Deciphering interferon functions in avian influenza using receptor knockout models in the natural host
- TUM School of Life Sciences, Weihenstephan, Department of Molecular Life Sciences, Reproductive Biotechnology, Technical University of Munich, Germany
- Department of Veterinary Sciences, Ludwig-Maximilians-Universität München, Germany
- Institute of Virology, Freie Universität Berlin, Germany
- Bavarian Animal Health Service, Department of Pathology, Germany
- Clinic for Poultry, University of Veterinary Medicine Hannover, Germany
- EW GROUP GmbH, Germany
- Center for Infection Prevention (ZIP), Technical University of Munich, Germany
Figures
Figure 1
Targeting strategy and genotype for knockout (KO) 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 homology-directed repair (HDR) technology. The IFNAR1 KO clone has a 7 bp deletion and produces a stop codon after 21 bp. (b) For IFNLR1-positive primordial germ cell (PGC) 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. Labeled molecular weight markers are shown in panel d.
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Figure 1—source data 1
Labeled uncropped gel image for the IFNLR1 PCR genotyping assay shown in Figure 1d, indicating the relevant lanes and expected bands.
- https://cdn.elifesciences.org/articles/107855/elife-107855-fig1-data1-v1.zip
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Figure 1—source data 2
Original uncropped gel image file for the IFNLR1 PCR genotyping assay displayed in Figure 1d.
- https://cdn.elifesciences.org/articles/107855/elife-107855-fig1-data2-v1.zip
Figure 2
Growth and successful generation of genetically modified chickens.
(a) Body weight development of WT, IFNLR1−/−, and IFNAR1−/− chickens (n=12 per genotype). Data are presented as mean ± SEM. (b) Western blot confirms successful knockout of IFNAR1: WT, IFNAR1+/−, and IFNAR1−/− chicken embryonic fibroblast cells (n=3) were cultured in a six-well plate and stimulated with recombinant IFN-α (500 U/ml) for 12 hr. For the control groups, chicken embryo fibroblast (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-PAGE 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. β-Actin was used as loading control and detected at 42 kDa. (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 hr 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 hr post-infection by titration on Madine-Darby canine kidney (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. Complementary DNA (cDNA) was transcribed via GoScript Transcription Mix and 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 a 2% TBE gel to visualize the 108 bp IL-28Rα amplicon.
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Figure 2—source data 1
Labeled uncropped western blot and RT-PCR gel images for Figure 2b and d, indicating the relevant lanes, treatments, bands, and molecular weight markers.
- https://cdn.elifesciences.org/articles/107855/elife-107855-fig2-data1-v1.zip
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Figure 2—source data 2
Original uncropped image files for the western blot and RT-PCR gel analyses displayed in Figure 2b and d.
- https://cdn.elifesciences.org/articles/107855/elife-107855-fig2-data2-v1.zip
Figure 3 with 2 supplements
Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) and splenocytes of WT, IFNLR1−/−, and IFNAR1−/− in 1-month-old chickens.
(a) PBMCs and (b) splenocytes were isolated and analyzed for differential immune cell populations, including monocytes/macrophages, B cells, αβ TCR2,3+ or γδ TCR1+T cells, and associated CD4 and CD8 T cell subpopulations. Data are presented as mean ± SEM. 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).
Figure 3—figure supplement 1
Representation of the gating strategy to quantify populations of peripheral blood mononuclear cells (PBMCs) isolated from 1-month-old chicks.
Steps 1, 2, and 3 are common for all. All individual cell analyses were gated from step 3 except for the subpopulation of γδ and αβ T cells, which were gated from step 4. (a) Detection of monocytes and B cells. (b) Detection of γδ, αβ T cells, and their CD4 and CD8 subpopulations. (c) Secondary antibody control used for this experiment.
Figure 3—figure supplement 2
Microbiome analysis of the cecal contents from 1-month-old IFNLR1−/− chicks and their WT siblings.
