Gray Wayne L, Mahalingam Ravi
Department of Microbiology and Immunology, Slot 511, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205, USA. :
J Virol Methods. 2005 Dec;130(1-2):89-94. doi: 10.1016/j.jviromet.2005.06.013. Epub 2005 Jul 25.
Simian varicella is a natural varicella-like disease of nonhuman primates. The etiologic agent, simian varicella virus (SVV), is genetically related to varicella-zoster virus (VZV) and SVV infection of nonhuman primates is a useful model to investigate VZV pathogenesis and latency. In this study, we report development of a cosmid-based genetic system to generate SVV mutant viruses. SVV subgenomic DNA fragments (32-38kb) that span the viral genome were cloned into cosmid vectors. Co-transfection of Vero cells with four overlapping cosmid clones representing the entire SVV genome resulted in recombination and generation of infectious virus. SVV mutants were produced by manipulation of one cosmid and substitution into the genetic system. This genetic approach was used to insert a site-specific mutation within the SVV open reading frame 14 which encodes the nonessential glycoprotein C gene. In a subsequent experiment, the green fluorescent protein (GFP) gene was inserted into the SVV genome within ORF 14. These SVV mutants replicate as efficiently as wild-type SVV in cell culture. This cosmid-based genetic system will be useful to investigate the effect of viral mutations on SVV pathogenesis and latency and also to develop and evaluate recombinant varicella vaccines that express foreign antigens.
猴水痘是一种非人类灵长类动物的自然水痘样疾病。其病原体猴水痘病毒（SVV）在基因上与水痘带状疱疹病毒（VZV）相关，非人类灵长类动物的SVV感染是研究VZV发病机制和潜伏的有用模型。在本研究中，我们报告了一种基于黏粒的遗传系统的开发，用于产生SVV突变病毒。跨越病毒基因组的SVV亚基因组DNA片段（32 - 38kb）被克隆到黏粒载体中。用代表整个SVV基因组的四个重叠黏粒克隆共转染Vero细胞，导致重组并产生感染性病毒。通过操纵一个黏粒并将其替换到遗传系统中来产生SVV突变体。这种遗传方法用于在编码非必需糖蛋白C基因的SVV开放阅读框14内插入一个位点特异性突变。在随后的实验中，绿色荧光蛋白（GFP）基因被插入到ORF 14内的SVV基因组中。这些SVV突变体在细胞培养中与野生型SVV一样高效复制。这种基于黏粒的遗传系统将有助于研究病毒突变对SVV发病机制和潜伏的影响，也有助于开发和评估表达外源抗原的重组水痘疫苗。
