Ropsacitinib – SSR signal

Molecular cloning and functional characterization of duck TYK2

A B S T R A C T
Tyrosine kinase 2 (TYK2), a member of Janus kinase family, has been identiﬁed as a crucial protein in signal transduction initiated by interferons or interleukins in mammals. However, the function of avian TYK2 in innate immune response remains largely unknown. In this study, the full-length duck TYK2 (duTYK2) cDNA was cloned for the ﬁrst time, which encoded a putative protein of 1187 amino acid residues and showed the high sequence similarity with bald eagle, crested ibis, and white-tailed tropicbird TYK2s. The duTYK2 was widely expressed in all examined tissues of healthy ducks and showed diﬀuse cytoplasmic localization in duck embryo ﬁbroblasts (DEFs). Overexpression of duTYK2 signiﬁcantly enhanced ISRE promoter activity and induced the expression of viperin, PKR, 2′,5′-OAS, MX and ZAP in DEFs. The C-terminal kinase domain of duTYK2 is essential for duTYK2- mediated ISRE promoter activation. Furthermore, knockdown of duTYK2 dramatically decreased duck Tembusu virus (DTMUV)-, duck enteritis virus (DEV)-, poly(I:C)- or poly(dA:dT)-induced ISRE promoter activation. Additionally, duTYK2 expression exhibited antiviral activity against DTMUV. These results indicated that duTYK2 played a critical role in duck antiviral innate immunity.

1.Introduction
The interferon (IFN)-mediated innate immune response serves as the ﬁrst line of host defense against invading viruses (Akira et al., 2006; Mogensen, 2009). Following pathogen detection and subsequent IFN production, IFN molecules bind to their cell-surface receptors and ra- pidly initiate a signaling cascade via the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway, leading to the in- duction of expression of a wide variety of IFN-stimulated genes (ISGs) (Kisseleva et al., 2002; Schneider et al., 2014). Upon IFN binding, IFN receptors undergo conformational changes and subsequently form di- meric or oligomeric complexes, which lead to JAK apposition and transphosphorylation on speciﬁc tyrosine residues, releasing their in- trinsic catalytic activity (Levy and Darnell, 2002; Piehler et al., 2012). The activated JAKs in turn phosphorylate tyrosine residues within the cytoplasmic domains of IFN receptors, providing binding sites for the Src-homology-2 (SH2) domain of the STAT proteins (Bousoik and Montazeri Aliabadi, 2018). After binding to the receptors, STAT pro- teins are phosphorylated by JAKs. Phosphorylated STATs dissociate from the receptor-JAK complexes, form homo- or heterodimers, and then translocate to the nucleus where they complex with other nuclear proteins and bind to IFN-stimulated response element (ISRE) and
gamma-activated sequence (GAS) to regulate hundreds of ISGs ex- pression (Sands et al., 2006). JAK-STAT signaling not only plays an important role in antiviral innate immunity, but also primes and shapes the adaptive immune response (Fleming, 2016; Villarino et al., 2015).

JAKs are non-receptor tyrosine kinases and are widely found in many species, including mammals, birds, insects, and ﬁshes (Sobhkhez et al., 2013; Yamaoka et al., 2004). In mammals, there are four JAK members: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2) (Yamaoka et al., 2004). TYK2 was the ﬁrst JAK family member identiﬁed that plays a crucial role in cytokine signaling (Velazquez et al., 1992). Structure predictions showed that TYK2 contains four distinct domains: N-terminal 4.1, Ezrin, RadiXin, Moesin (FERM), the Src homology 2 (SH2) domains, the pseudokinase domain, and the C-terminal kinase domain (Kreins et al., 2015; Leitner et al., 2017; Wallweber et al., 2014). The FERM and SH2 domains are responsible for distinct receptor interactions, whereas the pseudokinase, possessing a canonical kinase domain but lacking catalytical function despite binding ATP, is im- portant for regulating the activity of kinase domain (Ferrao and Lupardus, 2017; Hammarén et al., 2015). TYK2 kinase activity was found to be essential for functional type I interferon responses (Prchal- Murphy et al., 2012). Using expression clones, the TYK2 gene was identiﬁed by its ability to restore the IFN response in a defect human cell line that is not responsive to IFN-α (Barbieri et al., 1994; Yeh et al., 2000), indicating that TYK2 is a critical component in type I interferon signaling pathway. In addition, TYK2 is also involved in several other TYK2s has been well clariﬁed (Sobhkhez et al., 2013; Yamaoka et al., 2004), the biological role of avian TYK2 remains largely unknown. Here we report the cloning and characterization of duck TYK2 (duTYK2) gene for the ﬁrst time. DuTYK2 mRNA was widely distributed in healthy duck tissues and exhibited tissue-speciﬁc expression patterns. Overexpression duTYK2 signiﬁcantly activated ISRE promoter in duck embryo ﬁbroblasts (DEFs), whereas duTYK2 knockdown blocked dsRNA/dsDNA-induced ISRE activity. Finally, we found that duTYK2 possessed antiviral activity against duck Tembusu virus (DTMUV) in- fection.

