Differential Proteome And Transcriptome Analysis Of Splenic Tissues Of Chickens Infected With H5N1 Subtype Avian Influenza Virus

Avian influenza(AI) is an infectious disease caused by avian influenza virus(AIV) which belongs to the family Orthomyxoviridae. AIV can infect all domestic birds and wild birds. Wild waterfowls are thought to act as a natural reservoir of influenza A virus. The highly pathogenic H5N1 AIV has attracted widespread concern because of its threat to poultry production and potential to cross species barriers and cause human infections or even a new human pandemic. Influenza virus infection and transmission are relied not only on itself but host factors. Recently, a number of studies have been conducted to investigate the mechanism of pathogenesis and transmission of influenza virus, and several virulence factors and key amino acid residues have been identified. The genome of AIV contains eight segments, and encodes 11 different proteins. As an obligate parasite, AIV must extensively exploit the translational, enzymatic and other machineries, and component of host cells for its efficient replication, synthesis and assembly of viral components, and release of progeny viruses. On the other hand, specific alterations were also induced by virus infection to the host cells. These alterations occurred at transcriptome level as well as proteome level, which might be important for us to search for potential targets for further studies and better understand the pathogenesis of AIV. However, posttranscriptional and translational processing might regulate the distribution and quantity of proteins in vivo and in vitro. An integrate transcriptomic and proteomic analysis in biological studies may provide more comprehensive molecular characterization. To investigate the host cellular response alterations following AIV infection, different analysis have provided important information regarding host response to influenza virus infection and accelerated the better understanding on the mechanism of influenza virus pathogenesis. However, most of the related studies in this field are focused on the host responses in mammalian species, and such studies are substantially lacking in avian species.The spleen, as the main peripheral immune organ, is responsible for initiating immunity to blood-borne pathogens and for filtering the blood of foreign material and damaged red blood cells and plays anti-infection and immune response function in the viral infection. However, studies regarding splenic proteome alteration in chicken after AIV infection have not been reported.To investigate the host response against AIV infections, an integrative analysis of transcriptome and proteomic in splenic tissues of chickens infected with H5N1 subtype AIV was performed using chicken genome arrays, two dimensional gel electrophoresis(2-DE) and mass spectroscopy. Gene ontology(GO) and KEGG annotation are used to analysis differentially expressed proteins and genes based on biological processes, molecular functions and pathway. In addition, real-time quantitative PCR(RT-q PCR) was used to confirm the m RNA transcript levels of differentially expressed proteins and genes from 2-DE and genome array analysis. To further confirm the alterations of protein expression during H5N1 subtype AIV infection, western blot analysis was carried out. The main results were as followed:(1) Virus titer, pathological and immunohistochemistry analysis of H5N1 subtyp AIV-infected chickensBrain, lung, thymus, trachea, heart, liver, spleen, kidney, and bursa of fabricius were collected from chickens 24 h post inoculation with H5N1 subtype AIV or PBS as a control. The virus titers of infected chicken tissues were determined by 50% egg infectious dose(EID50) assay with SPF eggs. Replication of the virus was detected in all nine organs of AIV-infected chickens, and the control chicken tissue homogenates did not cause the death of the embryonated SPF eggs.Brain, lung, thymus, trachea, heart, liver, spleen, kidney, and bursa of fabricius tissues were analysised by histological examination. The results showed degeneration, necrocytosis, erythrocytes effusion, macrophage infiltration and other pathological changes presented in the above organ.Immunohistochemical staining displayed that viral antigens were detected in all nine organs in AIV-infected chickens. The results were consistent with those of virus titer assay.