Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok, Thailand.
BMC Microbiol. 2010 Nov 16;10:290. doi: 10.1186/1471-2180-10-290.
With both shrimp and commercial insects such as honey bees, it is known that stable, persistent viral infections characterized by absence of disease can sometimes shift to overt disease states as a result of various stress triggers and that this can result in serious economic losses. The main research interest of our group is to understand the dynamics of stable viral infections in shrimp and how they can be destabilized by stress. Since there are no continuous cell lines for crustaceans, we have used a C6/36 mosquito cell line infected with Dengue virus to test hypotheses regarding these interactions. As a result, we accidentally discovered two new cytokine-like substances in 5 kDa extracts from supernatant solutions of acutely and persistently infected mosquito cells.
Naïve C6/36 cells were exposed for 48 h to 5 kDa membrane filtrates prepared from the supernatant medium of stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Subsequent challenge of naïve cells with a virulent stock of Dengue virus 2 (DEN-2) and analysis by confocal immunofluorescence microscopy using anti-DEN-2 antibody revealed a dramatic reduction in the percentage of DEN-2 infected cells when compared to control cells. Similar filtrates prepared from C6/36 cells with acute DEN-2 infections were used to treat stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Confocal immunofluorescence microscopy revealed destabilization in the form of an apoptosis-like response. Proteinase K treatment removed the cell-altering activities indicating that they were caused by small polypeptides similar to those previously reported from insects.
This is the first report of cytokine-like substances that can alter the responses of mosquito cells to Dengue virus. This simple model system allows detailed molecular studies on insect cytokine production and on cytokine activity in a standard insect cell line.
与虾和商业昆虫（如蜜蜂）一样，人们知道，稳定、持续的病毒感染有时会由于各种应激触发而从无明显疾病状态转变为明显疾病状态，并且这可能导致严重的经济损失。我们小组的主要研究兴趣是了解虾中稳定病毒感染的动态，以及它们如何因应激而变得不稳定。由于没有虾的连续细胞系，我们使用感染登革热病毒的 C6/36 蚊细胞系来测试这些相互作用的假设。结果，我们在急性和持续性感染蚊细胞的上清液中 5 kDa 提取物中意外发现了两种新的细胞因子样物质。
幼稚的 C6/36 细胞在 5 kDa 膜滤器中暴露 48 小时，该滤器由稳定感染登革热病毒的 C6/36 蚊细胞培养物的上清液中的上清液制备。随后用登革热病毒 2 （DEN-2）的毒力株对幼稚细胞进行挑战，并通过使用抗 DEN-2 抗体的共聚焦免疫荧光显微镜分析，与对照细胞相比， DEN-2 感染的细胞百分比明显降低。使用从急性 DEN-2 感染的 C6/36 细胞中制备的类似滤器处理稳定感染登革热病毒的 C6/36 蚊细胞培养物。共聚焦免疫荧光显微镜显示，细胞不稳定，呈凋亡样反应。蛋白激酶 K 处理去除了改变细胞活性的物质，表明它们是由类似于以前从昆虫中报道的小多肽引起的。
这是首次报道可以改变蚊细胞对登革热病毒反应的细胞因子样物质。这种简单的模型系统允许对昆虫细胞因子产生和标准昆虫细胞系中细胞因子活性进行详细的分子研究。
