Genetic Basis of Drug Resistant Mutants of B Virus

描述(申请人提供)：猴B病毒(BV)是猕猴的正常病原体，与人类单纯疱疹病毒(HSV)关系密切。虽然BV在猕猴中不是一种严重的感染，但当它传播给人类时，它很容易入侵中枢神经系统，如果不治疗，死亡率约为80%。由于BV感染的严重性，该病毒已被列入CDC精选制剂名单，成为潜在的生物恐怖主义制剂。化疗是控制人类BV感染的唯一选择，但人们对各种药物治疗BV的疗效知之甚少。目前对BV感染的治疗基本上是利用已知对HSV安全有效的药物，假设它们对BV也有效，因为病毒是相关的。我们最近测试了一些核苷类似物对BV的疗效。在这些研究过程中，分离出了两个自然产生的耐药变异体(PCVr和GCVr)。在单纯疱疹病毒中，所有与环状核苷耐药相关的突变都发生在病毒胸苷激酶(TK)和/或DNA聚合酶基因中。对这两个BV突变体的TK基因和DNA pol基因进行了测序，结果表明它们与亲本野生型病毒基因的序列完全相同。因此，其他一些病毒基因(S)似乎参与了这些BV分离株的耐药性决定。这项应用将识别导致这些BV分离株耐药的病毒基因(S)。通过共转染的方法，我们将构建重组病毒来定位病毒基因组中包含耐药基因的区域。一旦该区域被定位，DNA测序将识别定义耐药性的准确序列差异。了解参与BV耐药性的基因(S)将使我们能够对相关机制做出一些预测，并为进一步的研究检验这些假说打开大门。所获得的知识也可能最终导致更好地了解BV对有效对抗HSV的药物的相对耐药性，并开发更有效的治疗BV感染的药物。

DESCRIPTION (provided by applicant): Monkey B virus (BV) is a normal pathogen of macaque monkeys that is closely related to human herpes simplex virus (HSV). Although BV is not a serious infection in macaques, when transmitted to humans it readily invades the central nervous system and, left untreated, has a fatality rate of approximately 80%. Due to the severity of BV infections, this virus has been included on the CDC Select Agent list as a potential bioterrorism agent. Chemotherapeutic treatment is the only option for controlling human BV infections, yet little is know about the efficacy of various drugs against BV. Current treatment for BV infections essentially utilizes drugs known to be safe and effective against HSV, the assumption being that they are also effective against BV since the viruses are related. We recently tested the efficacy of a number of nucleoside analogs against BV. In the course of these studies, two naturally occurring drug-resistant variants (PCVr & GCVr) were isolated. In HSV, all mutations conferring resistance to acyclo-nucleosides occur in the viral thymidine kinase (TK) and/or DNA polymerase genes. Sequencing of the TK and DNA pol genes of these two BV mutants revealed that they were identical to the sequence of the parental wild-type virus genes. Thus, some other virus gene(s) appears to be involved in determining the drug resistance of these BV isolates. This application will identify the viral gene(s) that are responsible for the drug resistance of these BV isolates. Using a co-transfection procedure, we will construct recombinant viruses to locate the region of the viral genome containing the drug resistance locus. Once this region has been localized, DNA sequencing will identify the precise sequence difference that defines drug resistance. Knowing the gene(s) involved in drug resistance of BV will allow some predictions to be made as to the mechanism involved and open the door to additional studies to test these hypotheses. The knowledge gained may also eventually lead to a better understanding of the comparative resistance of BV to drugs effective against HSV and to the development of more effective drugs for treatment of BV infections.