RESUMO
Highly pathogenic avian influenza viruses (HPAIV) emerge from low-pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse genetics-engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. In contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variant interactions in viral evolution. IMPORTANCE Highly pathogenic avian influenza viruses represent a threat to poultry production systems and to human health because of their impact on food security and because of their zoonotic potential. It is therefore crucial to better understand how these viruses emerge. Using a within-host competition model between high- and low-pathogenic avian influenza viruses, we provide evidence that highly pathogenic avian influenza viruses could be more likely to emerge in chickens than in ducks. These results have important implications for highly pathogenic avian influenza virus emergence prevention, and they underscore the importance of within-host viral variant interactions in virus evolution.
Assuntos
Galinhas , Suscetibilidade a Doenças , Patos , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H5N8/fisiologia , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Animais , Biomarcadores , Biópsia , Células Cultivadas , Coinfecção , Genótipo , Imuno-Histoquímica , Influenza Aviária/metabolismo , Influenza Aviária/patologia , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/patologia , RNA Viral , Especificidade da Espécie , Carga Viral , Virulência , Replicação ViralRESUMO
Mammalian cells utilize a wide spectrum of pathways to antagonize the viral replication. These pathways are typically regulated by antiviral proteins and can be constitutively expressed but also exacerbated by interferon induction. A myriad of interferon-stimulated genes (ISGs) have been identified in mounting broad-spectrum antiviral responses. Members of the interferon-induced transmembrane (IFITM) family of proteins are unique among these ISGs due to their ability to prevent virus entry through the lipid bilayer into the cell. In the current study, we generated transgenic chickens that constitutively and stably expressed chicken IFITM1 (chIFITM1) using the avian sarcoma-leukosis virus (RCAS)-based gene transfer system. The challenged transgenic chicks with clinical dose 104 egg infective dose 50 (EID50) of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 (clade 2.2.1.2) showed 100% protection and significant infection tolerance. Although challenged transgenic chicks displayed 60% protection against challenge with the sub-lethal dose (EID50 105), the transgenic chicks showed delayed clinical symptoms, reduced virus shedding, and reduced histopathologic alterations compared to non-transgenic challenged control chickens. These finding indicate that the sterile defense against H5N1 HPAIV offered by the stable expression of chIFITM1 is inadequate; however, the clinical outcome can be substantially ameliorated. In conclusion, chIFITM proteins can inhibit influenza virus replication that can infect various host species and could be a crucial barrier against zoonotic infections.
Assuntos
Antígenos de Diferenciação/genética , Proteínas Aviárias/genética , Galinhas/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/genética , Animais , Animais Geneticamente Modificados/genética , Galinhas/virologia , Técnicas de Transferência de Genes , Influenza Aviária/patologia , Influenza Aviária/virologiaRESUMO
The influenza A virus (IAV) PB2 subunit modulates viral polymerase activity, replication kinetics and pathogenicity. Here we identified novel PB2 substitutions at position 283 of H5 subtype IAV and evaluated their biological characteristics and virulence. The substitution PB2-M283L enhanced the growth capacity and polymerase activity in human and mammalian cells in comparison to the rWT virus. The substitution PB2-M283L displayed high virulence, resulting in a greater virus load in different tissues, more severe histopathological lesions and proinflammatory cytokines burst in mice. The substitution PB2-M283I had an opposite phenotype. Our data extend the important role of PB2 substitutions in the adaptation of H5 subtype IAVs to mammalian hosts.
