Role of Cherry Valley duck IRF1 mediated signal pathway in host anti-duck Tembusu virus.

China is the country with the largest amount of duck breeding as well as duck meat and egg production. In recent years, the emergence and spread of duck Tembusu virus (DTMUV) has become one of the important factors in reducing the amount of duck slaughter, which seriously endangers the duck breeding industry in our country. In-depth research on the mechanism of duck innate immunity facilitates the exploration of new models for the treatment of DTMUV infection. IRF1 can induce the expression of many antiviral immune factors in the animal organism and play an important role in the innate immune response. In this study, we used interfering RNA to knock down the IRF1 gene in DEF cells and then the cells were infected with DTMUV. We found that knockdown of IRF1 promoted DTMUV replication at an early stage and caused downregulation of the expression of several major pattern recognition receptors (PRRs), interleukins (IL), interferons (IFN), antiviral proteins, and MHC molecules by assay, showing that the duIRF1-mediated signaling pathway plays an extremely important role in DTMUV-induced host innate immunity. In addition, we constructed the recombinant expression plasmid pET32a(+)-duIRF1-His, and finally prepared the polyclonal antibody of duIRF1 with good specificity, hoping to provide a detection means for research on the mechanism of IRF1 in innate immunity in our laboratory and in this field.

The role of duck LGP2 in innate immune response of host anti-tembusu virus.

Laboratory of Genetics and Physiology 2 (LGP2), along with Retinoic Acid Induced Gene-I (RIG-I) and Melanoma Differentiation Associated Gene 5, are members of the retinoic acid-inducible gene-I-like receptors (RLRs) in pattern recognition receptors, playing an important role in the host's innate immunity. Due to lacking a caspase activation and recruitment domain, LGP2 is controversially regarded as a positive or negative regulator in the antiviral response. This study aimed to explore how duck LGP2 (duLGP2) participates in duck innate immunity and its role in countering the duck Tembusu virus (DTMUV). In duck embryo fibroblast cells, the overexpression of duLGP2 significantly reduced the cell's antiviral capacity by inhibiting type I interferon (IFN) production and the expression of downstream IFN-stimulated genes. Conversely, duLGP2 knockdown had the opposite effect. For the first time, we introduced the LGP2 gene fragment into duck embryos using a lentiviral vector to ensure persistent expression and generated gene-edited ducks with LGP2 overexpression. We demonstrated that duLGP2 facilitates DTMUV replication in both in vitro and in vivo experiments, leading to robust inflammatory and antiviral responses. Interestingly, the repressive effects of duLGP2 on type I IFN production were only observed in the early stage of DTMUV infection, with type I IFN responses becoming enhanced as the viral load increased. These results indicate that duLGP2 acts as a negative regulator during the resting state and early stages of DTMUV infection. This study provides a theoretical basis for further research on duck RLRs and developing new anti-DTMUV drugs or vaccine adjuvants.

Research Note: Genetic characterization of novel duck reoviruses from Shandong Province, China in 2022.

Since 2005, novel duck reoviruses have been outbreaks in duck breeding areas such as central China and South China. In recent years, the incidence rate of this disease is still increasing, bringing serious economic losses to waterfowl breeding industry. This study isolated 3 novel duck reoviruses (NDRV-SDLS, NDRV-SDWF, and NDRV-SDYC) from sick ducks in 3 local duck farms in Shandong Province. The study aimed to investigate the characteristics of these viruses. The virus is inoculated into duck embryo fibroblasts, where the virus replicates to produce syncytium and dies within 3 to 5 d. The viruses were also isolated from infected ducks, and RT-PCR amplified the whole genomes after passage purification in duck embryos. The resulting whole genome was analyzed for genetic evolution. The total length of the gene sequencing was 23,418 bp, divided into 10 fragments. Gene sequence comparison showed that the 3 strains had high similarity with novel duck reoviruses (NDRV) but low similarity with chicken-origin reovirus (chicken ARV) and Muscovy duck reovirus (MDRV), especially in the σC segment. Phylogenetic analysis of the 10 fragments showed that the 3 isolates constituted the same evolutionary clade as other DRV reference strains and were far related to ARV and MDRV in different evolutionary clades. The results of all 10 segments indicate that the isolates are in the evolutionary branch of NDRV, suggesting that the novel waterfowl reovirus is the dominant circulating strain in Shandong. This study complements the gene bank information of NDRV and provides references for vaccine research and disease prediction of NDRV in Shandong.

