MOLECULAR GENETICS OF VARICELLA ZOSTER VIRUS

水痘带状疱疹病毒(VZV)引起水痘和带状疱疹。我们有 开发了一种通过转染组织来产生重组VZV的系统 用四个重叠的VZV粘粒DNA培养细胞。重组VZV 将有助于研究单个VZV基因在感染中的作用。 突变已经被改造成宇宙体，产生VZV突变体 不能表达病毒胸苷合成酶，糖蛋白V 核糖核苷酸还原酶和ORF10基因。病毒已删除，用于 核糖核苷酸还原酶基因的生长速度慢于野生型病毒 在体外，对阿昔洛韦比亲本病毒更敏感。而当 ORF10的HSV-1同源物VP16是HSV-1复制所必需的 细胞培养，VZV中ORF10的缺失不会导致损害 在体外生长。选定的VZV突变体将用于接种几内亚 猪来确定病毒是否已经失去了建立 中枢神经系统的潜伏期。 大肠杆菌β-半乳糖苷酶基因已插入VZV 基因组，由此产生的病毒产生的斑块被X染成蓝色 盖尔。正在给豚鼠接种这种病毒，以确定 能够支持病毒复制和 建立潜伏期。单纯疱疹病毒(HSV)糖蛋白D 编码一种主要中和抗原的(GD)基因已插入到 VZV和由此产生的病毒在表面表达高水平的HSV gD 被感染的细胞。该VZV突变株接种豚鼠的实验研究 导致了针对HSV-1的中和抗体的产生。当 动物们被用活的单纯疱疹病毒挑战，他们发现病毒减少了 与对照动物相比，单纯疱疹病毒感染的严重程度。 表达人Fab片段的两个大肠杆菌组合文库 确认VZV已生成并筛选出中和活性 对抗VZV。

Varicella-zoster virus (VZV) causes chickenpox and herpes zoster. We have developed a system to generate recombinant VZV by transfecting tissue culture cells with four overlapping VZV cosmid DNAs. The recombinant VZV will be useful to study the role of individual VZV genes in infection. Mutations have been engineered into cosmids that yield VZV mutants that are unable to express the viral thymidylate synthetase, glycoprotein V ribonucleotide reductase, and ORF10 genes. Virus deleted for the ribonucleotide reductase gene grows at a slower rate than wild-type virus in vitro and is more sensitive to acyclovir than parental virus. While the HSV-1 homolog of ORF10, VP16, is essential for replication of HSV-1 in cell culture, deletion of ORF10 from VZV does not result in impaired growth in vitro. Selected VZV mutants will be used to inoculate guinea pigs to determine if the viruses have lost the ability to establish latency in the central nervous system. The E. coli beta-galactosidase gene, has been inserted into the VZV genome, and the resultant virus produces plaques that stain blue with X gal. Guinea pigs are being inoculated with this virus to determine the types of cells that are able to support viral replication and establishment of latency. The herpes simplex virus (HSV) glycoprotein D (gD) gene, encoding a major neutralizing antigen, has been inserted into VZV and the resulting virus expresses high levels of HSV gD on the surface of infected cells. Inoculation of guinea pigs with this VZV mutant resulted in development of neutralizing antibodies to HSV-1. When the animals were challenged with live herpes simplex virus they showed reduced severity of herpes simplex infection compared with control animals. Two combinatorial libraries of E. coli expressing human Fab fragments that recognize VZV have been generated and screened for neutralizing activity against VZV.