2.Materials and methods
serum (FBS, Gibco). Various tissues for detecting duTYK2 mRNA ex- pression proﬁles, including thymus, heart, liver, spleen, lung, kidney, cerebellum, cerebrum, windpipe, muscle, glandular stomach, muscular stomach, duodenum, cecum, and bursa of Fabricius, were collected from one-month old healthy Cherry Valley Ducks. After three washes with phosphate buﬀered saline (PBS), all tissues were submerged im- mediately in liquid nitrogen and stored at −80 °C for RNA isolation. Sendai virus (SeV) was obtained from the Centre of Virus Resource and Information (Wuhan Institute of Virology, Chinese Academy of Sciences). DTMUV strain MC (GenBank number: KX452096) was iso- lated previously from an egg-production drop duck farm in 2014. Duck enteritis virus (DEV) strain HB-10 was a gift from Dr. Xueying Hu at Huazhong Agricultural University. Three pairs of small interfering RNAs (siRNAs) targeting the duTYK2 gene was synthesized by Shanghai GenePharma Co., LTD (Shanghai, China). Poly(I:C) and poly(dA:dT) were purchased from Sigma (St Louis, MO, USA).According to the predicted sequence of duTYK2 from the NCBI Nucleotide Database (GenBank accession number: XM_013109258), a pair of gene-speciﬁc primers duTYK2-5′GSP and duTYK2-3′GSP(Table 1) were designed to amplify 5′ and 3′ terminal regions of duTYK2 using SMARTer RACE 5’/3′ Kit (Takara) according to the manufacturer’s instructions. After sequencing and analysis of the full- length cDNA sequence of duTYK2, one set of primers (duTYK2-F and duTYK2-R, Table 1) was designed to amplify the coding sequence of duTYK2 by RT-PCR using total RNA extracted from DEFs. The PCR product was digested with KpnI and NheI and subsequently cloned into the pCAGGS-Flag vector.The full-length mRNA sequence of duTYK2 was submitted to the GenBank database under the accession number MK472064. Amino acid sequences were aligned using the ClustalW method (http://www.ebi. ac.uk/Tools/msa/clustalw2) and the phylogenetic tree was constructed by MEGA 7 using neighbor-joining method. The functional domains of duTYK2 were predicated by the Simple Modular Architecture Research Tool (SMART) (http://smart.emblheidelberg.de/).
Cherry Valley Ducks using TRIzol reagent (Invitrogen) and subse- quently reverse-transcribed to cDNA by iScript cDNA Synthesis Kit (Bio- Rad) according to the manufacturer’s instructions. The quantitative real-time PCR was employed to detect the tissue distribution of duTYK2 using the SYBR® Green supermiX (Bio-Rad) with a pair of primers qduTYK2-F and qduTYK2-R (Table 1). As a reference gene, duck GAPDH gene was also ampliﬁed with primers qGAPDH-F and qGAPDH- R (Table 1).