Research Note: Complete genome cloning and genetic evolution analysis of four Cherry Valley duck circovirus strains in China in 2022.

In recent years, with the expansion of duck breeding industry in China, the infection rate of duck circovirus (DuCV) in duck and the mixed infection rate of DuCV with other diseases increased significantly, which seriously endanger the development of duck breeding industry. To study the epidemic status of duck circovirus in China, analyze the virus's genetics and evolution, and establish a foundation for scientific prevention and control of duck circovirus, our laboratory collected 4 disease materials preliminarily diagnosed as duck circovirus infections. Conventional PCR was used to amplify 4 strains of duck circovirus with a full length of 1993bp, and their sequences were compared and analyzed. The analysis showed that the 4 DuCVs had typical circovirus characteristics, including 3 major ORFs: ORFV1 (Rep protein), ORFC1 (Cap protein), ORFC2 (apoptosis-related protein), and a stem ring structure. The 4 strains were compared with 22 other reference strains, and the results revealed that all 4 strains belonged to the DuCV-I type represented by the German strain AY228555. Furthermore, the homology between the 4 DuCVs and the reference strains was up to 98.6%, which help us to understand the genotype and genetic variation of DuCV in these regions and provide a reference for the prevention and control of DuCV.

Duck LGP2 Downregulates RIG-I Signaling Pathway-Mediated Innate Immunity Against Tembusu Virus.

In mammals, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) has been demonstrated to play a critical role in activating downstream signaling in response to viral RNA. However, its role in ducks' antiviral innate immunity is less well understood, and how gene-mediated signaling is regulated is unknown. The regulatory role of the duck laboratory of genetics and physiology 2 (duLGP2) in the duck RIG-I (duRIG-I)-mediated antiviral innate immune signaling system was investigated in this study. In duck embryo fibroblast (DEF) cells, overexpression of duLGP2 dramatically reduced duRIG-I-mediated IFN-promotor activity and cytokine expression. In contrast, the knockdown of duLGP2 led to an opposite effect on the duRIG-I-mediated signaling pathway. We demonstrated that duLGP2 suppressed the duRIG-I activation induced by duck Tembusu virus (DTMUV) infection. Intriguingly, when duRIG-I signaling was triggered, duLGP2 enhanced the production of inflammatory cytokines. We further showed that duLGP2 interacts with duRIG-I, and this interaction was intensified during DTMUV infection. In summary, our data suggest that duLGP2 downregulated duRIG-I mediated innate immunity against the Tembusu virus. The findings of this study will help researchers better understand the antiviral innate immune system's regulatory networks in ducks.

Identification and antiviral effect of Cherry Valley duck IRF4.

Interferon regulatory factor 4 (IRF4) is a multifunctional transcription factor that plays an important regulatory role in the interferon (IFN) signaling. IRF4 participates in the process of antivirus, Th cell differentiation and B cell maturation by regulating the expression of IFN and some lymphokines. In this study, Cherry Valley duck IRF4 (duIRF4) was cloned and its cDNA was analyzed. Expression of duIRF4 in a wide variety of tissues and changes in duIRF4 expression due to viral infection also was detected by quantitative real-time PCR. The results show that duIRF4 contains 1,341 bp of ORF encoding a protein with 446 amino acids and contains 3 domains: DNA-binding domain (DBD), IRF-association domain (IAD) and nuclear localization signal (NLS). Quantitative real-time PCR analysis showed that duIRF4 was evenly expressed in all tissues examined, with the highest expression in the spleen, followed by the bursa of Fabricius, and lower in the skin and brain. In addition, expression of duIRF4 in the brain and spleen was significantly upregulated after being infected by duck plague virus, duck Tembusu virus, and novel duck reovirus. These data suggest that duIRF4 may be involved in innate immune response.

Structure and expression identification of Cherry Valley duck IRF8.

