Pathogenesis and host responses in lungs and kidneys following Canadian 4/91 infectious bronchitis virus (IBV) infection in chickens.

The infectious bronchitis virus (IBV) 4/91 was one of the common IBV variants isolated in Eastern Canada between 2013 and 2017 from chicken flocks showing severe respiratory and production problems. We designed an in vivo experiment, using specific pathogen free (SPF) chickens, to study the pathogenesis of, and host response to, Canadian (CAN) 4/91 IBV infection. At one week of age, the chickens were infected with 4/91 IBV/Ck/Can/17-038913 isolate. Swab samples were collected at predetermined time points. Five birds from the infected and the control groups were euthanized at 3, 7- and 10-days post-infection (dpi) to collect lung and kidney tissues. The results indicate IBV replication in these tissues at all three time points with prominent histological lesions, significant immune cell recruitment and up regulation of proinflammatory mediators. Overall, our findings add to the understanding of the pathogenesis of 4/91 infection and the subsequent host responses in the lungs and kidneys following experimental infection.

Repeated MDA5 Gene Loss in Birds: An Evolutionary Perspective.

Two key cytosolic receptors belonging to the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family sense the viral RNA-derived danger signals: RIG-I and melanoma differentiation-associated protein 5 (MDA5). Their activation establishes an antiviral state by downstream signaling that ultimately activates interferon-stimulated genes (ISGs). While in rare cases RIG-I gene loss has been detected in mammalian and avian species, most notably in the chicken, MDA5 pseudogenization has only been detected once in mammals. We have screened over a hundred publicly available avian genome sequences and describe an independent disruption of MDA5 in two unrelated avian lineages, the storks (Ciconiiformes) and the rallids (Gruiformes). The results of our RELAX analysis confirmed the absence of negative selection in the MDA5 pseudogene. In contrast to our prediction, we have shown, using multiple dN/dS-based approaches, that the MDA5 loss does not appear to have resulted in any compensatory evolution in the RIG-I gene, which may partially share its ligand-binding specificity. Together, our results indicate that the MDA5 pseudogenization may have important functional effects on immune responsiveness in these two avian clades.

Effects of Salmonella enterica ser. Enteritidis and Heidelberg on host CD4+CD25+ regulatory T cell suppressive immune responses in chickens.

Poultry infected with Salmonella mount an immune response initially, however the immune responses eventually disappear leading the bird to be a carrier of Salmonella. The hypothesis of this study is that Salmonella infection induces T regulatory cell numbers and cytokine production and suppress host T cells locally in the gut to escape the host immune responses. An experiment was conducted to comparatively analyze the effect of S. enterica ser. Enteritidis (S. Enteritidis) and S. enterica ser. Heidelberg (S. Heidelberg) infection on CD4+CD25+ T regulatory cell properties in chickens. A total of 144 broiler chicks were randomly distributed into three experimental groups of non-infected control, S. Enteritidis infected and S. Heidelberg infected groups. Chickens were orally inoculated with PBS (control) or 5x106 CFU/mL of either S. Enteritidis or S. Heidelberg at 3 d of age. Each group was replicated in six pens with eight chickens per pen. Chickens infected with S. Enteritidis had 6.2, 5.4, and 3.8 log10 CFU/g, and chickens infected with S. Heidelberg had 7.1, 4.8, and 4.1 log10 CFU/g Salmonella in the cecal contents at 4, 11, and 32 dpi, respectively. Both S. Enteritidis and S. Heidelberg were recovered from the liver and spleen 4 dpi. At 4, 11, and 32 dpi, chickens infected with S. Enteritidis and S. Heidelberg had increased CD4+CD25+ cell numbers as well as IL-10 mRNA transcription of CD4+CD25+ cells compared to that in the control group. CD4+CD25+ cells from S. Enteritidis- and S. Heidelberg-infected chickens and restimulated with 1 µg antigen in vitro, had higher (P < 0.05) IL-10 mRNA transcription than the CD4+CD25+ cells from the non-infected controls Though at 4dpi, chickens infected with S. Enteritidis and S. Heidelberg had a significant (P < 0.05) increase in CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, the CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, were comparable to that in the control group at 11 and 32dpi identifying that the host inflammatory response against Salmonella disappears at 11 dpi. It can be concluded that S. Enteritidis and S. Heidelberg infection at 3 d of age induces a persistent infection through inducing CD4+CD25+ cells and altering the IL-10 mRNA transcription of CD4+CD25+ cell numbers and cytokine production in chickens between 3 to 32 dpi allowing chickens to become asymptomatic carriers of Salmonella after 18 dpi.

