Effect of CRISPR-Cas9 mediated knockout of IRF3 gene in BHK-21 cells on immune gene expression and foot-and-mouth disease virus replication

IF 3.5 3区医学 Q3 IMMUNOLOGY

Microbial pathogenesis Pub Date : 2025-09-12 DOI:10.1016/j.micpath.2025.108022

Lekshmi J. Das, Gnanavel Venkatesan, Narayanan Krishnaswamy, Indu Shekhawat, Umapathi Vijayapillai, M. Priyanka, H.J. Dechamma

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引用次数: 0

Abstract

Foot-and-mouth disease (FMD) is an acute highly contagious viral disease of cloven-hoofed animals. Currently, FMD vaccine production and research mainly depend on the BHK-21 cell line. BHK-21 is highly sensitive to FMD virus, however, still a lot of room for improvement that can result in higher antigen yield in vaccine production facilities. IRF3 (Interferon regulatory factor 3) is a key transcription regulatory factor involved in the interferon (IFN) pathway, the immediate antiviral response of the cells. In this study, IRF3 knock-out (KO) BHK-21 cells were established using the CRISPR-Cas9 method. The KO cell line was stable in growth and morphological characteristics, unveiled in growth curve analysis. After infection with FMDV, the viral copy number, virus titer, and plaque forming units (PFU) were significantly increased in KO cells than those in parental BHK-21 (NC) cells. The relative gene expression of type I IFNs and ISGs such as IRF3, IRF7, viperin, Mx1 and ISG15 upon FMDV infection was severely reduced in KO cells. Results preliminarily reveal the role of IRF3 in cellular antiviral immune response, and the IRF3-KO cell line could also serve as a useful tool for FMDV research and vaccine production.

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CRISPR-Cas9介导的敲除BHK-21细胞IRF3基因对免疫基因表达和口蹄疫病毒复制的影响

口蹄疫是偶蹄动物的一种急性高传染性病毒性疾病。目前，口蹄疫疫苗的生产和研究主要依靠BHK-21细胞系。BHK-21对口蹄疫病毒高度敏感，但仍有很大的改进空间，可以提高疫苗生产设施的抗原产量。IRF3（干扰素调节因子3）是参与干扰素（IFN）途径的关键转录调节因子，是细胞的直接抗病毒反应。本研究利用CRISPR-Cas9方法构建了IRF3敲除（KO） BHK-21细胞。生长曲线分析显示，KO细胞株生长稳定，形态特征稳定。感染FMDV后，KO细胞的病毒拷贝数、病毒滴度和斑块形成单位（PFU）明显高于亲本BHK-21 （NC）细胞。在FMDV感染后，KO细胞中I型ifn和isg如IRF3、IRF7、viperin、Mx1、ISG15的相对基因表达量严重降低。结果初步揭示了IRF3在细胞抗病毒免疫应答中的作用，IRF3- ko细胞系也可作为FMDV研究和疫苗生产的有用工具。

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来源期刊

Microbial pathogenesis 医学-免疫学

CiteScore

7.40

自引率

2.60%

发文量

472

审稿时长

56 days

期刊介绍： Microbial Pathogenesis publishes original contributions and reviews about the molecular and cellular mechanisms of infectious diseases. It covers microbiology, host-pathogen interaction and immunology related to infectious agents, including bacteria, fungi, viruses and protozoa. It also accepts papers in the field of clinical microbiology, with the exception of case reports. Research Areas Include: -Pathogenesis -Virulence factors -Host susceptibility or resistance -Immune mechanisms -Identification, cloning and sequencing of relevant genes -Genetic studies -Viruses, prokaryotic organisms and protozoa -Microbiota -Systems biology related to infectious diseases -Targets for vaccine design (pre-clinical studies)