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. 2025 Aug 8;17(8):1097.
doi: 10.3390/v17081097.

Deficiency of IFNAR1 Increases the Production of Influenza Vaccine Viruses in MDCK Cells

Qi Wang  1 , Tuanjie Chen  1 , Mengru Feng  1 , Mei Zheng  1 , Feixia Gao  1 , Chenchen Qiu  1 , Jian Luo  1 , Xiuling Li  1
Affiliations

Deficiency of IFNAR1 Increases the Production of Influenza Vaccine Viruses in MDCK Cells

Qi Wang et al. Viruses. .

Abstract

Cell culture-based influenza vaccines exhibit comparable safety and immunogenicity to traditional egg-based vaccines. However, improving viral yield remains a key challenge in optimizing cell culture-based production systems. Madin-Darby canine kidney (MDCK) cells, the predominant cell line for influenza vaccine production, inherently activate interferon (IFN)-mediated antiviral defenses that restrict viral replication. To overcome this limitation, we employed CRISPR/Cas9 gene-editing technology to generate an IFN alpha/beta receptor subunit 1 (IFNAR1)-knockout (KO) adherent MDCK cell line. Viral titer analysis demonstrated significant enhancements in the yield of multiple vaccine strains (H1N1, H3N2, and type B) in IFNAR1-KO cells compared to wild-type (WT) cells. Transcriptomic profiling revealed marked downregulation of key interferon-stimulated genes (ISGs)-including OAS, MX2, and ISG15-within the IFNAR1-KO cells, indicating a persistent suppression of antiviral responses that established a more permissive microenvironment for influenza virus replication. Collectively, the engineered IFNAR1-KO cell line provides a valuable tool for influenza virus research and a promising strategy for optimizing large-scale MDCK cell cultures to enhance vaccine production efficiency.

Keywords: CRISPR/Cas9; IFNAR1; MDCK cells; influenza vaccine; interferon.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effects of IFNAR1 knockdown on IAV replication. (A) RT-qPCR showing IFNAR1 knockdown by siRNA. (B) Relative levels of NP expression in cells. (C) Virus titers in supernatants. Canine GAPDH was used as the reference gene. * p < 0.05, *** p < 0.001 (Student’s t-test).
Figure 2
Figure 2
Construction of IFNAR1-KO MDCK cell lines. (A) Schematic map of the gRNA target sites on the genomic regions of IFNAR1. (B) Agarose gel electrophoretic analysis of genomic DNA fragmentation in WT and IFNAR1-KO cells. (C) Sequence alignment analysis results. (D) Relative transcription levels of IFNAR1 in WT and IFNAR1-KO cells. Canine GAPDH was used as the reference gene. *** p < 0.001 (Student’s t-test). (E) Western blot detection of IFNAR1 protein. (F) Cell density curves of WT and IFNAR1-KO cells.
Figure 3
Figure 3
Transcriptome analysis of IFNAR1-KO vs. WT cells. (A) Heatmap of DEGs between IFNAR1-KO and WT cells. (B) Volcano plot shows DEGs between IFNAR1-KO and WT cells. (C) GO analysis of DEGs between IFNAR1-KO and WT cells. (D) Top 20 KEGG pathway analyses of DEGs between IFNAR1-KO and WT cells.
Figure 4
Figure 4
Transcription level of IFN-related genes verified by RT-qPCR. Total RNAs from WT and KO cells were extracted and RT-qPCR was carried out to examine the expression of genes including DDX58, OAS1, OAS2, ISG15, MX2, IFI44L, RTP4 and IFNB1. The experiments were repeated three times independently. The mRNA level of WT cells was set as 1. Canine GAPDH was used as the reference gene. ** p < 0.01, *** p < 0.001 (Student’s t-test).
Figure 5
Figure 5
Comparison of the replication of different virus subtypes in WT and IFNAR1-KO cells. (A) QPCR detection of NP mRNA levels of H1N1 and H3N2 in different cells. (B) Western blot analysis of NP expression. (C) Viral titers of H1N1 in different cells. (D) Viral titers of H3N2 in different cells. (E) qPCR detection of NP mRNA levels of B/Vic in different cells. (F) Viral titers of B/vic in different cells. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t-test).
Figure 6
Figure 6
Transcriptome analysis of IFNAR1-KO vs. WT cells after viral infection. (A) Volcano plot shows DEGs between WT and IFNAR1-KO cells after viral infection. (B) Top 20 KEGG pathway analyses of DEGs between WT and IFNAR1-KO cells after viral infection.
Figure 7
Figure 7
Transcription level of IFN-related genes verified by RT-qPCR. Total RNAs from WT and KO cells after viral infection were extracted and RT-qPCR was carried out to examine the expression of IFN-related genes. The experiments were repeated three times independently. The mRNA level of WT cells was set as 1. Canine GAPDH was used as the reference gene. ** p < 0.01, *** p < 0.001 (Student’s t-test).
Figure 8
Figure 8
Schematic model of IFNAR1 KO to affect IAV replication in MDCK cells.
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