Cytokine 113 (2019) 31–38 Contents lists available at ScienceDirect Cytokine journal homepage: www.elsevier.com/locate/cytokine Duck interferon regulatory factor 7 (IRF7) can control duck Tembusu virus (DTMUV) infection by triggering type I interferon production and its signal T transduction pathway Shun Chena,b,c,1, Tao Wanga,1, Peng Liua, Chao Yanga, Mingshu Wanga,b,c, Renyong Jiaa,b,c, ⁎ Dekang Zhub,c, Mafeng Liua,b,c, Qiao Yanga,b,c, Ying Wua,b,c, Xinxin Zhaoa,b,c, Anchun Chenga,b,c, a Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China b Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China c Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China ARTICLE INFO ABSTRACT Keywords: Human interferon regulatory factor 7 (IRF7) plays an important role in the innate antiviral immune response. To Duck date, the characteristics and functions of waterfowl IRF7 have not been clarified. This study reports the cDNA IRF7 sequence, tissue distribution, and antiviral function of duck IRF7. The duck IRF7 gene has a 1536-bp open read Tembusu virus frame (ORF) and encodes a 511-amino acid polypeptide. IRF7 is highly expressed in the blood and pancreas of 5- Type I interferon day-old ducklings and in the small intestine, large intestine and liver of 60-day-old adult ducks. Indirect im- Innate immune response munofluorescence assay (IFA) showed that over-expressed duck IRF7 was located in both the cytoplasm and nucleus of transfected duck embryo fibroblasts (DEFs), which was also observed in poly(I:C)-stimulated or duck Tembusu virus (DTMUV)-infected DEFs. Titres and copies of DTMUV were significantly reduced in DEFs over- expressing IRF7. Moreover, overexpression of duck IRF7 significantly induced IFNα/β, but not IFNγ, mRNA expression, and transcription of downstream interferon-stimulated genes (ISGs), such as MX, OASL and IL-6, which were significantly induced by poly(I:C) co-stimulation, was enhanced. Additionally, duck IRF7 over- expression can significantly activate the IFNβ promoter in DEFs. Collectively, duck IRF7 plays an important role in host anti-DTMUV immune regulation, which depends on type I interferons and associated signal transduction pathway(s). 1. Introduction IRF3 having critical roles in type I IFN-mediated innate immunity. Recognition of pathogens by PRRs results in activation of IRF3 and/or The innate immune response provides the first line of host defence IRF7 [11,12]. Phosphorylation activates IRF7, inducing dimerization during infection. Interferons (IFNs) are critical factors of the innate and exposure of the nuclear localization signal (NLS) [2,8]. Activated immune response [1,2], the initiation of which response depends on IRF7/IRF3 then translocates to the nucleus and forms an enhanceosome recognition of pathogen-associated molecular patterns (PAMPs) by with activating protein 1 (AP-1) and nuclear factor kappa B (NF-κB). pattern recognition receptors (PRRs) [3]. Toll-like receptors (TLRs) and These proteins bind to their respective positive regulatory domains, retinoic-acid-inducible gene I (RIG-I) are key PRRs in the host anti- leading to recruitment of co-factors, resulting in transcription of type I dsRNA viral response [4,5] and can recruit downstream factors when IFNs [2]. activated, resulting in production of interferon and transcription of First identified in 2010, duck Tembusu virus (DTMUV) is a newly interferon-stimulated genes (ISGs) [2,6]. emerging virus that leads to acute egg-drop syndrome in ducks [13]. First cloned in 1997 [7], human IRF7 is the common downstream DTMUV belongs to the Flavivirus genus and has a single positive- factor in both the TLR and RIG-I signalling pathways [8,9]. All IRF7s stranded RNA genome [14]. A recent report demonstrated that DTMUV contain a conserved N-terminal DNA-binding domain (DBD) and an can activate the host innate immune response through the MDA5 and IRF-associated domain (IAD) [10], with IRF family members IRF7 and TLR signalling pathways [15]. However, it remains unknown whether ⁎ Corresponding author at: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, No. 211 Huimin Road, Wenjiang District, Chengdu city, Sichuan province 611130, China. E-mail address: [email protected] (A. Cheng). 