(a) Rarefaction curves of all samples show that sequencing depth was sufficient. (b) A phylogram based on Ward’s minimum variance method shows the hierarchical clustering of samples. The sample distance is shown on the x-axis. The IFNLR1−/− chicks are abbreviated as L, and their WT siblings as WL. (c) Multidimensional scaling plots of microbial profiles. Groups were compared pairwise. The scale bar indicates the distance between samples (d=0.1 describes 10%). (d) Richness as an index for α-diversity. Means are displayed as bold bars. (e) Taxonomic differences between the groups at the phylum level. Means are indicated as bold bars. The number at the bottom shows the number of observations in the groups.
Figure 4 with 3 supplements
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 keyhole limpet hemocyanin (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 2 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 enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± 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). # Indicates significant difference (p≤0.05) for IFNAR1−/− vs. WT and IFNLR1−/−.
Figure 4—figure supplement 1
In experiment 2, 5-week-old WT, IFNLR1−/−, and IFNAR1−/− chicks were immunized via intramuscular injection with 300 µg keyhole limpet hemocyanin (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 2 weeks after the initial immunization. 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). The total concentration of IgM, IgY, and levels of KLH antigen-specific IgM and IgY were assessed using enzyme-linked immunosorbent assay (ELISA). Peripheral blood mononuclear cells (PBMCs) were isolated on day 3 post-PP, and splenocytes were isolated on day 5 PB for fluorescence-activated cell sorting (FACS) analysis of immune and MHC-positive cells. T cells of the spleen were used to study T cell receptor (TCR) repertoire. At least 6 animals per group were included.
Figure 4—figure supplement 2
Flow cytometry analysis of immune cells and their MHC-positive subpopulations in the blood and spleen of WT, IFNLR1−/−, and IFNAR1−/− chicks immunized with keyhole limpet hemocyanin (KLH).
(a) Peripheral blood mononuclear cells (PBMCs) were isolated at day 3 post-primary immunization and analyzed for differential immune cell populations, including monocytes, B cells, αβ TCR2,3+ or γδ TCR1+T cells, MHCI, MHCII, MHCII+B cells, and MHCII+monocytes. (b) Splenocytes were isolated at day 5 post-booster immunization and analyzed for differential immune cell populations and associated MHC subpopulations. Data are presented as mean ± SEM of 6 birds per genotype. One-way ANOVA was used to analyze the difference between the groups (*, p≤0.05).
Figure 4—figure supplement 3
Representation of gating strategy quantifying immune cells and their MHC-positive subpopulations.
Steps 1 and 2 are common for all. All individual cell analyses were gated from step 2 except for MHCII+monocytes. (a) Immune cells and their subpopulation positive for MHCII. (b) Fluorescence minus one (FMO) controls and secondary antibody control were used for this experiment.
Figure 5 with 3 supplements
Overall similar splenic TCRβ repertoire across genotypes with reduced TRBV2-1 responses in keyhole limpet hemocyanin (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. Data are presented as mean ± SD.
Figure 5—figure supplement 1
Relative frequency of Vα-Jα genes.
Mean V-J pairing in TCRα CDR3s in IFNAR1−/−, IFNLR1−/−, and WT animals in the steady state (CTR) or at day 5 post-keyhole limpet hemocyanin (KLH) booster immunization. Data are presented as mean values.
Figure 5—figure supplement 2
Relative frequency of Vβ-Jβ genes.
Mean V-J pairing in TCRβ CDR3s in IFNAR1−/−, IFNLR1−/−, and WT animals in the steady state (CTR) or at day 5 post-keyhole limpet hemocyanin (KLH) booster immunization. Data are presented as mean values.
Figure 5—figure supplement 3
Relative frequency distribution of TCRβ CDR3s.
Distribution of CDR3 clonotypes color-coded by their relative frequencies in IFNAR1−/−, IFNLR1−/−, and WT animals in the steady state (CTR) or at day 5 post-keyhole limpet hemocyanin (KLH) booster immunization.
