Ji Chun-Miao, Wang Bin, Zhou Jiyong, Huang Yao-Wei
Key Laboratory of Animal Virology of Ministry of Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China.
Key Laboratory of Animal Virology of Ministry of Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China.
Virology. 2018 Apr;517:16-23. doi: 10.1016/j.virol.2018.02.019. Epub 2018 Mar 2.
A monkey cell line Vero (ATCC CCL-81) is commonly used for porcine epidemic diarrhea virus (PEDV) propagation in vitro. However, it is still controversial whether the porcine aminopeptidase N (pAPN) counterpart on Vero cells (Vero-APN) confers PEDV entry. We found that endogenous expression of Vero-APN was undetectable in the mRNA and the protein levels in Vero cells. We cloned the partial Vero-APN gene (3340-bp) containing exons 1 to 9 from cellular DNA and subsequently generated two APN-knockout Vero cell lines by CRISPR/Cas9 approach. PEDV infection of two APN-knockout Vero cells had the same efficiency as the Vero cells with or without neuraminidase treatment. A Vero cells stably expressing pAPN did not increase PEDV production. SiRNA-knockdown of pAPN in porcine jejunum epithelial cells had no effects on PEDV infection. The results suggest that there exists an additional cellular receptor on Vero or porcine jejunal cells independent of APN for PEDV entry.
猴细胞系Vero（ATCC CCL - 81）常用于猪流行性腹泻病毒（PEDV）的体外增殖。然而，Vero细胞上的猪氨肽酶N（pAPN）对应物（Vero - APN）是否赋予PEDV进入细胞的能力仍存在争议。我们发现Vero细胞中Vero - APN的内源性表达在mRNA和蛋白质水平均无法检测到。我们从细胞DNA中克隆了包含外显子1至9的部分Vero - APN基因（3340 bp），随后通过CRISPR/Cas9方法构建了两个APN基因敲除的Vero细胞系。两种APN基因敲除的Vero细胞被PEDV感染的效率与经或未经神经氨酸酶处理的Vero细胞相同。稳定表达pAPN的Vero细胞并未增加PEDV的产量。在猪空肠上皮细胞中通过小干扰RNA（SiRNA）敲低pAPN对PEDV感染没有影响。结果表明，Vero细胞或猪空肠细胞上存在独立于APN的其他细胞受体用于PEDV进入。