Interferon regulatory factor 8 (IRF8) is also known as interferon (IFN) consensus sequence binding protein (ICSBP), which plays an important role in IFN signal transduction. In this study, we cloned the full-length coding sequence of Cherry Valley duck IRF8 (duIRF8) and analyzed its structure. In addition, we tested the distribution of IRF8 in the tissues of healthy Cherry Valley ducks, and the changes in IRF8 expression levels in the tissues after virus infection. The results show that the open reading frame (ORF) of IRF8 is 1293 bp, encodes 430 amino acids, and have 3 conserved domains: the N-terminal DBD domain, the C-terminal IAD domain, and the NLS domain. Besides, from the analysis of the phylogenetic tree, it can be known that the duIRF8 has the highest homology with the anser cygnoides, and has less homology with the fish. Analyzing the distribution level of IRF8 in the tissues, it is found that the expression level of IRF8 in the liver of Cherry Valley duck is the highest. However, after infection with duck Tambusu virus, novel duck reovirus, and duck plague virus, the expression of IRF8 in the spleen and brain all showed up-regulation. These data indicate that IRF8 is involved in the host's innate immune response against virus in Cherry Valley duck.

Regulation of MDA5-dependent anti-Tembusu virus innate immune responses by LGP2 in ducks.

Melanoma differentiation associated factor 5 (MDA5), which belongs to the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) family, has been proved to be a key pattern recognition receptor of innate antiviral signaling in duck, which plays an important role in anti-Tembusu virus (TMUV) infection. However, laboratory of genetics and physiology 2 (LGP2), the third member of RLRs family, the regulatory function on antiviral innate immunity of MDA5 is currently unclear. In this study, we investigated the subcellular localization of duck LGP2 (duLGP2) and confirmed that it is an important regulator of the duMDA5-mediated host innate antiviral immune response. The present experimental data demonstrate that the overexpression of duLGP2 inhibits duMDA5 downstream transcriptional factor (IRF-7, IFN-ß, and NF-κB) promoter activity, and duMDA5-mediated type I IFNs and ISGs expression were significantly suppressed by duLGP2 regardless of viral infection in vitro. The inhibition of duLGP2 on the antiviral activity of duMDA5 ultimately leads to an increase in viral replication. However, the overexpression of duLGP2 promotes expression of mitochondrial antiviral-signaling protein (MAVS) and duMDA5-mediated proinflammatory cytokines. This study provides a new rationale support for the duLGP2 regulates duMDA5-mediated anti-viral immune signaling pathway theory in duck.

Identification, cloning, and characterization of Cherry Valley duck CD4 and its antiviral immune responses.

CD4 protein is a single chain transmembrane glycoprotein and has a broad functionality beyond cell-mediated immunity. In this study, we cloned the full-length coding sequence (CDS) of duck CD4 (duCD4) and analyzed its sequence and structure, and expression levels in several tissues. It consists of 1,449 nucleotides and encodes a 482 amino acid protein. The putative protein of duCD4 consisted of an N-terminal signal peptide, three immunoglobulins and one immunoglobulins-like domain in its central, one terminal transmembrane region, and a C-terminal domain of the CD4 T cell receptor. The duCD4 also has the typical signature "CXC" of CD4s. The multiple sequence alignment suggests duCD4 has four potential N-glycosylation sites and the phylogenetic analysis suggests duCD4 shares greater similarity with avian than other vertebrates. Quantitative real-time PCR analysis showed that duCD4 mRNA transcripts are widely distributed in the healthy Cherry Valley duck, and the highest level in the thymus. During the virus infection, the obvious change of duCD4 expression was observed in the spleen, lung and brain, which suggesting that duCD4 could be involved in the host's immune response to multiple types of viruses. Our research studied the characterization, tissue distribution, and antiviral immune responses of duCD4.

Cloning, analysis, and anti-duck Tembusu virus innate immune response of Cherry Valley duck tripartite motif-containing 32.