Figure 6 with 2 supplements
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 chorioallantoic membrane (CAM) were sampled 24 hr post-infection. The viral titers of WSN33, H3N1, and H9N2 were quantified by titration on Madine-Darby canine kidney (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-transcribed with GoScript Transcription Mix and Random Primers, and qPCR was done via GoTaq. 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. Data are presented as mean ± SEM. Individual symbols represent individual embryos; the number of embryos varies by genotype and virus group and is shown by the individual symbols in each panel. Statistical differences between groups are indicated by asterisks (*, p≤0.05).
Figure 6—figure supplement 1
Design of experiment 3, 11-day-old embryos were infected with 1000 FFU of four distinct viral strains: a laboratory-adapted human influenza A virus (H1N1), two low pathogenic avian influenza A virus strains (H3N1 and H9N2), and infectious bronchitis virus (IBV)-Beaudette strain virus.
Allantoic fluid and chorioallantoic membrane (CAM) were sampled 24 hr post-infection. The experiment was performed blindly, and the genotype of embryos was determined, and only WT and homozygous embryos were selected for further analysis. The viral titers of WSN33, H3N1, and H9N2 were quantified by titration on Madine-Darby canine kidney (MDCK) cells. For the IBV, qPCR was used to measure the viral titer in the CAM. Relative mRNA expression of Mx and IFN-λ were studied in the CAM using qPCR. IFN-α/β concentration was quantified by titration of allantoic fluids on CEC-32 #511 cells. The probability of survival in the challenged groups was also investigated.
Figure 6—figure supplement 2
The probability of survival in WT, IFNLR1−/−, and IFNAR1−/− embryos.
On embryonic day 11, chicken embryos were infected with 1000 FFU of influenza A virus strains (WSN33/H3N1/H9N2) and infectious bronchitis virus (IBV)-Beaudette strain, and the probability of survival in the challenged groups was recorded. Statistical differences between groups are indicated by asterisks (*, p≤0.05).
Figure 7 with 1 supplement
In vivo challenge of WT, IFNLR1−/−, and IFNAR1 −/− hens with a low pathogenic avian influenza A virus (H3N1).
(a) Probability of survival in the challenged hens. (b) Chickens' body weight (kg) was measured before the viral challenge and at the end of the experiment (date of animal euthanization). (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 5–7 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. Comparisons were designed to represent equivalent phases of disease progression. For panel e, IFNAR1−/− hens at day 2 (high viral titer) were compared to WT and IFNLR1−/− hens at day 5 (comparable high titer). (f) Plasma IFN-α/β concentration was quantified by titration on CEC-32 #511 cells. For panel f, IFNAR1−/− hens (day 2, high titer) were compared to WT and IFNLR1−/− hens at day 3 (low titer) and days 5–7 (high titer), reflecting distinct infection phases. (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 severity regarding lymphoplasmacytic 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 5–7 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; original magnification, ×100. Scale bars, 100 µm. For panels with error bars, data are presented as mean ± SEM. Seven infected hens and three non-infected control hens were included per genotype. Individual symbols represent individual hens sampled at the indicated time points. Statistical differences between groups are indicated by asterisks (*, p≤0.05).
Figure 7—figure supplement 1
In experiment 4, 27-week-old WT, IFNLR1−/−, and IFNAR1−/− hens were challenged with H3N1 avian influenza (106 FFU) in 0.2 ml PBS per bird and distributed via nasal and tracheal routes.
Seven birds were used for the infection experiment, and three others were used as negative controls per group. The IFNAR1−/− hens showed severe sickness symptoms and were euthanized at day 2 post-infection, whereas WT, IFNLR1−/− hens showed similar symptoms and were euthanized in the period between days 5 and 7 post-infection. Birds were monitored daily for clinical symptoms. Tracheal and cloacal swabs were collected to analyze the viral RNA loads by qPCR. Plasma was collected to estimate IFN-α/β concentration through luciferase assay. The spleen was collected for an immune-related gene expression study through qPCR. The female reproductive scoring was also studied to evaluate the impact of type I and type III IFNs on H3N1 pathogenesis.
