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.

中文翻译：

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猪流行性腹泻病毒不依赖氨肽酶-N进入Vero或猪小肠上皮细胞。

猴细胞系Vero（ATCC CCL-81）通常用于猪流行性腹泻病毒（PEDV）的体外繁殖。但是，在Vero细胞（Vero-APN）上的猪氨肽酶N（pAPN）对应物是否允许PEDV进入仍然有争议。我们发现Vero-APN的内源性表达在Vero细胞的mRNA和蛋白质水平中无法检测到。我们从细胞DNA克隆了含有外显子1至9的部分Vero-APN基因（3340 bp），随后通过CRISPR / Cas9方法生成了两个APN敲除Vero细胞系。PEDV感染两个APN基因敲除Vero细胞的效率与经过或未经过神经氨酸酶处理的Vero细胞相同。稳定表达pAPN的Vero细胞不会增加PEDV的产生。猪空肠上皮细胞中pAPN的siRNA敲低对PEDV感染没有影响。