Tripartite motif-containing 32 (TRIM32) is an E3 ubiquitin ligase with multiple functions. In this study, we amplified TRIM32 gene from the Cherry Valley duck, and its cDNA sequence contained an open reading frame of 1,950 bp that encodes 649 amino acids. Duck TRIM32 (duTRIM32) mRNA was expressed in all tissues tested. A series of immune-related genes that were induced by viral infection, including interferon alfa, IL-1ß, retinoic acid-inducible gene-I, Mx, and OAS, were regulated by duTRIM32 expression. DuTRIM32 overexpression inhibits duck Tembusu virus (DTMUV) replication in the early stages of viral infection. Knockdown of duTRIM32 expression by siRNA reduced the ability of duck embryo fibroblast cells to mount a type Ⅰ interferon response to DTMUV. Therefore, our results suggest that the duTRIM32-mediated signal pathway plays an essential role in DTMUV infection-induced innate immune response.

DDX1 from Cherry valley duck mediates signaling pathways and anti-NDRV activity.

Novel duck reovirus (NDRV) causes severe economic losses to the duck industry, which is characterized by hemorrhagic spots and necrotic foci of the livers and spleens. DEAD-box helicase 1 (DDX1) plays a critical role in the innate immune system against viral infection. However, the role of duck DDX1 (duDDX1) in anti-RNA virus infection, especially in the anti-NDRV infection, has yet to be elucidated. In the present study, the full-length cDNA of duDDX1 (2223 bp encode 740 amino acids) was firstly cloned from the spleen of healthy Cherry valley ducks, and the phylogenetic tree indicated that the duDDX1 has the closest relationship with Anas platyrhynchos in the bird branch. The duDDX1 mRNA was widely distributed in all tested tissues, especially in the duodenum, liver, and spleen. Overexpression of duDDX1 in primary duck embryo fibroblast (DEF) cells triggered the activation of transcription factors IRF-7 and NF-κB, as well as IFN-ß expression, and the expression of the Toll-like receptors (TLR2, TLR3, and TLR4) was significantly increased. Importantly, after overexpressing or knocking down duDDX1 and infecting NDRV in DEF cells, duDDX1 inhibits the replication of NDRV virus and also regulates the expression of pattern recognition receptors and cytokines. This indicates that duDDX1 may play an important role in the innate immune response of ducks to NDRV. Collectively, we first cloned DDX1 from ducks and analyzed its biological functions. Secondly, we proved that duck DDX1 participates in anti-NDRV infection, and innovated new ideas for the prevention and control of duck virus infection.

Duck HMGB2 Mediates Signaling Pathways in the Innate Immunity of Hosts Against Viral Infections.

High-mobility group box 2 (HMGB2) belongs to the HMG-box family that participates in a variety of biologic processes. Recent studies have suggested that HMGB2 plays an important role in the innate immunity of fish. Cherry Valley duck is the main duck bred for meat consumption in China, but there is limited research available on the impact of duck HMGB2 (duHMGB2) in antiviral innate immunity. Here, duHMGB2 genes were first cloned and analyzed from the spleen of Cherry Valley ducks. We show that duHMGB2 is widely distributed in most tissues of healthy ducks, and duHMGB2 was differentially expressed in three organs (the spleen, brain, and lung) of ducks during different viral infections. duHMGB2 is mainly expressed in the nucleus of duck embryo fibroblast (DEF) cells. However, duHMGB2 is released into the cytoplasm after viral infection. DuHMGB2 induced expression of several genes that regulate the immune response. Moreover, duHMGB2 activated and upregulatede transcription factor NF-κB promoter activity. We also used single gene manipulations (knockout or overexpression) to confirm that duHMGB2 can inhibit the replication of duck plague virus, duck Tembusu virus, and the novel duck reovirus in DEF cells. These data show that duHMGB2 can activate the antiviral innate immunity of the host. Thus, duHMGB2 may be considered an immune adjuvant against infectious diseases in duck.

Cherry Valley Duck Galectin-2 Plays an Essential Role in Avian Pathogenic Escherichia coli Infection-Induced Innate Immune Response.

