Resistance to pseudorabies virus by knockout of nectin1/2 in pig cells
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ORIGINAL ARTICLE
Resistance to pseudorabies virus by knockout of nectin1/2 in pig cells Yaoqiang Huang1 · Zicong Li1 · Changxu Song1 · Zhenfang Wu1 · Huaqiang Yang1 Received: 27 November 2019 / Accepted: 1 September 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract 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.
Introduction Pseudorabies virus (PRV) is a herpesvirus belonging to the subfamily Alphaherpesvirinae. The natural reservoir hosts of PRV are pigs and wild boars [1], but it has also been reported to infect other farm animals (such as cattle and sheep), pets (such as cats and dogs), birds, and humans [2–4]. PRV infection in pigs can cause respiratory distress, fever, fatal neurological symptoms, and abortions. Adult pigs can survive a PRV infection, but piglets have a high mortality rate, giving rise to considerable economic losses to the pig industry worldwide [5, 6]. The precise mechanism by which PRV infects host cells is not fully understood. Most previous studies have been expanded based on results obtained with other alphaherpesviruses, including herpes simplex viruses 1 and 2 (HSV-1, HSV-2). In alphaherpesvirus infections, the interaction of the viral envelope glycoprotein D (gD) with a host receptor Handling Editor: Graciela Andrei. * Zhenfang Wu [email protected] * Huaqiang Yang [email protected] 1
National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou, China
is crucial for entry of the virus into the host cell [7]. Multiple gD receptors have been identified, including herpesvirus entry mediator (HVEM, renamed HveA), nectin1 (also known as HveC or poliovirus receptor-related 1 [PVRL1]), nectin2 (also known as HveB or poliovirus receptor-related 2 [PVRL2]), and poliovirus receptor (Pvr, also known as HveD or CD155) [8–11]. The receptors involved in
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