The role of ORF3 protein in the molecular pathogenesis of porcine circovirus 2 infection

176 276 0
The role of ORF3 protein in the molecular pathogenesis of porcine circovirus 2 infection

Đang tải... (xem toàn văn)

Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống

Thông tin tài liệu

THE ROLE OF ORF3 PROTEIN IN THE MOLECULAR PATHOGENESIS OF PORCINE CIRCOVIRUS INFECTION ANBU KUMAR KARUPPANNAN NATIONAL UNIVERSITY OF SINGAPORE 2011 THE ROLE OF ORF3 PROTEIN IN THE MOLECULAR PATHOGENESIS OF PORCINE CIRCOVIRUS INFECTION ANBU KUMAR KARUPPANNAN (B.V.Sc., Madras Veterinary College, India, M.Sc., University of Kentucky, USA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 2011 TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE i ACKNOWLEDGEMENTS I am most thankful to my supervisor Professor Jimmy Kwang for providing me the precious opportunity to work in his lab, his valuable guidance and support throughout my stay in his laboratory. His wide knowledge, experience and interesting ideas have always amazed, educated and motivated me. His constant encouragement has always made me confident and has been a guiding beacon towards the goals of my work. I would like to thank my thesis committee, Dr. Vincent Chow, Dr. Cai Yu and Dr Toshiro Ito for their valuable comments and suggestions. Their diverse backgrounds and guidance has led my work in the proper direction. I also express my sincere thanks to all the past and current members of the Animal health biotechnology lab, especially, Dr. Liu Jue, Jennifer Lau, Zhu Yu, Jia Qiang, Dr. He Fang, Sumathy, Dr. Beau Fenner, Meng Tao, Dr. Syed Musthaq, TLL animal facility, TLL microscopy unit for their help, technical inputs and support in various aspects. All of them were always there when I needed help and support. I would like to express my appreciation to Song Yu, Kian Hong, Peck Junwei, and Reetu for stimulating scientific discussions and friendship. I also thank my family, especially my wife, for enduring me during the course of my study. Above all, I thank the Temasek Life Sciences Laboratory and National University of Singapore for providing me the opportunity and privilege of this education and training. ii TABLE OF CONTENTS TITLE…………………………………………………………………………… i ACKNOWLEDGEMENTS…………………………………………………… . ii TABLE OF CONTENTS……………………………………………………… . iii SUMMARY………………………………………………………………………. vii LIST OF TABLES……………………………………………………………… ix LIST OF FIGURES……………………………………………………………… x LIST OF SYMBOLS AND ABBREVIATIONS………………………………. xii LIST OF PUBLICATIONS…………………………………………………… . xiv Chapter1: INTRODUCTION…………………………… .…………… . 1.1 Introduction…………………………………………………………. 1.2 PCV2 associated disease conditions (PCVAD) …………………… 1.3 Morphology and Replication cycle………………………………… 1.4 Transcriptome and proteome of PCV……………………………… 1.5 Morphogenesis ……………………………………………………. 1.6 Epidemiological history of porcine circovirus infections………… 1.7 Evolutionary aspects of circoviruses……………………………… 1.8 Transmission of the virus…………………………………………… 1.9 Model of the PCVAD development………………………………… 2 10 14 15 16 18 18 1.10 Host Virus interaction………………………………………………. 20 1.11 Thesis outline……………………………………………………… 24 Chapter 2: Ablation of ORF3 expression from porcine circovirus leads to the attenuation of its pathogenicity in SPF piglets …………… 2.1 Introduction………………………………………………………… 2.2 Materials and methods………………………………………………. 26 27 29 iii 2.2.1 Viruses and cell culture……………………………………… 29 2.2.2 Generation of Mutant viruses and their characterization.…… 29 2.2.3 Yeast two hybrid assays…………………………………… 30 2.2.4 Antibodies and recombinant proteins………………………. 30 2.2.5 Experimental design………………………………………… 30 2.2.6 Serological analysis…………………………………………. 32 2.2.7 Quantitative real time PCR…………………………………. 32 2.2.8 Histology…………………………………………………… 33 2.2.9 Flow cytometry……………………………………………… 2.3 34 Results……………………………………………………………… 35 2.3.1 Mutations and genetic stability of the virus.