臺灣博碩士論文加值系統

豬隻離乳後多系統消耗性症候群 (PMWS)是一種新興的豬隻疾病，並證實PCV2為其主要的病原。此病毒廣泛的分佈於世界各地的養豬場，然而在流行病學上卻呈現高感染率但低發病率的情形。在一些研究中發現，當豬隻受到免疫活化亦或有其他病原共同感染時，如細菌、病毒，則PMWS的發病率會大為增加。截至目前為止，PCV2的標的細胞大多被認為主要是以單核球與吞噬球系列的細胞為主，另有少數人提出淋巴球亦具有感受性。本實驗室之前的研究指出，豬隻肺泡巨噬細胞經革蘭氏陰性菌脂多醣 (LPS) 刺激之後，PCV2會進入到核內複製，此外周邊血液淋巴球經致裂原concanavalin A (Con A)刺激後，其PCV2的抗原含有率亦呈顯著的增加。為探討免疫活化在PCV2的致病機制上所扮演的角色，因此擬檢測單核球與淋巴球分別及同時受到活化時，PCV2的抗原及核酸含有率是否會有更顯著的差異，以期模擬豬隻同時受到免疫活化以及細菌共同感染時，PCV2病毒含量的變化。
本實驗分別使用革蘭氏陰性菌脂多醣 (LPS) 和致裂原 (Con A) 刺激周邊血液單核細胞 (PBMCs) (其中含有淋巴球以及單核球)，以間接免疫螢光染色的方式偵測抗原含有率，並利用細胞表面抗原的染色來分辨PCV2陽性的主要細胞群；另外，亦使用及時定量聚合酶鏈鎖反應和原位雜合聚合酶鏈鎖反應，來觀察病毒核酸含有率的變化，並以豬腎-15細胞株測定病毒力價。結果顯示，以革蘭氏陰性菌脂多醣 (LPS) 及/或致裂原 (Con A) 同時刺激豬之PBMCs，不論是在抗原及核酸含有率均較不處理組或LPS處理組來的高，且隨著培養時間增加而有上升的趨勢，在病毒力價方面LPS與Con A共同處理組於第三天時亦較其他處理組高，此外有三分之二的陽性細胞為SWC3－的細胞。根據目前結果，可以推測單核球與淋巴球之間有正向的交互作用存在，以促使PCV2抗原及核酸的增加，這些證據顯示當豬隻受到免疫活化時，或許因PCV2病毒量的增加，導致受感染豬隻發病率的增加。

Porcine circovirus type Ⅱ (PCV2) infection has been demonstrated to be an essential factor in the induction of the newly emerged disease, postweaning multisystemic wasting syndrome (PMWS), in pigs. PCV2 antibodies have been found in pigs worldwidely, usually with high seroprevalence. Although monocyte/macrophage lineage cells are considered as the major target cells, the role of lymphocytes on the disease development is still uncertain. Immune activation and co-factors such as bacteria or viruses have been suggested to be important factors in the induction of PMWS. Our previous studies have demonstrated that the PCV2 nucleic acid and antigens could be detected intranuclearly in bacterial lipopolysaccharide (LPS)-treated PCV2-inoculated swine alveolar macrophages (AMs) and in concanavalin A (Con A)-stimulated swine peripheral blood lymphocytes (PBLs). The objective of the present study was to further evaluate whether there is an enhancement effect on the PCV2-positive rate in either monocytes or lymphocytes of peripheral blood following monophasic or biphasic stimulation with LPS and/or Con A in healthy PCV2-carrier pigs. After stimulation with LPS and/or Con A, both PCV2 antigen- and nucleic acid-containing rates of the peripheral blood mononuclear cells (PBMCs) of healthy PCV2-carrier pigs measured by immunofluorescent assay (IFA), surface marker IFA, in situ hybridization-polymerase chain reaction (ISH-PCR), and real time PCR increased with time. The levels of the PCV2 antigen-containing rate in Con A-treated group and the group treated simultaneously with LPS and Con A ( (LPS + Con A)-treated groups) were significantly greater than those of the NT and LPS-treated groups. Significant difference was also seen among the LPS-, Con A, and (LPS + Con A)-treated groups. The viral titer of the (LPS + Con A)-treated group increased at 3 days post-incubation (DPI) as did antigen- and nucleic acid-containing rates. Two third of the PCV2-positive cells belonged to SWC3－ population; this implies that lymphocytes may also play an important role on PCV2 replication. The results indicate that interaction between PBMs and PBLs may exist and simultaneous activation of PBMs and PBLs may result in increased PCV2 load in both cells. The results further support that immune activation may increase the morbidity of PMWS in PCV2-infected pigs via the increase in viral load.

