RESUMO
H9N2 avian influenza virus (AIV) is the most widespread low pathogenic (LP) AIV in poultry and poses a serious zoonotic risk. Vaccination is used extensively to mitigate the economic impact of the virus. However, mutations were acquired after long-term circulation of H9N2 virus in poultry, particularly in the hemagglutinin (HA) proteolytic cleavage site (CS), a main virulence determinant of AIV. Compared to chickens, little is known about the genetic determinants for adaptation of H9N2 AIV to turkeys. Here, we describe 36 different CS motifs in Eurasian H9N2 viruses identified from 1966 to 2019. The European H9N2 viruses specify unique HACS with particular polymorphism by insertion of non-basic amino acids at position 319. Recombinant viruses carrying single HACS mutations resembling field viruses were constructed (designated G319, A319, N319, S319, D319 and K319). Several viruses replicated to significantly higher titers in turkey cells than in chicken cells. Serine proteases were more efficient than trypsin to support multicycle replication in mammalian cells. Mutations affected cell-to-cell spread and pH-dependent HA fusion activity. In contrast to chickens, mutations in the HACS modulated clinical signs in inoculated and co-housed turkeys. G319 exhibited the lowest virulence, however, it replicated to significantly higher titers in contact-turkeys and in vitro. Interestingly, H9N2 viruses, particularly G319, replicated in brain cells of turkeys and to a lesser extent in mammalian brain cells independent of trypsin. Therefore, the silent circulation of potentially zoonotic H9N2 viruses in poultry should be monitored carefully. These results are important for understanding the adaptation of H9N2 in poultry and replication in mammalian cells.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Perus/virologia , Replicação Viral/genética , Motivos de Aminoácidos , Aminoácidos/metabolismo , Animais , Encéfalo/virologia , Gatos , Bases de Dados Genéticas , Células HEK293 , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H9N2/metabolismo , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/enzimologia , Influenza Aviária/metabolismo , Mutação , Filogenia , Serina Proteases/metabolismo , Suínos/virologia , Tripsina/farmacologiaRESUMO
BACKGROUND: Influenza viruses must utilize host factors to complete their lifecycle. Species-specific differences in host factors between birds and mammals mean that avian influenza viruses (AIVs) replicate well in avian hosts but not in human hosts. Acidic nuclear phosphoprotein 32 family member A (ANP32A) has been identified as the host restriction factor for the viral polymerase (vPol) activity of AIVs. The ANP32A belongs to the conserved ANP32 family, the functional roles of which during viral replication remain unclear. METHODS: In this study, we targeted chicken ANP32A using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing to examine the functional roles of ANP32A and other members of the ANP32 family. RESULTS: We showed that chicken ANP32A only, not ANP32B and ANP32E, plays a pivotal role in supporting vPol activity of AIVs. Furthermore, we found that the human ANP32C, ANP32D, and ANP32E have suppressive effects on vPol activity in contrast to human ANP32A and ANP32B. CONCLUSIONS: Chicken and human ANP32 family members had different effects on vPol activity, suggesting that species-specific vPol activity of AIVs could be caused by the differential functions and overall competency of ANP32 family members.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Influenza A/enzimologia , Influenza Aviária/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Replicação Viral/genética , Animais , Galinhas , Cães , Técnicas de Silenciamento de Genes , Influenza Aviária/enzimologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular , Células Madin Darby de Rim Canino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de DNARESUMO
The non-structural protein 1 (NS1) of different influenza A virus (IAV) strains can differentially regulate the activity of c-Jun terminal kinase (JNK) and PI-3 kinase (PI3K). Whether varying JNK and PI3K activation impacts autophagy and IAV replication differently remains uncertain. Here we report that H5N1 (A/mallard/Huadong/S/2005) influenza A virus induced functional autophagy, as evidenced by increased LC3 lipidation and decreased p62 levels, and the presence of autolysosomes in chicken fibroblast cells. H9N2 (A/chicken/Shanghai/F/98) virus weakly induced autophagy, whereas H1N1 virus (A/PR/8/34, PR8) blocked autophagic flux. H5N1 virus activated JNK but inhibited the PI-3 kinase pathway. In contrast, N9N2 virus infection led to modest JNK activation and strong PI-3 kinase activation; whereas H1N1 virus activated the PI-3 kinase pathway but did not activate JNK. SP600125, a JNK inhibitor, inhibited H5N1 virus-induced autophagy and virus replication in a DF-1 chicken fibroblast cell line. Our study uncovered a previously unrecognized role of JNK in IAV replication and autophagy.
