Porcine deltacoronavirus induces apoptosis in swine testicular and LLC porcine kidney cell lines in vitro but not in infected intestinal enterocytes in vivo.
Porcine deltacoronavirus induces apoptosis in swine testicular and LLC porcine kidney cell lines in vitro but not in infected intestinal enterocytes in vivo.
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DOI：
10.1016/j.vetmic.2015.10.022
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发表时间：
2016
影响因子：
3.3
通讯作者：
Saif LJ
中科院分区：
文献类型：
--
作者：
Jung K;Hu H;Saif LJ
PDCoV 在体内和体外诱导细胞死亡的机制尚不清楚。体内 PDCoV 感染的肠细胞未出现 TUNEL 染色阳性。在受感染的 LLC-PK 和 ST 细胞中发现了 TUNEL 阳性信号（凋亡核碎片），这些信号也表现出细胞病变效应。 PDCoV 在体内不会诱导受感染的肠上皮细胞凋亡，但在体外会诱导 LLC-PK 和 ST 细胞凋亡。我们比较了猪德拉冠状病毒（PDCoV）在体内诱导受感染肠细胞死亡的机制以及在体外诱导受感染LLC猪肾（LLC-PK）和猪睾丸（ST）细胞死亡的机制。我们对四只 11 至 14 日龄的限生猪的小肠和大肠的单个或连续切割的组织切片进行了组织学分析和免疫荧光 (IF) 染色来检测 PDCoV 抗原，这些猪口服接种了 8.8–11.0 log10 基因组当量 (GE) 的美国 PDCoV 毒株 OH-FD22 或 OH-FD100 (n = 3)，或模拟 (n = 1)。在分别含有 2.5–10 μg/ml 胰蛋白酶和 1% 胰酶的细胞培养基中，对接种了细胞适应性 PDCoV 菌株 OH-FD22-P44（第 44 代）的 LLC-PK 和 ST 细胞进行了类似的比较测定。接种后第3-4天，受感染的猪表现出严重的水样腹泻和/或呕吐，主要是弥漫性、严重的萎缩性肠炎，萎缩的绒毛上皮内衬的肠细胞出现轻度至中度的细胞质空泡。通过 IF，PDCoV 抗原在绒毛或隐窝上皮细胞中很明显。无PDCoV抗原阳性，小肠和大肠绒毛或隐窝上皮细胞，其细胞质也呈空泡或形态正常，呈TUNEL染色阳性。相反，通过双重 IF 和 TUNEL 染色，在 PDCoV 抗原阳性的 LLC-PK 和 ST 细胞中发现了大部分 TUNEL 阳性信号（凋亡核碎片），这些信号也表现出细胞病变效应，例如细胞变圆、脱离和成簇聚集。二次膜联蛋白 V/碘化丙啶 (PI) 染色显示，与阴性对照相比，接种 21 小时后膜联蛋白 V 或 PI 阳性 LLC-PK 和 ST 细胞数量增加。因此，PDCoV不会在体内诱导受感染的肠上皮细胞凋亡，但会诱导猪源的两种受感染细胞系LLC-PK和ST细胞凋亡。
The mechanisms of PDCoV induced cell death in vivo and in vitro were unknown. No PDCoV-infected enterocytes in vivo showed positive TUNEL staining. TUNEL-positive signals (apoptotic nuclear fragmentation) were found in infected LLC-PK and ST cells that also showed cytopathic effects. PDCoV does not induce apoptosis in infected enterocytes in vivo, but in LLC-PK and ST cells in vitro. We compared the mechanisms of porcine delatacoronavirus (PDCoV) induced death of infected enterocytes in vivo and infected LLC porcine kidney (LLC-PK) and swine testicular (ST) cells in vitro. We conducted histologic analysis and immunofluorescence (IF) staining for the detection of PDCoV antigens, and TUNEL assay in singly or serially cut tissue sections from the small and large intestines of four, 11- to 14-day-old gnotobiotic pigs, inoculated orally with 8.8–11.0 log10 genomic equivalents (GE) of US PDCoV strains OH-FD22 or OH-FD100 (n = 3), or mock (n = 1). Similar comparative assays were done on LLC-PK and ST cells inoculated with the cell-adapted PDCoV strain OH-FD22-P44 (passage 44) in cell culture medium with 2.5–10 μg/ml of trypsin and 1% pancreatin, respectively. At post-inoculation days 3–4, infected pigs showed severe watery diarrhea and/or vomiting and mainly, diffuse, severe atrophic enteritis, with mild to moderate cytoplasmic vacuolation of the enteroctyes lining the atrophied villous epithelium. By IF, PDCoV antigens were evident in villous or crypt epithelial cells. No PDCoV antigen-positive, small and large intestinal villous or crypt epithelial cells, of which cytoplasm was also either vacuolated or morphologically normal, showed positive TUNEL staining. In contrast, by double IF and TUNEL staining, most of the TUNEL-positive signals (apoptotic nuclear fragmentation) were found in PDCoV antigen-positive LLC-PK and ST cells that also showed cytopathic effects, such as cell rounding, detachment and clumping in clusters. Secondary annexin V/propidium iodide (PI) staining revealed increased numbers of annexin V- or PI-positive LLC-PK and ST cells at 21 h after inoculation, compared to the negative controls. Thus, PDCoV does not induce apoptosis in the infected intestinal enterocytes in vivo, but in two infected cell lines of swine origin, LLC-PK and ST cells.
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