Chicken Interferon Research Articles

Construction of recombinant Lactococcus expressing thymosin and interferon fusion protein and its application as an immune adjuvant

BackgroundIn recent years, biosafety and green food safety standards have increased the demand for immune enhancers and adjuvants. In the present study, recombinant food-grade Lactococcus lactis (r-L. lactis-Tα1-IFN) expressing thymosin Tα1 and chicken interferon fusion protein was constructed.ResultsThe in vitro interactions with macrophages revealed a mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate both macrophage J774-Dual™ NF-κB and interferon regulator (IRF) signaling pathways. In vitro interactions with chicken peripheral blood mononuclear cells (PBMCs) demonstrated that a mixture of recombinant r-L. lactis-Tα1-IFN significantly enhanced the expression levels of interferon (IFN)-γ, interleukin (IL)-10, CD80, and CD86 proteins in chicken PBMCs. Animal experiments displayed that injecting a lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate the proliferation of T cells and antigen-presenting cells in chicken PBMCs. Moreover, 16S analysis of intestinal microbiota demonstrated that injection of the lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly improve the structure and composition of chicken intestinal microbiota, with a significant increase in probiotic genera, such as Lactobacillus spp. Results of animal experiments using the lysis mixture of recombinant r-L. lactis-Tα1-IFN as an immune adjuvant for inactivated chicken Newcastle disease vaccine showed that the serum antibody titers of the experimental group were significantly higher than those of the vaccine control group, and the expression levels of cytokines IFN-γ and IL-2 were significantly higher than those of the vaccine control group.ConclusionThese results indicate that food-safe recombinant r-L. lactis-Tα1-IFN has potential as a vaccine immune booster and immune adjuvant. This study lays the foundation for the development of natural green novel animal immune booster or immune adjuvant.

Cyclooxygenase-2/prostaglandin E2 pathway regulates infectious bronchitis virus replication in avian macrophages.

Infectious bronchitis virus (IBV) is a significant respiratory pathogen that affects chickens worldwide. As an avian coronavirus, IBV leads to productive infection in chicken macrophages. However, the effects of IBV infection in macrophages on cyclooxygenase-2 (COX-2) expression are still to be elucidated. Therefore, we investigated the role of IBV infection on the production of COX-2, an enzyme involved in the synthesis of prostaglandin E2 (PGE2) in chicken macrophages. The chicken macrophage cells were infected with two IBV strains, and the cells and culture supernatants were harvested at predetermined time points to measure intracellular and extracellular IBV infection. IBV infection was quantified as has been the COX-2 and PGE2 productions. We found that IBV infection enhances COX-2 production at both mRNA and protein levels in chicken macrophages. When a selective COX-2 antagonist was used to reduce the COX-2 expression in macrophages, we observed that IBV replication decreased. When IBV-infected macrophages were treated with PGE2 receptor (EP2 and EP4) inhibitors, IBV replication was reduced. Upon utilizing a selective COX-2 antagonist to diminish PGE2 expression in macrophages, a discernible decrease in IBV replication was observed. Treatment of IBV-infected macrophages with a PGE2 receptor (EP2) inhibitor resulted in a reduction in IBV replication, whereas the introduction of exogenous PGE2 heightened viral replication. Additionally, pretreatment with a Janus-kinase two antagonist attenuated the inhibitory effect of recombinant chicken interferon (IFN)-γ on viral replication. The evaluation of immune mediators, such as inducible nitric oxide (NO) synthase (iNOS), NO, and interleukin (IL)-6, revealed enhanced expression following IBV infection of macrophages. In response to the inhibition of COX-2 and PGE2 receptors, we observed a reduction in the expressions of iNOS and IL-6 in macrophages, correlating with reduced IBV infection. Overall, IBV infection increased COX-2 and PGE2 production in addition to iNOS, NO, and IL-6 expression in chicken macrophages in a time-dependent manner. Inhibition of the COX-2/PGE2 pathway may lead to increased macrophage defence mechanisms against IBV infection, resulting in a reduction in viral replication and iNOS and IL-6 expressions. Understanding the molecular mechanisms underlying these processes may shed light on potential antiviral targets for controlling IBV infection.