DEFs were cultured in 24-well plates on glass coverslips and transfected with pCAGGS-Flag-duTYK2 or empty vector until they reached approXimately 50% conﬂuence. After 24 h, cells were ﬁXed with 4% paraformaldehyde (Sigma) in PBS for 10 min, permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 10 min, and then blocked with 5% skimmed milk in PBS for 1 h. After three washes with PBS, cells were incubated with mouse anti-Flag antibody (MBL, Woburn, MA, USA) for 1 h and then treated with ﬂuorescein isothiocyanate (FITC)- labeled goat anti-mouse IgG secondary antibody (Invitrogen). Subsequently, the nuclei were stained with 4’,6-diamidino-2-phey- lindole (DAPI, Invitrogen) for 15 min. Fluorescence was detected by a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Zena, Germany). The DNA fragments encoding the open reading frame of duTYK2 was inserted into pCAGGS-Flag vector. SiX truncated mutants, duTYK2 (aa279-1187), duTYK2 (aa584-1187), duTYK2 (aa866-1187), duTYK2 (aa1-894), duTYK2 (aa1-589), and duTYK2 (aa1-445) were ampliﬁed using primers list in Table 1 and sub-cloned into the pCAGGS-Flag vector. ISRE luciferase reporter vector was purchased from Promega. DEFs were seeded into 48 well plates and cultured until reaching about 80% conﬂuence. Cells were co-transfected with 200 ng/well of various expression plasmids or an empty vector and 50 ng/well of reporter plasmids (ISRE-Luc) along with 50 ng/well of pGL4.74 as an internal control vector using lipofectamine 2000 (Invitrogen). The cells were harvested at the indicated time, and the relative ﬁreﬂy and Renilla luciferase activity were measured by the dual-luciferase reporter assay system (Promega, USA).DEFs were cultured in 24-well plates and transfected with pCAGGS- Flag-duTYK2 or empty vector until they reached approXimately 80% conﬂuence. DEFs were inoculated with SeV as the positive control. After transfection or stimulation at 24 h, cells were collected and total RNA was extracted using TRIzol reagent (Invitrogen). After reverse transcription (iScript cDNA Synthesis Kit, Bio-Rad), the quantitative real-time PCR was employed to detect the expression level of viperin, PKR, 2′,5′-OAS, MX and ZAP using the SYBR® Green supermiX (Bio-Rad) with primers listed in Table 1. Duck GAPDH gene was also ampliﬁed as a control.

DEFs were cultured in 60-mm dishes and grown until about 80% conﬂuence. Cells were transfected with various expression plasmids using lipofectamine 2000 reagent (Invitrogen). At 30 h post-transfec- tion, cells were lysed with 180 μL lysis buﬀer (65 mM Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate, 3% DL-dithiothreitol and 40% glycerol). The cell lysates were separated by 12% acrylamide SDS-PAGE and the separated proteins were electro-blotted onto polyvinylidene diﬂuoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and incubated with speciﬁc antibody. The membranes were then incubated with anti-Flag monoclonal anti- bodies (MBL) and horseradish peroXidase (HRP)-conjugated goat anti- mouse IgG (Sigma) was used as the secondary antibody. Signals were collected using the Super Signal West Pico Luminal kit (Pierce).DEFs were inoculated with DTMUV 24 h after transfection with pCAGGS-Flag-duTYK2 or siduTYK2. DTMUV-infected cells were col- lected at diﬀerent time points, and virus titers were examined by 50% cell culture infectious dose (TCID50) endpoint dilution assay. Viral titers were calculated by the method of Reed and Muench (Reed and Muench, 1938).Statistical signiﬁcance was analyzed using student’s t-test. P-va- lues < 0.05 were considered signiﬁcant. P-values < 0.01 were con- sidered extremely signiﬁcant.Quantitative analysis of duTYK2 mRNA in diﬀerent tissues. Relative duTYK2 mRNA levels were measured by qRT-PCR and normalized to GAPDH gene. Error bars indicated the standard deviation. These results were normal- ized to the expression level of thymus. (B) Subcellular localization of duTYK2. pCAGGS-Flag-duTYK2 or an empty vector was transfected into DEFs. After 24 h, the cells were stained with anti-Flag antibody and visualized with ﬂuorescein isothiocyanate-labeled goat anti-mouse IgG. Nuclei were stained with DAPI. Fluorescence was examined by a Zeiss LSM 510 confocal laser-scanning mi- croscope. 