Assuntos
Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Virulência/genética , Replicação Viral/genética , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Citocinas/metabolismo , Patos , Feminino , Humanos , Vírus da Influenza A/patogenicidade , Influenza Aviária/patologia , Influenza Humana/patologia , Pulmão/patologia , Pulmão/virologia , Metionina , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Organismos Livres de Patógenos Específicos , Carga ViralRESUMO
Abstract Vaccination is a good strategy for the prevention of avian influenza virus. In this research Gamma Irradiated Avian Influenza (Sub type H9N2) Vaccine (GAIV) was prepared by 30 kGy irradiation and used for vaccination of broiler chickens. The purpose was a comparison of immune responses in the two routes of administration for the GAIV vaccine; intranasal and subcutaneously, use of Montanide ISA70 and Trehalose accompanied with irradiated vaccine and compare with formalin vaccine. The Influenza Virus A/Chicken/IRN/Ghazvin/2001/H9N2 was irradiated and used for vaccine formulation, and formalin inactivated AIV was used as conventional vaccine. Chickens were vaccinated by GAIV with and without Trehalose, GAIV and formalin vaccines with ISA70, two routes of administration were intranasal and subcutaneously. All the vaccinated chickens showed a significant increase in antibody titration. The most significant increase of antibody titration was in irradiated vaccine plus Trehalose groups intranasal and subcutaneously. After the first and second intranasal vaccination, the amount of IFN-gamma increased in the irradiated vaccine plus Trehalose group compared to other groups. However, most of the vaccinated groups did not show any significant increase of IFN-α concentration. Histopathological examination revealed lymphocyte infiltration (++), foci dispersed of hemorrhage and edema in intranasal vaccination groups and in addition to these, thickening of alveolar septa was observed in the injection groups. GAIV vaccine can be a good candidate for vaccine preparation, and Trehalose as a stabilizer protects viral antigenic proteins, also makes more absorbance of antigen by the inhalation route. In vaccinated chickens the ulcers in injected vaccines were lower than intranasal vaccines.
Assuntos
Animais , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Aviária/patologia , Influenza Aviária/prevenção & controle , Galinhas , Influenza Aviária/imunologiaRESUMO
This study aimed to investigate whether intranasally coadministered four tandem copies of extracellular domains of M2 (M2e) and polyethyleneimine (PEI), a mucosal adjuvant, can protect chickens against H9N2 influenza A virus infection. Groups of chickens were intranasally vaccinated with M2e plus PEI adjuvant, M2e alone or PEI adjuvant, and antibody (serum IgG and mucosal IgA) and cellular (CD4+ T cells and IFN-γ levels) immune responses were measured post-vaccination. We demonstrated that the chickens vaccinated with M2e plus PEI adjuvant showed significantly (p < 0.05) higher M2e-specific systemic IgG and mucosal IgA responses compared to the chickens that received either M2e alone or PEI adjuvant. The IgA responses measured in lungs were almost comparable to that of the serum IgG levels. Upon restimulation of the vaccinated peripheral blood mononuclear cells (PBMCs) with M2e antigen, significantly (p < 0.05) higher IFN-γ levels were observed only in M2e plus PEI adjuvant vaccinated group. Lymphoproliferative and CD4+ T cell responses, as measured by MTT-based assay and flow cytometry, respectively, were also observed significantly (p < 0.05) higher in M2e plus PEI adjuvant vaccinated chickens. On challenge with the H9N2 virus (104TCID50) at 28th day post-vaccination, M2e plus PEI adjuvant vaccinated group exhibited lower lung inflammation and viral load compared to the chickens treated with either M2e alone or PEI adjuvant. In summary, we show that intranasally coadministered M2e and PEI adjuvant can elicit humoral and cell-mediated immune responses and can reduce viremia levels in chickens post H9N2 infection in chickens.
Assuntos
Galinhas , Vírus da Influenza A Subtipo H9N2 , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Doenças das Aves Domésticas/prevenção & controle , Proteínas da Matriz Viral/imunologia , Administração Intranasal , Animais , Feminino , Imunidade Celular , Imunidade Humoral , Imunidade nas Mucosas , Imunogenicidade da Vacina , Vacinas contra Influenza/administração & dosagem , Influenza Aviária/patologia , Influenza Aviária/virologia , Pulmão/patologia , Pneumopatias/patologia , Pneumopatias/veterinária , Polietilenoimina , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Domínios Proteicos , Distribuição Aleatória , Eliminação de Partículas ViraisRESUMO
The effect of depletion of macrophages using clodronate liposomes as well as macrophage response following viral infections have been studied in various mouse-virus infection models, but they have not been extensively studied in chickens relevant to virus infections. When we infected day 6 chickens with H4N6 low pathogenic avian influenza virus (LPAIV), we observed that H4N6 LPAIV infection increased the staining intensity of KUL01+ cells in trachea, lungs and duodenum of chickens at 3â¯days post-infection. Then, we used clodronate liposomes intra-abdominally in 5â¯day-old chickens and found significant reduction of staining intensity of KUL01+ cells in trachea and duodenum but not in lungs at 4â¯days post-treatment. When we infected the clodronate liposome and PBS liposome treated chickens with H4N6 LPAIV intra-nasally at day 6, we found no effect on H4N6 LPAIV genome loads in trachea, lungs and duodenum of chickens. This study indicates that although KUL01+ cell intensity are increased in respiratory and gastrointestinal tissues in chickens following H4N6 LPAIV infection, the decrease of KUL01+ cell intensity using clodronate liposomes did not change the H4N6 LPAIV genome loads in any of the examined tissues suggesting that KUL01+ cells may not be critical during H4N6 LPAIV infection in chicken.