ChIFNα regulates adventitious root development in Lotus japonicus via an auxin-mediated pathway

ABSTRACT Adventitious roots (ARs), developing from non-root tissue, play an important role in some plants. Here, the molecular mechanism of AR differentiation in Lotus japonicus L. (L. japonicus) with the transformed chicken interferon alpha gene (ChIFNα) encoding cytokine was studied. ChIFNα transgenic plants (TP) were identified by GUS staining, PCR, RT-PCR, and ELISA. Up to 0.175 μg/kg rChIFNα was detected in TP2 lines. Expressing rChIFNα promotes AR development by producing longer roots than controls. We found that the effect was enhanced with the auxin precursor IBA treatment in TP. IAA contents, POD, and PPO activities associated with auxin regulation were higher than wild type (WT) in TP and exogenous ChIFNα treatment plants. Transcriptome analysis revealed 48 auxin-related differentially expressed genes (DEGs) (FDR < 0.05), which expression levels were verified by RT-qPCR analysis. GO enrichment analysis of DEGs also highlighted the auxin pathway. Further analysis found that ChIFNα significantly enhanced auxin synthesis and signaling mainly with up-regulated genes of ALDH, and GH3. Our study reveals that ChIFNα can promote plant AR development by mediating auxin regulation. The findings help explore the role of ChIFNα cytokines and expand animal gene sources for the molecular breeding of growth regulation of forage plants.

Transcriptome analysis of pre-immune state induced by interferon gamma inhibiting the replication of H9N2 avian influenza viruses in chicken embryo fibroblasts.

Interferon (IFN), a critical antiviral cytokine produced by pathogens-induced cells, plays an important role in host innate immune system. In this study, to investigate the inhibition effect of IFN on avian influenza virus (AIV), Chicken Embryo Fibroblasts (CEFs) was infected by H9N2 AIV. The pre-immune state and transcriptome analysis have been observed and performed. The result showed chicken interferon gamma (chIFN-γ) have the most inhibitory effect on H9N2 virus among three types of chicken interferons (chIFNs). Inhibition of chIFN-γ on H9N2 virus was verified by indirect immunofluorescence, RT-qPCR and western blot. The possible signaling pathways induced by chIFN-γ with or without virus were analyzed by transcriptome. The transcriptome data were compared among H9N2-infected, chIFN-γ-treated, chIFN-γ+H9N2-treated, and Control groups. In summary, RNA-sequencing (RNA-seq) data suggested that H9N2 virus infection resulted in corresponding response of certain defensive, inflammatory and metabolism pathways to the virus replication in CEFs. Furthermore, while CEFs were treated with chIFN-γ, many immune-related signaling pathways in cells are affected and altered. Antiviral genes involved in these immune pathways such as interferon regulatory factors, chemokines, interferon-stimulated genes (ISGs) and transcription factors were significantly up-regulated, and showed significant antiviral responses. Compared with virus infected CEFs alone, pretreatment with IFN induced the expression of antiviral genes and activated related antiviral pathways, inhibited the viral replication as result. Our study provided functional annotations for antiviral genes and the basis for studying the mechanism of chIFN-γ mediated response against H9N2 AIV.

Genetic variation in chicken interferon signalling pathway genes in research lines showing differential viral resistance.

Avian viruses of economic interest are a significant burden on the poultry industry, affecting production traits and resulting in mortality. Furthermore, the zoonosis of avian viruses risks pandemics developing in humans. Vaccination is the most common method of controlling viruses; however current vaccines often lack cross‐protection against multiple strains of each virus. The mutagenicity of these viruses has also led to virulent strains emerging that can overcome the protection offered by vaccines. Breeding chickens with a more robust innate immune response may help in tackling current and emerging viruses. Understanding the genetic evolution of different lines will thus provide a useful tool in helping the host in the fight against pathogens. This study focuses on the interferon genes and their receptors in different chicken lines that are known to be more resistant or susceptible to particular avian viruses. Comparing genotypic differences in these core immune genes between the chicken lines may explain the phenotypic differences observed and aid the identification of causative variations. The relative resistance/susceptibility of each line to viruses of interest (Marek’s disease virus, infectious bursal disease, infectious bronchitis virus and avian influenza virus) has previously been determined. Here we identify single nucleotide polymorphisms in interferons and downstream genes. Functional prediction tools were used to identify variants that may be affecting protein structure, mRNA secondary structure or transcription factor and micro‐RNA binding sites. These variants were then considered in the context of the research lines and their distribution between phenotypes. We highlight 60 variants of interest in the interferon pathway genes that may account for susceptibility/resistance to viral pathogens.