Assuntos
Autofagia , Vírus da Influenza A/fisiologia , Influenza Aviária/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Replicação Viral , Animais , Antracenos/farmacologia , Autofagia/efeitos dos fármacos , Células Cultivadas , Galinhas , Ativação Enzimática/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/fisiologia , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Aviária/fisiopatologia , Influenza Aviária/virologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Especificidade da Espécie , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Previous research revealed the induction of chicken USP18 (chUSP18) in the lungs of chickens infected with highly pathogenic avian influenza viruses (HPAIVs). This activity was correlated with the degree of pathogenicity of the viruses to chickens. As mammalian ubiquitin-specific protease (USP18) is known to remove type I interferon (IFN I)-inducible ubiquitin-like molecules from conjugated proteins and block IFN I signalling, we explored the function of the chicken homologue of USP18 during avian influenza virus infection. With this aim, we cloned chUSP18 from cultured chicken cells and revealed that the putative chUSP18 ORF comprises 1137 bp. Comparative analysis of the predicted aa sequence of chUSP18 with those of human and mouse USP18 revealed relatively high sequence similarity among the sequences, including domains specific for the ubiquitin-specific processing protease family. Furthermore, we found that chUSP18 expression was induced by chicken IFN I, as observed for mammalian USP18. Experiments based on chUSP18 over-expression and depletion demonstrated that chUSP18 significantly enhanced the replication of a low-pathogenic avian influenza virus (LPAIV), but not an HPAIV. Our findings suggest that chUSP18, being similar to mammalian USP18, acts as a pro-viral factor during LPAIV replication in vitro.
Assuntos
Proteínas Aviárias/metabolismo , Vírus da Influenza A/fisiologia , Influenza Aviária/enzimologia , Doenças das Aves Domésticas/enzimologia , Proteases Específicas de Ubiquitina/metabolismo , Replicação Viral , Animais , Proteínas Aviárias/genética , Galinhas , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Aviária/genética , Influenza Aviária/virologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Proteases Específicas de Ubiquitina/genética , VirulênciaRESUMO
UNLABELLED: The influenza A virus genome possesses eight negative-strand RNA segments in the form of viral ribonucleoprotein particles (vRNPs) in association with the three viral RNA polymerase subunits (PB2, PB1, and PA) and the nucleoprotein (NP). Through interactions with multiple host factors, the RNP subunits play vital roles in replication, host adaptation, interspecies transmission, and pathogenicity. In order to gain insight into the potential roles of RNP subunits in the modulation of the host's innate immune response, the interactions of each RNP subunit with retinoic acid-inducible gene I protein (RIG-I) from mammalian and avian species were investigated. Studies using coimmunoprecipitation (co-IP), bimolecular fluorescence complementation (BiFc), and colocalization using confocal microscopy provided direct evidence for the RNA-independent binding of PB2, PB1, and PA with RIG-I from various hosts (human, swine, mouse, and duck). In contrast, the binding of NP with RIG-I was found to be RNA dependent. Expression of the viral NS1 protein, which interacts with RIG-I, did not interfere with the association of RNA polymerase subunits with RIG-I. The association of each individual virus polymerase component with RIG-I failed to significantly affect the interferon (IFN) induction elicited by RIG-I and 5' triphosphate (5'ppp) RNA in reporter assays, quantitative reverse transcription-PCR (RT-PCR), and IRF3 phosphorylation tests. Taken together, these findings indicate that viral RNA polymerase components PB2, PB1, and PA directly target RIG-I, but the exact biological significance of these interactions in the replication and pathogenicity of influenza A virus needs to be further clarified. IMPORTANCE: RIG-I is an important RNA sensor to elicit the innate immune response in mammals and some bird species (such as duck) upon influenza A virus infection. Although the 5'-triphosphate double-stranded RNA (dsRNA) panhandle structure at the end of viral genome RNA is responsible for the binding and subsequent activation of RIG-I, this structure is supposedly wrapped by RNA polymerase complex (PB2, PB1, and PA), which may interfere with the induction of RIG-I signaling pathway. In the present study, PB2, PB1, and PA were found to individually interact with RIG-Is from multiple mammalian and avian species in an RNA-independent manner, without significantly affecting the generation of IFN. The data suggest that although RIG-I binding by RNA polymerase complex is conserved in different species, it does not appear to play crucial role in the modulation of IFN in vitro.
