Characterization of porcine circovirus type 2 infection in immune cells and lymphoid organs by using in vitro and in vivo models
Date Issued
2012
Date
2012
Author(s)
Lin, Chun-Ming
Abstract
Porcine circovirus type 2 (PCV2) is a small, non-enveloped, single-stranded, circular DNA virus, and the infection of PCV2 is distributed in swine population worldwide. Experimental and field studies indicate that PCV2 is a required but insufficient causative agent of postweaning multisystemic wasting syndrome (PMWS), a disease that has a significant impact on swine production. The characteristic feature of PMWS is the presence of high levels of PCV2 antigens and nucleic acid in the lymphoid system along with variable lymphoid depletion and granulomatous inflammation. Until now, the precise mechanism regarding the replication of PCV2 in immune cells and the pathogenesis of PMWS are remained to be elucidated. Therefore, serial in vitro (Chapters II and III) and in vivo (Chapters IV, V and VI) models were performed in the present study to clarify above issues.
In the first portion of the present study, it is hypothesized that immune activation induces PCV2 replication in activated lymphocytes. Therefore, concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) was used as an in vitro model to clarify the susceptibility of swine lymphocytes to PCV2 (Chapter II). Subsequently, this model was further expanded by the addition of monocyte-derived dendritic cells (MoDCs) and recombinant cytokines, including interleukin 2 (IL-2), IL-4, and interferon γ, to determine the correlations between lymphocyte activation and PCV2 replication (Chapter III). The evidences of increase in cellular PCV2 antigen- and/or nucleic acid-containing rates, PCV2 genome copy number, and titer of infectious PCV2 in mitogen-stimulated, PCV2-infected PBLs support that PCV2 infects and replicates in activated swine lymphocytes. By phenotyping the PCV2 antigen-positive cells, it was revealed that T and B lymphocytes as well as monocytes were susceptible to PCV2 infection; however, IgM-positive B lymphocytes appeared to have a relatively higher PCV2-positive rate (Chapter II). While lymphocytic cells served as a major site of PCV2 replication, MoDCs harbored a significant amount of PCV2 antigens but did not actively sustain the replication of PCV2. Further study has demonstrated that IL-2 and the accessory cell function of DCs were key factors required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes (Chapter III).
In the second portion of the present study, serials in vivo studies were conducted to characterize the changes in inguinal LNs in the naturally PCV2-infected pigs (Chapters IV and V) and to infer the possible mechanism of PCV2-associated lymphoid lesion developed (Chapter V and VI). It is speculated that limited by their sensitivity, conventional in situ hybridization (ISH) and/or immunohistochemical (IHC) staining may not be sufficient to detect few copies of PCV2 contained in a portion of immune cells. Therefore, a more sensitive method, indirect in situ polymerase chain reaction PCR (ISPCR), was developed through the combination of PCR and ISH procedure (Chapter IV). Using the indirect ISPCR, signals of PCV2 nucleic acid, especially those in germinal centers, could be more effectively detected. In addition, a detailed grading system was proposed to categorize the pattern of natural PCV2 infection in lymphoid follicles (Chapter IV) and used as criteria for sample selection in the following studies (Chapters V and IV).
To obtain tissue-based information effectively, a new model of high-throughput tissue microarray (TMA) in conjunction with semi-quantified ISH/IHC staining and statistic analysis was established (Chapter V). The results of multiple regression analysis showed that each of PCV2, porcine respiratory and reproductive syndrome virus (PRRSV), and porcine parvovirus (PPV) had its own contributions on the development of lymphoid lesions in PMWS with PCV2 as the major causative agent. B lymphocyte depletion and macrophage proliferation and infiltration are the two hallmarks of PCV2-associated lymphoid lesions. Apart from blood recruitment, local T cell and macrophage proliferation may play a crucial role on the development of granulomatous inflammation (Chapter V). Furthermore, the expressions of 92 selected immune genes in 7 and 35 inguinal LNs obtained from healthy subclinically PCV2-infected and PMWS-affected pigs were assessed by the integration of several quantitative reverse transcription polymerase chain reaction experiments (Chapter IV). Using hierarchical cluster analysis, the gene expression profiles in these PCV2-infected LNs were generally compatible with the divergent functions of different immune cell populations. Aberrant immune activation and imbalanced Th1/Th2 orientation are considered to contribute to the development of PCV2 infection-associated lymphoid lesions. Principle component analysis of the expression profile of the 92 selected immune genes in the 42 above mentioned LNs revealed that 52.23% of the total data variants could be explained by the top-3 principle components, suggesting that the disease development of PCV2 infection may be associated with a few major and some minor factors (Chapter VI).