Chicken DDX1 Acts as an RNA Sensor to Mediate IFN-ß Signaling Pathway Activation in Antiviral Innate Immunity.

Chickens are the natural host of Newcastle disease virus (NDV) and avian influenza virus (AIV). The discovery that the RIG-I gene, the primary RNA virus pattern recognition receptor (PRR) in mammals, is naturally absent in chickens has directed attention to studies of chicken RNA PRRs and their functions in antiviral immune responses. Here, we identified Asp-Glu-Ala-Asp (DEAD)-box helicase 1 (DDX1) as an essential RNA virus PRR in chickens and investigated its functions in anti-RNA viral infections. The chDDX1 gene was cloned, and cross-species sequence alignment and phylogenetic tree analyses revealed high conservation of DDX1 among vertebrates. A quantitative RT-PCR showed that chDDX1 mRNA are widely expressed in different tissues in healthy chickens. In addition, chDDX1 was significantly upregulated after infection with AIV, NDV, or GFP-expressing vesicular stomatitis virus (VSV-GFP). Overexpression of chDDX1 in DF-1 cells induced the expression of IFN-ß, IFN-stimulated genes (ISGs), and proinflammatory cytokines; it also inhibited NDV and VSV replications. The knockdown of chDDX1 increased the viral yield of NDV and VSV and decreased the production of IFN-ß, which was induced by RNA analog polyinosinic-polycytidylic acid (poly[I:C]), by AIV, and by NDV. We used a chicken IRF7 (chIRF7) knockout DF-1 cell line in a series of experiments to demonstrate that chDDX1 activates IFN signaling via the chIRF7 pathway. Finally, an in-vitro pulldown assay showed a strong and direct interaction between poly(I:C) and the chDDX1 protein, indicating that chDDX1 may act as an RNA PRR during IFN activation. In brief, our results suggest that chDDX1 is an important mediator of IFN-ß and is involved in RNA- and RNA virus-mediated chDDX1-IRF7-IFN-ß signaling pathways.

Evolution of innate and adaptive immune genes in a non-model waterbird, the common tern.

Toll-like receptors (TLRs) and the Major Histocompatibility Complex (MHC) are the key pathogen-recognition genes of vertebrate immune system and they have a crucial role in the initiation of innate and adaptive immune response, respectively. Recent advancements in sequencing technology sparked research on highly duplicated MHC genes in non-model species, but TLR variation in natural vertebrate populations has remained little studied and comparisons of polymorphism across both TLRs and MHC are scarce. Here, we aimed to compare variation across innate (four TLR loci) and adaptive (MHC class I and class II) immune genes in a non-model avian species, the common tern Sterna hirundo. We detected relatively high allelic richness at TLR genes (9-48 alleles per locus), which was similar to or even higher than the estimated per locus allelic richness at the MHC (24-30 alleles at class I and 13-16 alleles at class II under uniform sample sizes). Despite this, the total number of MHC alleles across all duplicated loci (four class I and three class II) was much higher and MHC alleles showed greater sequence divergence than TLRs. Positive selection targeted relatively more sites at the MHC than TLRs, but the strength of selection (dN/dS ratios) at TLRs was higher when compared to MHC class I. There were also differences in the signature of positive selection and recombination (gene conversion) between MHC class I and II (stronger signature at class II), suggesting that mechanisms maintaining variation at the MHC may vary between both classes. Our study indicates that allelic richness of both innate and adaptive immune receptors may be maintained at relatively high levels in viable avian populations and we recommend a transition from the traditional gene-specific to multi-gene approach in studying molecular evolution of vertebrate immune system.

OVA-Experienced CD4+ T Cell Transfer and Chicken Protein Challenge Affect the Immune Response to OVA in a Murine Model.

