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.

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.

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).

Proteomic Analysis of Differential Expression of Cellular Proteins in Response to Avian H9N2 Virus Infection of A549 Cells.

In this study, differentially expressed proteins in A549 cells (human lung adenocarcinoma epithelial cell line) infected with H9N2 avian influenza virus (AIV) were investigated by two-dimensional electrophoresis (2-DE). Sixteen different spots between the groups (ratio > 2, p < 0.05) were identified with mass spectrometry identification. Proteins located in the downstream of the NF-κB and IFN transcription factor pathways were identified, e.g., ISG15. Actin and keratin were also identified, suggesting that the cytoskeleton may plays an important role in the AIV infection of mammalian cells. These findings could provide insights into the interaction between host and influenza viruses and might provide valuable information for clarifying the pathogenesis of viral infections as well.

The mRNA and Proteins Expression Levels Analysis of TC-1 Cells Immune Response to H9N2 Avian Influenza Virus.

Since 1994, the H9N2 avian influenza virus (AIV) has spread widely in mainland China, causing great economic losses to the poultry industry there. Subsequently, it was found that the H9N2 AIV had the ability to infect mammals, which gave rise to great panic. In order to investigate the immune response of a host infected with H9N2 AIV, TC-1 cells were set as a model in this research. Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods were used to study the expression changes of pattern recognition receptors (PRRs), inflammatory cytokines, and chemokines in AIV-infected TC-1 cells. Our research found that TC-1 cells had similar susceptibility to both CK/SD/w3 (A/Chicken/Shandong/W3/2012) and CK/SD/w4 (A/Chicken/Shandong/W4/2012) H9N2 isolates, while the CK/SD/w3 isolate had a stronger capability of replication in the TC-1 cells. At the same time, the expression of PRRs (melanoma differentiation-associated gene 5, MDA-5), cytokines [interleukin (IL)-1ß and IL-6], and chemokines [regulated on activation, normal T cell expressed and secreted (RANTES) and interferon-γ-induced protein-10 kDa (IP-10)] were significantly up-regulated. These results indicated that MDA-5, IL-1ß, IL-6, RANTES, and IP-10 might play important roles in the host immune response to H9N2 AIV infection. This study provided useful information for further understanding the interaction between H9N2 virus infection and host immunity, and had certain guiding significance for the prevention and treatment of this disease.

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections.

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-ß promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1ß and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

Host immune responses of ducks infected with H5N1 highly pathogenic avian influenza viruses of different pathogenicities.

Our previous studies have illustrated three strains of duck-origin H5N1 highly pathogenic avian influenza viruses (HPAIVs) had varying levels of pathogenicity in ducks (Sun et al., 2011). However, the host immune response of ducks infected with those of H5N1 HPAIVs was unclear. Here, we compared viral distribution and mRNA expression of immune-related genes in ducks following infection with the two HPAIV (A/Duck/Guangdong/212/2004, DK212 and A/Duck/Guangdong/383/2008, DK383). DK383 could replicate in the tested tissue of ducks (brain, spleen, lungs, cloacal bursa, kidney, and pancreas) more rapid and efficiently than DK212 at 1 and 2 days post-inoculation. Quantitative real-time PCR analysis showed that the expression levels of TLR3, IL-6, IL-8, and MHC class II in brains were higher than those of respective genes in lungs during the early stage of post infection. Furthermore, the expression levels of IL-6 and IL-8 in the brain of ducks following infection with DK383 were remarkably higher than those of ducks infected with DK212, respectively. Our results suggest that the shift in the H5N1 HPAIVs to increased virulence in ducks may be associated with efficient and rapid replication of the virus, accompanied by early destruction of host immune responses. These data are helpful to understand the underlying mechanism of the different outcome of H5N1 HPAIVs infection in ducks.