Assuntos
RNA Helicases DEAD-box/metabolismo , Vírus da Influenza A Subtipo H9N2/enzimologia , Influenza Aviária/enzimologia , Influenza Humana/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Doenças dos Suínos/enzimologia , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Patos , Humanos , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/genética , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Camundongos , Infecções por Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Ligação Proteica , RNA Polimerase Dependente de RNA/genética , Receptores Imunológicos , Suínos , Doenças dos Suínos/genética , Proteínas Virais/genéticaRESUMO
Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA(1) allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses.
Assuntos
Furina/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H9N2/metabolismo , Influenza Aviária/enzimologia , Influenza Humana/enzimologia , Doenças das Aves Domésticas/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Galinhas , Furina/genética , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H9N2/química , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/genética , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Dados de Sequência Molecular , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Processamento de Proteína Pós-Traducional , Alinhamento de SequênciaRESUMO
In chicken, ST3 beta-galactoside alpha-2, 3-sialyltransferase III (ST3GAL III) is one of the key enzymes participating in the biosynthesis of avian influenza virus receptors. Knowledge about ST3Gal III could increase our understanding of its function in the occurrence and development of avian influenza. To date, no detailed data have been published about the expression pattern and histological distribution of ST3Gal III in chicken. This paper presents data on the nucleotide sequence, mRNA expression pattern and histological distribution of ST3Gal III protein in yellow chicken for the first time. The results show that the nucleotide homology of ST3Gal III among yellow chicken and turkey, chicken, cattle, humans, swine, mouse, rat and chimpanzee was 98%, 92%, 75%, 59%, 70%, 76% and 75%. The relative expression level of ST3Gal III in the heart, kidney and brain of yellow chicken was significantly higher than in other tissues examined for mRNA level (P < 0.05). The histological distribution of ST3Gal III in the heart, liver, spleen, lung, thymus and bursa of Fabricius was determined by immunohistochemical staining using rabbit anti-chicken ST3Gal III IgG prepared by us. Interestingly, the epithelial reticular cells in the immune organs proved to be positive, which may suggest that these cells are important immune cells playing a role in influenza virus infection. The results of this study provide basic data for further research on the functions of ST3Gal III in chicken.
Assuntos
Galinhas/genética , Galinhas/metabolismo , Regulação Enzimológica da Expressão Gênica , Sialiltransferases/genética , Sialiltransferases/metabolismo , Animais , Sequência de Bases , Galinhas/classificação , Galinhas/imunologia , Humanos , Soros Imunes/metabolismo , Imunoglobulina G/isolamento & purificação , Imuno-Histoquímica , Influenza Aviária/enzimologia , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido Nucleico , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Drug binding and unbinding are transient processes which are hardly observed by experiment and difficult to analyze by computational techniques. In this paper, we employed a cost-effective method called "pathway docking" in which molecular docking was used to screen ligand-receptor binding free energy surface to reveal possible paths of ligand approaching protein binding pocket. A case study was applied on oseltamivir, the key drug against influenza a virus. The equilibrium pathways identified by this method are found to be similar to those identified in prior studies using highly expensive computational approaches.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Virus da Influenza A Subtipo H5N1/enzimologia , Simulação de Acoplamento Molecular , Neuraminidase/metabolismo , Oseltamivir/farmacologia , Animais , Aves , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Influenza Aviária/tratamento farmacológico , Influenza Aviária/enzimologia , Influenza Aviária/virologia , Simulação de Acoplamento Molecular/economia , Ligação ProteicaRESUMO
Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or "snatched" from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors.