Chicken ISG12(2) attenuates Newcastle disease virus and enhances the efficiency of Newcastle disease vaccine via activating immune pathways.

Low virulence and strong immunogenicity are quite important for Newcastle disease virus (NDV) producing Newcastle disease (ND) living-attenuated vaccine. However, immunogenicity of NDV positively correlates to its virulence. Usually, the velogenic NDV induces stronger immune responses of poultry than the lentogenic strain, but virulent NDV poses a risk for chicken. In this study, we identified the chicken interferon (IFN)-stimulated gene 12-2 (ISG12(2)) not only attenuated NDV, but also increased immunogenicity of ND vaccine strain. First, we found that NDV infection or IFNs stimulation induced expression of chicken ISG12(2) that reinforced expression of IFNs. Over-expression or knock-down proved that chicken ISG12(2) inhibited NDV replication. Then, recombinant NDV LaSota strains (rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2)), expressing ISG12(2), were rescued. Pathogenicity tests showed that ISG12(2) expression attenuated NDV. RNA-seq or RT-qPCR demonstrated that, comparing to rLaSota/Fmut and rLaSota, rLaSota/Fmut/ISG12(2) and rLaSota/ISG12(2) induced hosts to produce cytokines enriching in innate and adaptive immune pathways in vitro and in vivo. Finally, we showed that rLaSota/ISG12(2) vaccination improved immune condition of chicken to quickly respond NDV infection and then enhance protection. These results suggest that chicken ISG12(2) is a potential novel molecular adjuvant to regulate immune responses, which decrease virulence and increase immunogenicity of NDV. The chicken ISG12(2) may contribute to the development of high efficient poultry vaccine.

Chicken interferon regulatory factor 7 (IRF7) can control ALV-J virus infection by triggering type I interferon production through affecting genes related with innate immune signaling pathway

In order to breed new birds with strong disease resistance, it is necessary to first understand the mechanism of avian antiviral response. Interferon regulatory factor 7 (IRF7) is not only a member of type I interferons (IFNs) regulatory factor (IRFs) family, but also a major regulator of the IFN response in mammals. However, whether IRF7 is involved in the host innate immune response remains unclear in poultry, due to the absence of IRF3. Here, we first observed by HE stains that with the increase of the time of ALV-J challenge, the thymus was obviously loose and swollen, the arrangement of liver cell was disordered, and the bursa of fabricius formed vacuolated. Real-time PCR detection showed that the expression level of IRF7 gene and related immune genes in ALV-J group was significantly higher than that in control group (P < 0.05). To further study the role of chicken IRF7 during avian leukosis virus subgroup J (ALV-J) infection, we constructed an induced IRF7 overexpression and interfered chicken embryo fibroblasts (CEFs) cell and performed in vitro infection using low pathogenic ALV-J and virus analog poly(I:C). In ALV-J and poly(I:C) stimulated CEFs cells, the expression level of STAT1, IFN-α, IFN-β, TLR3 and TLR7 were increased after IRF7 overexpressed, while the results were just the opposite after IRF7 interfered, which indicating that IRF7 may be associated with Toll-like receptor signaling pathway and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 is an important regulator of IFN and is involved in chicken anti-ALV-J innate immunity.