………………… 35 2.3.2 Molecular interaction between ORF3 and Pirh2…………… 39 2.3.3 Characterization of the double mutant virus in vivo………… 43 2.3.4 Serum viremia and virus specific antibody response……… 43 2.3.5 Lymphocyte counts………………………………………… 48 2.3.6 Histological findings………………………………………… 2.4 50 Discussion…………………………………………………………… 54 Chapter 3: Porcine circovirus type ORF3 protein competes with P53 in binding to Pirh2 and mediates the deregulation of P53 homeostasis… 3.1 Introduction………………………………………………………… 3.2 Materials and methods……………………………………………… 60 61 65 3.2.1 Cell culture and transient transfections………………………. 65 3.2.2 Plasmids and recombinant proteins…………………………… 65 3.2.3 Binding assays……………………………………………… 66 3.2.4 Co-immunoprecipitation assays……………………………… 67 iv 3.2.5 Immunofluorescence assays…………………………………. 68 3.2.6 Estimation of protein turnover rates………………………… 68 3.2.7 MTT assay for cell viability………………………………… 69 3.2.8 Flowcytometry……………………………………………… 69 3.2.9 In vitro ubiquitination assay………………………………… 3.3 70 Results………………………………………………………………… 71 3.3.1 The ORF3 protein prevents p53 from binding pPirh2 in vitro and in vivo…………………………………………………… 3.3.2 ORF3 protein alters the subcellular localization of pPirh2…. 71 76 3.3.3 Mapping of the minimal domain of ORF3 protein that binds with pPirh2………………………………………………… . 3.3.4 Interaction of ORF3 protein with pPirh2 up-regulates cellular p53 levels…………………………………………………… 3.3.5 ORF3 interferes with the in vitro ubiquitination of p53…… 3.4 80 86 88 Discussion……………………………………………………………. 90 Chapter 4: ORF3 of porcine circovirus enhances the in vitro and in vivo spread of the virus .……………………………………… 4.1 Introduction………………………………………………………… 4.2 94 95 Materials and methods……………………………………………… 98 4.2.1 Cell culture and viruses………………………………………. 98 4.2.2 Quantitative real-time PCR………………………………… 99 4.2.3 Plasmids and transfection……………………………………. 99 4.2.4 Western blot analysis………………………………………… 99 4.2.5 Assay for caspase activity……………………………………. 100 4.2.6 Mice infections studies………………………………………. 4.3 100 Results……………………………………………………………… 103 4.3.1 Growth kinetics of wild-type PCV2 and ORF3-deficient PCV2 …………………………………………………… … 4.3.2 Role of ORF3-induced apoptosis in the spread of the virus in cell culture……………………………………………………. 103 106 v 4.3.3 Mixed culture of ORF3-deficient PCV2 with a chimeric PCV1-2 virus………………………………………………… 4.3.4 Role of ORF3-induced apoptosis in the in vivo spread of the virus………………………………………………………… . 4.3.5 Role of macrophages in the spread of PCV2 viremia……… 4.4 109 112 114 Discussion…………………………………………………………… 118 Conclusion…………………………………………………………………… 123 5.1 The role of ORF3 in the pathogenicity of PCV2 infection and the molecular mechanism behind the cellular pathogenesis…………… . 124 Future directions……………………………………………………… 129 REFERENCES…………………………………………………………………. 131 5.2 vi SUMMARY Porcine circovirus (PCV2) of the Circoviridae family is a nonenveloped, single stranded DNA virus with a circular genome of 1.7 kilobases. It is a major pathogen of porcine species causing growth retardation, lymphadenopathy, multiorgan inflammation and immune suppression, especially affecting weanling piglets. The PCV2 open reading frame (ORF3) codes a 104 amino acid protein that causes apoptosis of PCV2 infected cells, and is not essential for virus replication. This thesis describes the characterisation of the role of ORF3 in the molecular and the systemic pathogenesis during the PCV2 infection in cell culture, mice model and in natural infection in piglets. Mutant PCV2 lacking the expression of ORF3 are infectious and replicate in cells in vitro, but not cause apoptosis of the infected cells. The ORF3 of PCV2 has been shown to be involved in the pathogenesis of the virus in mice model. In PCV2 infected piglets, B and CD4 T lymphocyte depletion and lymphoid organ destruction are generally observed; however, the ORF3 deficient PCV2 is attenuated in its pathogenicity in infected piglets. The mutant virus does not cause any observable disease or perturbation of the lymphocyte count in the inoculated piglets and elicits an efficient immune response. When