Assuntos
Galinhas , Ácido Clodrônico/farmacologia , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Macrófagos/metabolismo , Doenças das Aves Domésticas/virologia , Carga Viral/veterinária , Animais , Anticorpos Monoclonais/imunologia , Duodeno/patologia , Imunofluorescência/veterinária , Genoma Viral , Influenza Aviária/imunologia , Influenza Aviária/patologia , Pulmão/patologia , Macrófagos/patologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/patologia , Coloração e Rotulagem , Traqueia/patologiaRESUMO
Guineafowl of different ages were inoculated intravenously with a H6N2 wild waterfowl-origin low pathogenicity avian influenza virus (LPAIV). No clinical disease was observed. The infected birds had atrophy of the spleen, thymus, and cloacal bursa when compared with the noninfected control groups. The central and peripheral lymphoid tissues presented either lymphoproliferative or degenerative lesions that increased in intensity from 14 to 21 days postinoculation (DPI). Lymphoid depletion was present in the bursa, thymic lobes, and spleen T-dependent zone. In contrast, lymphoid proliferation was observed in liver, pancreas, and spleen B-dependent zone. Bronchus associated lymphoid tissue hyperplasia was observed in the lungs of the birds at 14 and 21 DPI. The virus was detected by virus isolation and reverse transcription PCR from both oropharyngeal and cloacal swabs with higher isolation rates from the latter. Most birds from the LPAIV inoculated groups shed virus up to 7 DPI. The virus was infrequently isolated from lung, kidney, liver, bursa, or spleen of infected birds until 14 DPI and from two samples (kidney and spleen, 1-yr-old birds) at 21 DPI. These data indicate that the wild bird-origin LPAIV used in this study caused pantropic infection in guineafowl when inoculated intravenously.
Assuntos
Galliformes/virologia , Vírus da Influenza A/fisiologia , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Eliminação de Partículas Virais , Animais , Animais Selvagens/virologia , Vírus da Influenza A/genética , Influenza Aviária/patologia , Fígado/patologia , Fígado/virologia , Baço/patologia , Baço/virologia , VirulênciaRESUMO
Multiple respiratory infections have a significant impact on health and economy. Pathogenesis of co-infecting viruses and bacteria and their interaction with mucosal surfaces are poorly characterized. In this study we established a co-infection model based on pre-incubation of tracheal organ cultures (TOC) with Mycoplasma (M.) gallisepticum and a subsequent infection with avian influenza virus (AIV). Mycoplasma gallisepticum modified the pathogenesis of AIV as demonstrated in TOC of two different avian species (chickens and turkeys). Co-infection promoted bacterial growth in tracheal epithelium. Depending on the interaction time of M. gallisepticum with the host cells, AIV replication was either promoted or suppressed. M. gallisepticum inhibited the antiviral gene expression and affected AIV attachment to the host cell by desialylation of α-2,3 linked sialic acids. Ultrastructural analysis of co-infected TOC suggests that both pathogens may attach to and possibly infect the same epithelial cell. The obtained results contribute to better understanding of the interaction dynamics between M. gallisepticum and AIV. They highlight the importance of the time interval between infections as well as the biological properties of the involved pathogens as influencing factors in the outcome of respiratory infections.
