Targeting strategy and genotype for knockout of chicken IFN-α/β receptor (IFNAR1) and IFN-λ receptor (IFNLR1).

a, Guide RNAs designed to target the coding region within exon 1 of the IFNAR1 chain of the IFN-α/β receptor and the epithelium-specific chain (IFNLR1) of the IFN-λ receptor. For IFNAR1, the INDEL was pre-designed using CRISPR-mediated HDR technology. The IFNAR1 KO clone has 7 bp deletion and produces a stop codon after 21 bp. b, For IFNLR1 positive PGCs clones, the clone with the maximum deletion (28 bp) was selected as it is suitable to design IFNLR1 KO specific PCR genotyping assay and produce a stop codon after 87 bp. c, TaqMan genotyping assay for IFNAR1. d, PCR genotyping assay for IFNLR1.

Growth, and successful generation of genetically modified chickens.

a, Body weight development of WT, IFNLR1−/− and IFNAR1−/− chickens. b, Western blot confirms successful knockout of IFNAR1: WT, IFNAR1+/−, and IFNAR1−/− chicken embryonic fibroblast cells (n = 3) were cultured in a 6-well plate and stimulated with recombinant IFN-α (500 U/ml) for 12 hours. For the control groups, CEF cells were treated with CEF medium without recombinant IFN-α. After stimulation, the control and treatment groups’ CEF cells were trypsinized and collected for an SDS-Gel and Western Blot experiment. The results demonstrate functional loss of type I IFN receptor (IFNAR1) in CEF IFNAR1−/− cells treated with recombinant IFN-α, as the Mx protein (75 kDa) was observed solely in the CEF WT and IFNAR1+/− cells treated with recombinant IFN-α. c, WSN33 viral titration in WT and IFNAR1−/− embryo pretreated with recombinant IFN-α. ED10 chicken embryos (blue columns) were stimulated with 1.5 × 105 U of recombinant IFN-α in 100 μl PBS 12 h before and at the time of infection. As controls (black columns) did not receive IFN-α stimulation. All the groups of embryos were infected with 1000 FFU of WSN33, and viral load in the allantoic fluid was analyzed 24 h post-infection by titration on MDCK cells. Number of embryos, for the control groups (WT = 4, IFNAR1−/− = 4), and treatment groups (WT = 4, IFNAR1−/− = 4). Significance was calculated by one-way ANOVA. Statistically different groups (P ≤ 0.05) are indicated with different superscript letters (a, b, c). d, RT-PCR confirms functional loss of IFNLR1 in homozygous embryos: expression analysis of IFN-λ receptor (IL-28Rα) was studied in different tissues of 18-day-old embryos challenged with H3N1 to ensure induced expression of IL-28Rα. RNA was isolated via the Trizol method after the genotyping of embryos. cDNA was transcribed via GoScript Transcription Mix, Random Primers. PCR was performed with β-actin primers as control and IL-28Rα primers to check the presence of IL-28Rα receptors in tissues. Nuclease-free water (NFW) was used as a negative control for both. A 1.5% tris-borate-EDTA (TBE) was used to visualize β-actin amplicon at 300 bp and 2% TBE to visualize 108 bp for IL-28Rα.

Flow cytometry analysis of PBMCs and splenocytes of WT, IFNLR1−/− and IFNAR1−/− in one-month-old chickens.

a, PBMCs and b, splenocytes were isolated and analyzed for differential immune cell populations, including monocytes, B cells, αβ TCR2,3+ or γδ TCR1 + T cells, and associated CD4 and CD8 T cells subpopulations. One-way ANOVA was used to analyze the difference between the groups (*, P ≤ 0.05). Number of animals for the analysis of PBMCs (WT = 5, IFNLR1−/− = 5, IFNAR1−/− = 4), and analysis of splenocytes (WT = 7, IFNLR1−/− = 5, IFNAR1−/− = 4).