Assuntos
Desenho de Fármacos , Endorribonucleases , Inibidores Enzimáticos/química , Virus da Influenza A Subtipo H5N1/enzimologia , Simulação de Acoplamento Molecular , RNA Polimerase Dependente de RNA , Proteínas Virais , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Cristalografia por Raios X , Cães , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/química , Inibidores Enzimáticos/farmacologia , Humanos , Influenza Aviária/tratamento farmacológico , Influenza Aviária/enzimologia , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/química , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/químicaRESUMO
Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1.
Assuntos
Antivirais/química , Antivirais/farmacologia , Vírus da Influenza A/enzimologia , Influenza Aviária/tratamento farmacológico , Neuraminidase/antagonistas & inibidores , Oseltamivir/análogos & derivados , Oseltamivir/farmacologia , Animais , Antivirais/síntese química , Sítios de Ligação , Aves/virologia , Vírus da Influenza A Subtipo H7N1/química , Vírus da Influenza A Subtipo H7N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H7N1/enzimologia , Vírus da Influenza A Subtipo H7N3/química , Vírus da Influenza A Subtipo H7N3/efeitos dos fármacos , Vírus da Influenza A Subtipo H7N3/enzimologia , Vírus da Influenza A/química , Vírus da Influenza A/efeitos dos fármacos , Influenza Aviária/enzimologia , Modelos Moleculares , Neuraminidase/química , Neuraminidase/metabolismo , Oseltamivir/síntese químicaRESUMO
Avian influenza viruses (AIV) raise worldwide veterinary and public health concerns due to their potential for zoonotic transmission. While infection with highly pathogenic AIV results in high mortality in chickens, this is not necessarily the case in wild birds and ducks. It is known that innate immune factors can contribute to the outcome of infection. In this context, retinoic acid-inducible gene I (RIG-I) is the main cytosolic pattern recognition receptor known for detecting influenza A virus infection in mammalian cells. Chickens, unlike ducks, lack RIG-I, yet chicken cells do produce type I interferon (IFN) in response to AIV infection. Consequently, we sought to identify the cytosolic recognition elements in chicken cells. Chicken mRNA encoding the putative chicken analogs of CARDIF and LGP2 (chCARDIF and chLGP2, respectively) were identified. HT7-tagged chCARDIF was observed to associate with mitochondria in chicken DF-1 fibroblasts. The exogenous expression of chCARDIF, as well as of the caspase activation and recruitment domains (CARDs) of the chicken melanoma differentiation-associated protein 5 (chMDA5), strongly activated the chicken IFN-ß (chIFN-ß) promoter. The silencing of chMDA5, chCARDIF, and chIRF3 reduced chIFN-ß levels induced by AIV, indicating their involvement in AIV sensing. As with mammalian cells, chLGP2 had opposing effects. While overexpression decreased the activation of the chIFN-ß promoter, the silencing of endogenous chLGP2 reduced chIFN-ß induced by AIV. We finally demonstrate that the chMDA5 signaling pathway is inhibited by the viral nonstructural protein 1. In conclusion, chicken cells, including DF-1 fibroblasts and HD-11 macrophage-like cells, employ chMDA5 for sensing AIV.