compared with the wildtype virus infection, the ORF3 mutant PCV2 infection is characterized by mild viremia and absence of pathological lesions. In infected cells, the ORF3 protein interacts with the porcine homologue of Pirh2 (pPirh2), a p53-induced ubiquitin-protein E3 ligase and causes the accumulation of p53 by disrupting the physiological association of p53 and pPirh2. The ORF3 protein competes with p53 in binding to pPirh2. The amino vii acid residues 20 to 65 of the ORF3 protein are essential in this interaction of ORF3 protein with pPirh2, which leads to an alteration in the cellular localization and a significant reduction in the stability of pPirh2. These events contribute to the deregulation of p53 by pPirh2, leading to increased p53 levels and apoptosis of the infected cells. In addition to its role in causing the apoptosis of the immune cells, characteristic of the PCV2 infection associated disease conditions, the ORF3 also plays a role in the systemic dissemination of the PCV2 infection. The ORF3 expedites the spread of the virus by inducing the early release of the virus from the infected cells. Further, in PCV2 infected mice, the ORF3 induced apoptosis also aids in recruiting macrophages to phagocytise the infected apoptotic cells leading to the systemic dissemination of the infection. The apoptotic activity of the ORF3 of PCV2 hence lends advantage to the spread of the virus. viii List of Tables Table 1. Functional domains of ORF1……………… ………………………….11 Table 2. List of cellular proteins reported to interact with PCV2 proteins… … 23 Table 3. List of ORF3 mutations in the mutant PCV2………………… …….…37 Table 4. List of ORF3 fragments used for yeast two hybrid assays with pPirh2 .42 ix Krakowka, S., Ellis, J.A., McNeilly, F., Ringler, S., Rings, D.M., Allan, G., 2001. Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2). Vet. Pathol. 38, 31–42. Krakowka, S., Ellis, J.A., McNeilly, F., Gilpin, D., Meehan, B., McCullough, K., Allan, G., 2002. Immunologic features of porcine circovirus type infection. Viral Immunol. 15, 567–582. Krakowka, S., Ellis, J., McNeilly, F., Waldner, C., Rings, D.M., Allan, G., 2007. Mycoplasma hyopneumoniae bacterins and porcine circovirus type (PCV2) infection: induction of postweaning multisystemic wasting syndrome (PMWS) in the gnotobiotic swine model of PCV2-associated disease. Can. Vet. J. 48, 716– 724. Kubbutat, M.H.G., Jones, S.N., Vousden, K.H., 1997. Regulation of p53 stability by Mdm2. Nature 87, 299–303. Ladekjaer-Mikkelsen, A.S., Nielsen, J., Stadejek, T., Storgaard, T., Krakowka, S., Ellis, J., McNeilly, F., Allan, G., Bøtner, A., 2002. Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and nonimmunostimulated 3- week-old piglets experimentally infected with porcine circovirus type (PCV2). Vet. Microbiol. 89, 97–114. 146 Leng, R.P., Lin, Y., Ma,W.,Wu, H., Lemmers, B., Chung, S., Parant, J.M., Lozano, G., Hakem, R., Benchimol, S., 2003. Pirh2, a p53-induced ubiquitin– protein ligase, promotes p53 degradation. Cell 112, 779–791. Lefebvre DJ, Meerts P, Costers S, Misinzo G, Barbé F, Van Reeth K, Nauwynck HJ. 2008. Increased porcine circovirus type replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation. Vet Microbiol. 132, 74-86. Levine, A.J., 1997. p53, the cellular gate keeper for growth and division. Cell 88, 323–331. Littman DR, Rudensky AY. 2010. Th17 and regulatory T cells in mediating and restraining inflammation. Cell. 140, 845-58. Liu, J., Chen, I., Kwang, J., 2005. Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virusinduced apoptosis. J. Virol. 79, 8262–8274. Liu, J., Chen, I., Du, Q., Chua, H., Kwang, J., 2006. The ORF3 protein of porcine circovirus type is involved in viral pathogenesis in vivo. J. Virol. 80, 5065–5073. Liu, J., Zhu, Y., Chen, I., Lau, J., He, F., Lau, A.,Wang, Z., Karuppannan, A.K., Kwang, J., 2007. The ORF3 protein of porcine circovirus type interacts with 147 porcine ubiquitin E3 ligase Pirh2 and facilitates p53 expression in viral infection. J. Virol. 