Galectins play important roles in the host's innate immunity as pattern recognition receptors. In this study, the coding sequences of galectin-2 were identified from Cherry Valley ducks. Tissue distribution of duck galectin-2 (duGal-2) in healthy ducks and ducks infected with avian pathogenic Escherichia coli (APEC) was studied, respectively. The results showed that duGal-2 expression was higher in the gut, kidney, and liver tissue, and weakly expressed in the lung and brain, in healthy ducks; however, the expression level of duGal-2 was detected as being up-regulated after infection with APEC. In addition, knockdown or overexpression of duGal-2 in DEFs was achieved by small interference RNA (siRNA) transfection and plasmid transduction, respectively. The knockdown of duGal-2 led to a decrease in the expression of some inflammatory cytokines such as IL-1ß, IL-6, and IL-8, while the expression levels of anti-inflammatory factor IL-10 were up-regulated. At the same time, the bacterial load of APEC was increased after knockdown of duGal-2 in vitro. However, the opposite results were obtained in the duGal-2 overexpression group. Taken together, duGal-2 plays an important role in the host against APEC infection.

High-mobility group box 1 protein (HMGB1) from Cherry Valley duck mediates signaling pathways and antiviral activity.

High-mobility group box 1 protein (HMGB1) shows endogenous damage-associated molecular patterns (DAMPs) and is also an early warning protein that activates the body's innate immune system. Here, the full-length coding sequence of HMGB1 was cloned from the spleen of Cherry Valley duck and analyzed. We find that duck HMGB1(duHMGB1) is mostly located in the nucleus of duck embryo fibroblast (DEF) cells under normal conditions but released into the cytoplasm after lipopolysaccharide (LPS) stimulation. Knocking-down or overexpressing duHMGB1 had no effect on the baseline apoptosis rate of DEF cells. However, overexpression increased weakly apoptosis after LPS activation. In addition, overexpression strongly activated the IFN-I/IRF7 signaling pathway in DEF cells and significantly increased the transcriptional level of numerous pattern recognition receptors (PRRs), pro-inflammatory cytokines (IL-6, TNF-α), IFNs and antiviral molecules (OAS, PKR, Mx) starting from 48 h post-transfection. Overexpression of duHMGB1 strongly impacted duck virus replication, either by inhibiting it from the first stage of infection for novel duck reovirus (NDRV) and at late stage for duck Tembusu virus (DTMUV) or duck plague virus (DPV), or promoting replication at early stage for DTMUV and DPV infection. Importantly, data from duHMGB1 overexpression and knockdown experiments, time-dependent DEF cells transcriptional immune responses suggest that duHMGB1 and RIG-I receptor might cooperate to promote the expression of antiviral proteins after NDRV infection, as a potential mechanism of duHMGB1-mediated antiviral activity.

Molecular identification of duck DDX3X and its potential role in response to Tembusu virus.

ATP-dependent DEAD (Asp-Glu-Ala-Asp)-box RNA helicases not only regulate RNA metabolism, but also are involved in host antiviral innate immune responses. It is important to investigate the orthologs of this protein family to broaden our understanding of innate immunity and promote protective strategies against viral infections in ducks. In the current study, duck DDX3X (duDDX3X) was first cloned, which consists of 1959 bp encoding a protein of 652 amino acids. duDDX3X has the typical structure of this family, including nine motifs, DEAD and HELICc domains. The amino acid sequence of duDDX3X shares a high similarity with the DDX3Xs of avian and mammalian. Quantitative real-time PCR indicated that duDDX3X was ubiquitously expressed in nearly all tissues. Overexpression of duDDX3X could activate interferon (IFN)-ß and enhance the RIG-I-induced IFN-ß yield in duck embryo fibroblast cells. However, duDDX3X had no significant effect on the expression of proinflammatory cytokines such as IL-1ß, IL-6, and CXCL-8. Tembusu virus (TMUV) infection significantly downregulated duDDX3X. Overexpression and siRNA interference studies showed that duDDX3X inhibited the replication of TMUV through IFN-ß at the early stages of infection. Collectively, our results indicated that duDDX3X could positively modulate type I interferon and play an essential role in response to TMUV infection. This study will contribute to a better understanding of duDDX3X in the innate immune system of ducks and lay a solid foundation for further studies of duDDX3X in antiviral immunity.

Galectin-1 induces immune response and antiviral ability in Cherry Valley ducks after duck plague virus infection.