Tissue distribution and developmental changes of interferon regulatory factors in chickens and effects of infectious bursal disease virus infection

Interferon regulatory factors (IRFs) are a family of transcription factors that play a role in a variety of biological processes including immune regulation of interferon and expression of inflammatory cytokines. However, the data on IRFs are rather limited in chickens. In the present study, qRT-PCR was used to study the tissue distribution of IRFs in chickens at D15 (the 15th day of raising) and developmental changes of all chIRFs (Chicken interferon regulatory factors) in BF from E15 (the 15th day of incubation) to D15. The effects of IBDV infection with chickens on the transcriptional level of chIRFs were also investigated. The results showed: (1) chIRF1 mRNA was expressed much more abundantly in intestinal tract, chIRF2, chIRF6, chIRF7, chIRF8 and chIRF10 distributed mainly in liver or/and kidney. The expression of chIRF5 was mainly in spleen and chIRF4 distributed uniquely abundantly in BF. (2) The mRNA expression levels of chIRF5, chIRF7, chIRF8 and chIRF10 was low before hatching of chicken and at D1 and increased significantly from D5 till to the experiment end and the fold change of chIRF5 at D10 and chIRF7 at D5 reached 41.0-fold and 15.7-fold compared to that of E15, respectively (P < 0.05). ChIRF4 mRNA level was always high during the whole experiment except for E15 and it was 11.9-fold at the highest time point than that of E15 (the lowest time point). (3) When chicken was infected with IBDV, the expression levels of chIRF2, chIRF7 and chIRF10 mRNA had the tendency of increasing first and then decreasing but they peaked at 1dpi, 2 dpi, and 3dpi, respectively. The expression of chIRF5 mRNA was suppressed obviously during the whole experiment stage in IBDV-infected chicken. And chIRF4 expression was up-regulated transitorily at 1dpi and then was suppressed on a very low level till to the experiment end. Conclusion: The chIRFs were constitutively expressed in different tissues examined and has tissue-specific expression. Of them, chIRF2, chIRF4, chIRF5, chIRF7, chIRF8 and chIRF10 were related closely with the development or immune response of BF, and when chicken was infected with IBDV, some of them were activated, earlier or later on, some of them were suppressed. These findings would help to sieve out a few antiviral chIRF candidate gene to improve the host's innate immune and provide a foundation of the further exploiting a new vaccine adjuvant.

Serum cytokine concentration in native Nicobari fowl of Andaman and Nicobar Islands

Serological cytokine profile was studied in native Nicobari fowl of A&N Islands and its comparative evaluation was carried out in other local poultry population. Blood was collected from 150 poultry comprising 30 samples each of Nicobari fowl, commercial layer and broilers, desi bird and Vanaraja poultry. The concentration of cytokines viz. chicken interferon alpha and beta (IFN-α and IFN-β) (ng/L), chicken interleukin- 6 (IL-6) (ng/L), chicken interleukin-12 (IL-12) (pg/ml), chicken toll like receptor (TLR 4) (ng/ml) and chicken major histo-compatibility complex MHC (ng/ml) were determined by immunoenzymatic assay (ELISA). Results revealed that the concentrations IFN-α and IFN-β in serum is significantly higher in commercial layers that was statistically at par with Nicobari fowl. The cytokine, IL- 6 was significantly lowest in native Nicobari fowl and desi birds and highest in broilers and vanaraja birds. Significantly higher serum concentration of IL-12 was recorded in Nicobari fowl and it was statistically comparable with desi birds and broilers. Level of TLR4 in serum was significantly highest in broilers followed by commercial layers and Nicobari fowl. Commercial broilers, layers and vanaraja birds have significantly higher level of MHC followed by Nicobari fowl. Hence it can be concluded that the higher concentrations of serum IFN-α and β, IL-12, TLR4 and MHC in native Nicobari fowl indicates their innate immunological balance that maintain immune homeostasis and enhances immune function and resistance to disease by stimulating T and B lymphocyte formation modulating cytokine secretion profiles.

Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

A construction strategy for a baculovirus-silkworm multigene expression system and its application for coexpression of type I and type II interferons.

