COXSACKIEVIRUS 3B PERSISTENCE AND REACTIVATION IN VIVO

柯萨奇病毒与许多不同的人类疾病有关，包括急性和慢性心肌炎、病毒诱导的胰岛素依赖型糖尿病(IDDM)、胰腺炎、慢性炎症性肌病和慢性疲劳综合征。这项拟议的研究将使用表达增强型绿色荧光蛋白(EGFP)的重组病毒来研究柯萨奇病毒B3(CVB3)感染小鼠的持久性机制。我们的实验室从以前对CVB3持久性和致病机制的研究中，已经建立了坚实的分子和免疫学技术和试剂基础。对重组EGFP-CVB3的初步研究表明，培养中的特定群体的Hela RW细胞不能支持生产性感染，包括静止细胞(G0)和被阻断在细胞周期的G2/M期的细胞。以下实验将检验这样一种假设，即CVB3在小鼠体内的持久性可能依赖于无法支持病毒复制的静止细胞的感染；以及随后细胞周期状态的改变可能导致病毒重新激活，并在宿主中进一步引起病毒/免疫介导的病理(包括心肌炎)。具体目的：1.检测持续或潜伏CVB3RNA的稳定性。感染了EGFP-CVB3的静止细胞，在无血清培养中不传代，将在多个时间点监测病毒复制(空斑试验)和病毒RNA的稳定性(RT-PCR)。用10%胎牛血清刺激细胞后，观察病毒拯救(GFP、RT-PCR和空斑试验)。2.探讨哪些因素或刺激可能参与了CVB3的再激活。组织学和GFP荧光显示，在心脏组织中表达SV40T抗原的转基因小鼠在感染EGFP-CVB3后具有更强的致病能力。来自持续感染小鼠的刺激的PBL将通过GFP表达来检测CVB3的重新激活。

Coxsackieviruses have been implicated in a number of different human diseases, including acute and chronic myocarditis, viral-induced insulin-dependent diabetes mellitus (IDDM), pancreatitis, chronic inflammatory myopathy, and chronic fatigue syndrome. The proposed research will examine the mechanisms of persistence in Coxsackievirus B3 (CVB3) infected mice, using a recombinant virus expressing the enhanced green fluorescent protein (eGFP). Our laboratory has established a solid foundation of molecular and immunological techniques and reagents from previous studies of CVB3 persistence and pathogenesis. Preliminary studies with recombinant eGFP-CVB3 indicate that a certain population of Hela RW cells in culture cannot support a productive infection, including quiescent cells (G0) and cells blocked at the G2/M phase of the cell cycle. The following experiments will test the hypothesis that persistence of CVB3 in mice may rely on infection of quiescent cells incapable of supporting viral replication; and that a subsequent change in the cell-cycle status may lead to virus reactivation and further viral/immune mediated pathology (including myocarditis) in the host. SPECIFIC AIMS: 1. To examine the stability of persistent or latent CVB3 RNA. Quiescent cells infected with eGFP-CVB3 and maintained in serum free media without passage will be monitored at multiple time points for viral replication (plaque assay) and for stability of viral RNA (RT-PCR). Virus rescue will be observed (GFP, RT-PCR, and plaque assay) after cell stimulation with 10 % FBS. 2. To investigate what factors or stimuli may be involved in reactivation of CVB3. Transgenic mice expressing SV40 T antigen in cardiac tissues will be characterized for greater pathogenesis following infection with eGFP-CVB3 by histology and GFP fluorescence. Stimulated PBLs from persistently infected mice will be examined for CVB3 reactivation by GFP expression.