Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.
PLoS One. 2009 Sep 28;4(9):e7214. doi: 10.1371/journal.pone.0007214.
Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.
爱泼斯坦-巴尔病毒（EBV）与大量人类癌症的发展密切相关。为了开发一种监测与 EBV 感染相关的早期细胞和病毒事件的系统，我们使用自我重组 BAC 构建体，该构建体包含 EBV 基因组的 172-kb，命名为 MD1 BAC（Chen 等人，2005 年，J.Virology），通过同源重组引入绿色荧光蛋白（GFP）表达盒，所得 BAC 克隆 BAC-GFP-EBV 被转染到 HEK 293T 上皮细胞系中。通过来自稳定的 HEK 293T BAC-GFP-EBV 细胞系的化学诱导剂，诱导重组 GFP EBV 产生子代病毒，并通过绿色荧光和原代 B 细胞的生长监测 GFP EBV 对人原代 B 细胞的永生化作用。通过流式细胞术监测 GFP EBV 引起的感染、B 细胞激活和细胞增殖，通过 B 细胞表面抗原 CD5、CD10、CD19、CD23、CD39、CD40、CD44 和细胞内增殖标志物 Ki-67 的表达进行监测。结果显示 Ki-67 的表达显著增加，并且在感染后 6-7 天继续增加。同样，CD40 信号逐渐增加，而 CD23 信号在感染后 6-12 小时增加，最大程度在第 3 天，然后减少。监测病毒基因表达模式显示早期裂解基因表达的爆发。在早期感染过程中，潜伏基因之前早期裂解基因表达的上调强烈表明 EBV 以裂解基因表达的早期爆发感染原代 B 细胞，并且产生的子代病毒能够感染新的原代 B 细胞。这个过程可能在细胞转化之前建立潜伏期至关重要。新感染的原代 B 细胞可以进一步分析，以研究 EBV 感染引起的 B 细胞激活。
