Department of Microbiology, Harbin Medical University, China.
Intervirology. 2011;54(5):268-75. doi: 10.1159/000321351. Epub 2011 Jan 14.
To evaluate the stability of coxsackievirus B (CVB) genome integrated with the enhanced green fluorescent protein gene (egfp) and provide valuable information for the use of the recombinant CVB variant.
A CVB3 variant expressing eGFP was constructed by insertion of the egfp open-reading frame (ORF) at the 5' end of CVB3 ORF. The recombinant virus CVB3-eGFP was serially passaged in HeLa cells. The deletions in the CVB3-eGFP genome around egfp were examined by reverse transcription polymerase chain reaction and sequencing.
Genomic deletions of CVB3-eGFP could be observed as early as the 2nd passage. Sequencing showed that the genomic deletions caused either viral ORF shifts or partial deletions of the viral VP4 coding sequence. The 6th passage of CVB3-eGFP was checked by plaque assay for eGFP expression. All plaque-like foci showed eGFP expression. eGFP expression was also viewed in HeLa cells infected with plaque-forming viruses.
The insertion of egfp destabilized the CVB3 genome. The genomic deletions led to lethal mutations because of the termination of viral protein synthesis due to viral ORF shift and loss of partial viral gene. These findings imply that experimental data based on CVB integrated with the reporter gene should be interpreted with caution.
评估与增强型绿色荧光蛋白基因（egfp）整合的柯萨奇病毒 B（CVB）基因组的稳定性，为重组 CVB 变体的应用提供有价值的信息。
通过将 egfp 开放阅读框（ORF）插入 CVB3 ORF 的 5'端，构建表达 eGFP 的 CVB3 变体。将重组病毒 CVB3-eGFP 连续传代于 HeLa 细胞。通过反转录聚合酶链反应和测序检测 egfp 周围 CVB3-eGFP 基因组中的缺失。
早在第 2 次传代时就可以观察到 CVB3-eGFP 的基因组缺失。测序显示，基因组缺失导致病毒 ORF 移位或病毒 VP4 编码序列的部分缺失。通过噬斑试验检查第 6 代 CVB3-eGFP 的 eGFP 表达。所有斑状焦点均显示 eGFP 表达。感染形成噬菌斑的病毒的 HeLa 细胞中也观察到 eGFP 表达。
egfp 的插入使 CVB3 基因组不稳定。基因组缺失导致致命突变，因为病毒 ORF 移位和部分病毒基因丢失导致病毒蛋白合成终止。这些发现表明，基于与报告基因整合的 CVB 的实验数据应谨慎解释。