Assuntos
Proteínas Aviárias/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , RNA Helicases DEAD-box/metabolismo , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/metabolismo , Doenças das Aves Domésticas/metabolismo , RNA Helicases/metabolismo , Transdução de Sinais , Animais , Proteínas Aviárias/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Linhagem Celular , Galinhas/metabolismo , Galinhas/virologia , RNA Helicases DEAD-box/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/enzimologia , Influenza Aviária/genética , Influenza Aviária/virologia , Interferon beta/genética , Interferon beta/metabolismo , Dados de Sequência Molecular , Doenças das Aves Domésticas/enzimologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , RNA Helicases/genéticaRESUMO
In mammals, Mda5 and RIG-I are members of the evolutionary conserved RIG-like helicase family that play critical roles in the outcome of RNA virus infections. Resolving influenza infection in mammals has been shown to require RIG-I; however, the apparent absence of a RIG-I homolog in chickens raises intriguing questions regarding how this species deals with influenza virus infection. Although chickens are able to resolve certain strains of influenza, they are highly susceptible to others, such as highly pathogenic avian influenza H5N1. Understanding RIG-like helicases in the chicken is of critical importance, especially for developing new therapeutics that may use these systems. With this in mind, we investigated the RIG-like helicase Mda5 in the chicken. We have identified a chicken Mda5 homolog (ChMda5) and assessed its functional activities that relate to antiviral responses. Like mammalian Mda5, ChMda5 expression is upregulated in response to dsRNA stimulation and following IFN activation of cells. Furthermore, RNA interference-mediated knockdown of ChMda5 showed that ChMda5 plays an important role in the IFN response of chicken cells to dsRNA. Intriguingly, although ChMda5 levels are highly upregulated during influenza infection, knockdown of ChMda5 expression does not appear to impact influenza proliferation. Collectively, although Mda5 is functionally active in the chicken, the absence of an apparent RIG-I-like function may contribute to the chicken's susceptibility to highly pathogenic influenza.
Assuntos
Galinhas/imunologia , RNA Helicases DEAD-box/imunologia , Regulação da Expressão Gênica/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/imunologia , Interferon beta/imunologia , Sequência de Aminoácidos , Animais , Galinhas/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Influenza Aviária/enzimologia , Dados de Sequência Molecular , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
BACKGROUND: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression. CONCLUSIONS/SIGNIFICANCE: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection.
Assuntos
Virus da Influenza A Subtipo H5N1 , Influenza Aviária/enzimologia , Óxido Nítrico Sintase Tipo II/análise , Índice de Gravidade de Doença , Animais , Galinhas , Patos , Inflamação , Influenza Aviária/patologiaRESUMO
The mitogen-activated protein kinase (MAPK) family is responsible for important signalling pathways which regulate cell activation, differentiation, apoptosis and immune responses. Studies have shown that influenza virus infection activates MAPK family members in mammals. While the extracellular signal-regulated kinase (ERK)1/2 is important for virus replication, activation of p38 controls the expression of RANTES, interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha. In this study, we report that avian influenza virus (AIV) activates ERK, p38 and Jun-N-terminal kinases in avian species. In chicken macrophages, while ERK was required for H9N2 AIV replication, ERK regulated proinflammatory cytokines IL-1beta, IL-6 and IL-8, which is distinct from what has been previously reported in mammalian cells. Moreover, ERK alone suppressed TNF-alpha and FasL and inhibited TNF-family-mediated extrinsic apoptosis in H9N2-infected chicken macrophages. Taken together, these findings suggest that ERK signalling may uniquely play important roles in avian host responses to AIV infection.