Chicken meat is often a major component of a modern diet. Allergy to chicken meat is relatively rare and occurs independently or in subjects allergic to ovalbumin (OVA). We examined the effect of adoptive transfer of OVA-CD4+ T cells on the immune response to OVA in mice fed chicken meat. Donor mice were injected intraperitoneally with 100 µg of OVA with Freund's adjuvant two times over a week, and CD4+ T cells were isolated from them and transferred to naïve mice (CD4+/OVA/ChM group), which were then provoked with OVA with FA and fed freeze-dried chicken meat for 14 days. The mice injected with OVA and fed chicken meat (OVA/ChM group), and sensitized (OVA group) and healthy (PBS group) mice served as controls. Humoral and cellular response to OVA was monitored over the study. The CD4+/OVA/ChM group had lowered levels of anti-OVA IgG and IgA, and total IgE. There were significant differences in CD4+, CD4+CD25+, and CD4+CD25+Foxp3+ T cells between groups. OVA stimulation decreased the splenocyte proliferation index and IFN-γ secretion in the CD4+/OVA/ChM group compared to the OVA group. IL-4 was increased in the OVA/ChM mice, which confirms allergenic potential of the egg-meat protein combination. Transfer of OVA-experienced CD4+ T cells ameliorated the negative immune response to OVA.

Avian antibodies (IgY) targeting spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibit receptor binding and viral replication.

BACKGROUND: The global pandemic of Coronavirus infectious disease 2019 (COVID-19), caused by SARS-CoV-2, has plunged the world into both social and economic disarray, with vaccines still emerging and a continued paucity of personal protective equipment; the pandemic has also highlighted the potential for rapid emergence of aggressive respiratory pathogens and the need for preparedness. Avian immunoglobulins (IgY) have been previously shown in animal models to protect against new infection and mitigate established infection when applied intranasally. We carried out a proof-of-concept study to address the feasibility of using such antibodies as mucosally-applied prophylaxis against SARS-CoV-2. METHODS: Hens were immunized with recombinant S1 spike glycoprotein of the virus, and the resulting IgY was evaluated for binding specificity, inhibition of glycoprotein binding to angiotensin converting enzyme-2 (ACE2) protein (the requisite binding site for the virus), and inhibition of viral replication in Vero cell culture. RESULTS: Titers of anti-S1 glycoprotein IgY were evident in yolks at 14 days post-immunization, peaking at 21 days, and at peak concentrations of 16.8 mg/ml. IgY showed strong and significant inhibition of S1/ACE2 binding interactions, and significantly inhibited viral replication at a concentration of 16.8 mg/ml. Four weeks' collection from eggs of two hens produced a total of 1.55 grams of IgY. CONCLUSIONS: In this proof-of-concept study we showed that avian immunoglobulins (IgY) raised against a key virulence factor of the SARS-CoV-2 virus successfully inhibited the critical initial adhesion of viral spike glycoproteins to human ACE2 protein receptors and inhibited viral replication in vitro, in a short period using only two laying hens. We conclude that production of large amounts of IgY inhibiting viral binding and replication of SARS-CoV-2 is feasible, and that incorporation of this or similar material into an intranasal spray and/or other mucosal protecting products may be effective at reducing infection and spread of COVID-19.

Heat stress inhibits expression of the cytokines, and NF-κB-NLRP3 signaling pathway in broiler chickens infected with salmonella typhimurium.

High ambient temperature has potential influence on oxidative stress, or systemic inflammation affecting poultry production and immune status of chickens. Heat stress (HS) induces intestinal inflammation and increases susceptibility of harmful pathogens, such as Salmonella and Escherichia coli. Intestinal inflammation is a common result of body immune dysfunction. Therefore, we designed an experiment to analyze the effects of 35 ± 2 °C HS on salmonella infection in chickens through regulation of the immune responses. 40 broiler chickens were randomly divided into 4 groups: control group, heat stress (HS) group, salmonella typhimurium (ST) group and model group (heat stress + salmonella typhimurium, HS + ST). Birds in HS and model group were treated with 35 ± 2 °C heat stress 6 h a day and for 14 continuous days. Then, ST and model group birds were orally administrated with 1 mL ST inoculum (109 cfu/mL). Chickens were sacrificed at the 4th day after ST administration and ileum tissues were measured. We observed that heat stress decreased ileum TNF-α and IL-1ß protein expressions. Concomitantly heat stress decreased NLRP3 and Caspase-1 protein levels. The protein expressions of p-NF-κB-p65 and p-IκB-α in ileum. Heat stress also inhibited IFN-α, p-IRF3 and p-TBK1, showing a deficiency in the HS + ST group birds. Together, the present data suggested that heat stress suppressed intestinal immune activity in chickens infected by salmonella typhimurium, as observed by the decrease of immune cytokines levels, which regulated by NF-κB-NLRP3 signaling pathway.

