Proteomic Analysis Of Feather Pulp And Skin Of Chickens Infected With Marek's Disease Virus

Marek's disease (MD) is a prevalent, contagious lymphoma in chickens caused by a herpesvirus called Marek's disease virus (MDV), usually characterized by mononuclear cellular infiltrates in peripheral nerves, various other organs and tissues including iris and skin. MD is the first virus-induced tumor disease, which could be prevented by vaccine. MD has become a good nature model for understanding oncogenism of human cancer.MDV is a highly cell-associated a-herpes virus that replicates in both genetically resistant and susceptible chickens. The enveloped, cell-free virus is shed by the skin and feather debris only, which is the source of infection for other chickens via the respiratory route. However, little is known about the gene networks and host-virus interaction involved the production and release of virus particles. In this study we obtained two-dimensional gel electrophoresis (2-DE) profile of chicken skin and feather pulp total soluble proteins and identified some differently expressed proteins between MDV infected and specific pathogen free (SPF) chicken by matrixassociated laser dissociation/ionization time of flightmass spectrometry (MALDI-TOF-MS). The different level proteins or new proteins were analyzed with bio-informatics. This is the first proteomics analysis of host responses to MDV infection in chicken skin and feather pulp. Our results will provide some new data for investigating production of cell-free virus and pathogenesis of MDV.1. SPF chickens infected with MDV and pathological observation SPF Chickens (n=56) were inoculated intra-abdominally with 1000 plaque-forming unit (PFU) of RB1B at 1 day old. Chickens (n=40) injected with DMEM were served as negative controls. Four chickens from each group were killed every 7 days post inoculation (dpi). The samples such as serum, feather, skin, and viscera were collected and frozen at -70℃. Clinical symptoms including weakness, anorexia, depression gradually was observed about 14dpi. Pp38 gene of the virus was also amplified by polymerase chain reaction (PCR) from feather of infected group at the same time. Paralysis was found in some of chickens infected group after 3 weeks infection. In addition, enlargement of liver, spleen and kidney were observed after dissection but atrophy for bursa of Fabricius and thymus. Gross lesions and death occur after four to five weeks post infection. The visceral tumors in liver, spleen and kidney were found. The pathological section of skin and feather were revealed intense infiltration of macrophages and lymphocytes. Agar gel precipition (AGP) tests showed that infected chickens produced MDV antibodies in serum and antigen in feather. All the results indicate that the infected chickens have been developed MD. The animal experiment provides abundant materials for further studies on proteome of feather pulp and skin of chickens infected with MDV.2. Establishment of the Two-Dimensional Electrophoresis for Feather Pulp of SPF ChickensThe feather follicle and feather pulp of birds are important sites of replication and release of some avian virus. The feather provides a convenient living tissue for studying the virus replication, release, virus genes expression, virus-host interaction, and so on. In the study, the total proteins were extracted from the feather pulp of SPF chicken, and separated by two-dimensional electrophoresis. The 2-DE maps were optimized with different factors such as lysis buffer, pH range of immobilized pH gradient (IPG) gel strip, IEF, stain, etc. Finally, the best procedure for 2-DE profile of chicken feather pulp proteome with high reproducibility and resolution was developed. The results could be better when loading 400μg proteins with buffer I (8M urea,2%chaps,50mMDTT, 0.2%bio-lyte 3/10,0.001%bromphenol blue, 1mM PMSF) to 17cm, pH5-8 IPG strip, focusing on 60000 volt hrs, staining with commassie brilliant blue G-250. Analysis with PDQuest 8.0.1, more than 700 resoluble polypeptide spots were detected on each image, and the repeatability of samples from different chickens is higher than 92%. The method provides a basis for further application of feather pulp to investigating pathogenesis of MDV.3. Proteomics analysis of feather pulp from chickens infected with Marek's disease virusFeather follicle epithelium (FFE) and feather are sites which produce and release enveloped infectious Marek's disease virus. However, little is known about the gene networks and host-virus interaction during the production and release of virus particles. The present study aimed to obtain 2-DE profile of total soluble proteins of chicken feather infected with MDV and find differential expression of proteins between MDV infected and uninfected specific pathogen free (SPF) chickens.Feather pulp was extracted from feather tips collected from chickens infected with MDV and the control. The 2-DE maps of soluble proteins were obtained as described in section 2, and analyzed with PDQuest 8.0.1. The results showed that 41 spots, which expression level changed above two fold, were detected.25 of these spots were up-regulated,7 spots down-regulated,9 spots newly induced expression in group infected with MDV.25 spots changed significantly were further analyzed by MALDI-TOF-MS.21 spots, corresponding to 20 proteins, were successfully identified. These differently expressed proteins are apolipoprotein AI, Immunoglobulin lambda chain, Actin, cytoplasmic type 5,Calponin-1,14-3-3 sigma (two spots are the same protein), ATP synthase alpha subunit, S100 calcium binding protein A 11, Ovotransferrin precursor, triosephosphate isomerase 1(TPI1), RCJMB04₂91737 kDa protein, STMN1 13 kDa protein, PSMC6 46 kDa protein, Eukaryotic translation initiation factor 5A-1, ETFA 34 kDa protein, TXN Thioredoxin, PKM2 58 kDa protein, Superoxidedismutase (SOD2), ACPI Low molecular weight phosphotyrosine protein phosphatase. Bioinformatics study indicates these differential proteins are mainly associated with metabolism (40%), cell proliferation (25%), immuno-related(15%) and cytoskeleton(10%).4. Proteomics analysis of chicken skins infected with Marek's disease virusTotal proteins were extracted from skin of chickens infected with RB1B at 28th days post infection (dpi) and analyzed by two-dimensional electrophoresis (2-DE) with 17cm,pH5-8 IPG strips. Twenty three protein spots changed above two fold were found, in which 16 spots corresponding to 14 unique proteins were successfully identified by further MS. Among of the identified proteins, three newly induced in infected group wereβ2-microglobulin, Heat shock protein 25 and Creatine kinase M-type.10 up-regulated include, Apolipoprotein A-I (apoA-I), putative uncharacterized protein, Serum albumin, Myosin light chainl (cardiac muscle), Myosin light chain 1 (skeletal muscle isoform),β-enolase, Triosephosphate isomerase (TPI1), Phosphoglycerate kinase (PGK1), Adipocyte fatty acid binding protein and similar to retinoid binding proteins (RBP7).1 down-regulated was transthyretin (TTR) 15kD protein. Based on bioinformatics analysis, the differently expressed proteins could be functionally classified into 5 groups as following:immune-related proteins, cell regulatory proteins, skeleton proteins, metabolism-related proteins and transport proteins. In order to verify the proteomics results, the mRNA level of ApoA I beta-2-microglobulin andβ-actin as internal control were detected by semi-quantitative RT-PCR. The protein level of ApoA I was detected by western blot. The results of mRNA and western blot were consistent to those of 2-DE. These results indicate that expression of these proteins may relate to the disorder of cell metabolism and proliferation, and finally induce transformation of lymphocytes. Our study provides a base for further understand of pathogenesis, diagnosis, and prevention of MDV.