Galectin-1, as a typical animal galactose-binding protein, it is found on the cell surface and in the extracellular matrix. Cloning the full-length coding sequence of galectin-1 from the spleens of Cherry Valley ducks revealed that the coding sequence of duck galectin-1 (duGal-1) comprises 405 bp, encoding 134 amino acids. Homologic analysis revealed its amino acid sequence is most identical to that of Anas platyrhynchos (98.8%) followed by Gallus gallus. Quantitative real-time PCR analysis indicated that duGal-1 mRNA is broadly expressed in healthy Cherry Valley duck tissues, primarily in the heart and trachea but minimally in the lung and skin. Meanwhile, the duGal-1 expression is slightly upregulated in the infected liver and spleen. Furthermore, the expression levels of ISGs (Mx, PKR, OAS) and some cytokines such as IFN-α, IL-1ß, IL-2, are up-regulated to varying degrees after overexpression the duGal-1, In contrast, Knockdown of duGal-1 found that the expression levels of ISGs and some inflammatory cytokines were down-regulated. Antiviral assay showed that duGal-1 could inhibit viral replications early during infection. This is the first study of the cloning, tissue distribution, and antiviral immune responses of duGal-1, and findings imply it is involved in the early stages of antiviral innate immune responses to duck plague virus infections in ducks.

Dissemination of Escherichia coli carrying plasmid-mediated quinolone resistance (PMQR) genes from swine farms to surroundings.

Different samples were collected from three swine farms in China to investigate the spread of antibiotic-resistant Escherichia coli. A total of 130 E. coli isolates were obtained from feces, air, river water, silt, and soil samples and characterized. The susceptibility of the E. coli isolates to 19 antibiotics was tested. The results revealed that the resistance rates of the E. coli isolates against 9 antibiotics were high. The minimum inhibitory concentration (MIC) values of ciprofloxacin, ofloxacin, and nalidixic acid were mainly in the ranges of 2-64, 8-64, and 8-64 µg/ml. The plasmid-mediated quinolone resistance (PMQR) genes qnr, aac(6')-Ib-cr, qepA, and oqxAB were detected by polymerase chain reaction (PCR), and the similarity of E. coli from different samples was identified by pulsed-field gel electrophoresis (PFGE). The detection rates of the qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA, and oqxAB genes in the E. coli isolates from three swine farms were in the range of 10.87-23.08%, 13.04-20.51%, 40.00-43.48%, 30.43-38.46%, 6.52-12.82%, and 7.69-17.39%, respectively. The PFGE result showed that 49% (49/100) of isolates originating from air, river water, soil, and silt samples had ≥85% similarity to fecal-obtained isolates, and 40.82% (20/49) of them shared the same PMQR genes with fecal-obtained isolates. This indicated that E. coli carrying PMQR genes and originating from feces in swine farms could spread to the external environment, which could be a potential threat to the public environment and human health.

Characterization and Roles of Cherry Valley Duck NLRP3 in Innate Immunity During Avian Pathogenic Escherichia coli Infection.

The nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that is involved in host innate immunity and located in the cytoplasm. In the present study, the full-length cDNA of Cherry Valley duck NLRP3 (duNLRP3) (2,805 bp encode 935 amino acids) was firstly cloned from the spleen of healthy Cherry Valley ducks, and the phylogenetic tree indicated that the duNLRP3 has the closest relationship with Anas platyrhynchos in the bird branch. According to quantitative real-time PCR analysis, the duNLRP3 mRNA has a broad expression spectrum in healthy Cherry Valley duck tissues, and the highest expression is in the pancreas. There was significant up-regulation of duNLRP3 mRNA expression in the liver and down-regulation in the spleen after infection with avian pathogenic Escherichia coli (APEC) O1K1, especially at 3 days after the infection. Ducks hatched from NLRP3-lentiviral vector-injected eggs had significantly higher duNLRP3 mRNA expression in the liver, spleen, brain, and cecum, which are tissues usually with lower background expression. The mRNA expression levels of inflammatory cytokines IL-1ß, IL-18, and TNF-α significantly increased after the APEC infection in those tissues. The bacterial content in the liver and spleen decreased significantly compared with the NC-lentiviral vector-injected ducks. In addition, in the duck embryo fibroblasts, both of the overexpression and knockdown of duNLRP3 can trigger the innate immune response during the E. coli infection. Specifically, overexpression induced antibacterial activation, and knockdown reduced the antibacterial activity of the host cells. The IL-1ß, IL-18, and TNF-α mRNA expressions showed up-regulation or down-regulation. The results demonstrate that duNLRP3 has a certain antibacterial activity during E. coli infection. These findings also contribute to better understanding the importance of duNLRP3 in regulating the inflammatory response and the innate immune system of ducks.

