Resistance to pseudorabies virus by knockout of nectin1/2 in pig cells

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.