Pseudorabies virus (PRV) is a pig pathogen that causes substantial economic losses to the pig industry. Infection of host cells by PRV is mediated by the membrane proteins nectin1 and nectin2, which are presumed to be receptors for PRV infection. Here, we generated nectin1/2 knockout (KO) cells with the aim of establishing a PRV-resistant cell model. Nectin1 and 2 were ablated in PK15 cells by CRISPR/Cas9-mediated gene targeting. PRV infection in either nectin1 or nectin2 KO cells showed a significant reduction in viral growth compared with wild-type (WT) cells. We further simultaneously deleted nectin1 and nectin2 in PK15 cells and found that double KO cells showed no further increase in resistance to PRV compared with single gene-KO cells, despite being more resistant than WT. By investigating the cell entry steps of PRV infection, we found that nectin1 or/and nectin2 KO did not greatly affect virus attachment or internalization to cells but blocked cell-to-cell spread. Our results demonstrate that KO of either nectin1 or nectin2 confers PRV resistance to PK15 cells. This strategy could be applied to establish PRV-resistant pigs with nectin1/2 modifications to benefit the pig industry.

伪狂犬病病毒（PRV）是一种猪病原体，会给养猪业造成巨大的经济损失。PRV感染宿主细胞是由膜蛋白nectin1和nectin2介导的，这些蛋白被认为是PRV感染的受体。在这里，我们生成了nectin1 / 2基因敲除（KO）细胞，目的是建立PRV耐药细胞模型。Nectin1和2通过CRISPR / Cas9介导的基因靶向消融在PK15细胞中。与野生型（WT）细胞相比，nectin1或nectin2 KO细胞中的PRV感染显示病毒生长显着降低。我们进一步同时删除了PK15细胞中的nectin1和nectin2，发现双KO细胞与单基因KO细胞相比，对PRV的耐药性没有进一步增加，尽管比WT耐药。通过研究PRV感染的细胞进入步骤，我们发现nectin1或/和nectin2 KO不会极大地影响病毒对细胞的附着或内在化，但会阻止细胞之间的传播。我们的结果表明，nectin1或nectin2的KO赋予PRV对PK15细胞的抗性。该策略可用于建立具有nectin1 / 2修饰的抗PRV的猪，从而有利于养猪业。