Xiao Yan, An Tong-Qing, Tian Zhi-Jun, Wei Tian-Chao, Jiang Yi-Feng, Peng Jin-Mei, Zhou Yan-Jun, Cai Xue-Hui, Tong Guang-Zhi
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, CAAS, No. 427 Maduan Street, Nangang District, Harbin, 150001, China.
Arch Virol. 2015 Mar;160(3):649-62. doi: 10.1007/s00705-014-2309-7. Epub 2014 Dec 12.
Since the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) variant emerged in 2006, it has caused death in more than 20 million pigs in China and other Southeast Asian countries, making it the most destructive swine pathogen currently in existence. To characterize the cellular responses to HP-PRRSV infection, the gene expression profile of porcine alveolar macrophage (PAM) cells, the primary target cells of PRRSV, was analyzed in HP-PRRSV-infected and uninfected PAMs by suppression subtractive hybridization. After confirmation by Southern blot, genes that were differentially expressed in the HP-PRRSV-infected and uninfected PAMs were sequenced and annotated. Genes that were upregulated mainly in HP-PRRSV-infected PAM cells were related to immunity and cell signaling. Among the differentially expressed genes, Mx1 and HSP70 protein expression was confirmed by western blotting, and IL-8 expression was confirmed by ELISA. In PAM cells isolated from HP-PRRSV-infected piglets, the differential expression of 21 genes, including IL-16, TGF-beta type 1 receptor, epidermal growth factor, MHC-I SLA, Toll-like receptor, hepatoma-derived growth factor, FTH1, and MHC-II SLA-DRB1, was confirmed by real-time PCR. To our knowledge, this is the first study to demonstrate differential gene expression between HP-PRRSV-infected and uninfected PAMs in vivo. The results indicate that HP-PRRSV infection excessively stimulates genes involved in the innate immune response, including proinflammatory cytokines and chemokines.
自2006年高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)变异株出现以来,它已在中国和其他东南亚国家导致超过2000万头猪死亡,成为目前存在的最具破坏性的猪病原体。为了表征细胞对HP-PRRSV感染的反应,通过抑制性消减杂交分析了PRRSV的主要靶细胞——猪肺泡巨噬细胞(PAM)在感染和未感染HP-PRRSV时的基因表达谱。经Southern印迹确认后,对在感染和未感染HP-PRRSV的PAM中差异表达的基因进行测序和注释。主要在感染HP-PRRSV的PAM细胞中上调的基因与免疫和细胞信号传导有关。在差异表达的基因中,通过蛋白质免疫印迹法确认了Mx1和HSP70蛋白的表达,通过酶联免疫吸附测定法确认了IL-8的表达。在从感染HP-PRRSV的仔猪分离的PAM细胞中,通过实时PCR确认了包括IL-16、转化生长因子β1型受体、表皮生长因子、MHC-I SLA、Toll样受体、肝癌衍生生长因子、FTH1和MHC-II SLA-DRB1在内的21个基因的差异表达。据我们所知,这是第一项在体内证明感染和未感染HP-PRRSV的PAM之间存在差异基因表达的研究。结果表明,HP-PRRSV感染过度刺激了参与先天免疫反应的基因,包括促炎细胞因子和趋化因子。