Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, 72205, USA.
Arch Virol. 2011 May;156(5):739-46. doi: 10.1007/s00705-010-0889-4. Epub 2011 Apr 13.
Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid, and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectious SVV (rSVV-BAC). The mini-F vector sequences flanked by loxP sites were removed by co-infection of Vero cells with rSVV-BAC and adenovirus expressing Cre-recombinase. Recombinant SVV generated using the SVV-BAC genetic system has similar molecular and in vitro replication properties as wild-type SVV. To demonstrate the utility of this approach, a SVV ORF 10 deletion mutant was created using two-step Red-mediated recombination. The results indicate that SVV ORF 10, which encodes a homolog of the HSV-1 virion VP-16 transactivator protein, is not essential for in vitro replication but is required for optimal replication in cell culture.
猿猴水痘病毒（SVV）与人类水痘-带状疱疹病毒密切相关，可导致非人类灵长类动物出现水痘和带状疱疹样疾病。在这项研究中，将微型 F 复制子插入 SVV 粘粒中，通过共转染重叠的 SVV 粘粒，在 Vero 细胞中生成感染性 SVV。SVV 全基因组作为细菌人工染色体（BAC）进行克隆，在大肠杆菌中连续传代可稳定增殖。将 pSVV-BAC DNA 转染至 Vero 细胞可产生感染性 SVV（rSVV-BAC）。通过 rSVV-BAC 和表达 Cre 重组酶的腺病毒共感染 Vero 细胞，可去除两侧带有 loxP 位点的微型 F 载体序列。使用 SVV-BAC 遗传系统产生的重组 SVV 具有与野生型 SVV 相似的分子和体外复制特性。为了证明该方法的实用性，使用两步 Red 介导的重组创建了 SVV ORF10 缺失突变体。结果表明，SVV ORF10 编码 HSV-1 病毒粒子 VP-16 转录激活蛋白的同源物，其对于体外复制并非必需，但对于细胞培养中的最佳复制是必需的。
