Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai, PR China.
PLoS One. 2013 Aug 2;8(8):e69997. doi: 10.1371/journal.pone.0069997. Print 2013.
Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3'-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.
猪流行性腹泻病毒（PEDV）在中国和其他亚洲国家的养猪业中造成了严重的经济损失。感染通常会导致仔猪急性、常致命的腹泻。尽管该疾病具有重大影响，但目前尚无操纵病毒基因组的系统，这严重阻碍了对该病毒的研究。我们已经建立了一种基于靶向 RNA 重组的 PEDV 反向遗传学系统，该系统允许修饰编码结构蛋白和 ORF3 蛋白的病毒基因组 3'末端。使用该系统，我们从病毒基因组中完全删除了 ORF3 基因，并表明 ORF3 蛋白对于病毒在体外的复制不是必需的。此外，我们在病毒基因组的两个位置插入了异源基因（即 GFP 和 Renilla 荧光素酶基因），要么作为额外的表达盒，要么作为 ORF3 基因的替代品。我们通过建立一种方便、快速的病毒中和测定法，证明了 GFP 和 Renilla 荧光素酶的表达以及这些病毒的应用。新的 PEDV 反向遗传学系统将能够对结构蛋白和辅助 ORF3 蛋白进行功能研究，并能够合理设计和开发下一代 PEDV 疫苗。