Table of Contents
口試委員審定書
致謝
中文摘要 ...……………………………………………………….................. Ⅰ
Abstract ………………………………………………………………............ Ⅲ
Table of Contents ……………………………………………………............... Ⅴ
Figures ………………………………………………………………........... Ⅷ
1 Introduction 1
2 Review of Literature 3
2.1 Porcine Circovirus 3
2.1.1 Taxonomy 3
2.1.2 Structure 3
2.1.3 Replication and Infection 5
2.2 Postweaning Multisystemic Wasting Syndrome (PMWS) 6
2.2.1 Introduction 6
2.2.2 Clinical Signs and Pathology 7
2.2.3 Target Cells and Tissue Distribution 8
2.2.4 Pathogenesis of PMWS 10
2.2.4.1 Mechanism of PCV2 Entering Host Cells 10
2.2.4.2 Roles of Immunoactivation and Co-infection on PMWS Development 11
2.2.4.3 Alterations in Immune System of PMWS-Affected Pigs 12
2.3 Introduction to Lipopolysaccharide (LPS) 14
2.4 Introduction to Mitogens 16
3 Materials and Methods 19
3.1 Experimental Design 19
3.1.1 Experimental Flow Chart 20
3.2 Experimental Animals 21
3.3 Materials 21
3.3.1 Preparation of Peripheral Blood Mononuclear Cells (PBMCs) 21
3.3.1.1 Method of Isolation 21
3.3.1.2 Culture Medium 22
3.3.1.3 Concentration of Lipopolysaccharide (LPS) 24
3.3.1.4 Concentration of Concanavalin A (Con A) 25
3.3.1.5 Antibodies 25
3.4 Methods 25
3.4.1 Cell Viability Detection 25
3.4.1.1 Trypan Blue Dye Exclusion Assay 25
3.4.1.2 Annexin V/ Propidium Iodide (AV/PI) 26
3.4.2 Indirect Immunofluorescent Assay (IFA) 27
3.4.2.1 Fluorescent Microscopy (Fixed Cells and IFA) 27
3.4.2.2 Flow Cytometry (Suspended Cell Fixation and IFA) 28
3.4.2.3 Cell Surface Marker and PCV2 Antigen (Suspended Cell Fixation and IFA) 29

3.4.3 Quantitative Real-Time Polymerase Chain Reaction (Real-time PCR) 29
3.4.4 In Situ Hybridization Polymerase Chain Reaction (ISH-PCR) 31
3.4.5 Viral Titration 32
3.5 Statistical Analysis 33
4 Results 34
4.1 Cell Viability 34
4.1.1 Trypan Blue Dye Exclusion Test 34
4.1.2 Annexin V(AV)/Propidium Iodide (PI) 34
4.2 PCV2 Antigen-Containing Rate (ACR) 35
4.2.1 Fluorescent Microscopy 35
4.2.1.1 Polyclonal Antibody 35
4.2.1.2 Monoclonal Antibody (mAb) 36
4.2.2 Flow Cytometry 37
4.3 PCV2 and Surface Marker 37
4.4 PCV2 Nucleic Acid-Containing Rate 38
4.4.1 Quantitative Real-Time Polymerase Chain Reaction 38
4.4.2 In Situ Hybridization Polymerase Chain Reaction 39
4.5 Viral Titration 39
5 Discussion 54
6 References 66

Alexander, C. and Rietschel, E.T., 2001. Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res 7, 167-202.
Allan, G.M. and Ellis, J.A., 2000. Porcine circoviruses: a review. J Vet Diagn Invest 12, 3-14.
Allan, G.M., Kennedy, S., McNeilly, F., Foster, J.C., Ellis, J.A., Krakowka, S.J., Meehan, B.M. and Adair, B.M., 1999. Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. J Comp Pathol 121, 1-11.
Allan, G.M., McNeilly, F., Ellis, J., Krakowka, S., Botner, A., McCullough, K., Nauwynck, H., Kennedy, S., Meehan, B. and Charreyre, C., 2004. PMWS: experimental model and co-infections. Vet Microbiol 98, 165-8.