Reduced IFNLR1−/− and IFNAR1−/− chicken ability to produce IgM and IgY in response to immunization.

Five-week-old chickens were immunized via intramuscular injection with 300 µg KLH, mixed 1:1 with incomplete Freund’s adjuvant. A booster injection of 300 µg KLH, mixed 1:1 with Freund’s incomplete adjuvant, was administered two weeks after the initial immunization. Blood plasma samples were collected from all groups before immunization (day 0), on day 5 post-primary immunization (PP), and on days 3 and 5 post-booster immunization (PB). a-d, Total concentration of IgM, IgY, and KLH antigen-specific IgM and IgY levels were assessed using ELISA. Data are presented as mean and SEM of at least 6 birds per genotype, with the same animals, tracked over time. Statistical differences between groups are indicated by asterisks (*, P ≤ 0.05). # Indicate significant difference (P ≤ 0.05) for IFNAR1−/− vs. WT and IFNLR1−/−.

Overall similar splenic TCRβ repertoire across genotypes with reduced TRBV2-1 responses in KLH-immunized IFNAR1−/− chickens.

TCRβ CDR3 clonotypes in IFNAR1−/−, IFNLR1−/−, and WT animals in the steady state (CTR) or at day 5 post-KLH booster immunization. a, CDR3 Spectratype; b, Vβ gene usage; c, Counts of the 20 most prevalent 3-mers within the top 100 TRBV2-1 clonotypes for each sample, with individual samples indicated by black boxes. Mean ± SD.

Viral titration, IFN-α/β concentration, IFN-λ, and Mx mRNA expression in WT, IFNLR1−/− and IFNAR1−/− embryos.

a, embryonic day 11 chicken embryos were infected with 1000 FFU of influenza A virus strains (WSN33/H3N1/H9N2) and IBV- Beaudette strain, and allantoic fluid and chorioallantois membrane (CAM) were sampled 24 hours post-infection. The viral titers of WSN33, H3N1, and H9N2 were quantified by titration on MDCK cells. For the IBV, qPCR was used to measure the viral titer in the CAM, and the housekeeping gene 28S was used to normalize target gene expression. b, IFN-α/β concentration was quantified by titration of allantoic fluids on CEC-32 #511 cells. c, Relative mRNA expression of IFN-λ in the CAM. d, Relative mRNA expression of Mx in the CAM. For the qPCR study, RNA was isolated by Trizol and reverse transcripted with GoScript Transcription Mix, Random Primers, and qPCR was done via Go Taq. The WT control group was used as a calibrator for Mx and IFN-λ expression, and the housekeeping gene 18S was used to normalize target gene expression. Relative quantification of the target gene was performed using the 2^ΔΔCT method. A no-template control was also included. Statistical differences between groups are indicated by asterisks (*), with significance at P ≤ 0.05.

In vivo challenge of WT, IFNLR1−/− and IFNAR1 −/− hens with a low pathogenic avian influenza A virus (H3N1).

a, Chickens’ body weight (kg) was measured before the viral challenge and at the end of the experiment (date of animal euthanization). b, Probability of survival in the challenged hens. c-d, H3N1 titer in the tracheal and cloacal swabs using qPCR. The IFNAR1 hens were sampled on day 2 post-infection. WT and IFNLR1−/− hens were sampled on day 3 and days 5 to 7 post-infection based on the days of euthanization. The housekeeping gene 28S was used to normalize target gene expression. e, Relative mRNA expression of IFN-λ in the spleen. f, Plasma IFN-α/β concentration was quantified by titration on CEC-32 #511 cells. g, Scoring of lesions in the reproductive tract in all in vivo H3N1 challenged groups. The scoring is on a scale of 0-3, where 0 indicates normal and 3 maximum severities regarding lymphoplasmacellular salpingitis with mucosa atrophy. The IFNAR1−/− hens were euthanized and scored on day 2 post-infection. WT and IFNLR1−/− hens were euthanized and scored on days 5 to 7 post-infection based on the days of euthanization. h, Histological assessment of the reproductive tract on day 5 post infection. WT control: normal histological appearance of the infundibulum and magnum; WT H3N1: moderate acute inflammation of the infundibulum and magnum with hyperemia, edema, loss of epithelial cells (arrowheads), and perivascular infiltration of lymphocytes (arrow); IFNLR1−/−: milder acute inflammation of the infundibulum and magnum with hyperemia, edema, and loss of epithelial cells in the magnum (arrowhead). Hematoxylin-eosin (HE) stain; 100x, respectively. Statistical differences between groups are indicated by asterisks (*, P ≤ 0.05).