Figure 8 with 1 supplement
Schematic representation of H3N1 host cell interaction and interferon (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 excessive 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.
Figure 8—figure supplement 1
Relative mRNA expression of various pro- and anti-inflammatory cytokines (a), interferon-stimulated genes (b), and interferon signaling inhibitors (c) in the spleen of H3N1 challenged hens.
For the qPCR study, RNA was isolated by TRIzol and reverse-transcribed with GoScript, and qPCR was done via GoTaq. The WT control group was used as a calibrator for the expression of all the studied genes, and the housekeeping gene 18S was used to normalize target gene expression. Relative quantification of the studied gene was performed using the 2^ΔΔCT method. A no-template control was also included. The IFNAR1−/− hens were euthanized on day 2 post-infection. WT and IFNLR1−/− hens were euthanized on days 5–7 post-infection based on the days of euthanization. Only spleen samples collected on day 5 post-infection were included for WT and IFNLR1−/− hens for the gene expression study. Data are presented as mean ± SEM; n=3 hens per genotype. Individual symbols represent individual hens. Statistical differences between groups are indicated by asterisks (*, p≤0.05).
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Gallus gallus) | IFNAR1 | NCBI Gene | GeneID: 395665 | Target gene encoding the type I IFN receptor chain (IFNAR1) studied in this work. |
| Gene (Gallus gallus) | IFNLR1; IL-28Rα | NCBI Gene | GeneID: 419694 | Target gene encoding the epithelium-specific type III IFN receptor chain (IFNLR1) studied in this work. |
| Strain, strain background (Gallus gallus) | Lohman selected leghorn chickens | Lohman | SPF hatching eggs used to derive animals; adult in vivo H3N1 challenge experiments used hens; housed at TUM Animal Research Center. | |
| Genetic reagent (Gallus gallus) | IFNAR1−/− chicken line | This paper | Newly generated CRISPR/HDR IFNAR1 knockout line; 7 bp deletion causing a stop codon after 21 bp; maintained at Technical University of Munich; The line is available upon reasonable request from the Reproductive Biotechnology, TUM School of Life Sciences, Technical University of Munich | |
| Genetic reagent (Gallus gallus) | IFNLR1−/− chicken line | This paper | Newly generated CRISPR/Cas9 IFNLR1 knockout line; 28 bp deletion causing a stop codon after 87 bp; maintained at Technical University of Munich; The line is available upon reasonable request from the Reproductive Biotechnology, TUM School of Life Sciences, Technical University of Munich | |
| Genetic reagent (Gallus gallus) | IFNAR1 knockout PGC clone | This paper | Primordial germ cell clone carrying the IFNAR1 knockout allele used to generate germline chimeras. PGC-derived material was generated from male PGCs. | |
| Genetic reagent (Gallus gallus) | IFNLR1 knockout PGC clone | This paper | Primordial germ cell clone carrying the IFNLR1 knockout allele used to generate germline chimeras. PGC-derived material was generated from male PGCs. | |
| Cell line (Gallus gallus) | Chicken embryonic fibroblast cells, CEFs | This paper | Primary cultures derived from ED10 embryos; genotyped as WT, IFNAR1+/− or IFNAR1−/− before IFN-α stimulation and western blot analysis. | |
| Cell line (Coturnix coturnix) | CEC-32 #511 IFN reporter cells | Quail reporter cells expressing firefly luciferase under control of the chicken Mx promoter; maintained with G418 (250 µg/ml). | ||