Allan, G.M., McNeilly, F., Kennedy, S., Meehan, B., Ellis, J. and Krakowka, S., 2000a. Immunostimulation, PCV-2 and PMWS. Vet Rec 147, 170-1.
Allan, G.M., McNeilly, F., Meehan, B.M., Ellis, J.A., Connor, T.J., McNair, I., Krakowka, S. and Kennedy, S., 2000b. A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation. J Vet Med B Infect Dis Vet Public Health 47, 81-94.
Bratanich, A.C. and Blanchetot, A., 2002. PCV2 replicase transcripts in infected porcine kidney (PK15) cells. Virus Genes 25, 323-8.
Brunborg, I.M., Moldal, T. and Jonassen, C.M., 2004. Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR. J Virol Methods 122, 171-8.
Chae, C., 2004. Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology. Vet J 168, 41-9.
Chae, C., 2005. A review of porcine circovirus 2-associated syndromes and diseases. Vet J 169, 326-36.
Chang, H.W., Jeng, C.R., Liu, J.J., Lin, T.L., Chang, C.C., Chia, M.Y., Tsai, Y.C. and Pang, V.F., 2005. Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha. Vet Microbiol 108, 167-77.
Chang, H.W., Pang, V.F., Chen, L.J., Chia, M.Y., Tsai, Y.C. and Jeng, C.R., 2006. Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages. Vet Microbiol 115, 311-9.
Cheung, A.K., 2003. Transcriptional analysis of porcine circovirus type 2. Virology 305, 168-80.
Chianini, F., Majo, N., Segales, J., Dominguez, J. and Domingo, M., 2003. Immunohistochemical characterisation of PCV2 associate lesions in lymphoid and non-lymphoid tissues of pigs with natural postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol 94, 63-75.
Darwich, L., Segales, J., Domingo, M. and Mateu, E., 2002. Changes in CD4(+), CD8(+), CD4(+) CD8(+), and immunoglobulin M-positive peripheral blood mononuclear cells of postweaning multisystemic wasting syndrome-affected pigs and age-matched uninfected wasted and healthy pigs correlate with lesions and porcine circovirus type 2 load in lymphoid tissues. Clin Diagn Lab Immunol 9, 236-42.
Darwich, L., Segales, J. and Mateu, E., 2004. Pathogenesis of postweaning multisystemic wasting syndrome caused by Porcine circovirus 2: An immune riddle. Arch Virol 149, 857-74.
Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M.A., Lassota, P. and Traganos, F., 1992. Features of apoptotic cells measured by flow cytometry. Cytometry 13, 795-808.
Dobrovolskaia, M.A., Vogel, S.N., 2002. Toll receptors, CD14, and macrophages activation and deactivation by LPS. Microbes and Infection 4, 903-914.
Ellis, J.A., Bratanich, A., Clark, E.G., Allan, G., Meehan, B., Haines, D.M., Harding, J., West, K.H., Krakowka, S., Konoby, C., Hassard, L., Martin, K. and McNeilly, F., 2000. Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome. J Vet Diagn Invest 12, 21-7.
Equils, O., Faure, E., Thomas, L., Bulut, Y., Trushin, S. and Arditi, M., 2001. Bacterial lipopolysaccharide activates HIV long terminal repeat through Toll-like receptor 4. J Immunol 166, 2342-7.
Fenaux, M., Halbur, P.G., Haqshenas, G., Royer, R., Thomas, P., Nawagitgul, P., Gill, M., Toth, T.E. and Meng, X.J., 2002. Cloned genomic DNA of type 2 porcine circovirus is infectious when injected directly into the liver and lymph nodes of pigs: characterization of clinical disease, virus distribution, and pathologic lesions. J Virol 76, 541-51.
Gilpin, D.F., McCullough, K., Meehan, B.M., McNeilly, F., McNair, I., Stevenson, L.S., Foster, J.C., Ellis, J.A., Krakowka, S., Adair, B.M. and Allan, G.M., 2003. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system. Vet Immunol Immunopathol 94, 149-61.
Grasland, B., Loizel, C., Blanchard, P., Oger, A., Nignol, A.C., Bigarre, L., Morvan, H., Cariolet, R. and Jestin, A., 2005. Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus. Vet Res 36, 685-97.