3.Results Based on the predicated Anas platyrhynchos TYK2 partial gene se- quence (XM_013109258), the full-length duTYK2 cDNA was sequenced by the rapid ampliﬁcation of cDNA ends (RACE) PCR using the primers duTYK2-5′GSP and duTYK2-3′GSP (Table 1, Fig. 1A). After analyzing the open reading frames (ORFs) of duTYK2 gene, duTYK2 coding sequence was conﬁrmed and cloned by RT-PCR using the total RNA iso- lated from DEFs with a pair of primers duTYK2-F and duTYK2-R (Table 1, Fig. 1A). The full-length cDNA of duTYK2 was 3878bp and encoded a protein of 1187 amino acids, which was submitted to Gen- Bank (Accession number: MK472064) (Fig. 1A). According the NCBI's latest annotation, 20 exons located on an Unplaced Scaﬀold (NW_020866045.1) constituted the entire coding sequence of duTYK2 (Fig. 1B). Multiple alignments of amino acids sequence of TYK2s from dif- ferent species showed that duTYK2 presented 92% sequence identity with bald eagle, crested ibis, and white-tailed tropicbird TYK2s, whereas duTYK2 displayed relatively low identity with human (64%) and zebraﬁsh (53%) (Fig. 2A). Consistent with the results of the mul- tiple alignments, duTYK2 and other bird TYKs were grouped in the same cluster (Fig. 2B). Quantitative real-time RT-PCR was used to examine the abundance of duTYK2 mRNA in the duck tissues. As shown in Fig. 3A, duTYK2 was expressed in all examined tissues, indicating that duTYK2 was con- stitutively and ubiquitously expressed in various tissues. Speciﬁcally, duTYK2 mRNA expression was relatively high in liver and heart among all tested tissues, whereas relatively low abundance mRNAs of duTYK2 were detected in muscular stomach and spleen.To study the subcellular location of duTYK2, DEFs were transfected with the plasmid expressing Flag-tagged duTYK2 and stained with anti- Flag antibody for indirect immunoﬂuorescence. Not surprisingly, the subcellular distribution showed that duTYK2 was localized pre- dominantly in the cytoplasm in DEFs (Fig. 3B).Most of the classical antiviral genes contain ISRE motifs in their promoter regions, which are usually regulated by the kinase activity of TYK2 (Fink and GrandvauX, 2013). To explore whether duTYK2 in- creases the ISRE promoter activity, DEFs were transfected with duTYK2 expression plasmid along with ISRE-Luc reporter plasmid. As shown in Fig. 4A, overexpression of duTYK2 dramatically increased ISRE pro- moter activity in a dose-dependent manner in DEFs, compared to the vector group. Not surprisingly, duTYK2 expression also enhanced the expression of viperin, PKR, 2′,5′-OAS, MX and ZAP (Fig. 4B). Based on the SMART analysis results, duTYK2 was composed of a N-terminal four-point-one, ezrin, radiXin, moesin (FERM) domain (aa22- 279), an SH2-like domain (aa446-538), a pseudokinase domain (aa590- 866), and a kinase domain on its C-terminal end (aa895-1169) (Fig. 4C). According to the distribution of duTYK2 domains, siX deleted mutants of duTYK2 were generated to analyze their roles in duTYK2- mediated ISRE activation (Fig. 4C). The plasmids expressing these mutants were transfected into DEFs respectively and their expression was veriﬁed by Western blot (Fig. 4D). As shown in Fig. 4E, compared to wide-type duTYK2 group, overexpression of the duTYK2 mutants without the kinase domain completely lost the ability to induce ISRE activity, whereas duTYK2 deletion constructs lacking FERM and SH2 domains had little eﬀect on ISRE activity. Notably, overexpression of the duTYK2 (aa 866–1187) mutant, only possessing the kinase domain, also dramatically increased the activity of ISRE promoter, although to a less extent than wide-type duTYK2 or the duTYK2 (aa 584–1187) mu- tant containing both pseudokinase and kinase domains (Fig. 4E). It seems like that the kinase domain of duTYK2 played a pivotal role in the activation of ISRE promoter and the pseudokinase domain of duTYK2 also partially contributed to duTYK2-induced ISRE activation. To further study the role of duTYK2 in ISRE promoter activation, we designed three siRNAs that targeted coding region of duTYK2 mRNA (Table 2). SiduTYK2-1 was found to degrade endogenous duTYK2 mRNA most eﬀectively among three synthetic siRNAs when measuring knockdown of duTYK2 mRNA by qRT-PCR (Fig. 5A), and therefore si- duTYK2-1 was used in the following gene knockdown experiments.Overexpression of duTYK2 activated ISRE promoter and induced ISGs expression. (A) DEFs were transfected with 50, 100 or 200 ng of pCAGGS-Flag-duTYK2 along with ISRE-Luc (50ng/well) and pGL4.74 plasmid (50ng/well) using lipofectamine 2000. **P < 0.01 compared to empty vector. (B) DEFs were transfected with 500 ng of pCAGGS-Flag-duTYK2 or empty vector using lipofectamine 