Yan Mengfei, Zhu Liqi, Yang Qian
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
Virol J. 2014 Nov 19;11:193. doi: 10.1186/s12985-014-0193-0.
Porcine circovirus-associated disease (PCVAD) is caused by a small pathogenic DNA virus, Porcine circovirus type 2 (PCV2), and is responsible for severe economic losses. PCV2-associated enteritis appears to be a distinct clinical manifestation of PCV2. Most studies of swine enteritis have been performed in animal infection models, but none have been conducted in vitro using cell lines of porcine intestinal origin. An in vitro system would be particularly useful for investigating microfilaments, which are likely to be involved in every stage of the viral lifecycle.
We confirmed that PCV2 infects the intestinal porcine epithelial cell line IPEC-J2 by means of indirect immunofluorescence, transmission electron microscopy, flow cytometry and qRT-PCR. PCV2 influence on microfilaments in IPEC-J2 cells was detected by fluorescence microscopy and flow cytometry. We used Cytochalasin D or Cucurbitacin E to reorganize microfilaments, and observed changes in PCV2 invasion, replication and release in IPEC-J2 cells by qRT-PCR.
PCV2 infection changes the ultrastructure of IPEC-J2 cells. PCV2 copy number in IPEC-J2 cells shows a rising trend as infection proceeds. Microfilaments are polymerized at 1 h p.i., but densely packed actin stress fibres are disrupted and total F-actin increases at 24, 48 and 72 h p.i. After Cytochalasin D treatment, invasion of PCV2 is suppressed, while invasion is facilitated by Cucurbitacin E. The microfilament drugs have opposite effects on viral release.
PCV2 infects and proliferates in IPEC-J2 cells, demonstrating that IPEC-J2 cells can serve as a cell intestinal infection model for PCV2 pathogenesis. Furthermore, PCV2 rearranges IPEC-J2 microfilaments and increases the quantity of F-actin. Actin polymerization may facilitate the invasion of PCV2 in IPEC-J2 cells and the dissolution of cortical actin may promote PCV2 egress.
猪圆环病毒相关疾病（PCVAD）由一种致病性小DNA病毒——猪圆环病毒2型（PCV2）引起，会造成严重经济损失。PCV2相关性肠炎似乎是PCV2的一种独特临床表现。大多数关于猪肠炎的研究都是在动物感染模型中进行的，但尚未使用猪肠道来源的细胞系在体外开展研究。体外系统对于研究微丝特别有用，微丝可能参与病毒生命周期的各个阶段。
我们通过间接免疫荧光、透射电子显微镜、流式细胞术和qRT-PCR证实PCV2感染猪肠道上皮细胞系IPEC-J2。通过荧光显微镜和流式细胞术检测PCV2对IPEC-J2细胞中微丝的影响。我们使用细胞松弛素D或葫芦素E重组微丝，并通过qRT-PCR观察PCV2在IPEC-J2细胞中的入侵、复制和释放变化。
PCV2感染会改变IPEC-J2细胞的超微结构。随着感染的进行，IPEC-J2细胞中的PCV2拷贝数呈上升趋势。感染后1小时微丝聚合，但在感染后24、48和72小时，密集排列的肌动蛋白应激纤维被破坏，总F-肌动蛋白增加。细胞松弛素D处理后，PCV2的入侵受到抑制，而葫芦素E促进入侵。微丝药物对病毒释放有相反的作用。
PCV2在IPEC-J2细胞中感染并增殖，表明IPEC-J2细胞可作为PCV2致病机制的细胞肠道感染模型。此外，PCV2会重新排列IPEC-J2微丝并增加F-肌动蛋白的数量。肌动蛋白聚合可能促进PCV2在IPEC-J2细胞中的入侵，而皮质肌动蛋白的溶解可能促进PCV2释放。