Assuntos
Coinfecção/veterinária , Interações Hospedeiro-Patógeno , Vírus da Influenza A/patogenicidade , Influenza Aviária/patologia , Infecções por Mycoplasma/patologia , Mycoplasma gallisepticum/patogenicidade , Doenças das Aves Domésticas/patologia , Animais , Apoptose , Galinhas/microbiologia , Galinhas/virologia , Coinfecção/microbiologia , Coinfecção/virologia , Células Epiteliais/metabolismo , Epitélio/microbiologia , Epitélio/virologia , Influenza Aviária/microbiologia , Influenza Aviária/virologia , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/virologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácidos Siálicos/metabolismo , Traqueia/microbiologia , Traqueia/virologia , Perus/microbiologia , Perus/virologiaRESUMO
Since early 2014, several outbreaks involving novel reassortant highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been detected in poultry and wild bird species in Asia, Europe and North America. These viruses have been detected in apparently healthy and dead wild migratory birds, as well as in domestic chickens, turkeys, geese and ducks. In this study, we describe the pathology of an outbreak of H5N8 HPAIV in breeder ducks in the UK. A holding with approximately 6000 breeder ducks, aged approximately 60 weeks, showed a gradual reduction in egg production and increased mortality over a 7-day period. Post-mortem examination revealed frequent fibrinous peritonitis, with severely haemorrhagic ovarian follicles and occasional splenic and pancreatic necrosis and high incidence of mycotic granulomas in the air sacs and lung. Low-to-moderate levels of HPAI H5N8 virus were detected mainly in respiratory and digestive tract, with minor involvement of other organs. Although histopathological examination confirmed the gross pathology findings, intralesional viral antigen detection by immunohistochemistry was not observed. Immunolabelled cells were rarely only present in inflamed air sacs and serosa, usually superficial to granulomatous inflammation. Abundant bacterial microcolonies were observed in haemorrhagic ovaries and oviduct. The limited viral tissue distribution and presence of inter-current fungal and bacterial infections suggest a minor role for HPAIV H5N8 in clinical disease in layer ducks.
Assuntos
Surtos de Doenças/veterinária , Patos/virologia , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , Animais , Feminino , Vírus da Influenza A/classificação , Influenza Aviária/epidemiologia , Influenza Aviária/patologia , Doenças das Aves Domésticas/epidemiologia , Reino Unido/epidemiologia , VirulênciaRESUMO
UNLABELLED: PB1-F2 protein, the 11th influenza A virus (IAV) protein, is considered to play an important role in primary influenza virus infection and postinfluenza secondary bacterial pneumonia in mice. The functional role of PB1-F2 has been reported to be a strain-specific and host-specific phenomenon. Its precise contribution to the pathogenicity and transmission of influenza virus in mammalian host, such as swine, and avian hosts, such as turkeys, remain largely unknown. In this study, we explored the role of PB1-F2 protein of triple-reassortant (TR) H3N2 swine influenza virus (SIV) in pigs and turkeys. Using the eight-plasmid reverse genetics system, we rescued wild-type SIV A/swine/Minnesota/1145/2007 (H3N2) (SIV 1145-WT), a PB1-F2 knockout mutant (SIV 1145-KO), and its N66S variant (SIV 1145-N66S). The ablation of PB1-F2 in SIV 1145 modulated early-stage apoptosis but did not affect the viral replication in swine alveolar macrophage cells. In pigs, PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes, and lung pathology. On the other hand, in turkeys, SIV 1145-KO infected poults, and its in-contacts developed clinical signs earlier than SIV 1145-WT groups and also displayed more extensive histopathological changes in intestine. Further, turkeys infected with SIV 1145-N66S displayed poor infectivity and transmissibility. The more extensive histopathologic changes in intestine and relative transmission advantage observed in turkeys infected with SIV 1145-KO need to be further explored. Taken together, these results emphasize the host-specific roles of PB1-F2 in the pathogenicity and transmission of IAV. IMPORTANCE: Novel triple-reassortant H3N2 swine influenza virus emerged in 1998 and spread rapidly among the North American swine population. Subsequently, it showed an increased propensity to reassort, generating a range of reassortants. Unlike classical swine influenza virus, TR SIV produces a full-length PB1-F2 protein, which is considered an important virulence marker of IAV pathogenicity. Our study demonstrated that the expression of PB1-F2 does not impact the pathogenicity of TR H3N2 SIV in pigs. On the other hand, deletion of PB1-F2 caused TR H3N2 SIV to induce clinical disease early and resulted in effective transmission among the turkey poults. Our study emphasizes the continuing need to better understand the virulence determinants for IAV in intermediate hosts, such as swine and turkeys, and highlights the host-specific role of PB1-F2 protein.
