| Influenza A viruses are important pathogens that cause acute respiratory diseases and annual epidemics in humans and poultry, which have become serious health and economic concerns in China. Because proinflammatory cytokines are markedly elevated during highly pathogenic avian influenza virus infection, the cytokine storm is hypothesized to be the main cause of mortaily. Previous reports showed the guinea pig is susceptible to influenza virus. Further study showed that the virus failed to induce the cytokine storm in guinea pig, which is easily eliminated5-7dpi. These demonstrate that the guinea pig is a suitable animal model for host defense against influenza virus studies. This study is mainly divided into two parts. The first part is using guinea pigs as model animal, JH4and104C1as model cells, H5N1influenza viruses as model virus. The host factors affect resistant to infection with influenza A virus was evaluated. In this study, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach and Real-time PCR method was used to investigate the dynamic host response induced by H5N1influenza virus in vivo and in vitro. Based on the data from proteomics analysis, we probe the antivirus function of differentially expressed protein and identify the key pathway of anti-inflammatory activity. The second part is to evaluate the pathogenesis of humans to H9N2avian influenza virus (AIV). The pathogenicity analysis of H9N2influenza virus was determined in non-human primates.Part1:The global differential expressed protein and selected gene expression changes in lungs and JH4cell infected with H5N1AIV were investigated using iTRAQ proteomic approach and quantitative real-time PCR. The results showed that the H5N1AIV wasn’t supportive to induce early and sustained inflammatory in vivo and in vitro. Proteomic approach analysis showed that200proteins were significantly differential expression at1dpi, and63proteins changed at3dpi in lungs in response to H5N1AIV infection. In addition, the cell level showed that132proteins were changed at12hpi, and81proteins changed at24hpi in JH4cell. Using gene ontology and KEGG pathways analysis, the results indicate the differentially expressed proteins are mainly involved in interferon pathways, apoptosis and complement activation. The results suggest that the modulated proteins in infected lungs and cells were possibly involved in protective effects and cytokine regulation.In vitro anti-influenza virus function of RIG-I, MAVS, SPD, SPA, GBP-1, Mx-1, HSP70and HSP90were investigated using overexpression technology. The GBP-1gene was also investigated using RNAi technology. The results showed that protein expression to over-express RIG-I, MAVS and Mx-1gene decreased the titers of AIV in JH4cell and104C1cell. The inhibition ratio of RIG-I and MAVS are more significantly higher than Mx-1. The overexpression of GBP-1reduced the level of cytokine induced by AIV, however, it made antiviral effect by the synergic action of RIG-I.Complement C3, a member of the complement system of serum proteins, is a major component of the innate immune and inflammatory responses. In this study, we demonstrate that H5N1influenza virus infected guinea pig had increased levels of C3activation byproducts as compared to guinea pig infected with pandemic2009H1N1influenza viruses. Research in CVF-treated guinea pig demonstrated that C3was required for protection from influenza infection, proper viral clearance, and associated with changes in cellular infiltration. Interestingly, Treatment of influenza virus infected guinea pig with CVF, resulted in a reduction of cell apoptosis and robust IP10gene expression. The results suggest that the complement C3may help control an acute inflammatory response by inducing apoptosis.Part2:Several cases of humans infected with the H9N2avian influenza virus have been described since1999, however, the infectivity and pathogenicity of H9N2in humans is not well defined. A non-human primate model in rhesus macaques was developed to study H9N2virus infections as a means of better understanding the pathogenesis and virulence of this virus, in addition to testing antiviral drugs. Rhesus macaques inoculated with H9N2AIV presented with biphasic fever and viral pneumonia. H9N2was recovered from nasal washes and pharyngeal samples up to days7-9post infection followed by an increase in HI (hemagglutination inhibition) antibody titers. Tissue tropism and immunohistochemistry indicated that H9N2AIV replicated in the upper respiratory tract (turbinate, trachea, and bronchus) and in all lobes of the lung. Our data suggest that rhesus macaques are a suitable animal model to study H9N2influenza virus infections, particularly in the context of viral evolution and pathogenicity. |
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