Bacterial artificial chromosomes for HSV genomics

描述（由申请方提供）：单纯疱疹病毒（HSV）是一种重要的常见人类病原体。在美国，HSV是非创伤性失明的主要原因，伴随的眼部疾病包括树突状上皮角膜炎、结膜炎和睑缘炎，以及致盲坏死性基质角膜炎。此外，HSV引起唇疱疹、生殖器溃疡，并且是病毒性脑炎的主要原因。使用定义的遗传改变已经成为许多领域的标准，以深入了解基因的功能。这种遗传学方法通常很麻烦，产生基因改变的生物体通常比实际分析遗传功能本身要耗时得多。因此，需要应用新技术来加速突变体的产生。对于疱疹病毒来说，情况确实如此，其大的DNA基因组虽然可以通过同源重组进行反向遗传学，但使确定突变体的产生复杂化。 该提案的目标是利用细菌人工染色体（BAC）技术的力量，使HSV适合细菌遗传方法。对于其他一些疱疹病毒，BAC技术允许在不到一周的时间内产生几个突变体。这与允许在2-3个月内产生单个突变体的当前HSV重组方法相反。因此，将为HSV-1的三种主要实验室毒株和HSV-2的两种毒株中的每一种生成BAC。在将其用作诱变模板之前，将从这些BAC中的每一种再生病毒，并将其表型与其原始亲本菌株仔细比较。这将确保此类病毒如BAC的繁殖不会固有地引起任何这些病毒的基因表达谱、毒力或发病机制的任何不确定的变化。一旦建立和表征，这些试剂将被保藏在ATCC，以允许所有研究人员使用这种强大的技术。这项工作将代表该领域的一个重大进展，允许以标准化的方式快速产生HSV突变体，用于基础研究，以及疫苗，抗肿瘤剂和基因递送载体的开发。该提案的成功结果与R 03计划目标和NEI角膜疾病计划的战略目标一致，将对所有从事HSV感染及其致盲后遗症研究的实验室产生重大影响。

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV) is a significant and common human pathogen. HSV is a leading cause of nontraumatic blindness in the US with an accompanying ocular diseases ranging from dendritic epithelial keratitis, conjunctivits and blepharitis, to blinding necrotizing stromal keratitis. In addition, HSV causes cold sores, genital sores, and is a leading cause of viral encephalitis. The use of defined genetic alterations has become standard in many fields to gain insight into the functions of genes. Such genetic approaches -are often cumbersome, with the generation of genetically altered organisms often being far more time-consuming than the actual analysis of genetic function itself. There is, therefore, a need for the application of novel technologies to speed up the generation of mutants. This is certainly the case for the herpes viruses whose large DNA genomes, although amenable to reverse genetics by homologous recombination, complicate the generation of defined mutants. The goal of this proposal is to harness the power of bacterial artificial chromosome (BAC) technology to make HSV amenable to bacterial genetic approaches. For some other herpes viruses, BAC technology allows the generation of several mutants in less than a week. This is in contrast to current HSV recombination methodologies that allow generation of a single mutant in 2-3 months. BACs will therefore be generated for each of the three major laboratory strains of HSV-1 and two strains of HSV-2. Prior to their use as templates for mutagenesis, viruses will be regenerated from each of these BACs and their phenotypes compared carefully to their original parental strains. This will ensure that the propagation of such viruses as BACs does not inherently cause any undefined changes in the gene expression profiles, virulence, or pathogenesis of any of these viruses. Once established and characterized these reagents will be deposited with ATCC to allow all researchers access to this powerful technology. This work will represent a major advance in the field in allowing the rapid generation of HSV mutants in a standardized fashion for basic research, as well as for vaccine, anti-tumor agent, and gene delivery vector development. The successful outcome of this proposal, consistent with the R03 program objectives and strategic goals of the NEI's Corneal Diseases Program, will have a major impact on the research of all laboratories working on HSV infections and their blinding sequelae.