Assuntos
Apoptose , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Aviária/enzimologia , Influenza Aviária/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Linhagem Celular , Galinhas , Citocinas/genética , Citocinas/imunologia , Ativação Enzimática , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Aviária/fisiopatologia , Influenza Aviária/virologia , Macrófagos/citologia , Macrófagos/virologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The most foolproof way to promote survival in epidemics of potentially lethal influenza is to target, not highly mutable viral proteins, but rather intracellular signaling pathways which promote viral propagation or lung inflammation. NF-kappaB, activated in influenza-infected lung epithelial cells and macrophages, is one likely target in this regard, as it plays a role both in viral replication and in the excessive lung inflammation often evoked by influenza infection. Indeed, salicylates, which suppress NF-kappaB activation, have been shown to reduce the lethality of H5N1 avian-type influenza in mice. Another potential target is NADPH oxidase, as this may be a major source of influenza-evoked oxidant stress in lung epithelial cells as well as in phagocytes attracted to lung parenchyma. A number of studies demonstrate that oxidant stress contributes to overexuberant lung inflammation and lethality in influenza-infected mice. The documented utility of N-acetylcysteine, a glutathione precursor, for promoting survival in influenza-infected mice, and diminishing the severity of influenza-like infections in elderly humans, presumably reflects a key role for oxidative stress in influenza. The lethality of influenza is also reduced in mice pretreated with adenovirus carrying the gene for heme oxygenase-1; this benefit may be mediated, at least in part, by the ability of bilirubin to inhibit NADPH oxidase. It may be feasible to replicate this benefit clinically by administering biliverdin or its homolog phycocyanobilin, richly supplied by spirulina. If this latter speculation can be confirmed in rodent studies, a practical and inexpensive regimen consisting of high-dose salicylates, spirulina, and N-acetylcysteine, initiated at the earliest feasible time, may prove to have life-saving efficacy when the next killer influenza pandemic strikes.
Assuntos
Regulação Enzimológica da Expressão Gênica , Influenza Humana/enzimologia , Influenza Humana/mortalidade , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Animais , Aves , Surtos de Doenças , Humanos , Inflamação , Influenza Aviária/enzimologia , Influenza Aviária/mortalidade , Pulmão/patologia , Macrófagos/metabolismo , Camundongos , Modelos Teóricos , Ficobilinas/farmacologia , Ficocianina/farmacologia , Transdução de SinaisRESUMO
Amongst all the internal gene segments (PB2. PB1, PA, NP, M and NS), the avian PB1 segment is the only one which was reassorted into the human H2N2 and H3N2 pandemic strains. This suggests that the reassortment of polymerase subunit genes between mammalian and avian influenza viruses might play roles for interspecies transmission. To test this hypothesis, we tested the compatibility between PB2, PB1, PA and NP derived from a H5N1 virus and a mammalian H1N1 virus. All 16 possible combinations of avian-mammalian chimeric viral ribonucleoproteins (vRNPs) were characterized. We showed that recombinant vRNPs with a mammalian PB2 and an avian PB1 had the strongest polymerase activities in human cells at all studied temperature. In addition, viruses with this specific PB2-PB1 combination could grow efficiently in cell cultures, especially at a high incubation temperature. These viruses were potent inducers of proinflammatory cytokines and chemokines in primary human macrophages and pneumocytes. Viruses with this specific PB2-PB1 combination were also found to be more capable to generate adaptive mutations under a new selection pressure. These results suggested that the viral polymerase activity might be relevant for the genesis of influenza viruses of human health concern.
Assuntos
Adaptação Biológica , Vírus da Influenza A/enzimologia , Influenza Aviária/enzimologia , Mamíferos/virologia , Subunidades Proteicas/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Animais , Aves , Linhagem Celular , Galinhas , Citocinas/metabolismo , Cães , Genes Virais , Humanos , Mediadores da Inflamação/metabolismo , Vírus da Influenza A/genética , Luciferases/metabolismo , Mutação/genética , Seleção Genética , Inoculações SeriadasRESUMO
