1) and respiratory syncytial virus (RSV) in a male newborn. On the first day of life, the newborn required passive oxygen therapy, followed by respiratory support with nasal continuous positive airway pressure (nCPAP) due to respiratory insufficiency. As the newborn's respiratory effort was intensifying, he was intubated. In the second day of life, a nasopharyngeal swab was taken yielding the presence of H1N1 and RSV in the RT-PCR test. The child was isolated and given oseltamivir and empirical antibiotic therapy, which improved his condition. Other newborns who initially stayed with the sick child in the post-delivery room did not obtain oseltamivir prophylactically as their nasopharyngeal swabs were negative. The child's parents denied the occurrence of influenza-like symptoms within 14 days of delivery, which suggests a transplacental transmission of the child's infection or asymptomatic course of infection in the parents. In conclusion, this report confirms the possibility of viral coinfections in newborns, which points attention to considering a panel of respiratory viruses in the diagnostics. Symptoms of influenza in newborns may be atypical, including a fever-free course. Oseltamivir treatment in newborns with influenza seems an effective therapeutic measure.


Assuntos
Coinfecção , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Coinfecção/diagnóstico , Coinfecção/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/complicações , Influenza Humana/diagnóstico , Influenza Humana/tratamento farmacológico , Masculino , Infecções por Vírus Respiratório Sincicial/complicações , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico
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