Assuntos
Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus Reordenados/fisiologia , Proteínas Virais/metabolismo , Animais , Apoptose , Especificidade de Hospedeiro , Influenza Aviária/patologia , Influenza Aviária/transmissão , Influenza Aviária/virologia , Intestinos/patologia , Pulmão/patologia , Pulmão/virologia , Macrófagos/fisiologia , Macrófagos/virologia , Camundongos , América do Norte , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Vírus Reordenados/crescimento & desenvolvimento , Vírus Reordenados/patogenicidade , Genética Reversa/métodos , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Perus , Carga Viral , Virulência , Replicação Viral , Eliminação de Partículas ViraisRESUMO
Saliva proteins may protect older people from influenza, however, it is often noted that hospitalizations and deaths after an influenza infection mainly occur in the elderly population living with chronic diseases, such as diabetes and cancer. Our objective was to investigate the expression level of the terminal α2-3- and α2-6-linked sialic acids in human saliva from type 2 diabetes mellitus (T2DM), liver disease and gastric cancer (GC) patients and assess the binding activity of these linked sialic acids against influenza A viruses (IAV). We observed that the expression level of the terminal α2-3-linked sialic acids of elderly individuals with T2DM and liver disease were down-regulated significantly, and the terminal α2-6 linked sialic acids were up-regulated slightly or had no significant alteration. However, in the saliva of patients with GC, neither sialic acid was significantly altered. These findings may reveal that elderly individuals with chronic diseases, such as diabetes and liver disease, might be more susceptible to the avian influenza virus due to the decreased expression of terminal α2-3-linked sialic acids in their saliva.
Assuntos
Diabetes Mellitus Tipo 2/genética , Influenza Humana/genética , Hepatopatias/genética , Ácidos Siálicos/biossíntese , Neoplasias Gástricas/genética , Idoso , Animais , Aves , Doença Crônica , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/patologia , Humanos , Vírus da Influenza A/patogenicidade , Influenza Aviária/complicações , Influenza Aviária/genética , Influenza Aviária/patologia , Influenza Humana/complicações , Influenza Humana/patologia , Hepatopatias/complicações , Hepatopatias/patologia , Risco , Saliva/metabolismo , Ácidos Siálicos/genética , Neoplasias Gástricas/complicações , Neoplasias Gástricas/patologiaRESUMO
Many viruses infect and replicate in their host via the intestinal tract, e.g. many picornaviruses, several coronaviruses and avian influenza viruses of waterfowl. To analyze infection of enterocytes is a challenging task as culture systems for differentiated intestinal epithelial cells are not readily available and often have a life span that is too short for infection studies. Precision-cut intestinal slices (PCIS) from chicken embryos were prepared and shown that the epithelial cells lining the lumen of the intestine are viable for up to 4 days. Using lectin staining, it was demonstrated that α2,3-linked sialic acids, the preferred receptor determinants of avian influenza viruses, are present on the apical side of the epithelial cells. Furthermore, the epithelial cells (at the tips) of the villi were shown to be susceptible to infection by an avian influenza virus of the H9N2 subtype. This culture system will be useful to analyze virus infection of intestinal epithelial cells and it should be applicable also to the intestine of other species.
Assuntos
Células Epiteliais/virologia , Vírus da Influenza A Subtipo H9N2/crescimento & desenvolvimento , Influenza Aviária/patologia , Mucosa Intestinal/virologia , Experimentação Animal , Animais , Galinhas , Vírus da Influenza A Subtipo H9N2/isolamento & purificaçãoRESUMO
To understand human response to avian H9N2 influenza, we investigated the effects of the viral infection on A549, HepG2, and HeLa cells at low and high MOIs. To identify virus-host interplay, expression of Mx and NP genes was measured in the cells supernatants. Cell viability and apoptosis were evaluated by MTT assay, DNA fragmentation, and florescent staining. The virus titration and NP gene transcript levels indicate lower susceptibility of HeLa cell to H9N2 replication than other cells. Although H9N2 did produce a faster CPE in HepG2, high dose of the virus induced apoptosis within early stage of A549 infection. The DNA laddering was enhanced in the cell correlated with increase in virus transcripts. The undetectable to different regulation levels of Mx gene were observed in response to H9N2 infection suggesting that an insufficient antiviral defense in the noncompetent-IFN HepG2 cell promotes efficient viral replication. These results showed that the permissivity of HepG2 for H9N2 is comparable with A549; however, liver cells are not target tissue respond to the infection. These data revealed that the H9N2 virus induced apoptosis signaling via mitochondrial pathway in human alveolar epithelial cells, indicating that the induction may be associated with a dose-dependent manner.