In conclusion, evidences from the present study suggest that lymphocytes are indeed susceptible to PCV2 (Chapters II, III and IV). Immune activation modulated by DCs and cytokines triggers the replication of PCV2 in lymphocytes (Chapter III). Subsequently, the perturbation of immune regulation by PCV2 facilitates the co-infection of other pathogens. Due to the persistent or repeated episodes of antigenic stimulation, the balance between immune suppression and activation is further perturbed (Chapter VI). As the progression of PMWS development, lymphoid depletion and granulomatous inflammation occur in the LNs bearing heavy PCV2 load (Chapter V).
In the first portion of the present study, it is hypothesized that immune activation induces PCV2 replication in activated lymphocytes. Therefore, concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) was used as an in vitro model to clarify the susceptibility of swine lymphocytes to PCV2 (Chapter II). Subsequently, this model was further expanded by the addition of monocyte-derived dendritic cells (MoDCs) and recombinant cytokines, including interleukin 2 (IL-2), IL-4, and interferon γ, to determine the correlations between lymphocyte activation and PCV2 replication (Chapter III). The evidences of increase in cellular PCV2 antigen- and/or nucleic acid-containing rates, PCV2 genome copy number, and titer of infectious PCV2 in mitogen-stimulated, PCV2-infected PBLs support that PCV2 infects and replicates in activated swine lymphocytes. By phenotyping the PCV2 antigen-positive cells, it was revealed that T and B lymphocytes as well as monocytes were susceptible to PCV2 infection; however, IgM-positive B lymphocytes appeared to have a relatively higher PCV2-positive rate (Chapter II). While lymphocytic cells served as a major site of PCV2 replication, MoDCs harbored a significant amount of PCV2 antigens but did not actively sustain the replication of PCV2. Further study has demonstrated that IL-2 and the accessory cell function of DCs were key factors required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes (Chapter III).
In the second portion of the present study, serials in vivo studies were conducted to characterize the changes in inguinal LNs in the naturally PCV2-infected pigs (Chapters IV and V) and to infer the possible mechanism of PCV2-associated lymphoid lesion developed (Chapter V and VI). It is speculated that limited by their sensitivity, conventional in situ hybridization (ISH) and/or immunohistochemical (IHC) staining may not be sufficient to detect few copies of PCV2 contained in a portion of immune cells. Therefore, a more sensitive method, indirect in situ polymerase chain reaction PCR (ISPCR), was developed through the combination of PCR and ISH procedure (Chapter IV). Using the indirect ISPCR, signals of PCV2 nucleic acid, especially those in germinal centers, could be more effectively detected. In addition, a detailed grading system was proposed to categorize the pattern of natural PCV2 infection in lymphoid follicles (Chapter IV) and used as criteria for sample selection in the following studies (Chapters V and IV).
To obtain tissue-based information effectively, a new model of high-throughput tissue microarray (TMA) in conjunction with semi-quantified ISH/IHC staining and statistic analysis was established (Chapter V). The results of multiple regression analysis showed that each of PCV2, porcine respiratory and reproductive syndrome virus (PRRSV), and porcine parvovirus (PPV) had its own contributions on the development of lymphoid lesions in PMWS with PCV2 as the major causative agent. B lymphocyte depletion and macrophage proliferation and infiltration are the two hallmarks of PCV2-associated lymphoid lesions. Apart from blood recruitment, local T cell and macrophage proliferation may play a crucial role on the development of granulomatous inflammation (Chapter V). Furthermore, the expressions of 92 selected immune genes in 7 and 35 inguinal LNs obtained from healthy subclinically PCV2-infected and PMWS-affected pigs were assessed by the integration of several quantitative reverse transcription polymerase chain reaction experiments (Chapter IV). Using hierarchical cluster analysis, the gene expression profiles in these PCV2-infected LNs were generally compatible with the divergent functions of different immune cell populations. Aberrant immune activation and imbalanced Th1/Th2 orientation are considered to contribute to the development of PCV2 infection-associated lymphoid lesions. Principle component analysis of the expression profile of the 92 selected immune genes in the 42 above mentioned LNs revealed that 52.23% of the total data variants could be explained by the top-3 principle components, suggesting that the disease development of PCV2 infection may be associated with a few major and some minor factors (Chapter VI).
In conclusion, evidences from the present study suggest that lymphocytes are indeed susceptible to PCV2 (Chapters II, III and IV). Immune activation modulated by DCs and cytokines triggers the replication of PCV2 in lymphocytes (Chapter III). Subsequently, the perturbation of immune regulation by PCV2 facilitates the co-infection of other pathogens. Due to the persistent or repeated episodes of antigenic stimulation, the balance between immune suppression and activation is further perturbed (Chapter VI). As the progression of PMWS development, lymphoid depletion and granulomatous inflammation occur in the LNs bearing heavy PCV2 load (Chapter V).
Subjects
porcine circovirus type 2
lymphocyte
immune
Type
thesis
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