Schematic representation of H3N1 host cell interaction and IFN signaling pathway in WT and IFNAR1−/− at day 2 post-H3N1 challenge.

a, Immune signaling pathway in WT epithelial cells during an H3N1 viral infection. The cleaved hemagglutinin (HA) proteins of H3N1 bind to sialic acid receptors on the surface of epithelial cells, facilitating viral entry through endocytosis (1). The viral RNA inside the endosome is recognized by toll-like receptors (TLRs) (2), leading to the activation of the MYD88 signaling pathway (3) and melanoma differentiation-associated protein 5 (MDA5) and/or laboratory of genetics and physiology 2 (LGP2) (4). In the MDA5/LGP2 pathway, mitochondrial antiviral-signaling proteins (MAVS) are activated. The activation of MYD88 leads to the recruitment of IRAK1/4 and TRAF6/3, which then stimulate the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interferon regulatory factor 7 (IRF7) and activating protein 1 (AP-1) (5). These transcription factors induce the production of pro-inflammatory cytokines and type I and type III IFNs (6). Type I IFN (IFN-α/β) binds to its receptors (IFNAR1/2) by autocrine and paracrine signaling (7). This binding activates the JAK-STAT pathway, leading to the phosphorylation of signal transducers and activators of transcription (STATs). These STATs translocate to the nucleus and activate the transcription of interferon-stimulated response elements (ISREs). The activation of ISREs drives the expression of interferon-stimulated genes (ISGs), establishing a robust antiviral state that effectively halts viral replication and spread within the cell (8). Similarly, type III IFN (IFN-λ) contributes to the antiviral state by binding to its receptors (IL-28Rα /IL-10Rβ) and activating the JAK-STAT pathway in an autocrine or paracrine manner (9). The immune response is tightly regulated by negative feedback mechanisms, including suppressor of cytokine signaling (SOCS1) and Src Homology 2-containing Phosphatase 2 (SHP2), which inhibit further production of IFNs and prevent excessive inflammatory responses (10). This response ensures that any initial viral replication is quickly suppressed, protecting the cell from significant infection and excessing immune responses that could lead to cytokine storm. The viral replication is significantly halted since the cells are in an antiviral state. b, Immune signaling pathway in IFNAR1−/− epithelial cells. Virus entry to the cell, recognition of viral RNA by the host cell, and induction of pro-inflammatory cytokines and IFNs happen in a mechanism similar to the WT cell (steps 1-6). However, without IFNAR1, IFN-α/β cannot bind to its receptors, disrupting downstream signaling and feedback inhibition, leading to unregulated IFN production and poor antiviral state (7). Higher IFN-λ is produced to compensate for the low antiviral state but cannot compensate for the absence of IFN-α/β (8). This makes the infected and neighboring cells easy targets for the H3N1 virus that can replicate quickly without resistance. Once inside the cell in the endosome, the viral RNA is released and transported into the nucleus for replication and transcription (9). Newly formed viral particles assemble, bud off, and are released to infect additional cells (10). In the absence of IFNAR1 and an antiviral state, the autocrine feedback loop mediated by SOCS1 and SHP2 is disrupted, and thus, the production of IFN-α/β is continuously increasing (11). The extensive pro-inflammatory cytokines and IFN production exacerbate inflammatory responses and cell death.