比較豬瘟2.1與3.4基因型病毒株感染豬肺泡巨噬細胞後的免疫反應與基因表現差異

Abstract

豬瘟（Classical swine fever, CSF）是一種由豬瘟病毒（Classical swine fever virus, CSFV）引起的高度傳染性疾病，具有強致病性，對豬隻免疫系統造成嚴重抑制。在臺灣，自1996年以來，豬瘟病毒的基因型經歷了顯著轉變，從過去占主導地位的基因型3.4（G3.4）被國外引入的新興基因型2.1（G2.1）所取代。這種基因型的優勢轉變不僅限於臺灣，也在其他國家觀察到。然而，這一現象背後的潛在機制仍有待進一步探討。先前的研究顯示，無論是在體外還是體內實驗中，G2.1的病毒複製效率均顯著高於 G3.4。除了病毒複製能力的差異之外，不同豬瘟基因型對宿主免疫調節機制是否存在差異，仍需進一步深入探討。巨噬細胞是豬瘟病毒感染的主要標靶細胞之一，能分泌促發炎細胞激素、免疫調節因子及血管活性物質，進而調控受感染豬隻的免疫反應。本研究主旨在探討不同基因型豬瘟病毒株引發的免疫反應差異，以瞭解其調控機制。本研究利用免疫螢光染色技術觀察兩種豬瘟病毒基因型對豬肺臟巨噬細胞的感染率，並採用高通量多重細胞激素檢測平台同步測定感染巨噬細胞分泌的細胞激素。此外，透過RNA轉錄體分析全面探討基因表現變化，並利用多種富集分析方法找出可能引起兩者反應差異的相關路徑。最後，使用即時定量聚合酶連鎖反應進行驗證。研究結果顯示，豬瘟G2.1的病毒株對於豬肺臟巨噬細胞的感染率在感染後第一天時顯著高於G3.4，且在感染後的第一至三天，豬瘟G2.1誘發的促發炎細胞激素IL-18，和在感染後的第二天的TNF-α的基因表現量均顯著高於豬瘟G3.4。RNA轉錄體分析結果顯示，豬瘟G2.1感染誘導的功能基因數量多於豬瘟G3.4，且涉及更多與促發炎相關的免疫訊息傳遞路徑。特別是在G2.1感染的巨噬細胞中，有較多的基因表現參與在路徑JAK-STAT signaling pathway及Th17 cell differentiation中，且與促發炎反應、Th1型免疫以及招募免疫細胞相關。透過轉錄組分析篩選出參與多條顯著富集路徑的基因，例如與細胞貼附相關的ICAM1基因和促發炎的S100A9基因，在豬瘟G2.1中的表達量顯著高於豬瘟G3.4，這些基因的差異顯示出豬瘟G2.1的感染與促發炎反應的增強，以及感染持續性有正向關聯性。而在豬瘟G3.4感染巨噬細胞後，某些表現量上升的特定基因，如招募B細胞的CXCL13，以及具有抗發炎功能的前列腺素受體PTGIR，被推論有可能是豬瘟G3.4在感染宿主後較容易被清除，且引發較弱發炎反應的原因。我們的研究顯示，相較於豬瘟G3.4，豬瘟G2.1能誘導更強的免疫刺激反應，顯示兩病毒在免疫調節機制上存在差異性。這些發現有助於探討野外豬瘟病毒株基因型置換的機制，並凸顯不同豬瘟病毒基因型所採用的免疫逃避策略。

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease that induces significant immunosuppression in pigs. Since 1996, a genotypic shift has occurred in Taiwan, with the historically dominant Genotype 3.4 (G3.4) replaced by the emergent Genotype 2.1 (G2.1). Similar shifts have been observed elsewhere, though the underlying mechanisms remain unclear. Previous studies indicate that G2.1 exhibits higher replication efficiency than G3.4 in both in vitro and in vivo models. However, beyond replication capacity, the immunomodulatory differences between these genotypes warrant further investigation. Macrophages, a key CSFV target, regulate immune responses by releasing pro-inflammatory cytokines and immune-modulating factors. This study examines the differential immune responses triggered by G2.1 and G3.4 to elucidate their regulatory mechanisms. Pig alveolar macrophage (PAM) infection rates were assessed via immunofluorescence assay (IFA), while cytokine secretion was analyzed using a high-throughput multiplex platform. RNA transcriptome analysis provided a comprehensive gene expression profile, with key findings validated via qPCR. The results show that G2.1 infects PAMs more efficiently than G3.4 at 1 day post-infection (dpi). IL-18 levels were significantly higher in G2.1-infected PAMs at 1, 2, and 3 dpi, while TNF-α levels were also elevated at 2 dpi. Transcriptomic analysis revealed that G2.1-infected PAMs exhibited increased activation of immune pathways, including JAK-STAT signaling and Th17 cell differentiation, which are linked to pro-inflammatory responses and Th1 immunity. Genes such as ICAM1 (cell adhesion) and S100A9 (pro-inflammatory response) were significantly upregulated in G2.1-infected cells, suggesting enhanced inflammation and viral persistence. Conversely, G3.4 uniquely upregulated genes like CXCL13 (B-cell recruiter) and PTGIR (anti-inflammatory prostaglandin receptor), potentially mitigating excessive immune activation and reducing host damage. These findings highlight the critical role of genotype-specific gene regulation in CSFV pathogenesis. Our study revealed that G2.1 induces a stronger immunostimulatory response than G3.4, suggesting differences in their immunomodulatory mechanisms. These findings provide valuable insights into the factors driving genotypic shifts in field strains and highlight the immune evasion strategies employed by different CSFV genotypes.