Haruna, J., Hanna, P., Hurnik, D., Ikede, B., Miller, L. and Yason, C., 2006. The role of immunostimulation in the development of postweaning multisystemic wasting syndrome in pigs under field conditions. Can J Vet Res 70, 269-76.
Hino, S., 2002. TTV, a new human virus with single stranded circular DNA genome. Rev Med Virol 12, 151-8.
Hodge, G., Hodge, S. and Han, P., 2000. Increased levels of apoptosis of leukocyte subsets in cultured PBMCs compared to whole blood as shown by Annexin V binding: relevance to cytokine production. Cytokine 12, 1763-8.
Ilyina, T.V. and Koonin, E.V., 1992. Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res 20, 3279-85.
Karahashi, H. and Amano, F., 2000. Lipopolysaccharide (LPS)-induced cell death of C3H mouse peritoneal macrophages in the presence of cycloheximide: different susceptibilities of C3H/HeN and C3H/HeJ mice macrophages. J Endotoxin Res 6, 33-9.
Kim, J. and Chae, C., 2002. Double in situ hybridization for simultaneous detection and differentiation of porcine circovirus 1 and 2 in pigs with postweaning multisystemic wasting syndrome. Vet J 164, 247-53.
Kim, J., Choi, C., Han, D.U. and Chae, C., 2001. Simultaneous detection of porcine circovirus type 2 and porcine parvovirus in pigs with PMWS by multiplex PCR. Vet Rec 149, 304-5.
Kim, J., Ha, Y., Jung, K., Choi, C. and Chae, C., 2004. Enteritis associated with porcine circovirus 2 in pigs. Can J Vet Res 68, 218-21.
Kiupel, M., Stevenson, G.W., Choi, J., Latimer, K.S., Kanitz, C.L. and Mittal, S.K., 2001. Viral replication and lesions in BALB/c mice experimentally inoculated with porcine circovirus isolated from a pig with postweaning multisystemic wasting disease. Vet Pathol 38, 74-82.
Krakowka, S., Ellis, J.A., McNeilly, F., Gilpin, D., Meehan, B., McCullough, K. and Allan, G., 2002. Immunologic features of porcine circovirus type 2 infection. Viral Immunol 15, 567-82.
Krakowka, S., Ellis, J.A., McNeilly, F., Ringler, S., Rings, D.M. and 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., Meehan, B., Kennedy, S., McNeilly, F. and Allan, G., 2000. Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Vet Pathol 37, 254-63.
Kurita-Ochiai, T., Fukushima, K. and Ochiai, K., 1999. Lipopolysaccharide stimulates butyric acid-induced apoptosis in human peripheral blood mononuclear cells. Infect Immun 67, 22-9.
Kyriakis, S.C., Saoulidis, K., Lekkas, S., Miliotis Ch, C., Papoutsis, P.A. and Kennedy, S., 2002. The effects of immuno-modulation on the clinical and pathological expression of postweaning multisystemic wasting syndrome. J Comp Pathol 126, 38-46.
Ladekjaer-Mikkelsen, A.S., Nielsen, J., Stadejek, T., Storgaard, T., Krakowka, S., Ellis, J., McNeilly, F., Allan, G. and Botner, A., 2002. Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2). Vet Microbiol 89, 97-114.
Lin, C.M., 2006. The susceptibility of swine lymphocytes to porcine circovirus type Ⅱ., 58-71.
Liu, J., Chen, I., Du, Q., Chua, H. and Kwang, J., 2006. The ORF3 protein of porcine circovirus type 2 is involved in viral pathogenesis in vivo. J Virol 80, 5065-73.
Magar, R., Larochelle, R., Thibault, S. and Lamontagne, L., 2000. Experimental transmission of porcine circovirus type 2 (PCV2) in weaned pigs: a sequential study. J Comp Pathol 123, 258-69.
Mancek, M., Pristovsek, P. and Jerala, R., 2002. Identification of LPS-binding peptide fragment of MD-2, a toll-receptor accessory protein. Biochem Biophys Res Commun 292, 880-5.
Mandal, P., Novotny, M. and Hamilton, T.A., 2005. Lipopolysaccharide induces formyl peptide receptor 1 gene expression in macrophages and neutrophils via transcriptional and posttranscriptional mechanisms. J Immunol 175, 6085-91.
