以Bacterial lipopolysaccharide活化豬第二型環狀病毒複製之研究

Investigation of Porcine Circovirus Type 2 Replication by Bacterial Lipopolysaccharide Activation in vitro

陳亮君(Liang-Jiun Chen)

指導教授 ： 鄭謙仁

共同指導教授 ： 龐飛(Victor Fei Pang)

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00804

摘要

離乳後多系統消耗性症候群（Postweaning multisystemic wasting syndrome; PMWS）主要發生於5-12週齡的豬隻，其引起的病原為第二型豬環狀病毒（Porcine circovirus type 2；PCV2）；PCV2與其他細菌及病毒的混合感染或患畜經過免疫刺激後常會增加此疾病的嚴重程度。目前PCV2確切的致病機制及其感染和複製的標的細胞仍不清楚，雖然在活體內PCV2感染之標的細胞主要為單核/巨噬細胞及抗原呈現細胞，但在活體外的試驗卻無法證實PCV2會在此類細胞進行複製。本研究為瞭解PCV2複製之機制，以豬肺泡巨噬細胞（PAMs）及細菌的脂多醣（LPS）進行PCV2活體外複製之實驗；利用抗PCV2高力價豬血清及抗PCV2 ORF1抗體進行免疫螢光染色（IFA），結果於LPS刺激後之PCV2感染PAMs的細胞核內出現陽性訊號；使用反轉錄聚合酶鏈鎖反應亦可偵測出PCV2 spliced Cap mRNA，其細胞培養上清液也可見病毒力價的上升，但同樣情形並不見於未經LPS處理之PCV2感染之PAMs。本實驗結果顯示經由適當的LPS刺激後，PCV2可在感染PAMs細胞內進行複製。另外，於LPS處理之PCV2感染組的細胞培養上清液，有較多量的IL-1beta、IL-8、IL-10及TNF-alpha等蛋白質表現。雖然LPS如何啟動PCV2複製的機制仍不清楚，但報告指出脂多醣刺激人類免疫缺陷病毒（Human immunodeficiency virus；HIV）感染之單核巨噬細胞，是經由細胞內Nuclear factor-kappaB（NF-κB）的活化，引起不同的功能反應而增加HIV的複製。因此使用PK-15細胞作為一模式，瞭解NF-κB的活化與PCV2複製是否存著關連性。本實驗分別使用NF-κB activation inhibitor及Phorbol 12-myristate 13-acetate（PMA）促使NF-κB抑制及活化，經PMA及NF-κB activation inhibitor各別處理之PK-15細胞皆可輕微增加PCV2病毒力價，而當PMA及NF-κB activation inhibitor共同處理之PK-15細胞則可見明顯增加PCV2病毒力價。本實驗結果說明了革蘭氏陰性菌於PCV2複製中，可能扮演了一個很重要的角色，且亦可改變免疫反應前細胞激素相（proinflammatory cytokines profiles）。於PK-15細胞株細胞模式試驗中得知NF-κB的活化與PCV2複製並不具有直接關聯性，但NF-κB的活化或抑制似乎是會藉由間接作用影響PCV2複製，然其真正的活化機序則有待更進一步的實驗探討。

關鍵字

環狀病毒 ； 脂多醣

並列摘要

Porcine circovirus type 2（PCV2） is the necessary causative agent of postweaning multisystemic wasting syndrome（PMWS） in swine; however, a variety of co-factors including other infectious agents, are thought to be necessary for the full development of the disease. PCV2 nucleic acid and/or antigens are consistently observed in cells of monocyte/macrophage lineage and antigen-presenting cells in the lesions of PMWS-affected pigs; but no evidence of PCV2 replication was observed in these cells in vitro. To pursuit the question regarding the mechanism of PCV2 replication, porcine alveolar macrophages（PAMs）and lipopolysaccharide（LPS） were used in this study. The results revealed intranuclear signal of PCV2 and ORF1 protein were only detected in E. coli’s polysaccharide（LPS）-treated PCV2-inoculated PAMs. The PCV2 replication products corresponding to PCV2 spliced Cap mRNA and increased levels of infectious virus progeny were also successfully demonstrated in the LPS-treated PCV2-inoculated PAMs, but not in the mock LPS-treated PCV2-inoculated PAMs. The mechanism of LPS triggering PCV2 replication is not understood but may support the hypothesis that LPS is a stimulator of PCV2 replication which may contribute to the full development of PMWS in pigs during appropriate bacteria co-infection. In the HIV studies, LPS stimulation enhances the replication of HIV in monocytic-macrophage linage cells. In this study, IL-1beta、IL-8、IL-10 and TNF-alpha expressions were notably increased in the LPS-treated PCV2-inoculated PAMs when compared to the mock LPS-treated PCV2-inoculated PAMs. The stimulation of LPS is related to the activation of NF-κB which is a transcription factor with diverse functions. In the subsequent study, attempts to induce NF-κB activation and inhibition by PMA and NF-κB activation inhibitor, respectively, in PK-15 cells were proceed to investigate the possible involvement of NF-κB activation in PCV2 replication. The result showed that virus titer was mildly increased in the PMA-treated PCV2-infected PK-15 cells and NF-κB activation inhibitor-treated PCV2-infected PK-15 cells when compared to the mock-treated PCV2-infected PK-15 cells. The virus titer was notably increased in the PMA- and NF-κB activation inhibitor co-treated PCV2-infected PK-15 cells when compared to the PCV2-infected PK-15 cells. The findings suggest that Gram-negative bacteria may be an important factor in PCV2 replication. Additionally, the proinflammatory cytokine profiles are also affected by bacterial lipopolysaccharide. From the results of PK-15 cells, the inhibition of NF-κB activation may indirectly relate to the PCV2 replication.

並列關鍵字

lipopolysaccharide ； PCV2 ； circovirus

參考文獻

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