Hong J R, Lin T L, Hsu Y L, Wu J L
Institute of Zoology, Academia Sinica, Nankang, Taipei, 115, Taiwan.
Virology. 1998 Oct 10;250(1):76-84. doi: 10.1006/viro.1998.9347.
The current view of infectious pancreatic necrosis virus (IPNV) infection includes a necrotic process that relies primarily on the histological appearance of tissue after the degenerative process. We tested this view by examining the possibility that apoptosis is a component of double-stranded RNA virus (IPNV) that induces fish embryonic cell death. Four kinds of assays for apoptosis were used in analyzing IPNV-infected CHSE-214 cells: (1) assay with terminal deoxynucleotidyl transferase (TdT)-mediated end-labeling of DNA in nuclei of intact cells during virus infection, (2) assay for procoagulant activity, (3) assay for DNA ladders, and (4) electron microscopic assays for the ultrastructural changes in characteristic apoptotic cells. In all p.i. samples, both low and high m.o.i. groups contained apoptotic nuclei, according to TdT-mediated dUTP labeling of intact cells, but in control CHSE-214 cells, apoptotic nuclei were rare at all levels of incubation sampled by TdT-mediated dUTP labeling. Prenecrotic or postnecrotic cells were found to express phosphatidylserine on the surface by annexin V-FITC labeling, but normal cells did not. DNAs from both 4 h p.i. of high m.o.i. and 8 h p.i. of low m.o.i. were found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. The IPNV-infected CHSE-214 cells were analyzed with an electron microscope and showed a pattern of ultrastructural change, indicating that apoptosis appears before pathological changes of necrosis, including condensed chromatin, fragmented nuclei, nuclei with chromatin marginations, and secondary necrosis from prenecrotic cells in IPNV-infected CHSE-214 cells. Together, these findings show that apoptosis precedes any detectable necrotic change in CHSE-214 cells that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in host cells and is followed by necrotic processes.
目前对于传染性胰腺坏死病毒（IPNV）感染的观点认为，其坏死过程主要依赖于组织在退行性变后的组织学表现。我们通过研究凋亡是否是双链RNA病毒（IPNV）诱导鱼类胚胎细胞死亡的一个组成部分来验证这一观点。在分析IPNV感染的CHSE-214细胞时，使用了四种凋亡检测方法：（1）在病毒感染期间，用末端脱氧核苷酸转移酶（TdT）介导对完整细胞核中的DNA进行末端标记的检测方法；（2）促凝活性检测方法；（3）DNA梯状条带检测方法；（4）对特征性凋亡细胞超微结构变化进行的电子显微镜检测方法。在所有感染后的样本中，根据TdT介导的dUTP对完整细胞的标记，低感染复数和高感染复数组均含有凋亡细胞核，但在对照CHSE-214细胞中，通过TdT介导的dUTP标记在所有取样的孵育水平下凋亡细胞核都很少见。通过膜联蛋白V-FITC标记发现，坏死前或坏死后的细胞在表面表达磷脂酰丝氨酸，而正常细胞则不表达。发现高感染复数感染后4小时和低感染复数感染后8小时的DNA均被切割成片段，表明在核小体间位点存在优先切割。对IPNV感染的CHSE-214细胞进行电子显微镜分析，结果显示出超微结构变化模式，表明凋亡出现在坏死的病理变化之前，包括IPNV感染的CHSE-214细胞中染色质浓缩、细胞核碎片化、染色质边缘化的细胞核以及坏死前细胞的继发性坏死。总之，这些发现表明，在目前被视为坏死的CHSE-214细胞中，凋亡先于任何可检测到的坏死变化出现。因此，凋亡是宿主细胞病理变化的起始特征，随后是坏死过程。