Mankertz, A., Caliskan, R., Hattermann, K., Hillenbrand, B., Kurzendoerfer, P., Mueller, B., Schmitt, C., Steinfeldt, T. and Finsterbusch, T., 2004. Molecular biology of Porcine circovirus: analyses of gene expression and viral replication. Vet Microbiol 98, 81-8.
Mankertz, A., Domingo, M., Folch, J.M., LeCann, P., Jestin, A., Segales, J., Chmielewicz, B., Plana-Duran, J. and Soike, D., 2000. Characterisation of PCV-2 isolates from Spain, Germany and France. Virus Res 66, 65-77.
Mankertz, A. and Hillenbrand, B., 2001. Replication of porcine circovirus type 1 requires two proteins encoded by the viral rep gene. Virology 279, 429-38.
Mankertz, A., Persson, F., Mankertz, J., Blaess, G. and Buhk, H.J., 1997. Mapping and characterization of the origin of DNA replication of porcine circovirus. J Virol 71, 2562-6.
Maratheftis, C.I., Andreakos, E., Moutsopoulos, H.M. and Voulgarelis, M., 2007. Toll-like receptor-4 is up-regulated in hematopoietic progenitor cells and contributes to increased apoptosis in myelodysplastic syndromes. Clin Cancer Res 13, 1154-60.
Maris, N.A., Dessing, M.C., de Vos, A.F., Bresser, P., van der Zee, J.S., Jansen, H.M., Spek, C.A. and van der Poll, T., 2006. Toll-like receptor mRNA levels in alveolar macrophages after inhalation of endotoxin. Eur Respir J 28, 622-6.
Mattern, T., Flad, H.D., Brade, L., Rietschel, E.T. and Ulmer, A.J., 1998. Stimulation of human T lymphocytes by LPS is MHC unrestricted, but strongly dependent on B7 interactions. J Immunol 160, 3412-8.
McNeilly, F., Kennedy, S., Moffett, D., Meehan, B.M., Foster, J.C., Clarke, E.G., Ellis, J.A., Haines, D.M., Adair, B.M. and Allan, G.M., 1999. A comparison of in situ hybridization and immunohistochemistry for the detection of a new porcine circovirus in formalin-fixed tissues from pigs with post-weaning multisystemic wasting syndrome (PMWS). J Virol Methods 80, 123-8.
Meehan, B.M., McNeilly, F., Todd, D., Kennedy, S., Jewhurst, V.A., Ellis, J.A., Hassard, L.E., Clark, E.G., Haines, D.M. and Allan, G.M., 1998. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 79 ( Pt 9), 2171-9.
Meerts, P., Misinzo, G., McNeilly, F. and Nauwynck, H.J., 2005a. Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages. Arch Virol 150, 427-41.
Meerts, P., Van Gucht, S., Cox, E., Vandebosch, A. and Nauwynck, H.J., 2005b. Correlation between type of adaptive immune response against porcine circovirus type 2 and level of virus replication. Viral Immunol 18, 333-41.
Nawagitgul, P., Morozov, I., Bolin, S.R., Harms, P.A., Sorden, S.D. and Paul, P.S., 2000. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J Gen Virol 81, 2281-7.
Nielsen, H.B. and Pedersen, B.K., 1997. Lymphocyte proliferation in response to exercise. Eur J Appl Physiol Occup Physiol 75, 375-9.
Nielsen, J., Vincent, I.E., Botner, A., Ladekaer-Mikkelsen, A.S., Allan, G., Summerfield, A. and McCullough, K.C., 2003. Association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol 92, 97-111.
Oh, J., Chang, K.W. and Hughes, S.H., 2006. Mutations in the U5 sequences adjacent to the primer binding site do not affect tRNA cleavage by rous sarcoma virus RNase H but do cause aberrant integrations in vivo. J Virol 80, 451-9.
Onuki, A., Abe, K., Togashi, K., Kawashima, K., Taneichi, A. and Tsunemitsu, H., 1999. Detection of porcine circovirus from lesions of a pig with wasting disease in Japan. J Vet Med Sci 61, 1119-23.
Pushkarsky, T., Dubrovsky, L. and Bukrinsky, M., 2001. Lipopolysaccharide stimulates HIV-1 entry and degradation in human macrophages. J Endotoxin Res 7, 271-6.