The mechanism for the pathogenesis of H5N1 infection in humans remains unclear. This study reveals that cyclooxygenase-2 (COX-2) was strongly induced in H5N1-infected macrophages in vitro and in epithelial cells of lung tissue samples obtained during autopsy of patients who died of H5N1 disease. Novel findings demonstrated that COX-2, along with tumor necrosis factor alpha and other proinflammatory cytokines were hyperinduced in epithelial cells by secretory factors from H5N1-infected macrophages in vitro. This amplification of the proinflammatory response is rapid, and the effects elicited by the H5N1-triggered proinflammatory cascade are broader than those arising from direct viral infection. Furthermore, selective COX-2 inhibitors suppress the hyperinduction of cytokines in the proinflammatory cascade, indicating a regulatory role for COX-2 in the H5N1-hyperinduced host proinflammatory cascade. These data provide a basis for the possible development of novel therapeutic interventions for the treatment of H5N1 disease, as adjuncts to antiviral drugs.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/enzimologia , Animais , Aves , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2 , Humanos , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/imunologia , Influenza Aviária/virologia , Influenza Humana/enzimologia , Influenza Humana/virologiaRESUMO
A reverse genetics approach was applied to generate variants of avian influenza virus A/FPV/Ro/34 (H7N1) containing mutations in the caspase cleavage sites of NP and M2 proteins. Mutation Gly16 --> Asp in avian virus NP made this protein (NPgd) sensitive to caspases, like human virus NP, and permitted its cleavage in infected cells. Mutant recombinant virus NPgd was able to replicate and stably carried Gly --> Asp mutation during passages in cultured cells, chicken eggs, and chickens. This variant was found to have significantly decreased virulence for chickens comparatively to wild type recombinant virus (wtr). Virus variants characterized by deletion Gly16 in NP (NPdel) and mutated caspase cleavage site VDVDD87 --> VNVND87 in M2 (M2nn) protein were shown to lack intracellular caspase-dependent cleavage of NP and M2, respectively, and to retain their ability to replicate in different hosts. Variant NPdel, like wide type virus, displayed a high chicken virulence whereas M2nn, like NPgd one, was found to possess a low virulent phenotype. The findings suggest that the mutations altering natural caspase cleavage motifs in NP and M2 do not restrict virus replication ability but can significantly reduce the virulent potential of the mutant viruses. Recombinant virus variants with altered caspase cleavage motifs could be proposed as a matrix for the design of live recombinant vaccines.
Assuntos
Caspases/metabolismo , Vírus da Influenza A/metabolismo , Influenza Aviária/enzimologia , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas da Matriz Viral/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CACO-2 , Linhagem Celular , Galinhas , Cães , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Mutação , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA/genética , Proteínas do Core Viral/genética , Proteínas da Matriz Viral/genética , Replicação Viral/genéticaRESUMO
The main contributions of the author and collaborators about sialidase (EC 3.2.1.18) of influenza virus types A and B and O-acetylesterase (EC 3.1.1.53) of type C are summarized. After a short introduction on the topic, the negative results obtained by the author on inhibitors are commented. Then, the peculiarities of the three procedures assayed, based on the NADH determination as a measurement for the sialidase activity, are discussed. The spectrofluorimetric measurement of NADH concentration is a more sensitive and convenient procedure than that by spectrophotometry, although it is less sensitive than that based on bioluminiscence. Sialidase activity is generally higher in influenza virus type A than in type B; however, some differences have been found between the three sub-types A analysed. Furthermore, thermal stability and stability against changes in the pH values are higher for influenza virus from ducks, followed by those from humans and, finally, by those from pigs. O-acetylesterase of influenza virus type C shows a broad specificity; it acts on O-acetyl-containing compounds which may not be sialic acids. It seems that this enzyme might contribute to facilitate the action of sialidase of influenza virus types A and B. The peculiarities of influenza virus type C suggest to include this type as a new genus in the future classification of viruses.
Assuntos
Hidrolases de Éster Carboxílico/análise , Neuraminidase/análise , Orthomyxoviridae/enzimologia , Acetilesterase , Animais , Patos , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza B/enzimologia , Influenza Aviária/enzimologia , Influenza Humana/enzimologia , Gammainfluenzavirus/enzimologia , Infecções por Orthomyxoviridae/enzimologia , SuínosRESUMO
The infectivity of influenza A viruses like fowl plague virus (FPV) with a cleaved hemagglutinin (HA) is highly sensitive to treatment at pH 5, while strains like PR 8 or virus N with a noncleaved HA survive under this condition. After double infection of chick embryo cells with FPV and PR 8 or virus N, the yield of virus with the HA gene of FPV is greatly reduced. However, it can now survive treatment at pH 5, and the surviving FPV particles form plaques only in the presence of trypsin, indicating that they were coated by the HA of PR 8 or virus N, depending on the coinfecting virus. The results are discussed with respect to the build-up and maintenance of a large reservoir of nonpathogenic influenza A viruses with noncleavable HA in water fowl.