Modeling KSHV Infection and Pathogenesis in SCID Mice

描述(由申请人提供)：体内模型对于了解病毒性疾病的致病机制以及评估抗击这些疾病的治疗方法至关重要。KSHV是从艾滋病患者中发现的B细胞淋巴瘤中分离出来的，称为PEL和MCD。对KSHV感染的初始细胞靶点以及导致病毒在体内传播和潜伏的细胞的了解还不完全。我们推测KSHV感染祖细胞B细胞是启动肿瘤发生的关键过程。已在KS患者的造血祖细胞(CD34+)中检测到KSHV基因组序列。我们已经证明KSHV感染人CD34+细胞，并且病毒基因在体外分化后的CFU-GM克隆集落中保持表达。用感染的人CD34+细胞重建淋巴细胞后，在人(CD14+)单核细胞和NOD/SCID-Hu嵌合小鼠的B淋巴细胞中检测到KSHV基因组DNA和LANA-1的表达。虽然这个模型系统不能很好地代表正常的人类造血和/或KSHV感染的所有方面，但KSHV在NOD/SCID-Hu小鼠中的持久性和CD34+细胞的感染提供了一个实验系统来解决体内KSHV复制、潜伏期和病理生物学的许多问题。这一新的动物模型将用于检测AZT和干扰素-α在体内抑制KSHV感染的效果。本研究旨在进一步完善和发展NOD/SCID-Hu小鼠KSHV感染模型，更好地了解KSHV感染在CD34+造血祖细胞中的作用。具体目的是：(1)评价、优化和评估KSHV和rKSHV.219对CD34+细胞的体外新生感染作用。确定KSHV是否在人CD34+造血祖细胞中建立了潜伏感染，并表征了病毒感染在抑制造血方面的作用。(2)通过CD34+HPC感染KSHV和rKSHV.219的NOD/SCID小鼠的免疫重建，确定KSHV的细胞趋向性和致瘤潜能。评价和鉴定KSHV/HHV-8感染对体内造血的影响。(3)检测AZT和IFNα是否诱导感染rKSHV.219的原代造血细胞发生凋亡。建立NOD/SCID-HU小鼠作为临床前模型，用于抑制KSHV复制的治疗干预。

DESCRIPTION (provided by applicant): In vivo models are critical to the understanding of pathogenic mechanisms operating in viral diseases, and for evaluation of therapeutic approaches to combat these diseases. KSHV has been isolated from B cell derived lymphomas found in patients with AIDS, termed PEL and MCD. There is an incomplete understanding of the initial cellular targets of KSHV infection and cells that contribute to viral dissemination and latency in vivo. We speculate that KSHV infection of progenitor B cells is key process in the initiation of oncogenesis. KSHV genomic sequences have been detected in hematopoietic progenitor (CD34+) cells from KS patients. We have demonstrated that KSHV infects human CD34+ cells and that viral gene expression is maintained in CFU-GM clonogenic colonies derived from the differentiation of these cells in vitro. KSHV genomic DNA and LANA-1 expression was detected in human (CD14+) monocytes and in B lymphocytes from NOD/SCID-hu chimeric mice following reconstitution of lymphopoiesis with infected human CD34+ cells. Although this model system is not perfectly representative of normal human hematopoiesis and/or all aspects of KSHV infection, the combination of KSHV persistence in NOD/SCID-hu mice and infection of CD34+ cells provides an experimental system in which to address many of the issues of KSHV replication, latency and pathobiology in vivo. This novel animal model will be employed to assay the efficacy of AZT and IFN-alpha in suppressing KSHV infection in vivo. This proposal aims to further refine and develop the NOD/SCID-hu mouse model of KSHV infection and to better understand the role of KSHV infection in CD34+ hematopoietic progenitor cells. Specific Aims are: (1) Evaluate, optimize and assess the effects of KSHV and rKSHV.219 de novo infection of CD34+ cells in vitro. Determine if KSHV establishes a latent infection in human CD34+ hematopoietic progenitor cells and characterize the role of viral infection on suppression of hematopoiesis. (2) Determine KSHV cell tropism and tumorigenic potential by immune reconstitution of NOD/SCID mice with CD34+ HPCs infected with KSHV and rKSHV.219. Evaluate and characterize the effect of KSHV/HHV-8 infection on hematopoiesis in vivo. (3) Determine if AZT and IFNalpha induces apoptosis in primary hematopoietic cells infected with rKSHV.219. Develop the NOD/SCID-hu mouse as a pre-clinical model for therapeutic intervention to inhibit KSHV replication.