DEAD/DEAH-box helicase 5 is hijacked by an avian oncogenic herpesvirus to inhibit interferon beta production and promote viral replication.

DEAD-box helicase 5 (DDX5) plays a significant role in tumorigenesis and regulates viral replication of several viruses. An avian oncogenic herpesvirus, Marek's disease virus (MDV), is widely known to cause immunosuppression and lymphoma in chickens. However, the underlying mechanisms of how DDX5 plays a role in viral replication remain unclear. In this study, we show that MDV inhibits the production of interferon beta (IFN-ß) in chicken embryo fibroblasts (CEFs) by increasing the expression level and promoting the nuclear aggregation of DDX5. We further reveal how DDX5 down-regulates melanoma differentiation-associated gene 5/toll-like receptor 3 signaling through the fundamental transcription factor, interferon regulatory factor 1. MDV replication is suppressed, and the production of IFN-ß is promoted in the DDX5 absented CEFs. Taken together, our investigations demonstrate that MDV inhibits IFN-ß production by targeting DDX5-mediated signaling to facilitate viral replication, which offers a novel insight into the mechanism by which an avian oncogenic herpesvirus replicates in chicken cells.

Characterization of immunological properties of chicken chemokine CC motif ligand 5 using new monoclonal antibodies.

CCL5 (formerly RANTES) belongs to the CC (or ß) chemokine family and is associated with a plethora of inflammatory disorders and pathologic states. CCL5 is mainly produced and secreted by T cells, macrophages, epithelial cells, and fibroblasts and acts as a chemoattractant to recruit effector cells to the inflammation sites. Chicken CCL5 (chCCL5) protein is closely related to avian CCL5 orthologs but distinct from mammalian orthologs, and its modulatory roles in the immune response are largely unknown. The present work was undertaken to characterize the immunological properties of chCCL5 using the new sets of anti-chCCL5 mouse monoclonal antibodies (mAbs). Eight different mAbs (6E11, 6H1, 8H11, 11G1, 11G11, 12H1, 13D1, and 13G3) were characterized for their specificity and binding ability toward chCCL5. Two (13G3 and 6E11) of them were selected to detect native chCCL5 in chCCL5-specific antigen-capture ELISA. Using 13G3 and 6E11 as capture and detection antibodies, respectively, the ELISA system detected serum chCCL5 secretions in Clostridium perfringens- and Eimeria-infected chickens. The intracellular expressions of chCCL5 in primary cells or cell lines derived from chickens were validated in immunocytochemistry and flow cytometry assays using both 13G3 and 6E11 mAbs. Furthermore, 6E11, but not 13G3, neutralized chCCL5-induced chemotaxis in vitro using chicken PBMCs. These molecular characteristics of chCCL5 demonstrate the potential application of anti-chCCL5 mAbs and CCL5-specific antigen-capture detection ELISA for detecting native chCCL5 in biological samples. The availability of these new immunological tools will be valuable for fundamental and applied studies in avian species.

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-ß, 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.

Cloning and structural analysis of complement component 3d in wild birds provides insight into its functional evolution.