| Cell line (Canis lupus familiaris) | MDCK cells | Madine-Darby canine kidney cells used for influenza virus titration; laboratory stock. | ||
| Transfected construct (Gallus gallus) | PX458-Cas9-2A-eGFP-IFNAR1-sgRNA | This paper | Plasmid: PX458 | PX458-Cas9/eGFP backbone carrying IFNAR1-targeting sgRNA; sgRNA sequence provided in Supplementary file 1a. |
| Transfected construct (Gallus gallus) | PX458-Cas9-2A-eGFP-IFNLR1-sgRNA | This paper | Plasmid: PX458 | PX458-Cas9/eGFP backbone carrying IFNLR1-targeting sgRNA; sgRNA sequence provided in Supplementary file 1a. |
| Strain, strain background (Influenza A virus) | WSN/33 (H1N1) | Supplementary file 1d | Virus strain used for in ovo challenge; titer 4500 U/µl; infection dose 1000 U/100 µl. | |
| Strain, strain background (Influenza A virus) | A/Chicken/Belgium/460/2019 (H3N1) | Supplementary file 1d | Low pathogenic avian influenza strain used for in ovo and in vivo challenge; infection dose 1000 U/100 µl for in ovo experiments. | |
| Strain, strain background (Influenza A virus) | A/chicken/Saudi Arabia/CP7/1998 (H9N2) | Supplementary file 1d | Low pathogenic avian influenza strain used for in ovo challenge; infection dose 1000 U/100 µl. | |
| Strain, strain background (Infectious bronchitis virus) | IBV-Beaudette strain | Supplementary file 1d | Avian gamma coronavirus strain used for in ovo challenge; infection dose 1000 U/100 µl. | |
| Antibody | Anti-human MxA (mouse monoclonal) | Merck (Cat# MABF938) | Clone: M143 | Primary antibody for western blot detection of chicken Mx protein (75 kDa). |
| Antibody | Anti-chicken β-actin (mouse monoclonal) | Thermo Fisher Scientific (MA5-15739) | Clone: BA3R | Primary antibody for western blot loading control (42 kDa). |
| Antibody | HRP-conjugated anti-mouse IgG (donkey polyclonal) | Thermo Fisher Scientific (Cat# 715-035-151) | Secondary antibody for western blot detection. | |
| Antibody | Anti-chicken TCRγδ-BIOT (mouse monoclonal) | Biozol (Cat# SBA-8230-08) | Clone: TCR-1 | Flow cytometry (0.625 µg/ml); Supplementary file 1e. |
| Antibody | Anti-chicken TCRαβ/Vβ1-BIOT (mouse monoclonal) | Biozol (Cat# SBA-8240-08) | Clone: TCR-2 | Flow cytometry (2.5 µg/ml); Supplementary file 1e |
| Antibody | Anti-chicken TCRαβ/Vβ2-BIOT (mouse monoclonal) | Biozol (Cat# SBA-8250-08) | Clone: TCR-3 | Flow cytometry (2.5 µg/ml); Supplementary file 1e |
| Antibody | Anti-chicken Bu1-FITC (mouse monoclonal) | Biozol (Cat# SBA-8395-02) | Clone: AV20 | Flow cytometry (2.5 µg/ml); Supplementary file 1e |
| Antibody | Anti-chicken KUL01_UNLAB (mouse monoclonal) | Biozol (Cat# SBA-8420-01) | Clone: KUL01 | Flow cytometry (2.5 µg/ml); Supplementary file 1e |
| Antibody | Anti-chicken CD8α_Pacific Blue (mouse monoclonal) | Biozol (Cat# 8220-26) | Clone: CT-8 | Flow cytometry (0.625 µg/ml); Supplementary file 1e |
| Antibody | Anti-chicken CD8β_UNLAB (mouse monoclonal) | Biozol (Cat# 8280-01) | Clone: EP42 | Flow cytometry (0.625 µg/ml); Supplementary file 1e |
| Antibody | Anti-chicken CD4_FITC (mouse monoclonal) | Biozol (Cat# 8210-02) | Clone: CT-4 | Flow cytometry (0.625 µg/ml); Supplementary file 1e |
| Antibody | Anti-mouse IgG2a_PE (rat monoclonal) | Biozol (Cat# 1155-09) | Clone: SB84a | Secondary antibody for flow cytometry (0.125 µg/ml); Supplementary file 1e |
| Peptide, recombinant protein | Streptavidin_APC | VWR (Cat# 20-4317-U500) | Flow cytometry detection reagent for biotinylated antibodies (0.2 µg/ml); Supplementary file 1e. | |
