猪δ冠状病毒感染猪树突状细胞及对免疫因子分泌能力的影响

Effects of porcine deltacoronavirus on infection and secretion of immune factors in porcine dendritic cells

以猪树突状细胞(porcine dendritic cells,pDCs)为模型,探究猪δ冠状病毒(porcine deltacoronavirus,PDCoV)是否能够对pDCs进行有效感染及对抗病毒细胞因子分泌的影响。采集猪外周抗凝血,分离获得猪外周血单核细胞,经白细胞介素-4(interleukin-4,IL-4)和集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)诱导培养获得未成熟树突状细胞,利用倒置显微镜和透射电镜观察其生物学形态,通过蛋白质免疫印迹和激光共聚焦试验检测病毒是否可以对pDCs进行感染,qRT-PCR检测抗病毒因子IFN-α和ISG56的表达。结果显示,成功分离培养得到具有典型树突状细胞形态的pDCs,将PDCoV接种至分离培养得到的pDCs中,PDCoV可以有效感染,可以在pDCs中进行复制并产生感染性的子代病毒颗粒,病毒滴度达到4.4 lgTCID50/0.1 mL。同时检测PDCoV感染pDCs时对抗病毒因子表达的影响,结果表明,在病毒感染pDCs后48 h产生了高水平的IFN-α和ISG56。本研究成功诱导培养得到pDCs,首次证实PDCoV可以在体外感染pDCs,并在细胞中进行有效地复制,pDCs在病毒感染后促进IFN-α、ISG56的分泌来发挥抗病毒免疫效应,为后续研究PDCoV的致病机制提供了新的思路。

Porcine deltacoronavirus(PDCoV)is a new intestinal coronavirus that causes diarrhea in nursing piglets,which seriously harms the pig industry.This study aims to use porcine dendritic cells(pDCs)as a model to explore whether PDCoV can effectively infect pDCs and its effect on the secretion of anti-viral cytokines.Peripheral anticoagulant cells of porcine were first collected.Porcine peripheral blood mononuclear cells were then isolated and induced by interleukin-4(IL-4)and colony-stimulating factor(GM-CSF)to obtain immature dendritic cells,and their biological morphology was observed by inverted microscopy and transmission electron microscopy.Western blot and laser confocal assays were used to detect whether the virus could infect pDCs,and qRT-PCR were used to detect the expression of antiviral factors IFN-a and ISG56.The results showed that pDCs with typical dendritic cell morphology were successfully isolated and cultured,and PDCoV could effectively infect and replicate in pDCs and produce infectious progeny viral particles,with a viral titer of 4.41gTCID_(50)/0.1 mL.At the same time,the effect of PDCoV infection on the expression of antiviral factors in pDCs was detected,and the results showed that high levels of IFN-αand ISG56 were produced 48 h after infection in pDCs.In summary,we successfully obtained pDCs by induced culture,and confirmed for the first time that PDCoV can infect pDCs in vitro and effectively replicate in cells,and pDCs promote the secretion of IFN-αand ISG56 after viral infection to exert antiviral immune effects,which provides a novel idea for subsequent research on the pathogenic mechanism of PDCoV.