行政院國家科學委員會專題研究計畫期中進度報告:豬第二型環狀病毒單獨感染及混合豬生殖與呼吸綜合症病毒感染對豬隻免疫細胞之影響及其可能之作用機制(1/3)

離乳豬多系統消耗性症候群 (Postweaning multisystemic wasting syndrome, PMWS) 是由第二型豬環狀病毒(Porcine circovirus type 2, PCV2)引起，由於PCV2 患豬 常伴有繼發性病毒、細菌及黴菌感染，且PCV2 與豬生殖與呼吸綜合症病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)在豬場的混合感染情形極為普遍，加 上PCV2 及PRRSV 感染的標的細胞皆為單核 球及巨噬細胞，因此釐清PCV2 之致病機制及 混合PRRSV 感染對免疫細胞的影響，在對本 病之控制及減輕其危害上將有所助益。本實驗 以PCV2 與PRRSV 單獨感染或不同順序混合 感染豬肺臟巨噬細胞(AM)分為六組包括：對 照組(G1)、單獨PCV2 組(G2)、單獨PRRSV 組(G3)、先有PCV2 感染18 小時再以PRRSV 感染組(G4)、先有PRRSV 感染18 小時再以 PCV2 感染組(G5)、PCV2 與PRRSV 兩病毒同 時感染組(G6)進行研究。由PCV2 高陽性率可 知AM 為PCV2 重要保存細胞；而PRRSV 感 染率，除了G4 由5%逐漸降至1-2%外，其餘 各PRRSV 感染組均維持在5-10%。在存活率 方面，PCV2 單獨感染並不造成AM 死亡及凋 亡，然而各PRRSV 感染組，則有明顯細胞死 亡及凋亡之情形，且以先有PRRSV 感染之G5 及單獨PRRSV 感染之G3 最為嚴重，而先有 PCV2 感染之G4，呈現AM 暫時性的吞噬力 降低，但各PRRSV 感染組則有明顯AM 吞噬 力下降之情形，並以先有PRRSV 感染之G5 及單獨PRRSV 感染之G3 最為嚴重，此外， 並發現PCV2 及PRRSV 感染對AM 內呼吸風 暴(respiratory burst)產生之氧自由基(oxygen free radical) O2 -及H2O2 亦有顯著抑制之影響。 另外，單獨PCV2 感染與各PRRSV 感染組， 在殺菌力方面亦均明顯低於對照組，其中亦以 先有PRRSV 感染之G5 及單獨PRRSV 感染之 G3 最為嚴重。為了解釋是否PCV2 的存在有 抑制後續PRRSV 對AM 感染之影響，經進一 步分析發現PCV2 感染後能促使AM分泌大量 IFN-α，此可能為導致後續PRRSV 感染率下 降之原因。在其他細胞激素表現方面，發現 PCV2 及/或PRRSV 感染早期僅造成暫時的 IL-1β增加，但不論是在PCV2 及PRRSV 單獨 或共同感染組，相對於控制組皆有明顯增加 IL-8 分泌之情形。由不同PCV2 及PRRSV 感 染AM 次序發現其所造成影響並不一致，亦可 推測與現場共同感染PCV2 及PRRSV 豬隻所 呈現不同病徵有關。而PCV2 及PRRSV 所誘 導產生的IFN-α及TNF-α推測可能與PMWS 豬隻發現之間質性肺炎致病機制有關。Porcine circovirus 2 (PCV2) is currently considered the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). Pigs affected by PMWS often suffered from secondary viral, bacterial, and fungal infection. In addition, dual infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) in pigs is common. It is known that the target cells of both PCV2 and PRRSV are monocytes/macrophages. Thus, evaluating the immunological effects and possible mechanism of action of PCV2 and PRRSV infection should contribute to the control and limiting of the disease. The present study was to compare the effects of the infection of both viruses, singular or combined, on swine alveolar macrophages (AMs). Six groups were designated, including control (G1), PCV2 alone (G2), PRRSV alone (G3), PRRSV following 18 h of initial PCV2 infection (G4), PCV2 following 18 h of initial PRRSV infection (G5), and concurrent PCV2/PRRSV infection (G6). High antigen containing rate without cytocidal effect in G2 suggested that AMs were an important reservoir of PCV2. A low but constant infectious rate about 5-10% was noted in all PRRSV-infected groups except for G4. In G4, the PRRSV infectious rate was about 5% and gradually reduced and dropped to 1-2%. A transient decrease in phagocytosis but persistent significant reduction in microbial killing of Candida albicans in G2 indicated that PCV2-infected AMs might become dysfunction in against secondary pulmonary pathogens. In G3 and G5, there was a marked increase in AM death and apoptosis, and reduction in its phagocytic and microbicidal capacities. In addition, inhibition in the production of oxygen free radical, O2 - and H2O2, were also observed in both PCV2 and/or PRRSV infected groups. A lower PRRSV infectious rate, cell death, and apoptosis, and higher phagocytic capacity than G3 and G5 were seen in G4 and G6. The results suggest that in dual infection the pre-existing or co-existing PCV2 may reduce not only PRRSV replication but also PRRSV-related AM dysfunction. High levels of interferon-α (IFN-α) and increasing levels of tumor necrosis factor α (TNF-α) productions were seen in all PCV2-infected and in all PCV2 and/or PRRSV-infected groups, respectively. Further anti-IFN-α antibody blocking assay revealed that the PCV2-mediated reduction effect on PRRSV might be a result of PCV2’s strong potential of IFN-α induction. In other cytokines’ expression, PCV2 and/or PRRSV infection only caused a temporary increase in IL-1β but a significant increased in the secretion of IL-8. Different infection orders of PCV2 and/or PRRSV in swine AMs leading to different results may explain, at least partially, the prominent clinic variations of PMWS-affected pigs in the filed. The PCV2 and/or PRRSV-related IFN-α and TNF-α induction may also play a role in the pathogenesis of interstitial pneumonia in PMWS-affected pigs