The Bombyx mori nucleopolyhedrovirus (BmNPV) baculovirus expression system (BES) is a eukaryotic expression system. It possesses great capability for post‐translation modification in expression of foreign proteins. With the counterselection cassette rpsL‐neo and phage λ‐Red recombinase, the defective‐rescue BmNPV BES reBmBac can be employed for efficient heterologous multigene coexpression at different gene sites in one baculovirus genome. In the present study, a recombinant baculovirus, reBm‐Cαγ, carrying two types of chicken interferon (IFN) genes (chIFN‐α and chIFN‐γ) was constructed using the reBmBac system. The chIFN‐α and chIFN‐γ genes were inserted into the same baculovirus genome at the polyhedron and p10 gene sites, respectively. The recombinant baculovirus was capable of coexpressing both chIFN‐α and chIFN‐γ. The expression levels of the two types of IFN in the coexpression product were exponentially high, at approximately 1.7 and 2.5 times higher, respectively, than those in the corresponding single‐expression products. The increase in expression level corresponds to replacement of the nonessential p10 gene in the reBm‐Cαγ recombinant baculovirus. This coexpression of recombinant chicken IFNs showed superior antiviral activity.

AVIAN INNATE IMMUNITY WITH AN EMPHASIS ON CHICKEN MELANOMA DIFFERENTIATION-ASSOCIATED GENE 5 (MDA5)

Avian species have immune system to fight invading pathogens. The immune system comprises innate and adaptive immunity. Innate immunity relies on pattern recognition receptors to sense particular molecules present in pathogens, i.e. pathogen-associated molecular patterns (PAMPs), or danger signals in the environment, i.e. danger-associated molecular patterns (DAMPs). Cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) are the sensors recognizing cytoplasmic PAMP and/or DAMP. Among common avian species, chickens do not have RIG-I whereas ducks and finches do. Therefore, the other RLR member, melanoma differentiation-associated gene 5 (MDA5), is believed to play an important role to recognize intracellular pathogens in chickens. Chicken MDA5 has been identified and its function determined. Chicken MDA5 maintains the same domain architecture compared with MDA5 analogs in other animal species. The expression of chicken MDA5 was upregulated when a synthetic double-stranded RNA (dsRNA), polyriboinosinic:polyribocytidylic acids (poly(I:C)), was transfected into chicken cells, whereas that did not change when cells were incubated with poly(I:C). The enhanced expression of chicken MDA5 in chicken cells upregulated the expression of chicken interferon-[Formula: see text] (IFN-[Formula: see text]). The infection of dsRNA infectious bursal disease virus (IBDV) in non-immune cells triggered the activation of chicken MDA5 signaling pathway, leading to the production of IFN-[Formula: see text] and subsequent response of IFN-stimulated genes. Furthermore, in immune cells like macrophages, chicken MDA5 participated in sensing the infection of IBDV by activating downstream antiviral genes and molecules and modulating adaptive immunity.On the contrary, one of cytoplasmic NLR member, NLR family pyrin domain containing 3 (NLRP3), was cloned and functionally characterized in chicken cells. Chicken NLRP3 conserved the same domain architecture compared with NLRP3 analogs in other animal species. Chicken NLRP3 was highly expressed in kidney, bursa of Fabricius and spleen. The production of mature chicken interleukin 1 [Formula: see text] (IL-1[Formula: see text] in chicken macrophages was stimulated by lipopolysaccharide (LPS) treatment followed by short ATP exposure.In summary, chicken MDA5 was a cytoplasmic dsRNA sensor that mediated the production of type I IFN upon ligand engagement, whereas NLRP3 sensed danger signals, such as ATP, in the cytoplasm and cleaved pro-IL-1[Formula: see text] to produce mature IL-1[Formula: see text]. Chicken MDA5 was not only involved in the activation of innate immune responses in non-immune and immune cells, but it also participated in modulating adaptive immunity in immune cells. Chicken NLRP3 participated in the production of mature chicken IL-1[Formula: see text] upon ligand engagement.