Complement component 3 d (C3d) is the final cleavage product of the complement component C3 and serves as a crucial role in link innate and adaptive immunity, and increase B-cell sensitivity to an antigen by 1000-10000 fold. The crystal structure of human C3d revealed there are two distinct surfaces, a convex surface containing the thioester-constituting residues that mediate covalent binding to the target antigen, and a concave surface with an acidic pocket responsible for interaction with CR2. In this study, we cloned and sequenced cDNA fragment encoding C3d region from 15 wild bird species. Then, the C3d sequences from wild birds, chicken and mammals were aligned to construct phylogenetic trees. Phylogenetic tree displayed two main branches, indicating mammals and birds, but the bird C3d branch was divided into two main parts, with five wild birds (Ardeola bacchus, Zoothera, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus) clustering much closer to mammals. In addition, the C3d proteins of Ardeola bacchus, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus contained a Glu163 residue at the position at which Lys163 was found in other birds. However, Glu163 have the same charge polarity as Asp163, which is the key amino acid residue comprising the acidic pocket combined with CR2 found at this position in mammals, and Zoothera also possessed Asp163 at this position. Structure modeling analyses also verified that the C3ds of these five wild bird species exhibited the amino acid sequence and structure comprising the typical acidic pocket found in mammals that is required for combination with B cell surface receptors, which contribute electrostatic forces to interact with CR2. Our investigations indicate that some bird C3ds may already have the ability to bind with CR2 by electrostatic force, like mammals. As Ardeola bacchus, Zoothera, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus have more typical C3d concave acid pockets and thus a stronger ability to bind CR2, we speculate that these five wild birds may have a solider immunity against pathogens. Our phylogenetic and structural analyses of bird C3ds provide insights on the evolutionary divergence in the function of immune factors of avian and mammalian.

From Chickens to Humans: The Importance of Peptide Repertoires for MHC Class I Alleles.

In humans, killer immunoglobulin-like receptors (KIRs), expressed on natural killer (NK) and thymus-derived (T) cells, and their ligands, primarily the classical class I molecules of the major histocompatibility complex (MHC) expressed on nearly all cells, are both polymorphic. The variation of this receptor-ligand interaction, based on which alleles have been inherited, is known to play crucial roles in resistance to infectious disease, autoimmunity, and reproduction in humans. However, not all the variation in response is inherited, since KIR binding can be affected by a portion of the peptide bound to the class I molecules, with the particular peptide presented affecting the NK response. The extent to which the large multigene family of chicken immunoglobulin-like receptors (ChIRs) is involved in functions similar to KIRs is suspected but not proven. However, much is understood about the two MHC-I molecules encoded in the chicken MHC. The BF2 molecule is expressed at a high level and is thought to be the predominant ligand of cytotoxic T lymphocytes (CTLs), while the BF1 molecule is expressed at a much lower level if at all and is thought to be primarily a ligand for NK cells. Recently, a hierarchy of BF2 alleles with a suite of correlated properties has been defined, from those expressed at a high level on the cell surface but with a narrow range of bound peptides to those expressed at a lower level on the cell surface but with a very wide repertoire of bound peptides. Interestingly, there is a similar hierarchy for human class I alleles, although the hierarchy is not as wide. It is a question whether KIRs and ChIRs recognize class I molecules with bound peptide in a similar way, and whether fastidious to promiscuous hierarchy of class I molecules affect both T and NK cell function. Such effects might be different from those predicted by the similarities of peptide-binding based on peptide motifs, as enshrined in the idea of supertypes. Since the size of peptide repertoire can be very different for alleles with similar peptide motifs from the same supertype, the relative importance of these two properties may be testable.

Plasma Proteome Responses in Salmonid Fish Following Immunization.

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout (Oncorhynchus mykiss, Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.

Research on the Effect of Pediococcus pentosaceus on Salmonella enteritidis-Infected Chicken.