81, 9560–9567. Logan, I.R., Sapountzi, V., Gaughan, L., Neal, D.E., Robson, C.N., 2004. Control of human PIRH2 protein stability: involvement of TIP60 and the proteosome. J. Biol. Chem. 279, 11696–11704. Logan, I.R., Gaughan, L., McCracken, S.R., Sapountzi, V., Leung, H.Y., Robson, C.N., 2006. Human PIRH2 enhances androgen receptor signaling through inhibition of histone deacetylase and is overexpressed in prostate cancer. Mol. Cell. Biol. 26, 6502–6510. López-Soria S, Segalés J, Nofrarias M, Calsamiglia M, Ramírez H, Mínguez A, Serrano IM, Marín O, Callén A. 2004. Genetic influence on the expression of PCV disease. Vet Rec. 155, 504. López-Soria S, Nofrarías M, Calsamiglia M, Espinal A, Valero O, RamírezMendoza H, Mínguez A, Serrano JM, Marín O, Callén A, Segalés J. 2011. Postweaning multisystemic wasting syndrome (PMWS) clinical expression under field conditions is modulated by the pig genetic background. Vet Microbiol. 149, 3527. 148 Lucas, M., Stuart, L.M., Savill, J., Lacy-Hulbert, A., 2003. Apoptotic cells and innate immune stimuli combine to regulate macrophage cytokine secretion. J. Immunol. 171, 2610–2615. Lyoo KS, Kim HB, Joo HS. 2008. Evaluation of a nested polymerase chain reaction assay to differentiate between two genotypes of Porcine circovirus-2. J Vet Diagn Invest. 20, 283-8. Magar, R., Larochelle, R., Thibault, S., Lamontagne, L., 2000. Experimental transmission of porcine circovirus type (PCV2) in weaned pigs: a sequential study. J. Comp. Pathol. 123, 258–269. Mandrioli, L., Sarli, G., Panarese, S., Baldoni, S., Marcato, P.S., 2004. Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS). Vet. Immunol. Immunopathol. 97, 25–37. Mankertz A, Caliskan R, Hattermann K, Hillenbrand B, Kurzendoerfer P, Mueller B, Schmitt C, Steinfeldt T, Finsterbusch T. 2004. Molecular biology of Porcine circovirus: analyses of gene expression and viral replication. Vet Microbiol. 98, 81-8 Maruyama, S., Miyajima, N., Bohgaki, M., Tsukiyama, T., Shigemura, M., Nonomura, K., Hatakeyama, S., 2008. Ubiquitylation of epsilon-COP by PIRH2 and regulation of the secretion of PSA. Mol. Cell. Biochem. 307, 73–82. 149 Mason, P.W., Piccone, M.E., Mckenna, T.S., Chinsangaram, J., Grubman, M.J., 1997. Evaluation of a live-attenuated foot-and-mouth disease virus as a vaccine candidate. Virology 227, 96–102. Meehan, B.M., McNeilly, F., Todd, D., Kennedy, S., Jewhurst, V.A., Ellis, J.A., Hassard, L.E., Clark, E.G., Haines, D.M., Allan, G.M., 1998. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J. Gen. Virol. 79, 2171–2179. Misinzo, G., Delputte, P.L., Meerts, P., Lefebvre, D.J., Nauwynck, H.J., 2006. Porcine circovirus uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells. J. Virol. 80, 3487–3494. Momand, J., Zambetti, G.P., Olson, D.C., George, D., Levine, A.J., 1992. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53mediated transactivation. Cell 69, 1237–1245. Muhling J, Raye WS, Buddle JR, Wilcox GE. 2006. Genetic characterisation of Australian strains of porcine circovirus types and 2. Aust Vet J. 84, 421-5. Nayar GP, Hamel A, Lin L. Detection and characterization of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. 1997. Can Vet J. 38, 385-86. 150 Nielsen, J., Vincent, I.E., Bøtner, A., Ladekaer-Mikkelsen, A.S., Allan, G., Summerfield, A., McCullough, K.C., 2003. Association of lymphopenia with porcine circovirus type induced postweaning multisystemic wasting syndrome (PMWS). Vet. Immunol. Immunopathol. 92, 97–111. Noteborn, M.H., Todd, D., Verschueren, C.A., de Gauw, H.W., Curran,W.L., Veldkamp, S., Douglas, A.J., McNulty, M.S., van der Eb, A.J., Koch, G., 1994. A single chicken anemia virus protein induces apoptosis. J. Virol. 68, 346–351. Nussbaum, A.K., Whitton, J.L., 2004. The contraction phase of virus-specific CD8+ T cells is unaffected by a pan-caspase inhibitor. J. Immunol. 