Inhibition of the replication of H9N2 influenza virus in vivo by short-term repeated oral administration of chicken interferon α

To evaluate the optimal administration frequency for interferon-α (IFN-α) and the effect of its combined use with inactive virus on chicken flocks, the prokaryotic expression plasmid pET-22b-ChIFN-α was constructed and transferred into Escherichia coli BL21(DE3) host bacteria to induce the expression of chicken IFN-α and to harvest recombinant proteins inclusion bodies. The expression of recombinant chicken IFN-α was confirmed by SDS-PAGE, and the results demonstrated that the chicken IFN-α (20 kDa) was highly expressed using the prokaryotic expression vector with a concentration of 0.2 mg/mL in the medium. Chicken IFN-α was diluted to 2.5×10⁴ U/fowls and administered to immunized specific-pathogen-free chickens orally in combination with inactivated H9N2 subtype influenza virus. Chicken that received chicken IFN-α were safe after three repeated immunizations (96 h). In addition, chicken IFN-α could induce higher levels of antiviral-related inducible genes in peripheral blood, spleen, and thymus of chicken flocks. The results of a challenge assay revealed that the lowest detoxification rates of chicken IFN-α ranged from three to five days, suggesting a higher capacity to resist H9N2 subtype avian influenza virus. The present study obtained the optimal immune frequency and immunization period for chicken IFN-α to provide theoretical support for the optimal clinical application of IFN-α.

The cellular localization of avian influenza virus PB1-F2 protein alters the magnitude of IFN2 promoter and NFκB-dependent promoter antagonism in chicken cells.

The accessory protein, PB1-F2, of influenza A virus (IAV) functions in a chicken host to prolong infectious virus shedding and thus the transmission window. Here we show that this delay in virus clearance by PB1-F2 in chickens is accompanied by reduced transcript levels of type 1 interferon (IFN)-induced genes and NFκB-activated pro-inflammation cytokines. In vitro, two avian influenza isolate-derived PB1-F2 proteins, H9N2 UDL01 and H5N1 5092, exhibited the same antagonism of the IFN and pro-inflammation induction pathways seen in vivo, but to different extents. The two PB1-F2 proteins had different cellular localization in chicken cells, with H5N1 5092 being predominantly mitochondrial-associated and H9N2 UDL being cytoplasmic but not mitochondrial-localized. We hypothesized that PB1-F2 localization might influence the functionality of the protein during infection and that the protein sequence could alter cellular localization. We demonstrated that the sequence of the C-terminus of PB1-F2 determined cytoplasmic localization in chicken cells and this was linked with protein instability. Mitochondrial localization of PB1-F2 resulted in reduced antagonism of an NFκB-dependent promoter. In parallel, mitochondrial localization of PB1-F2 increased the potency of chicken IFN 2 induction antagonism. We suggest that mitochondrial localization of PB1-F2 restricts interaction with cytoplasmic-located IKKβ, reducing NFκB-responsive promoter antagonism, but enhances antagonism of the IFN2 promoter through interaction with the mitochondrial adaptor MAVS. Our study highlights the differential mechanisms by which IAV PB1-F2 protein can dampen the avian host innate signalling response.

Chicken endothelial cells are highly responsive to viral innate immune stimuli and are susceptible to infections with various avian pathogens