Assuntos
Apoptose/genética , Vírus da Influenza A Subtipo H9N2/genética , Influenza Aviária/genética , Animais , Aves/virologia , Sobrevivência Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação Viral da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Vírus da Influenza A Subtipo H9N2/patogenicidade , Influenza Aviária/patologia , Influenza Aviária/virologia , Proteínas do Core Viral/biossíntese , Replicação Viral/genéticaRESUMO
Our previous studies have illustrated three strains of duck-origin H5N1 highly pathogenic avian influenza viruses (HPAIVs) had varying levels of pathogenicity in ducks (Sun et al., 2011). However, the host immune response of ducks infected with those of H5N1 HPAIVs was unclear. Here, we compared viral distribution and mRNA expression of immune-related genes in ducks following infection with the two HPAIV (A/Duck/Guangdong/212/2004, DK212 and A/Duck/Guangdong/383/2008, DK383). DK383 could replicate in the tested tissue of ducks (brain, spleen, lungs, cloacal bursa, kidney, and pancreas) more rapid and efficiently than DK212 at 1 and 2 days post-inoculation. Quantitative real-time PCR analysis showed that the expression levels of TLR3, IL-6, IL-8, and MHC class II in brains were higher than those of respective genes in lungs during the early stage of post infection. Furthermore, the expression levels of IL-6 and IL-8 in the brain of ducks following infection with DK383 were remarkably higher than those of ducks infected with DK212, respectively. Our results suggest that the shift in the H5N1 HPAIVs to increased virulence in ducks may be associated with efï¬cient and rapid replication of the virus, accompanied by early destruction of host immune responses. These data are helpful to understand the underlying mechanism of the different outcome of H5N1 HPAIVs infection in ducks.
Assuntos
Patos/virologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/imunologia , Doenças das Aves Domésticas/imunologia , Animais , Patos/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Aviária/genética , Influenza Aviária/patologia , Influenza Aviária/virologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Pulmão/imunologia , Pulmão/patologia , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Reação em Cadeia da Polimerase em Tempo Real , Baço/imunologia , Baço/patologia , VirulênciaRESUMO
Differing sensitivity of influenza A viruses to antiviral effects of the Myxovirus resistance (Mx) protein implies varying global gene expression profiles in the host. The role of Mx protein during lethal avian influenza (AI) virus infection was examined using Mx1-deficient C57BL/6 (B6-Mx1(-/-)) and congenic Mx1-expressing (B6-Mx1(+/+)) mice infected with a virulent, mouse-adapted avian H5N2 Ab/Korea/ma81/07 (Av/ma81) virus. After infection, B6-Mx1(+/+) mice were completely protected from lethal AI-induced mortality, and exhibited attenuated clinical disease and reduced viral titers and pathology in the lungs, compared with B6-Mx1(-/-) mice. Transcriptional profiling of lung tissues revealed that most of the genes up-regulated after infection are involved in activation of the immune response and host defense. Notably, more abundant and sustained expression of cytokine/chemokine genes was observed up to 3 dpi in B6-Mx1(-/-) mice, and this was associated with excessive induction of cytokines and chemokines. Consequently, massive infiltration of macrophages/monocytes and granulocytes into lung resulted in severe viral pneumonia and potentially contributed to decreased survival of B6-Mx1(-/-) mice. Taken together, our data show that dysregulated gene transcriptional activity corresponded to persistent induction of cytokine/chemokines and recruitment of cytokine-producing cells that promote inflammation in B6-Mx1(-/-) mouse lungs. Thus, we provide additional evidence of the interplay of genetic, molecular, and cellular correlates governed by the Mx1 protein that critically determine disease outcome during lethal AI virus infection.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Inflamação/patologia , Influenza Aviária/prevenção & controle , Influenza Aviária/virologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Animais , Líquido da Lavagem Broncoalveolar , Galinhas , Citocinas/farmacologia , Cães , Proteínas de Ligação ao GTP/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/complicações , Inflamação/virologia , Vírus da Influenza A Subtipo H5N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H5N2/patogenicidade , Influenza Aviária/patologia , Interferons/farmacologia , Interleucinas/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Resistência a Myxovirus , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/patologia , Infecções por Orthomyxoviridae/genética , Virulência/efeitos dos fármacosRESUMO