173, 6611– 6618. O'Dea, M.A., Hughes, A.P., Davies, L.J., Muhling, J., Buddle, R., Wilcox, G.E., 2008. Thermal stability of porcine circovirus type in cell culture. J. Virol. Methods 147, 61–66. Okuda, Y., Ono, M., Yazawa, S., Shibata, I., 2003. Experimental reproduction of postweaning multisystemic wasting syndrome in cesarean-derived, colostrums deprived piglets inoculated with porcine circovirus type (PCV2): investigation of quantitative PCV2 distribution and antibody responses. J. Vet. Diagn. Invest. 15, 107–114. 151 Oliner, J.D., Pietenpol, J.A., Thiagalingam, S., Gyuris, J., Kinzler, K.W., Vogelstein, B., 1993. Oncoprotein MDM2 conceals the activation domain of tumor suppressor p53. Nature 362, 857–860. Onuki A, Abe K, Togashi K, Kawashima K, Taneichi A, Tsunemitsu H. 1999. Detection of porcine circovirus from lesions of a pig with wasting disease in Japan. J Vet Med Sci. 61, 1119-23. Pavet V, Portal MM, Moulin JC, Herbrecht R, Gronemeyer H. 2011. Towards novel paradigms for cancer therapy. Oncogene. 30, 1-20. Perez-Martin, E., Rovira, A., Calsamiglia, M., Mankertz, A., Rodriguez, F., Segales, J., 2007. A new method to identify cell types that support porcine circovirus type replication in formalin-fixed, paraffin-embedded swine tissues. J. Virol. Methods 146, 86–95. Perry AK, Chen G, Zheng D, Tang H, Cheng G. 2005. The host type I interferon response to viral and bacterial infections. Cell Res. 15, 407-22. Prives, C., Hall, P.A., 1999. The p53 pathway. J. Pathol. 187, 112–126. Qian, H., Wang, T., Naumovski, L., Lopez, C.D., Brachmann, R.K., 2002. Groups of p53 target genes involved in specific p53 downstream effects cluster into different classes of DNA binding sites. Oncogene 21, 7901–7911. 152 Ramamoorthy S, Meng XJ. Porcine circoviruses: a minuscule yet mammoth paradox. 2009.Anim Health Res Rev.10.1-20. Ramamoorthy S, Huang FF, Huang YW, Meng XJ. 2009. Interferon-mediated enhancement of in vitro replication of porcine circovirus type is influenced by an interferon-stimulated response element in the PCV2 genome. Virus Res. 145, 236-43. Resendes, A.R., Majó, N., Segalés, J., Mateu, E., Calsamiglia, M., Domingo, M., 2004. Apoptosis in lymphoid organs of pigs naturally infected by porcine circovirus type 2. J. Gen. Virol. 85, 2837–2844. Resendes AR, Majó N, van den Ingh TS, Mateu E, Domingo M, Calsamiglia M, Segalés J. 2011. Apoptosis in postweaning multisystemic wasting syndrome (PMWS) hepatitis in pigs naturally infected with porcine circovirus type (PCV2). Vet J. 189, 72-6. Riley, T., Sontag, E., Chen, P., Levine, A., 2008. Transcriptional control of human p53- regulated genes. Nat. Rev. Mol. Cell Biol. 9, 402–412. Rodríguez-Cariño C, Duffy C, Sánchez-Chardi A, McNeilly F, Allan GM, Segalés J. 2011, Porcine circovirus type morphogenesis in a clone derived from the L35 lymphoblastoid cell line. J Comp Pathol. 144, 91-102. 153 Roulston, A., Marcellus, R.C., Branton, P.E., 1999. Viruses and apoptosis. Annu. Rev. Microbiol. 53, 577–628. Sanchez Jr., R.E., Meerts, P., Nauwynck, H.J., Ellis, J.A., Pensaert, M.B., 2004. Characteristics of porcine circovirus-2 replication in lymphoid organs of pigs inoculated in late gestation or postnatally and possible relation to clinical and pathological outcome of infection. J. Vet. Diagn. Invest. 16, 175–185. Sarkar,A., Hall,M.W., Exline,M., Hart, J., Knatz, N.,Gatson, N.T.,Wewers, M.D., 2006. Caspase-1 regulates Escherichia coli sepsis and splenic B cell apoptosis independently of interleukin- 1beta and interleukin-18. Am. J. Respir. Crit. Care Med. 174, 1003–1010. Sato F, Tsuchiya S, Meltzer SJ, Shimizu K. MicroRNAs and epigenetics. 2011, FEBS J. 278, 1598-609. Schwartz, J.A., Brittle, E.E., Reynolds, A.E., Enquist, L.W., Silverstein, S.J., 2006. UL54-null pseudorabies virus is attenuated in mice but productively infects cells in culture. J. Virol. 80, 769–784. Segales, J., Domingo, M., 2002. Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review. Vet. Q. 24, 109–124. 154 Segales, J., Domingo, M., Chianini, F., Majo, N., Dominguez, J., Darwich, L., Mateu, E., 2004. Immunosuppression in postweaning multisystemic wasting syndrome affected pigs. Vet. Microbiol. 