(2) The proteome analysis of spleen from AIV infected chickensTo identify the differentially expressed proteins in splenocyte of chickens infected with H5N1 AIV, the total proteins of the splenic cells were extracted from the AIV infected or control chickens,separated by 2-DE and identified by MALDI-TOF/TOF. The results showed that 36 differential expresses protein spots(Fold change >1.5) were identified in AIV-infected chicken spleen in comparison with PBS control spleen and verified by MALDI-TOF/TOF.GO annotation indicated that these proteins were divided into 34 functional categories, including reproduction, cell morphogenesis, immune process, m RNA processing, translation, protein folding, protein complex assembly, cellular protein modification process, lipid metabolic process, transport, nucleocytoplasmic transport, response to stress, cell cycle, cell adhesion, catabolic process, biosynthetic process, ribonucleoprotein complex assembly, cell differentiation, cellular nitrogen compound metabolic process, nucleobase-containing compound catabolic process, growth, ribosome biogenesis, homeostatic process, small molecule metabolic process, symbiosis, anatomical structure development, cell division and macromolecular complex assembly of biological process. Most of the differentially expressed proteins involved in immune responses. Molecular function classification found most of differentially expressed proteins had cytoskeletal component, enzyme activity and ion binding function.(3) The transcriptional change analysis of spleen from AIV infected chickensIn order to investigate transcriptional change of chicken splenic tissues 24 h post inoculation with H5N1 AIV or PBS as a control, The Gene Chip® Chicken Genome Arrays were obtained from Affymetrix. The backrounds of microarray in AIV infected group and control group were respectively 38.93922 and 36.17032. 2457 genes had an altered expression pattern by a factor of 2 or greater and the call flag of present in one group or all groups, of which 1284 were up-regulated and 1173 down-regulated in the spleen of infected chickens compared with those of control chickens.GO category based on biological process showed that 24 categories were involved including reproduction, cell killing, immune process, metabolic process, cellular process, anatomical structure formation, viral reproduction, death, reproductive process, biological adhesion, multicellular organismal process, developmental process, growth, locomotion, pigmentation, biological rhythmic process, response to stimulus, localization, establishment of localization, multi-organism process, biological regulation, positive regulation of biological process, negitave regulation of biological process and regulation of biological process.To further define differentially expressed genes functions in chicken spleens after AIV infection, the KEGG database was used to analyze pathways. The results showed that 137 pathways were founded involving with differentially expressed genes. A probability(p)-value < 0.05 was used as a threshold. 35 pathways were involved, of which cell adhesion molecules, cytokine-cytokine receptor interaction, EMC-receptor interaction and focal adhesion showed the highest significance. Metabolic pathway, Toll like receptor, PPAR and P53 signal pathways also presented higher significance.(4) Validation of the differential expression proteins and genesRT-q PCR was performed to validate the proteome and transcriptome data for 16 selected differentially expressed genes which related to infection and immune response. The mRNA levels of tissue transglutaminase 2(TGM2) and desmin(DES) agreed with the proteomic analysis data. Inaddition, ACTR3, YWHAB, RPSA and B2 M agreed with the proteomic analysis data. The different of m RNA levels of LY86 showed the opposite trend. The mRNA levels of TGM2, IL1, IL6, IFN-γ, IFN-β, MMP1, SAMD3, PCSK5 and IL2 showed agreement on both up-and downregulated expression of the genes. The genes for TGM2 and B2 M gene show differential expression in 2-DE and microarray. The trend of TGM2 and B2 M showed agreement with RTq PCR, 2-DE and microarray analysis.Equal amounts of highly pathogenic H5N1 AIV infected and control splenic tissue proteins were examined by Western blot analysis with specific antibodies to TGM2, DES and GAPDH. In Western blot analysis, the TGM2 and DES demonstrated up regulation. These data agreed with the expression changes by the 2-DE analysis. The expression of TGM2 gene changes in microarray, 2-DE, RT-q PCR and Western blot. The trends of TGM2 showed agreement in four different tests.These results provided comprehensive knowledge regarding the host response to AIV infection in chicken splenic tissues at protein and gene level, which might advance a better understanding of pathogenic mechanism of AIV infection and extend the knowledge of the nature of virus-host interactions. In addition, the obtained data would advance the search for potential protein targets for further studies and provide clues for the development of antiviral therapeutics against H5N1 AIV.