OBJECTIVE: We developed subunit vaccines against H5 or H9 subtype avian influenza viruses (AIV) and infectious bursal disease viruses (IBDV). Viral protein 2 (VP2) of IBDV was used as cargo protein to display a 12-amino-acid (aa) immunodominant epitope derived from N-terminal M2 extracelluar domain (nM2e) of H5 or H9 subtype AIV. METHODS: The aa and nucleotide sequence of nM2e was determined by comparing the available avian influenza vaccine strains and alignment the AIV sequence available in GenBank. One copy of H5 or H9 nM2e was inserted into P(BC) region of VP2 origin from IBDV B87 vaccine strain by fusion polymerase chain reaction. The VP2(BC)nM2e recombinants were cloned into Bac-to-Bac expression system and transfected to Sf9 cell. The expressed chimeric protein was characterized by indirect immunofluorescence assay and Western blotting, and subsequently was used as antigen to develop vaccine. The non-immunized chicken was given two injections with the vaccine at a 4-week interval. Serum against VP2 and nM2e was tested by indirect ELISA and virus neutralization in chick embryo fibroblast. RESULTS: Both VP2(BC)nM2e recombinants were successfully constructed and expressed in Sf9 cell. Both chimeric proteins elicited antibody against VP2 and nM2e. The antibody level elicited by VP2(BC)nM2e(H5) vaccine was higher than that of VP2(BC)nM2e(H9). CONCLUSION: Both chimeric proteins were immunigenic, and the efficacy of VP2(BC)nM2e(H5) was higher than VP2(BC)nM2e(H9) chicken.
Assuntos
Vírus da Doença Infecciosa da Bursa/imunologia , Vírus da Influenza A/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Virais/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Animais , Formação de Anticorpos/imunologia , Infecções por Birnaviridae/virologia , Linhagem Celular , Embrião de Galinha , Galinhas , Fibroblastos/imunologia , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/farmacologia , Influenza Aviária/imunologia , Influenza Aviária/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Vacinas Virais/farmacologiaRESUMO
Ducks have played an important role in the emergence of H5N1 subtype of highly pathogenic avian influenza (HPAI), and the development of an effective vaccine against HPAI in ducks is a top priority. It has been shown that a recombinant fowlpox virus (FPV)-vectored vaccine can provide protection against HPAI in ducks. In this study, a recombinant fowlpox virus (rFPV-AIH5AIL6) coexpressing the haemagglutinin (HA) gene of the H5N1 subtype of the avian influenza virus (AIV) and chicken interleukin 6 gene was constructed and tested in Gaoyou and cherry valley ducks to evaluate the immune response in ducks. These animal studies demonstrated that rFPV-AIH5AIL6 induced a higher anti-AIV HI antibody response, an enhanced lymphocyte proliferation response, an elevated immune protection, and a reduction in virus shedding compared to a recombinant fowlpox virus expressing the HA gene alone (rFPV-SYHA). These data indicate that rFPV-AIH5AIL6 may be a potential vaccine against the H5 subtype of avian influenza in ducks and chicken interleukin 6 may be an effective adjuvant for increasing the immunogenicity of FPV-vectored AIV vaccines in ducks.
Assuntos
Portadores de Fármacos , Vírus da Varíola das Aves Domésticas/genética , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Interleucina-6/imunologia , Animais , Anticorpos Antivirais/sangue , Proliferação de Células , Galinhas , Modelos Animais de Doenças , Patos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Aviária/patologia , Influenza Aviária/virologia , Interleucina-6/genética , Linfócitos/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Eliminação de Partículas ViraisRESUMO
Avian influenza viruses (AIV) are an important emerging threat to public health. It is thought that sialic acid (sia) receptors are barriers in cross-species transmission where the binding preferences of AIV and human influenza viruses are sias α2,3 versus α2,6, respectively. In this study, we show that a normal fully differentiated, primary human bronchial epithelial cell model is readily infected by low pathogenic H5N1, H5N2 and H5N3 AIV, which primarily bind to sia α2,3 moieties, and replicate in these cells independent of specific sias on the cell surface. NHBE cells treated with neuraminidase prior to infection are infected by AIV despite removal of sia α2,3 moieties. Following AIV infection, higher levels of IP-10 and RANTES are secreted compared to human influenza virus infection, indicating differential chemokine expression patterns, a feature that may contribute to differences in disease pathogenesis between avian and human influenza virus infections in humans.