Protective Role of Rabbit Nucleotide-Binding Oligomerization Domain-2 (NOD2)-Mediated Signaling Pathway in Resistance to Enterohemorrhagic Escherichia coli Infection.

Nucleotide-binding oligomerization domain 2 (NOD2), a member of the NOD-like receptors (NLRs) family that is well-known to play a key role in innate immune responses and is involved in innate antibacterial responses. In this study, rabbit NOD2 (rNOD2) was cloned from rabbit kidney (RK) cells. It was distributed in various tissues, and the highest level of rNod2 was detected in spleen. Moreover, the expression of rNod2 was significantly upregulated in the heart, liver, and spleen induced by enterohemorrhagic Escherichia coli (EHEC). Overexpression of rNOD2 induced the expression of pro-inflammatory cytokine, including Il1ß, Il6, Ifn-γ, and Tnf, as well as defensins, including Defb124, Defb125, and Defb128 through the nuclear factor (NF)-κB signaling pathway. Furthermore, overexpression of rNOD2 inhibited the growth of EHEC, and knockdown of rNOD2 or inhibition of the NF-κB pathway promoted its replication. In addition, our results suggest that rNOD2 can significantly activate NF-κB signaling and trigger antibacterial defenses to increase the expression of pro-inflammatory cytokine and defensins after stimulation by EHEC. These findings are useful to further understanding the innate immune system of rabbits and providing a new perspective for the prevention of bacterial diseases in rabbits.

Fowl Adenovirus Serotype 4 SD0828 Infections Causes High Mortality Rate and Cytokine Levels in Specific Pathogen-Free Chickens Compared to Ducks.

Hydropericardium syndrome and inclusion body hepatitis, together called hydropericardium-hepatitis syndrome, are acute infectious diseases found in chickens. These diseases are caused primarily by fowl adenovirus serotype 4 (FAdV-4) strains. In this study, we isolated a FAdV-4 strain (SD0828) from clinically diseased chickens and phylogenetically analyzed the L1 loops of the hexon protein sequences in 3-week-old specific pathogen-free chickens and ducks infected intramuscularly and orally, determining differences in the pathogenicity by observing clinical signs and gross and histological lesions. We also detected the viral load in tissue samples. Postinfection necropsy showed that all chickens but no ducks exhibited typical necropsy lesions. Additionally, all chickens infected intramuscularly died within 2 days postinfection (dpi), and all those infected orally died within 5 dpi, whereas no infected ducks died before 28 dpi. Quantitative real-time polymerase chain reaction analysis was used to determine the viral load in the tissues of hearts, livers, spleens, lungs, and kidneys and in cloacal cotton swabs from infected chickens and ducks at 1, 2, 3, 5, 7, 14, 21, and 28 dpi. The greatest number of viral DNA copies was found in the livers of infected chickens, yet no virus was found in any samples from infected ducks. In addition, the viral load increased over time in both chicken and duck embryo fibroblasts (CEFs and DEFs, respectively); in the former, replication speed was significantly greater than in the latter. Innate immune responses were also studied, both in vivo and in vitro. In CEFs, DEFs, and chickens infected intramuscularly, but not in infected ducks, mRNA expression levels of proinflammatory cytokines (interleukin-6 and -8) and interferon-stimulated genes (Mx and OAS) were significantly upregulated. Although some cytokines showed significant upregulation in the oral chickens, most did not change significantly. Finally, the duck retinoic acid-inducible gene I and its caspase activation and recruitment domain both had significant antiviral functions in CEFs, particularly after 24 h postinfection. Taken together, this research provides new insights into the interactions between FAdV-4 and the innate immune systems of studied hosts (chickens and ducks).