ABSTRACTIt is well established that the endothelium plays a prominent role in the pathogenesis of various infectious diseases in mammals. However, little is known about the role of endothelial cells (EC) as targets for avian pathogens and their contribution to the pathogenesis of infectious diseases in galliform birds. First, we explored the innate immune response of primary chicken aortic endothelial cells (pchAEC), obtained from 18-day-old embryos, to stimulation with pathogen-associated molecular patterns or recombinant chicken interferons (type I, II and III IFNs). In spite of the abundant expression of a number of innate immune receptors, marked cytokine responses to stimulation with pathogen-associated molecular patterns were only seen in pchAEC treated with the TLR3 agonist polyI:C (pI:C) and the MDA5 agonist liposome-complexed polyI:C (L-pI:C), as was assessed by quantitative PCR and luciferase-based IFN-I/NFκB reporter assays. Treatments of pchAEC with IFN-α, IFN-γ and IFN-λ resulted in STAT1-phosphorylation/activation, as was revealed by immunoblotting. Next, we demonstrated that pchAEC are susceptible to infection with a variety of poultry pathogens, including Marek’s disease virus (MDV), infectious bursal disease virus (IBDV), avian pathogenic Escherichia coli (APEC) and Eimeria tenella. Our data highlight that chicken EC are potential targets for viral, bacterial and protozoan pathogens in gallinaceous poultry and may partake in the inflammatory and antimicrobial response. The pchAEC infection model used herein will allow further studies interrogating avian pathogen interactions with vascular EC.RESEARCH HIGHLIGHTSUse of a well-defined primary chicken aortic endothelial cell (pchAEC) culture model for studying avian host–pathogen interactions.pchAEC are responsive to innate immune stimulation with viral pathogen-associated molecular patterns and chicken type I, II and III interferons.pchAEC are susceptible to infections with economically important poultry pathogens, including MDV, IBDV, APEC and Eimeria tenella.

Enhancing Effect of 100.414-kHz Electromagnetic Field Produced by Defender's Pulse Generator on the ChIFN γ-Like Molecule Inducing Capacity of Lens culinaris Agglutinin and 10% PBS Washouts of Different Holocene Minerals.

Macrophages play key role in host defense and tissue repair, and thus understanding regulation of their function is important. For instance, our previous results have shown that in chicken macrophage system (CoMA cell line), application of a pulse of electromagnetic fields of frequencies 0.618, 1.054, 5.229, and 100.414 kHz induces production of interferon γ-like molecules. In this study, we have shown that the electromagnetic field of 100.414 kHz is the most effective in inducing synthesis of chicken interferon γ and chicken interferon γ-like molecules in CoMA cells, especially when combined with Lens culinaris agglutinin and 10% phosphate-buffered saline washouts of different Holocene minerals. A 2-minute pulse of electromagnetic field was produced by Defender’s pulse generator. Both chicken interferon γ and chicken interferon γ-like molecules from the cell supernatant were evaluated by an antiviral assay and were also analyzed with reverse-phase high-performance liquid chromatography on Phenomenex, Aeris peptide columns. Our results show that application of a single inducing factor (Lens culinaris agglutinin, 100.414 kHz electromagnetic field, 10% phosphate buffer saline washout) or combined usage of 2 of them moderately stimulated production of chicken interferon γ-like molecules (from 1.550 to 48.028 IU/mL), whereas the combination of 10% phosphate-buffered saline washout of Koprivnica rock + Lens culinaris agglutinin + 100.414 kHz/9 V resulted in an output of 162.122 IU/mL. Hence, we may conclude that a combined use of electromagnetic field, Holocene minerals, and Lens culinaris agglutinin greatly stimulates synthesis of chicken interferon γ-like molecules in CoMA cells.

Chicken interferon gamma fold expression level and tracheal lesion score differences between commercial broiler chickens vaccinated with live and vectored ILT vaccines

Infectious laryngotracheitis (ILT) is a respiratory tract disease affecting chickens around the world. The disease generates severe production losses due to increased mortality, decreased egg production, delayed body weight gain, and a predisposition to other respiratory pathogens causing enormous economic losses to the intensive poultry industry. Two licensed vaccines, comprising live or vectored ILT, are available to control the disease. The present trial was conducted to determine changes in chicken interferon gamma fold expression levels in chicken spleens and to evaluate tracheal lesion scores before and after vaccination with live and vectored infectious laryngotracheitis vaccines. Broilers were kept under commercial conditions until 35 days of age. Spleen and trachea samples were taken at 14, 28, and 35 days of age. Tracheas were stained with H&amp;E, histopathology was performed, and the INF-γ fold expression level in spleen samples was analyzed. In the spleens of birds vaccinated with the live ILT vaccine, the increase in INF-γ expression levels was statistically significantly (p &lt; 0.01) lower at 28 days of age, or 7 days post vaccination, (trial 1) and higher at 35 days of age, or 14 days post vaccination, (both trials). At 7 days post vaccination with the live ILT vaccine, the mean tracheal lesion score was significantly higher (p &lt; 0.01) than it was in the control group (both trials). At 14 days post vaccination with the live ILT vaccine, the mean tracheal lesion score was significantly higher (p &lt; 0.05) than it was in the control group 3 (trial 1). No significant differences were found between the control group 3 and group 2, which was vaccinated with the vectored HVT/LT vaccine (both trials). The results of the histology of trachea lesions indicate that vaccination with live ILT may have induced early local immunity. The INF-γ analysis results might indicate that the birds in group 2 (HVT/LT) did not develop local immunity until 35 days of age From the practical point of view, the vectored ILT vaccine is more beneficial because of its ease of administration in the hatchery, lower labor cost, and the absence of clinical signs post vaccination. Its potential drawback, however, is poor local immunity and the slow onset of optimal immunity in commercial broiler chickens