Quintana, J., Segales, J., Rosell, C., Calsamiglia, M., Rodriguez-Arrioja, G.M., Chianini, F., Folch, J.M., Maldonado, J., Canal, M., Plana-Duran, J. and Domingo, M., 2001. Clinical and pathological observations on pigs with postweaning multisystemic wasting syndrome. Vet Rec 149, 357-61.
Ramirez-Mendoza, H., Martinez, C., Mercado, C., Castillo-Juarez, H., Hernandez, J. and Segales, J., 2006. Porcine circovirus type 2 antibody detection in backyard pigs from Mexico City. Res Vet Sci.
Rodriguez-Arrioja, G.M., Segales, J., Calsamiglia, M., Resendes, A.R., Balasch, M., Plana-Duran, J., Casal, J. and Domingo, M., 2002. Dynamics of porcine circovirus type 2 infection in a herd of pigs with postweaning multisystemic wasting syndrome. Am J Vet Res 63, 354-7.
Rosell, C., Segales, J., Plana-Duran, J., Balasch, M., Rodriguez-Arrioja, G.M., Kennedy, S., Allan, G.M., McNeilly, F., Latimer, K.S. and Domingo, M., 1999. Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs. J Comp Pathol 120, 59-78.
Sanchez, R.E., Jr., Meerts, P., Nauwynck, H.J., Ellis, J.A. and 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, 175-85.
Sanchez, R.E., Jr., Meerts, P., Nauwynck, H.J. and Pensaert, M.B., 2003. Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life. Vet Microbiol 95, 15-25.
Segales, J., Allan, G.M. and Domingo, M., 2005. Porcine circovirus diseases. Anim Health Res Rev 6, 119-42.
Segales, J., Alonso, F., Rosell, C., Pastor, J., Chianini, F., Campos, E., Lopez-Fuertes, L., Quintana, J., Rodriguez-Arrioja, G., Calsamiglia, M., Pujols, J., Dominguez, J. and Domingo, M., 2001. Changes in peripheral blood leukocyte populations in pigs with natural postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol 81, 37-44.
Segales, J. and Domingo, M., 2002. Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review. Vet Q 24, 109-24.
Segales, J., Rosell, C. and Domingo, M., 2004. Pathological findings associated with naturally acquired porcine circovirus type 2 associated disease. Vet Microbiol 98, 137-49.
Shibahara, T., Sato, K., Ishikawa, Y. and Kadota, K., 2000. Porcine circovirus induces B lymphocyte depletion in pigs with wasting disease syndrome. J Vet Med Sci 62, 1125-31.
Summerfield, A., Knotig, S.M. and McCullough, K.C., 1998. Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death. J Virol 72, 1853-61.
Timmusk, S., Fossum, C. and Berg, M., 2006. Porcine circovirus type 2 replicase binds the capsid protein and an intermediate filament-like protein. J Gen Virol 87, 3215-23.
Tischer, I., Peter, D., Rasch, R., Pociuli, S., , 1987. Replication of porcine circovirus: induction by glucosamine and cell cycle dependence. Arch Virol 96, 39-57.
Tischer, I., Peters, D., Rasch, R. and Pociuli, S., 1987. Replication of porcine circovirus: induction by glucosamine and cell cycle dependence. Arch Virol 96, 39-57.
Tischer I., P.D., Rasch R., Pociuli S., , 1987. Replication of porcine circovirus: induction by glucosamine and cell cycle dependence. Arch Virol 96, 39-57.
Tizard, I.R., 2000. Cytokines and the Immune system. Saunders, USA.
Vincent, I.E., Carrasco, C.P., Herrmann, B., Meehan, B.M., Allan, G.M., Summerfield, A. and McCullough, K.C., 2003. Dendritic cells harbor infectious porcine circovirus type 2 in the absence of apparent cell modulation or replication of the virus. J Virol 77, 13288-300.
Wang, C., Huang, T.S., Huang, C.C., Tu, C., Jong, M.H., Lin, S.Y. and Lai, S.S., 2004. Characterization of porcine circovirus type 2 in Taiwan. J Vet Med Sci 66, 469-75.
Yu, S., Opriessnig, T., Kitikoon, P., Nilubol, D., Halbur, P.G. and Thacker, E., 2007a. Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs. Vet Immunol Immunopathol 115, 261-72.
Yu, S., Vincent, A., Opriessnig, T., Carpenter, S., Kitikoon, P., Halbur, P.G. and Thacker, E., 2007b. Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro. Vet Microbiol.