98, 151–158. Shackelford J, Pagano JS. 2005. Targeting of host-cell ubiquitin pathways by viruses. Essays Biochem. 41:139-56. Sheng, Y., Laister, R.C., Lemak, A., Wu, B., Tai, E., Duan, S., Lukin, J., Sunnerhagen, M., Srisailam, S., Karra, M., Benchimol, S., Arrowsmith, C.H., 2008. Molecular basis of Pirh2-mediated p53 ubiquitylation. Nat. Struct. Mol. Biol. 15, 1334–1342. Shi, K.C., Guo, X., Ge, X.N., Liu, Q., Yang, H.C., 2010. Cytokine mRNA expression profiles in peripheral blood mononuclear cells from piglets experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2. Vet. Microbiol. 140, 155–160. Shibahara, T., Sato, K., Ishikawa, Y., Kadota, K., 2000. Porcine circovirus induces B lymphocyte depletion in pigs with wasting disease syndrome. J. Vet. Med. Sci. 62,1125–1131. Shifrin AL, Chirmule N, Zhang Y, Raper SE. 2005. Macrophage ablation attenuates adenoviral vector-induced pancreatitis. Surgery. 137, 545-51. 155 Shimada M, Kitagawa K, Dobashi Y, Isobe T, Hattori T, Uchida C, Abe K, Kotake Y, Oda T, Suzuki H, Hashimoto K, Kitagawa M., 2009. High expression of Pirh2, an E3 ligase for p27, is associated with low expression of p27 and poor prognosis in head and neck cancers. Cancer Sci. 100, 866-72. Shuai J, Fu L, Zhang X, Zhu B, Li X, He Y, Fang W. 2011. Functional exchangeability of the nuclear localization signal (NLS) of capsid protein between PCV1 and PCV2 in vitro: Implications for the role of NLS in viral replication. Virol J. 8, 341. Sipos,W.,Duvigneau, J.C., Pietschmann, P., Schilcher, F.,Hofbauer,G., Hartl, R.T., Schmoll, F., 2005. Porcine dermatitis and nephropathy syndrome (PDNS) is associated with a systemic cytokine expression profile indicative of proinflammation and a Th1 bias. Vet. Immunol. Immunopathol. 107, 303–313. Steinfeldt T, Finsterbusch T, Mankertz A. 2001. Rep and Rep' protein of porcine circovirus type bind to the origin of replication in vitro. Virology. 291, 152-60. Steinfeldt T, Finsterbusch T, Mankertz A. 2007. Functional analysis of cis- and trans-acting replication factors of porcine circovirus type 1. J Virol. 81, 5696-704. Stevenson, G.W., Kiupel, M., Mittal, S.K., Kanitz, C.L., 1999. Ultrastructure of porcine circovirus in persistently infected PK-15 cells. Vet. Pathol. 36, 368–378. 156 Stevenson, G.W., Kiupel, M., Mittal, S.K., Choi, J., Latimer, K.S., Kanitz, C.L., 2001. Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors. J. Vet. Diagn. Invest. 13, 57–62. Stevenson LS, McCullough K, Vincent I, Gilpin DF, Summerfield A, Nielsen J, McNeilly F, Adair BM, Allan GM. 2006. Cytokine and C-reactive protein profiles induced by porcine circovirus type experimental infection in 3-week-old piglets. Viral Immunol. 19,189-95. Stevenson, L.S., Gilpin, D.F., Douglas, A., McNeilly, F., McNair, I., Adair, B.M., Allan, G.M., 2007. T lymphocyte epitope mapping of porcine circovirus type 2. Viral. Immunol. 20, 389–398. Timmusk, S., Fossum, C., Berg, M., 2006. Porcine circovirus type replicase binds the capsid protein and an intermediate filament-like protein. J. Gen. Virol. 87, 3215–3223. Timmusk, S., Wallgren, P., Brunborg, I.M., Wikström, F.H., Allan, G., Meehan, B.,McMenamy, M., McNeilly, F., Fuxler, L., Belák, K., Põdersoo, D., Saar, T., Berg, M., Fossum, C., 2008. Phylogenetic analysis of porcine circovirus type (PCV2) pre- and post-epizootic postweaning multisystemic wasting syndrome (PMWS). Virus Genes 36, 509–520. 157 Tischer I, Gelderblom H, Vettermann W, Koch MA. 1982. A very small porcine virus with circular single-stranded DNA. Nature. 295, 64-6. Tischer, I., Mields, W., Wolff, D., Vagt, M., Griem, W., 1986. Studies on epidemiology and pathogenicity of porcine circovirus. Arch. Virol. 91, 271–276. Vega-Rocha S., Byeon I.-J.L., Gronenborn B., Gronenborn A.M., Campos-Olivas R. 2007.Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2. J. Mol. Biol. 367:473-487 Victoria, J.G., Wang, C., Jones, M.S., Jaing, C., McLoughlin, K., Gardner, S., Delwart, E.L., 2010. Viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus. J. Virol. 