| Antibody | Anti-mouse IgG (H+L)-APC (goat polyclonal) | Biozol (Cat# SBA-1031-11L) | Polyclonal | Secondary antibody for flow cytometry (0.625 µg/ml); Supplementary file 1e |
| Antibody | Anti-chicken Bu-1-AF647 (mouse monoclonal) | Biozol (Cat# SBA-8395-31) | Clone: AV20 | Flow cytometry (1:500); Supplementary file 1e |
| Antibody | Anti-chicken MHC I (mouse monoclonal) | Southern Biotech (Cat# 8345-01) | Clone: F21-2 | Flow cytometry (1:200); Supplementary file 1e |
| Antibody | Anti-chicken MHC II-AF488 (mouse monoclonal) | Biozol (Cat# 8350-30) | Clone: 2G11 | Flow cytometry (1:1000); Supplementary file 1e |
| Sequence-based reagent | IFNAR1 ssODN part 1; IFNAR1 ssODN part 2 | Integrated DNA Technologies; this paper | HDR repair templates for IFNAR1 knockout; sequences provided in Supplementary file 1a. | |
| Sequence-based reagent | IFNLR1 sgRNA; IFNAR1 sgRNA | This paper | CRISPR/Cas9 sgRNAs; sequences and PAMs provided in Supplementary file 1a. | |
| Sequence-based reagent | IFNAR1 and IFNLR1 genotyping primers and probes | This paper | Primer/probe sequences provided in Supplementary file 1b. | |
| Sequence-based reagent | Sexing, RT-PCR, IBV qPCR and 28S qPCR primers/probes | This paper | Primer/probe sequences provided in Supplementary file 1c. | |
| Sequence-based reagent | qPCR primers for cytokines, ISGs, and IFN signaling genes | This paper | Primer sequences, amplicon sizes, and accessions provided in Supplementary file 1f. | |
| Sequence-based reagent | TCR amplicon sequencing primers | Früh et al., 2024 | TCR constant gene-specific 5' RACE and semi-nested/step-out PCR primers; see Materials and methods. | |
| Peptide, recombinant protein | Recombinant chicken IFN-α | Schultz et al., 1995 | Used for stimulation of CEFs and ED10 embryos; 500 U/ml for CEF stimulation; 1.5×105 U for embryo stimulation. | |
| Peptide, recombinant protein | Keyhole limpet hemocyanin; KLH | Sigma-Aldrich | Model T cell-dependent antigen used for immunization; 300 µg per injection. | |
| Commercial assay or kit | 5x HOT Firepol Probe qPCR Mix Plus | Solis Biodyne | Used for TaqMan genotyping of IFNAR1 KO animals. | |
| Commercial assay or kit | Firepol DNA Polymerase | Solis Biodyne | Used for endpoint PCR genotyping and RT-PCR assays. | |
| Commercial assay or kit | GoScript Reverse Transcription Mix | Promega | Cat#A5001 | Used for cDNA synthesis. |
| Commercial assay or kit | GoTaq qRT-PCR kit | Promega | Cat#A6002 | Used for qRT-PCR gene expression analysis. |
| Commercial assay or kit | Histopaque-1077 | Sigma-Aldrich | Cat#10771 | Used for PBMC and splenic mononuclear cell isolation. |
| Commercial assay or kit | SV Total RNA Isolation System | Promega | Cat#Z3100 | Used for RNA extraction before TCR amplicon sequencing. |
| Commercial assay or kit | Fixable Viability Dye eFluor 780 | eBioscience; Thermo Fisher Scientific | Cat#65-0865-14 | Flow cytometry viability dye; 1:1000; Supplementary file 1e. |
| Commercial assay or kit | Maxisorp non-immuno ELISA plates | Thermo Fisher Scientific | Used for IgM/IgY and KLH-specific ELISA assays. | |
| Chemical compound, drug | TRIzol reagent | VWR | Used for RNA isolation from CAM, spleen, and tissues. | |
| Chemical compound, drug | Incomplete Freund’s adjuvant | Sigma-Aldrich | Mixed 1:1 with KLH for primary and booster immunization. | |
| Chemical compound, drug | Geneticin; G418 | Thermo Fisher Scientific | Used at 250 µg/ml to maintain CEC-32 #511 reporter cells under selection pressure. | |