1 These authors contributed equally as co-first authors of this work. https://doi.org/10.1016/j.cyto.2018.06.001 Received 4 January 2018; Received in revised form 2 May 2018; Accepted 1 June 2018 Available online 06 June 2018 1043-4666/ © 2018 Elsevier Ltd. All rights reserved. S. Chen et al. Cytokine 113 (2019) 31–38 duck IRF7 is involved in those pathways. sacrificed, and tissues (the small intestine, large intestine, muscle, bursa Interestingly, IRF3 is absent in the avian genome [16], yet IRF7 may of Fabricius, thymus gland, liver, Harderian glands, spleen, pancreas, complement a portion of the IRF3 function [17]. Although chicken IRF7 brain, trachea, glandular stomach, gizzard, kidney, heart, lung, blood, has been identified [18], the characteristics and functions of IRF7 in and lymph) were collected. Total RNA form the tissues was extracted waterfowl have not yet been elucidated. In this study, duck IRF7 was using RNAiso Plus according to the manufacturer’s instructions (Takara, identified and cloned, and its tissue distribution in ducklings and ducks Japan). For quantitative reverse transcription-PCR (qRT-PCR), 1 μgof was examined. Duck IRF7 can activate the IFNβ promoter to induce total RNA was reverse transcribed following the standard protocol of expression of type I IFNs and ISGs. Duck IRF7 can also inhibit DTMUV PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Japan). The replication in vitro, a process that plays an important role in viral in- qRT-PCR reaction system included 0.4 μL of cDNA, 0.3 μL of qPCR- fection. IRF7-F (Table 1), 0.3 μL of qPCR-IRF7-R (Table 1), 4 μL of RNAase-free ddH2O and 5 μL of 2× EvaGreen qPCR Premix (Abcom, USA); the 2. Materials and methods following cycling conditions were used: 95 °C for 10 min, 95 °C for 15 s, and 63 °C for 1 min. 2.1. Experimental animals, virus, reagents 2.5. Western blot analysis of duck IRF7 expression Healthy ducklings and ducks were purchased from a farm in Chengdu City, China. The duck Tembusu virus (DTMUV), CQW1 strain Duck embryo fibroblasts (DEFs) were cultured in 6-well plates and (NCBI accession No. KM233707.1) (50% tissue culture infective dose transfected with 4 μg of pCAGGS-IRF7. After removing the culture 6 (TCID50) = 2.5 × 10 /mL), was provided by the Research Center of medium, the cells were lysed with 500 μL of RIPA Lysis and Extraction Avian Disease, Sichuan Agricultural University. Poly(I:C) was pur- Buffer (Thermo Fisher, USA). The cell lysates were separated by 12% chased from Sigma-Aldrich (USA). polyacrylamide gel electrophoresis, and the separated proteins were electroblotted onto polyvinylidene difluoride (PVDF) membranes 2.2. Sequence amplification and plasmid construction (Millipore, USA). Membranes were blocked overnight in 5% evaporated milk at 4 °C and incubated for 1 h with a 1:2000-diluted mouse anti- The reverse primer, IRF7-R (Table 1), for amplification of duck IRF7 Flag (ProteinTech, China) or mouse anti-β-actin (Ruiying Biological, was designed according to the predicted sequence of duck IRF7 (NCBI China) antibody at 37 °C. The membranes were then incubated for 1.5 h accession No. XM_021270589.1); the forward primer, IRF7-F (Table 1), with 1:2000-diluted horseradish peroxidase (HRP)-conjugated goat was designed according the conserved domain between chicken and anti-mouse IgG (Earthox, USA). The duck IRF7 protein was observed by goose IRF7. The conditions used were 98 °C for 2 min, 98 °C for 10 s, enhanced chemiluminescence (Bio-Rad, USA), and images were ac- 55 °C for 15 s, 72 °C for 20 s, and 72 °C for 5 min. The purified fragment quired using ChemiDoc M (Bio-Rad, USA). was TA cloned into the pMD19-T simple vector (pMD™19-T Vector Cloning Kit, Takara, Japan). pMD19-T-IRF7 was used as the template 2.6. Cellular localization of duck IRF7 in DEFs and pCAGGS-IRF7-F and pCAGGS-IRF7-R as primers (Table 1) for polymerase chain reaction (PCR) amplification. Then, the fragment was DEFs were cultured overnight in Dulbecco's modified Eagle's β fi fl cloned into pCAGGS vector with a Flag tag. IFN- -Luc expresses re y medium (DMEM) with 10% foetal bovine serum (FBS) and 5% CO2 at luciferase under the control of the duck IFN-β promoter (−96 to +1), 37 °C, and 2 μg/ml of plasmid of pCAGGS-IRF7 was transfected into which was constructed by our lab and described in a previous report cells at 70–90% confluence (Transintro EL transfection reagent, [19]. pRL-TK was purchased from Promega (Wisconsin, USA). Transgene, China). At 24 h post-transfection, the DEFs were stimulated with DTMUV (25,000 TCID50) or 50 ng/mL poly(I:C) for 12 h, washed 2.3. Sequence analysis and phylogenetic analysis with phosphate-buffered saline/Tween 20 (PBST), fixed with 4% par- aformaldehyde, permeabilized with 0.25% Triton-X 100 in PBS, and The amino acid sequence of duck IRF7 was analysed using tools on blocked with 0.5% bovine serum albumin (BSA) for overnight. After the SMART website (http://smart.embl-heidelberg.de/). Amino acid three washes with PBS, the cells were incubated with mouse anti-Flag sequence alignment including Gallus gallus (NP_990703.1), Homo sa- antibodies (Ruiying Biological, Suzhou, China) for 12 h at 4 °C. The cells piens (NP_001563.2) and Mus musculus (NP_058546.1) was conducted were treated with goat anti-mouse Alexa 568 (Life Technologies) for 1 h by Geneious software.