2000. Cells infected with SeV as a positive control. After transfection or stimulation, the expression level of viperin, PKR, 2′,5′-OAS, MX and ZAP was measured by qRT-PCR and normalized to GAPDH. Error bars indicated the standard deviation. **P < 0.01 compared to empty vector. (C) Schematic diagram presenting the structure of wild-type duTYK2 and its deletion mutants. (D) Western blot analysis of the expression of wild-type duTYK2 and deleted mutants in DEFs. Lanes 1–8 represented cells transfected with the empty vector, duTYK2, duTYK2 (aa279-1187), duTYK2 (aa584-1187), duTYK2 (aa866-1187), duTYK2 (aa1-894), duTYK2 (aa1-589), and duTYK2 (aa1-445), respectively. (E) DEFs were transfected with various expression plasmids of duTYK2 or empty vector together with the reporter plasmid ISRE-Luc and pGL4.74. All luciferase assays were repeated at least three times and data shown are means ± SD (n = 3). **P < 0.01 compared to wild-type duTYK2. DEFs were transfected with siduTYK2-1 or siNegative control, along with the ISRE-Luc reporter plasmid, followed by stimulation with DTMUV, DEV, poly(I:C) or poly(dA:dT). As shown in Fig. 5B, the ISRE promoter activity induced by DTMUV, DEV, poly(I:C) or poly(dA:dT) was signiﬁcantly inhibited by knockdown of duTYK2 gene, compared to that in the siNegative groups. These results indicated that duTYK2 was involved in dsRNA/dsDNA-induced ISRE promoter activation.(Tang et al., 2012). To explore the antiviral activity of duTYK2 against DTMUV, DEFs were transfected with the plasmid expressing duTYK2 or synthesized siduTYK2-1, followed by DTMUV infection. At diﬀerent time points post-infection, cultures were collected and titrated by TCID50 assay. DTMUV proliferation was signiﬁcantly reduced by overexpression of duTYK2 in DEFs (Fig. 6A); whereas duTYK2 knock- down dramatically promoted viral replication (Fig. 6B). 4.Discussion Previous studies revealed TYK2 is a critical member of the JAK fa- mily of protein tyrosine kinases shown to be essential for IFN-α/β signaling in mammals (Yamaoka et al., 2004). Recently, Sobhkhez et al. identiﬁed four diﬀerent isoforms of Atlantic salmon Tyk2 and found that overexpression of full-length salmon TYK2 enhances the transcript DuTYK2 knockdown inhibited DTMUV-, DEV-, poly(I:C)- or poly (dA:dT)-induced the ISRE promoter activity. (A) DEFs were transfected with 1 μg/well siduTYK2-1, siduTYK2-2, siduTYK2-3, or siNegative control using Lipofectamine 2000. Cells were collected at 30 h post-transfection for detecting endogenous duTYK2 mRNA by real-time RT-PCR. DuTYK2 mRNA expression was normalized to GAPDH mRNA. **P < 0.01 compared to siNegative control. (B) DEFs were transfected with 1 μg/well of siduTYK2-1 or siNegative control, along with ISRE-Luc (50ng/well) and pGL4.74 (50ng/well). At 24 h after transfection, the cells were transfected with poly(I:C) or poly(dA:dT), or in- fected with DTMUV or DEV. Luciferase experiments analysis were performed at 16 h post-stimulation. All luciferase assays were repeated at least three times and the data shown are means ± SD (n = 3). **P < 0.01 compared to siNegative control levels of the IFN-induced MX gene, indicating the involvement of salmon TYK2 in the salmon type I IFN signaling (Sobhkhez et al., 2013). However, the role of avian TYK2 in IFN signaling pathways has not been elucidated yet. In this study, we cloned the duTYK2 gene for the ﬁrst time and investigated the role of duTYK2 in duck IFN signal pathway. Bioinformatics analysis and RACE-PCR experiments showed that the duTYK2 gene has a predicted translation of 1187 amino acids, which had the same size with human TYK2 but was slightly longer than zeb- raﬁsh TYK2. Multiple sequence alignment of TYK2s sequences from several diﬀerent species showed that the amino acid sequences of TYK2 in waterfowls, including bald eagle, crested ibis, and white-tailed tro- picbird, appeared to be extremely conserved with at least 90% identity. However, duTYK2 shared only 76%, 64% and 53% amino acid identity to chicken, human and zebraﬁsh TYK2s, respectively. Four critical do- mains previously identiﬁed in mammalian and ﬁsh TYK2s can also be found in duTYK2. The N-terminal of duTYK2 consisted of FERM and SH2 domains, while the pseudokinase and kinase domains were located in the C-terminal. Among them, the kinase domain of TYK2 in diﬀerent species was high conserved with about 80% identity. Collectively, the evolutionary conservation and the retention of critical domains