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性糖尿病（IDDM）、胰腺炎、慢性炎性肌病和慢性疲劳综合征。该研究将使用表达增强型绿色荧光蛋白（eGFP）的重组病毒，研究柯萨奇病毒B3（CVB 3）感染小鼠的持久性机制。我们的实验室已经建立了一个坚实的基础，分子和免疫学技术和试剂从以前的研究CVB 3的持久性和发病机制。用重组eGFP-CVB 3进行的初步研究表明，培养中的某些Hela RW细胞群体不能支持生产性感染，包括静止细胞（G 0）和在细胞周期的G2/M期阻断的细胞。以下实验将检验以下假设：小鼠中CVB 3的持续存在可能依赖于不能支持病毒复制的静止细胞的感染;随后细胞周期状态的变化可能导致病毒再活化和宿主中进一步的病毒/免疫介导的病理（包括心肌炎）。具体目标：1.检测持久性或潜伏性CVB 3 RNA的稳定性。将在多个时间点监测用eGFP-CVB 3感染并维持在无血清培养基中而不传代的静止细胞的病毒复制（噬斑测定）和病毒RNA的稳定性（RT-PCR）。在用10% FBS刺激细胞后观察病毒拯救（GFP、RT-PCR和空斑测定）。2.探讨哪些因素或刺激可能参与CVB 3的再激活。在心脏组织中表达SV 40 T抗原的转基因小鼠将通过组织学和GFP荧光表征在用eGFP-CVB 3感染后更大的发病机制。将通过GFP表达检查来自持续感染小鼠的刺激的PBL的CVB 3再活化。

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.