Salmonella enteritidis can cause significant morbidity and mortality in humans and economic loss in the animal industry. Improving the innate immunity is an effective method to prevent S. enteritidis infection. Pediococcus pentosaceus is a Gram-positive coccus which had probiotics properties. Numerous previously published studies reported that probiotics were beneficial to gut microbiota by changing the intestinal flora structure and inhibiting the harmful microbial growth to enhance the innate immunity. We investigated the immunological effects of P. pentosaceus on Salmonella-infected chickens by the following experiment. A total of 120 broilers from AA line were fed and divided into 2 groups (treated and control groups) for the experiment from day 1. The control group was fed with the basic diet, while the treated group was fed with the basic diet adding P. pentosaceus microcapsule with the bacterial concentration of 1 g/kg in the feed and bacterial counts 2.5 × 109 CFU/g. All the birds were given with 0.5 ml of S. enteritidis bacterial suspension (109 CFU/ml) through oral cavity at day 9. The number of dead birds was recorded and used in the analysis. The bacterial culture method and quantitative real-time PCR analysis were used to evaluate the effects of P. pentosaceus on chickens infected with S. enteritidis and to ascertain the mechanism of the effect. The results showed that the P. pentosaceus could restrain the pathogenicity of S. enteritidis and reduce the death rate from 44.4% to 23.3%. The flora in the caecum exhibited "rising-declining" trends, and the gene (TLR4, MyD88, TRAF6 NF-κB, IFN-ß, TNF-a, IL6, and IL8) expression pattern was different between the experimental and control group. P. pentosaceus as a probiotic may competitively inhibit the growth of S. enteritidis and control the inflammatory response through regulating the gene expression which involved in the toll-like receptor pathway and inflammation pathway.

hnRNPH2 as an Inhibitor of Chicken MDA5-Mediated Type I Interferon Response: Analysis Using Chicken MDA5-Host Interactome.

RIG-I and MDA5 are two key pattern recognition receptors that sense the invasion of RNA viruses and initiate type I interferon (IFN) response. Although these receptors are generally conserved in vertebrates, RIG-I is absent in chickens, whereas MDA5 is present. Chicken MDA5 (chMDA5) plays a pivotal role in sensing the invasion of RNA viruses into cells. However, unlike mammalian MDA5, where there are in-depth and extensive studies, regulation of the chMDA5-mediated signaling pathway remains unexplored. In this study, we performed a pulldown assay and mass spectrometry analysis to identify chicken proteins that could interact with the N terminal of chMDA5 (chMDA5-N) that contained two CARDs responsible for binding of the well-known downstream adaptor MAVS. We found that 337 host proteins could potentially interact with chMDA5-N, which were integrated to build a chMDA5-N-host association network and analyzed by KEGG pathway and Gene Ontology annotation. Results of our analysis revealed that diverse cellular processes, such as RNA binding and transport and protein translation, ribosome, chaperones, and proteasomes are critical cellular factors regulating the chMDA5-mediated signaling pathway. We cloned 64 chicken genes to investigate their effects on chMDA5-mediated chicken IFN-ß production and confirmed the association of chicken DDX5, HSPA8, HSP79, IFIT5, PRDX1, and hnRNPH2 with chMDA5-N. In particular, we found that chicken hnRNPH2 impairs the association between chMDA5-N and MAVS and thus acts as a check on the chMDA5-mediated signaling pathway. To our knowledge, this study is the first to analyze the chicken MDA5-host interactome, which provides fundamental but significant insights to further explore the mechanism of chicken MDA5 signaling regulation in detail.

The immunomodulatory effect of cathelicidin-B1 on chicken macrophages.

Cathelicidins (CATHs) play an important role in the innate immune response against microbial infections. Among the four chicken cathelicidins, CATH-B1 is studied the least. In this study, the effect of CATH-B1 on the macrophage response towards avian pathogenic E. coli (APEC) and bacterial ligands was investigated. Our results show that APEC induced CATH-B1 gene expression in both a chicken macrophage cell line (HD11 cells) and primary macrophages, while expression of the other three CATHs was virtually unaffected. While the antimicrobial activity of CATH-B1 is very low under cell culture conditions, it enhanced bacterial phagocytosis by macrophages. Interestingly, CATH-B1 downregulated APEC-induced gene expression of pro-inflammatory cytokines (IFN-ß, IL-1ß, IL-6 and IL-8) in primary macrophages. In addition, CATH-B1 pre-incubated macrophages showed a significantly higher gene expression of IL-10 after APEC challenge, indicating an overall anti-inflammatory profile for CATH-B1. Using isothermal titration calorimetry (ITC), CATH-B1 was shown to bind LPS. This suggests that CATH-B1 reduces toll like receptor (TLR) 4 dependent activation by APEC which may partly explain the decreased production of pro-inflammatory cytokines by macrophages. On the contrary, direct binding of CATH-B1 to ODN-2006 enhanced the TLR21 dependent activation of macrophages as measured by nitric oxide production. In conclusion, our results show for the first time that CATH-B1 has several immunomodulatory activities and thereby could be an important factor in the chicken immune response.