Assuntos
Aves/virologia , Brônquios/patologia , Células Epiteliais/virologia , Vírus da Influenza A/fisiologia , Influenza Aviária/virologia , Influenza Humana/virologia , Ácido N-Acetilneuramínico/metabolismo , Adolescente , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Quimiocinas/metabolismo , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cílios/patologia , Cães , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Aviária/patologia , Influenza Humana/patologia , Masculino , Neuraminidase/farmacologia , Receptores de Superfície Celular/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia , Eliminação de Partículas Virais/efeitos dos fármacos , Eliminação de Partículas Virais/fisiologiaRESUMO
Avian influenza virus can be divided into two groups, highly pathogenic avian influenza virus (HPAI) and low pathogenic avian influenza virus (LPAI) based on their difference in virulence. To investigate if the difference in clinical outcome between LPAI and HPAI in chickens is due to immunological host responses in the lung within the first 24 hours post infection (hpi), chickens were infected with LPAI or HPAI of subtype H7N1. Virus was found in the caudal and cranial part of the lung. With LPAI, virus was localised around the intrapulmonary bronchus and secondary bronchi. In sharp contrast, HPAI was detected throughout the whole lung. However, based on viral RNA levels, no quantitative difference was observed between LPAI and HPAI infected birds. In infected areas of the lungs, an influx of CD8α+ cells as well as KUL01+ macrophages and dendritic cells (DC) occurred as fast as 8 hpi in both infected groups. No major difference between LPAI and HPAI infected birds in the induction of cytokines and interferons at mRNA level in lung tissue was found.In conclusion, the differences in lethality for chickens infected with LPAI or HPAI could be ascribed to difference in location of the virus. However similar amounts of viral RNA, similar cytokine mRNA levels, and similar influxes of CD8α+ and KUL01+ macrophages and DC were found between HPAI and LPAI in the lungs. A cytokine storm at mRNA level as described for mammals was not observed in the lungs of HPAI infected birds within 24 hpi.
Assuntos
Citocinas/genética , Células Dendríticas/imunologia , Vírus da Influenza A Subtipo H7N1/patogenicidade , Influenza Aviária/imunologia , Pulmão/imunologia , Macrófagos/imunologia , Doenças das Aves Domésticas/imunologia , Animais , Galinhas , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/patologia , Vírus da Influenza A Subtipo H7N1/genética , Influenza Aviária/patologia , Influenza Aviária/virologia , Pulmão/patologia , Macrófagos/patologia , Masculino , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , VirulênciaRESUMO
The avian influenza virus H9N2 subtype has circulated in wild birds, is prevalent in domestic poultry, and has successfully crossed the species boundary to infect humans. Phylogenetic analyses showed that viruses of this subtype appear to have contributed to the generation of highly pathogenic H5N1 viruses. Little is known about the host responses to H9N2 viruses in human airway respiratory epithelium, the primary portal for viral infection. Using an apically differentiated primary human tracheobronchial epithelial (TBE) culture, we examined host immune responses to infection by an avian H9N2 virus, in comparison with a human H9N2 isolate. We found that IFN-ß was the prominent antiviral component, whereas interferon gamma-induced protein 10 kDa (IP-10), chemokine (C-C motif) ligand (CCL)-5 and TNF-α may be critical in proinflammatory responses to H9N2 viruses. In contrast, proinflammatory IL-1ß, IL-8, and even IL-6 may only play a minor role in pathogenicity. Apparently Toll-like receptor (TLR)-3, TLR-7, and melanoma differentiation-associated gene 5 (MDA-5) contributed to the innate immunity against the H9N2 viruses, and MDA-5 was important in the induction of IFN-ß. We showed that the avian H9N2 virus induced apoptosis through the mitochondria/cytochrome c-mediated intrinsic pathway, in addition to the caspase 8-mediated extrinsic pathway, as evidenced by the cytosolic presence of active caspase 9 and cytochrome c, independent of truncated BH3 interacting domain death agonist (Bid) activation. Further, we demonstrated that FLICE-like inhibitory protein (FLIP), an apoptotic dual regulator, and the p53-dependent Bcl-2 family members, Bax and Bcl-x(s), appeared to be involved in the regulation of extrinsic and intrinsic apoptotic pathways, respectively. The findings in this study will further our understanding of host defense mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.