Generation of porcine PK-15 cells lacking theIfnar1gene to optimize the efficiency of viral isolation

Abstract Because pigs are intermediate or amplifying hosts for several zoonotic viruses, the pig-derived PK-15 cell line is an indispensable tool for studying viral pathogenicity and developing treatments, vaccines, and preventive measures to mitigate the risk of disease outbreaks. However, we must consider the possibility of contamination by type I interferons (IFNs), such as IFNα and IFNβ, or IFN-inducing substances, such as virus-derived double-stranded RNA or bacterial lipopolysaccharides, in clinical samples, leading to lower rates of viral isolation. In this study, we aimed to generate a PK-15 cell line that can be used to isolate viruses from clinical samples carrying a risk of contamination by IFN-inducing substances. To this end, we depleted the IFN alpha and beta receptor subunit 1 ( Ifnar1 ) gene in PK-15 cells using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 method. Treatment of PK-15 cells lacking Ifnar1 with IFNβ resulted in no inhibitory effects on viral infection by a lentiviral vector, influenza virus, and Akabane virus. These results demonstrate that PK-15 cells lacking Ifnar1 could represent a valuable and promising tool for viral isolation, vaccine production, and virological investigations.