Lactobacillus johnsonii-activated chicken bone marrow-derived dendritic cells exhibit maturation and increased expression of cytokines and chemokines in vitro.

Lactobacillus species are typical members of gut microflora that immunomodulatory effects and can regulate a variety of immune cells, such as dendritic cells (DCs). Notably, DCs possess the unique ability to initiate primary immune responses. Notably, DCs possess the unique ability to initiate primary immune responses. In this study, we investigated the effects of Lactobacillus johnsonii (L. johnsonii) on the maturation and activation of chicken bone marrow-derived dendritic cells (chBM-DCs). The chBM-DCs generated from chicken bone marrow monocytes were stimulated using lethally irradiated L. johnsonii. L. johnsonii-stimulated chBM-DCs upregulated the expression of major histocompatibility complex class II (MHC-II), CD40, and CD86, decreased phagocytosis, and increased the ability to induce the proliferation of allogeneic T cells, which displayed a mature phenotype and function. Upon maturation with L. johnsonii, the expression of Th1-type cytokines [interleukin (IL)-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α)], a Th2-type cytokine (IL-10), pro-inflammatory cytokines (IL-1ß and IL-6), and chemokines (CXCLi1 and CXCLi2) greatly increased; however, a high expression of IL-10 was only observed at mid-late time points for chBM-DCs stimulated with high doses of L. johnsonii. Moreover, L. johnsonii upregulated the mRNA levels of TLR2 and TLR5. These results reveal that L. johnsonii plays a potentially important role in modulating the immunological functions of chBM-DCs, suggesting that it influences and mediates immune responses in vitro.

Development and characterization of specific anti-Usutu virus chicken-derived single chain variable fragment antibodies.

Usutu virus belongs to the Japanese encephalitis serogroup within the Flaviviridae family. Mammals may become incidental hosts after the bite of an infected mosquito while birds act as the main reservoir. Human cases have become more common recently and elicit various outcomes ranging from asymptomatic to severe illness including encephalitis. Problematically, antisera against Usutu virus cross-react with other flaviviruses such as the co-circulating West Nile virus. As an approach to generate Usutu virus-specific antibodies, we immunized chickens with purified Usutu virus envelope protein domain III, isolated the spleen mRNA and generated an scFv phage display library. The most potent binders for Usutu virus domain III were selected via biopanning and their affinity to domain III was examined using SPR. Four scFvs bound the domain III of Usutu virus in the nanomolar region; two bound the protein over 40 times more strongly than West Nile virus domain III. We further characterized these scFv antibodies for suitability in standard laboratory tests such as western blots, ELISA, and neutralization tests. Four specific and one cross-reactive antibody performed well in western blots with domain III and the full-length envelope protein of Usutu virus and West Nile virus. All antibodies bound in virus ELISA assays to Usutu virus strain Vienna-2001. However, none of the antibodies neutralized either Usutu virus or West Nile virus. These antibody candidates could be crucial in future diagnostic tests to distinguish Usutu virus from other flaviviruses and might even offer virus neutralization after a conversion to Fab or IgG.

Immunomodulatory effects of avian ß-defensin 5 in chicken macrophage cell line.

Antimicrobial peptides (AMPs) protect host from pathogens as first line of defense. Especially, ß-defensins shows antimicrobial activity and immune modulation effects. Avian species also have ß-defensins as avian ß-defensins (AvBDs) from AvBD1 to AvBD14. In this study, we characterized chicken AvBD5 and demonstrated its immune modulatory functions in chicken macrophage cell line (HD11). Chicken AvBD5 is composed of a signal, pro, and mature peptides containing one α-helix, four ß-sheet, and three disulfide bonds. Here, we also showed that chicken AvBD5 induced Th1, Th2, and Th17 cytokines in chicken macrophage cell line and stimulated MAPK signaling pathways through ERK1/2 and p38 molecules. In addition, AvBD5 stimulated MyD88 and CD40 to regulate immune systems. Taken together, chicken AvBD5 can modulate host immune systems by inducing cytokines expression and stimulating MAPK signaling pathway.