84, 6033–6040. Vincent, I.E., Carrasco, C.P., Herrmann, B., Meehan, B.M., Allan, G.M., Summerfield, A., McCullough, K.C., 2003. Dendritic cells harbor infectious porcine circovirus type in the absence of apparent cell modulation or replication of the virus. J. Virol. 77, 13288–13300. Vincent IE, Carrasco CP, Guzylack-Piriou L, Herrmann B, McNeilly F, Allan GM, Summerfield A, McCullough KC. 2005, Subset-dependent modulation of dendritic cell activity by circovirus type 2. Immunology. 115, 388-98. 158 Vincent IE, Balmelli C, Meehan B, Allan G, Summerfield A, McCullough KC. 2007. Silencing of natural interferon producing cell activation by porcine circovirus type DNA. Immunology. 120, 47-56. Wallgren, P., Brunborg, I.M., Blomqvist, G., Bergstrom, G., Wikstrom, F., Allan, G., Fossum, C., Jonassen, C.M., 2009. The index herd with PMWS in Sweden: presence of serum amyloid A, circovirus viral load and antibody levels in healthy and PMWS-affected pigs. Acta Vet. Scand. 51, 13. Wang XM, Yang LY, Guo L, Fan C, Wu F., 2009. p53-induced RING-H2 protein, a novel marker for poor survival in hepatocellular carcinoma after hepatic resection. Cancer. 115, 4554-63. Welch J, Bienek C, Gomperts E, Simmonds P. 2006. Resistance of porcine circovirus and chicken anemia virus to virus inactivation procedures used for blood products. Transfusion. 46, 1951-8. Wiederkehr, D.D., Sydler, T., Buergi, E., Haessig, M., Zimmermann, D., Pospischil, A., Brugnera, E., Sidler, X., 2009. A new emerging genotype subgroup within PCV- 2b dominates the PMWS epizooty in Switzerland. Vet. Microbiol. 136,27–35. 159 Wikström FH, Meehan BM, Berg M, Timmusk S, Elving J, Fuxler L, Magnusson M, Allan GM, McNeilly F, Fossum C., 2007, Structure-dependent modulation of alpha interferon production by porcine circovirus oligodeoxyribonucleotide and CpG DNAs in porcine peripheral blood mononuclear cells. J Virol. 81,4919-27. Wikström FH, Fossum C, Fuxler L, Kruse R, Lövgren T. 2011. Cytokine induction by immunostimulatory DNA in porcine PBMC is impaired by a hairpin forming sequence motif from the genome of Porcine Circovirus type (PCV2). Vet Immunol Immunopathol. 139, 156-66. Yu, S., Opriessnig, T., Kitikoon, P., Nilubol, D., Halbur, P.G., Thacker, E., 2007. Porcine circovirus type (PCV2) distribution and replication in tissues and immune cells in early infected pigs. Vet. Immunol. Immunopathol. 115, 261–272. Yang, Y., Li, C.C., Weissman, A.M., 2004. Regulating the p53 system through ubiquitination. Oncogene 23, 2097–2106. Yao, K., Goodwin, M.A., Vakharia, V.N., 1998. Generation of a mutant infectious bursal disease virus that does not cause bursal lesions. J. Virol. 72, 2647–2654. Zheng, G., Ning, J., Yang, Y.C., 2007. PLAGL2 controls the stability of Pirh2, an E3 ubiquitin ligase for p53. Biochem. Biophys. Res. Commun. 364, 344–350. 160 Zhu, Y., Lau, A., Lau, J., Jia, Q., Karuppannan, A.K., Kwang, J., 2007. Enhanced replication of porcine circovirus type (PCV2) in a homogeneous subpopulation of PK15 cell line. Virology 369, 423–430. Zuckermann, F.A., Husmann, R.J., 1996. Functional and phenotypic analysis of porcine peripheral blood CD4/CD8 double-positive T cells. Virology 87, 500– 512. 161 [...]... status of herds, this study also reveals that certain aminoacids variations in ORF3 are also associated with PMWS in the herds Especially the positions 14, 29 , 41 and 1 02 of the ORF3 varied between the PCV2a and PCV2b groups This study lends clue to the role of the ORF3 protein in the pathogenesis of PCV2 Yet another interesting association with regard to the polymorphisms in ORF2 is the presence of the. .. proteins involved in the replication of the virus (Replicase proteins; Rep), of which the Rep and Rep’ are essential for the replication of the virus (Fig.5 & 6) (Steinfeldt et al., 20 01) The ratio of the Rep and the Rep’ transcripts and proteins are found to vary during the course of infection of the PCV1 and PCV2 virus, with a transient increase of the Rep’ compared to the Rep (Mankertz et al., 20 04... on the growth kinetics of PCV2 108 Figure 26 Role of ORF3 in the release of PCV2 from infected cells……… …111 Figure 27 Role of ORF3 induced apoptosis on the In Vivo spread of PCV… 113 Figure 28 Role of Macrophages in the In Vivo spread of PCV2… ………… 116 Figure 29 Induction of TNFα expression in Macrophages by ORF3 …… 117 xi List of Symbols and Abbreviations CD – Cluster of Differentiation CMV- Cytomegalovirus... for the Rep proteins The numbering of the nucleotide begins in the site of the “nicking” by Rep proteins and is followed as a convention in the field 9 1.4 Transcriptome and proteome of PCV The transcriptome of PCV1 and PCV2 have been thoroughly analysed in a series of studies using 5’ and 3’ RACE (Rapid amplification of cDNA ends) cloning (Cheung A K., 20 03 a, b) The transcription profiling of the. .. genome, and the ORF2 in the antisense strand, which codes for the capsid protein A third ORF, the ORF3, is ensconced in the antisense strand of the ORF1 The replication of the genome takes place in the nucleus The genome has a stem-loop forming sequence at the origin of replication, in between the two genes, ORF1 and ORF2, and a short intergenic region is present between the end of the ORFs (Fig 4) The stemloop... infection. ……….……………………… 52 Figure 15 InVitro Competetive binding assay: ORF3, p53, Pirh2………… … 73 Figure 16 InVivo Competetive binding: ORF3, p53, Pirh2……………… ……74 Figure 17 Effect of ORF3 protein on the subcellular-localization of pPirh2 ….77 Figure 18 Effect of ORF3 protein on the turnover of Pirh2…………… …… 78 Figure 19 Apoptosis induction by truncated and deletion mutants of ORF3 ….81 Figure 20 In Vitro and In Vivo... Particles xiii List of publications 1 Karuppannan AK, Kwang J ORF3 of porcine circovirus 2 enhances the in vitro and in vivo spread of the virus Virology 20 11 Feb 5;410(1) :24 8-56 2 Meng T, Jia Q, Liu S, Karuppannan AK, Chang CC, Kwang J Characterization and epitope mapping of monoclonal antibodies recognizing Nterminus of Rep of porcine circovirus type 2 J Virol Methods 20 10 May;165 (2) :22 2-9 3 Karuppannan... avian species, e.g Porcine circovirus, Bovine circovirus, Beak and feather disease virus, Pigeon circovirus, Goose circovirus, Canary circovirus, Starling circovirus, Finch circovirus, e.t.c (Firth et al., 20 09) To date there are two reported circoviruses that can infect porcine species, namely, the Porcine circovirus 1 (PCV1) and Porcine circovirus 2 (PCV2) The non-pathogenic PCV1 was initially identified... other hand, the PCV2 has been classified into many phylogroups based on the polymorphisms in the ORF2 gene coding for the capsid proteins (Cheung et al., 20 07, Firth et al., 20 10, Hughes et al., 20 08) Of these, the two major phylogroups are the PCV2a and PCV2b Cheung et al., (20 07) has defined short amino acid motifs which could be used to distinguish the PCV2a and PCV2b The motifs located from amino... of ORF3 deletion mutants……… ……83 Figure 21 Induction of cellular p53 by ORF3 mutants…………… ………… 87 Figure 22 Effect of ORF3 on the In Vitro ubiquitination of p53 by pPirh2 … 89 Figure 23 Accumulation kinetics of cell free and cell associated PCV2 virus 104 x Figure 24 Effect of caspase inhibitor zVAD on apoptosis induced by PCV2 107 Figure 25 Effect of caspase inhibitor zVAD on the growth kinetics of . This thesis describes the characterisation of the role of ORF3 in the molecular and the systemic pathogenesis during the PCV2 infection in cell culture, mice model and in natural infection in. PCV2 107 Figure 25 . Effect of caspase inhibitor zVAD on the growth kinetics of PCV2 108 Figure 26 . Role of ORF3 in the release of PCV2 from infected cells……… …111 Figure 27 . Role of ORF3 induced. causing the apoptosis of the immune cells, characteristic of the PCV2 infection associated disease conditions, the ORF3 also plays a role in the systemic dissemination of the PCV2 infection. The

Ngày đăng: 10/09/2015, 08:38

Từ khóa liên quan

Tài liệu cùng người dùng

Tài liệu liên quan