in duck TYK2 suggested its importance in IFN signaling. DuTYK2 genes showed ubiquitous tissue expression in healthy ducks. The higher expression levels were detected in liver and heart, and the expression in muscular stomach and spleen was relatively low. Similarly, mammals and Atlantic salmon TYK2 transcripts were also detected in all tested tissues (Sobhkhez et al., 2013; Yamaoka et al., 2004). According to data of tissue expression in the BioGPS (http:// biogps.org), human TYK2 mRNA is expressed abundantly in lymphoid organs and immune cells, and Atlantic salmon TYK2 gene was highly expressed in spleen, head kidney and muscle (Sobhkhez et al., 2013). These results suggested that TYK2 expression pattern was species-spe- ciﬁc, despite its ubiquitous tissue expression in various species. Fur- thermore, duTYK2 protein was found to be distributed mainly in the cytoplasm in DEFs, indicating that duTYK2 probably acts as an im- portant cytosolic kinase involved in duck IFN signaling. Recently, Sobhkhez et al. reported that expressing the intact kinase domain of ﬁsh TYK2 leads to its spontaneous phosphorylation and in- creased transcription of MX gene (Sobhkhez et al., 2013). In our study, overexpression of duTYK2 signiﬁcantly enhanced the ISRE promoter activity in DEFs, and expressing only the kinase domain of duTYK2 also dramatically activated the ISRE promoter, although to a less extent than wide-type duTYK2. It seems like that the expression of duTYK2 kinase domain could lead to the phosphorylation and activation of STATs, which subsequently translocate to the nucleus and activate ISRE pro- moter. The pseudokinase domain adopts a canonical protein kinase fold but lacks key catalytic residues, and is thought to regulate the activity of the kinase domain (Ungureanu et al., 2011). Up to now, the mole- cular mechanisms of how the TYK2 pseudokinase regulates the activity of the kinase domain are still not fully understood. Recently, Lupardus et al. reported the structure of the pseudokinase-kinase module of TYK2 and found that the pseudokinase domain of TYK2 exhibits an auto- inhibitory function on the kinase domain (Lupardus et al., 2014). Sur- prisingly, overexpression of the duTYK2 mutant with both pseudoki- nase and kinase domains was slightly more active than expressing the mutant containing the kinase domain alone in activating ISRE pro- moter. Further studies are needed to identify the role of duTYK2 pseudokinase domain in duck IFN signaling.DuTYK2 exhibited antiviral activity against DTMUV infection. DEFs were transfected with 1 μg/well pCAGGS-Flag-duTYK2 or an empty vector using Lipofectamine 2000. (B) DEFs were transfected with siduTYK2-1 or siNegative control. At 24 h after transfection, the cells were inoculated with 0.01 MOI DTMUV. At 12 h, 18 h, and 24 h post-infection, re- spectively, cells were collected to measure the virus titers by TCID50 using Reed-Muench method. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 compared to vector group or siNegative control group.Previous study showed that human TYK2-deﬁcient ﬁbrosarcoma cell lines are completely unresponsive to type I IFN (Velazquez et al., 1992). In this study, knockdown of duTYK2 signiﬁcantly reduced DTMUV-, DEV-, poly(I:C)- or poly(dA:dT)-induced the ISRE promoter activity, indicating that duTYK2 was involved in dsRNA/dsDNA-in- duced ISRE promoter activation. Recently, Prchal-Murphy et al. re- ported that TYK2 deﬁciency increases susceptibility to VSV and EMCV infection in mice (Prchal-Murphy et al., 2012). Our results showed that overexpression of duTYK2 signiﬁcantly inhibited DTMUV replication in DEFs, whereas knockdown of duTYK2 promoted the viral multi- plication. These results suggested that duTYK2 might be involved in type I IFN-mediated antiviral activities. In summary, the amino acids of duTYK2 shared high similarity to TYK2s from other species, especially to other bird TYK2s. DuTYK2 was ubiquitously expressed in various duck tissues and mainly was located in cytoplasm. Overexpression of duTYK2 in DEFs signiﬁcantly activated ISRE promoter, and the kinase domain of duTYK2 played a dominant role in ISRE promoter activation. Knockdown of duTYK2 dramatically reduced DTMUV-, DEV-, poly(I:C)- or poly(dA:dT)-induced ISRE pro- moter activity. To Ropsacitinib our knowledge, the current study is the ﬁrst report to characterize the function of duck TYK2, which may provide new in- sights on avian antiviral innate immune response.