Department of Cell and Molecular Biology, University of Sao Paulo School of Medicine, 3900, Av Bandeirantes, Ribeirão Preto, SP 14049-900, Brazil.
Virus Res. 2010 Apr;149(1):56-63. doi: 10.1016/j.virusres.2009.12.013. Epub 2010 Jan 18.
Oropouche (OROV) is a single-stranded RNA arbovirus of the family Bunyaviridae, genus Orthobunyavirus, which has caused over half a million cases of febrile illness in Brazil in the past 30 years. OROV fever has been registered almost exclusively in the Amazon region, but global warming, deforestation and redistribution of vectors and animal reservoirs increases the risk of Oropouche virus emergence in other areas. OROV causes a cytolytical infection in cultured cells with characteristic cytopathic effect 48h post-infection. We have studied the mechanisms of apoptosis induced by OROV in HeLa cells and found that OROV causes DNA fragmentation detectable by gel electrophoresis and by flow cytometric analysis of the Sub-G1 population at 36h post-infection. Mitochondrial release of cytochrome C and activation of caspases 9 and 3 were also detected by western blot analysis. Lack of apoptosis induced by UV-inactivated OROV reveals that virus-receptor binding is not sufficient to induce cell death. Results obtained in cells treated with chloroquine and cycloheximide indicated that viral uncoating and replication are required for apoptosis induction by OROV. Furthermore, treatment of the cells with pan-caspase inhibitor prevented OROV-induced apoptosis without affecting virus progeny production. The results show that OROV infection in vitro causes apoptosis by an intracellular pathway involving mitochondria, and activated by a mechanism dependent on viral replication and protein synthesis.
奥罗普切病毒（OROV）是一种单链 RNA 虫媒病毒，属于布尼亚病毒科，正布尼亚病毒属，在过去 30 年中，在巴西已导致超过 50 万例发热病例。OROV 热几乎只在亚马逊地区登记过，但全球变暖、森林砍伐以及媒介和动物宿主的重新分布增加了其他地区奥罗普切病毒出现的风险。OROV 在培养细胞中引起细胞溶解性感染，具有特征性细胞病变效应，感染后 48 小时即可观察到。我们研究了 OROV 在 HeLa 细胞中诱导细胞凋亡的机制，发现 OROV 在感染后 36 小时可通过凝胶电泳和 Sub-G1 群体的流式细胞术分析检测到 DNA 片段化。还通过 Western blot 分析检测到线粒体细胞色素 C 的释放和半胱天冬酶 9 和 3 的激活。经紫外线灭活的 OROV 诱导的细胞凋亡缺失表明，病毒受体结合不足以诱导细胞死亡。用氯喹和环己酰亚胺处理细胞获得的结果表明，病毒脱壳和复制是 OROV 诱导细胞凋亡所必需的。此外，用泛半胱天冬酶抑制剂处理细胞可预防 OROV 诱导的细胞凋亡，而不影响病毒后代的产生。结果表明，OROV 体外感染通过涉及线粒体的细胞内途径诱导细胞凋亡，并通过依赖于病毒复制和蛋白质合成的机制激活。