| Chemical compound, drug | Homemade ECL substrate | This paper | Prepared in-house; luminol-based HRP chemiluminescent substrate used for western blot signal development. | |
| Software, algorithm | GraphPad Prism | Dotmatics | Version:9.3.1; RRID:SCR_002798 | Used for graph generation. |
| Software, algorithm | SPSS Statistics | IBM | Version:28.0.1.1; RRID:SCR_002865 | Used for statistical analyses. |
| Software, algorithm | FlowJo | FlowJo; Ashland | Version:10.8.1; RRID:SCR_008520 | Used for flow cytometry data analysis. |
| Software, algorithm | QuantStudio Design & Analysis Software | Thermo Fisher Scientific | Version:1.5.2 | Used for qRT-PCR data acquisition/analysis. |
| Software, algorithm | FastQC | Babraham Bioinformatics | Version:0.12.1; RRID:SCR_014583 | Used for raw read quality assessment. |
| Software, algorithm | MultiQC | Ewels et al., 2016 | Version:1.23 | Used to summarize sequencing quality-control results. |
| Software, algorithm | MiXCR | Bolotin et al., 2015 | Version:4.7.0; RRID:SCR_018725 | Used for TCR analysis and annotation. |
| Software, algorithm | R | R Foundation for Statistical Computing | Version:4.4.1; RRID:SCR_001905 | Used for TCR repertoire and statistical analyses. |
| Software, algorithm | tidyverse | R package | Version:2.0.0 | Used for data plotting in R. |
| Software, algorithm | MASS | R package | Version:7.3–61 | Used for negative binomial generalized linear models. |
| Software, algorithm | emmeans | R package | Version:1.10.3 | Used for post hoc pairwise comparisons. |
| Software, algorithm | IMNGS2 | Lagkouvardos et al., 2016 | Used for processing 16S rRNA gene amplicon sequencing data. | |
| Software, algorithm | RHEA | Lagkouvardos et al., 2017 | Used for microbial profiling based on 16S rRNA gene amplicons. | |
| Other | Illumina MiSeq | Illumina | Sequencing instrument used for 16S rRNA gene amplicon sequencing and TCR amplicon sequencing. | |
| Other | AttuneNXT flow cytometer | Thermo Fisher Scientific | Instrument used for flow cytometry. | |
| Other | Vilber Lourmat Fusion Fx system | Vilber Lourmat | Imaging system used for western blot signal detection. | |
| Other | SpeedMill Homogenizer | Analytik Jena | Used for tissue homogenization. | |
| Other | BioSpec-nano spectrophotometer | Shimadzu | Used for RNA concentration/quality assessment. | |
| Other | 2100 Bioanalyzer | Agilent Technologies | Used for RNA integrity assessment. | |
| Other | FluoStar Omega microplate reader | BMG LABTECH | Instrument used for ELISA absorbance measurements at 450 nm with 620 nm reference filter; software version 5.91/5.70R2. | |
| Other | QuantStudio 5 system | Thermo Fisher Scientific | Instrument used for qRT-PCR data acquisition. | |
| Other | NanoDrop ND-1000 | Thermo Fisher Scientific | Instrument used for RNA quantification before TCR amplicon sequencing. |
Additional files
-
Supplementary file 1
Key experimental resources used in this study, including gene-editing constructs, primers, probes, virus strains, and antibodies.
- https://cdn.elifesciences.org/articles/107855/elife-107855-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/107855/elife-107855-mdarchecklist1-v1.docx
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Deciphering interferon functions in avian influenza using receptor knockout models in the natural host
eLife 14:RP107855.
https://doi.org/10.7554/eLife.107855.3