Chicken interferon regulatory factor 1 (IRF1) involved in antiviral innate immunity via regulating IFN-β production

Interferon regulatory factors (IRFs) is an important family for IFN expression regulating while viral infection. IRF1, IRF3, and IRF7 are the primary regulators that trigger type I IFN response in mammals. However, IRF3, which has been identified as the most critical regulator in mammals, is absent in chickens, and it is unknown whether IRF1 is involved in type I IFN signaling pathways in IRF3-deficient chicken cells. Here, we identified chicken IRF1 (chIRF1) as a critical IFN-β mediator in response to viral infection. Overexpression of chIRF1 activated IFN-β intensively and suppressed AIV and NDV viral replication. Moreover, the mRNA levels of IFN-β and ISGs increased during chIRF1 overexpression. In addition, deletion mutant analysis revealed that the first four domains of chIRF1 are indispensable for IFN-β induction. Together, our studies demonstrate that chIRF1 is an important regulator of IFN-β and is involved in chicken antiviral innate immunity.

Induction of innate immune response following introduction of infectious bronchitis virus (IBV) in the trachea and renal tissues of chickens

Infectious bronchitis (IB) is a highly contagious respiratory disease of chickens, which is caused by the infectious bronchitis virus (IBV). The innate immune response is crucial for antiviral infections and revealing the pathogenic mechanisms of IBV. In this study, we presents an evaluation of interferon (I, II and III IFNs) in renal and tracheal samples from chickens experimentally infected previously vaccinated or not. The results suggest differential expression of chicken interferon, among them type I IFN elaborate a major role in fighting off virus. And vaccine confers greater induction ability of innate immunity thereby vaccination prior infection occurs might be necessary. Above all, we found that IFN-λ also have an effect on IBV infection in trachea besides many other respiratory virus. This study provides the first comprehensive analysis of host-virus interactions of IBV with chicken innate immune response mediated by interferon in various groups.

Chicken IFN Kappa: A Novel Cytokine with Antiviral Activities

Interferons (IFNs) are essential components of the host innate immune system and define first-line of defence against pathogens. In mammals, several type I IFNs are identified, however, only limited data is available on the repertoire of IFNs in avian species. Here we report the characterization of chicken IFN-κ (chIFN-κ) near the type I IFN locus on the sex-determining Z chromosome. Genetic, evolutionary and syntenic analyses indicate that chIFN-κ is a type I IFN with conserved genetic features and promoter binding sites. chIFN-κ regulated the IFN-stimulated response element signalling pathways and activated a panel of IFN-regulated genes, antiviral mediators and transcriptional regulators. Priming of chicken primary fibroblasts and tracheal organ cultures with chIFN-κ imparted cellular protections against viral infections both in vitro and ex vivo. To determine whether chIFN-κ defines the antiviral state in developing chicken embryos, we used replication-competent retroviral RCAS vector system to generate transgenic chicken embryos that constitutively and stably expressed chIFN-κ. We could demonstrate that chIFN-κ markedly inhibited the replication of avian RNA viruses in ovo. Collectively, these results shed the light on the repertoire of IFNs in avian species and provide functional data on the interaction of the chIFN-κ with RNA viruses of poultry and public health importance.