Comparing the molecular evolution and recombination patterns of predominant PRRSV-2 lineages co-circulating in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses widespread epidemics in swine herds, yet the drivers underlying lineage replacements/fitness dynamics remain unclear. To delineate the evolutionary trajectories of PRRSV-2 lineages prevalent in China, we performed a comprehensive longitudinal phylodynamic analysis of 822 viral sequences spanning 1991-2022. The objectives encompassed evaluating lineage dynamics, genetic diversity, recombination patterns and glycosylation profiles. A significant shift in the dominance of PRRSV-2 sub-lineages has been observed over the past 3 decades, transitioning from sub-lineage 8.7 to sub-lineage 1.8, followed by extensive diversification. The analysis revealed discordant recombination patterns between the two dominant viral sub-lineages 1.8 and 8.7, underscoring that modular genetic exchanges contribute significantly to their evolutionary shaping. Additionally, a strong association was found between recombination breakpoint locations and transcriptional regulatory sequences (TRSs). Glycosylation patterns also demonstrated considerable variability across sub-lineages and temporally, providing evidence for immune-driven viral evolution. Furthermore, we quantified different evolutionary rates across sub-lineages, with sub-lineage 1.8 uniquely displaying the highest nucleotide substitution rates. Taken together, these findings provide refined insight into the evolutionary mechanisms underpinning cyclic shifts in dominance among regionally circulating PRRSV sub-lineages.

Differential innate immune responses to fowl adenovirus serotype 4 infection in Leghorn male hepatocellular and chicken embryo fibroblast cells.

Fowl adenovirus serotype 4 (FAdV-4) infections result in substantial economic losses in the poultry industry. Recent findings have revealed that FAdV-4 significantly suppresses the host immune response upon infection; however, the specific viral and host factors contributing to this immunomodulatory activity remain poorly characterized. Moreover, diverse cell types exhibit differential immune responses to FAdV-4 infection. To elucidate cell-specific host responses, we performed transcriptomic analysis of FAdV-4 infected leghorn male hepatocellular (LMH) and chicken embryo fibroblast (CEF) cells. Although FAdV-4 replicated more efficiently in LMH cells, it provoked limited interferon-stimulated gene induction. In contrast, FAdV-4 infection triggered robust antiviral responses in CEF cells, including upregulation of cytosolic DNA sensing and interferon-stimulated genes. Knockdown of key cytosolic DNA sensing molecules enhanced FAdV-4 replication in LMH cells while reducing interferon-stimulated gene expression. Our findings reveal cell-specific virus-host interactions that provide insight into FAdV-4 pathogenesis while identifying factors that mediate antiviral immunity against FAdV-4.

The H9N2 avian influenza virus increases APEC adhesion to oviduct epithelia by viral NS1 protein-mediated activation of the TGF-ß pathway.

H9N2 avian influenza is a low-pathogenic avian influenza circulating in poultry and wild birds worldwide and frequently contributes to chicken salpingitis that is caused by avian pathogenic Escherichia coli (APEC), leading to huge economic losses and risks for food safety. Currently, how the H9N2 virus contributes to APEC infection and facilitates salpingitis remains elusive. In this study, in vitro chicken oviduct epithelial cell (COEC) model and in vivo studies were performed to investigate the role of H9N2 viruses on secondary APEC infection, and we identified that H9N2 virus enhances APEC infection both in vitro and in vivo. To understand the mechanisms behind this phenomenon, adhesive molecules on the cell surface facilitating APEC adhesion were checked, and we found that H9N2 virus could upregulate the expression of fibronectin, which promotes APEC adhesion onto COECs. We further investigated how fibronectin expression is regulated by H9N2 virus infection and revealed that transforming growth factor beta (TGF-ß) signaling pathway is activated by the NS1 protein of the virus, thus regulating the expression of adhesive molecules. These new findings revealed the role of H9N2 virus in salpingitis co-infected with APEC and discovered the molecular mechanisms by which the H9N2 virus facilitates APEC infection, offering new insights to the etiology of salpingitis with viral-bacterial co-infections.IMPORTANCEH9N2 avian influenza virus (AIV) widely infects poultry and is sporadically reported in human infections. The infection in birds frequently causes secondary bacterial infections, resulting in severe symptoms like pneumonia and salpingitis. Currently, the mechanism that influenza A virus contributes to secondary bacterial infection remains elusive. Here we discovered that H9N2 virus infection promotes APEC infection and further explored the underlying molecular mechanisms. We found that fibronectin protein on the cell surface is vital for APEC adhesion and also showed that H9N2 viral protein NS1 increased the expression of fibronectin by activating the TGF-ß signaling pathway. Our findings offer new information on how AIV infection promotes APEC secondary infection, providing potential targets for mitigating severe APEC infections induced by H9N2 avian influenza, and also give new insights on the mechanisms on how viruses promote secondary bacterial infections in animal and human diseases.

Fowl adenovirus serotype 4 enters leghorn male hepatocellular cells via the clathrin-mediated endocytosis pathway.

Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype-4 (FAdV-4) has caused large economic losses to the world poultry industry in recent years. HHS is characterized by pericardial effusion and hepatitis, manifesting as a swollen liver with focal necroses and petechial haemorrhage. However, the process of FAdV-4 entry into hepatic cells remains largely unknown. In this paper, we present a comprehensive study on the entry mechanism of FAdV-4 into leghorn male hepatocellular (LMH) cells. We first observed that FAdV-4 internalization was inhibited by chlorpromazine and clathrin heavy chain (CHC) knockdown, suggesting that FAdV-4 entry into LMH cells depended on clathrin. By using the inhibitor dynasore, we showed that dynamin was required for FAdV-4 entry. In addition, we found that FAdV-4 entry was dependent on membrane cholesterol, while neither the knockdown of caveolin nor the inhibition of a tyrosine kinase-based signalling cascade affected FAdV-4 infection. These results suggested that FAdV-4 entry required cholesterol but not caveolae. We also found that macropinocytosis played a role, and phosphatidylinositol 3-kinase (PI3K) was required for FAdV-4 internalization. However, inhibitors of endosomal acidification did not prevent FAdV-4 entry. Taken together, our findings demonstrate that FAdV-4 enters LMH cells through dynamin- and cholesterol-dependent clathrin-mediated endocytosis, accompanied by the involvement of macropinocytosis requiring PI3K. Our work potentially provides insight into the entry mechanisms of other avian adenoviruses.

ß-catenin facilitates fowl adenovirus serotype 4 replication through enhancing virus-induced autophagy.

ß-catenin is a key component of the Wnt/ß-catenin signal transduction cascade which is a highly conserved signaling pathway in eukaryotes. Increasing evidence suggests that the Wnt/ß-catenin signaling pathway is involved in the infection of many viruses. However, its role in fowl adenovirus serotype 4 (FAdV-4) replication remains unclear. In the present study, we showed that FAdV-4 infection increased the expression of ß-catenin and promoted the nuclear translocation of ß-catenin. Overexpression of ß-catenin and LiCl treatment stimulated the accumulation of ß-catenin in the nucleus, and then facilitated FAdV-4 replication. Conversely, repression of ß-catenin by inhibitors and siRNA significantly inhibited FAdV-4 replication. Furthermore, inhibition of autophagy by 3-Methyladenine (3-MA) suppressed the FAdV-4 replication, and repression of ß-catenin inhibited the FAdV-4-triggered autophagy. In conclusion, the nuclear translocation of ß-catenin benefits FAdV-4 replication, and suppression of ß-catenin limits FAdV-4 production by inhibiting FAdV-4-induced autophagy. These findings indicated that ß-catenin is an important regulator of FAdV-4 replication which can serve as a potential target for anti-FAdV-4 agents.

Evaluation of the protective efficacy of six major immunogenic proteins of Mycoplasma Synoviae.

Mycoplasma synoviae (MS) is a primary avian pathogen prevalent worldwide that causes airsacculitis and synovitis in birds. Vaccination is recommended as the most cost-effective strategy in the control of MS infection. Novel alternative vaccines are needed for eradicating and controlling MS infection in flocks. DnaK, enolase, elongation factor Tu (EF-Tu), MSPB, NADH oxidase and LP78 are the major immunogenic antigens of MS and are promising targets for subunit vaccine candidates. In the present study, genes encoding DnaK, enolase, EF-Tu, MSPB, LP78, and NADH oxidase were cloned and expressed in Escherichia coli. Enzyme-linked immunosorbent assay showed that the six recombinant proteins were recognized by convalescent sera, indicating that they were expressed during infection. Two injections of the six subunit vaccines induced a robust antibody response and increased the concentrations of IFN-γ and IL-4, especially rEnolase and rEF-Tu. The proliferation of peripheral blood lymphocytes was enhanced in all of the immunized groups. Chickens immunized with rEnolase, rEF-Tu, rLP78, and rMSPB conferred significant protection against MS infection, as indicated by significantly lower DNA copies in the trachea, lower scores of air sac lesions, and lesser tracheal mucosal thickness than that in the challenge control. Especially, rEnolase provided the best protective efficacy, followed by rEF-Tu, rMSPB, and rLP78. Our finds demonstrate that the subunit vaccines and bacterin can only reduce the lesions caused by MS infection, but not prevent colonization of the organism. Our findings may contribute to the development of novel vaccine agents against MS infection.

Mycoplasma synoviae LP78 is a fibronectin/plasminogen binding protein, putative adhesion, and potential diagnostic antigen.

Mycoplasma synoviae (M. synoviae) is one of the major poultry pathogens causing infectious synovitis, airsacculitis, a high incidence of shell breakage, and egg production loss. However, the pathogenesis of M. synoviae remains unclear. Adhesion of mycoplasmas to host cells is a crucial step in infection and colonization. The purpose of this study was to determine the adhesive function of a putative P80 family lipoprotein (LP78) and evaluate its application in the detection of antibodies against M. synoviae. Recombinant LP78 (rLP78) was expressed in the supernatant component of Escherichia coli and mouse anti-rLP78 serum was prepared. Bioinformatic analysis and western blotting results revealed that LP78 was conservative among M. synoviae strains. It was distributed not only in the cytoplasm but also on the membrane of M. synoviae through western blotting and indirect immunofluorescence (IFA). The adherence of M. synoviae to DF-1 cells was significantly inhibited by mouse anti-rLP78 serum (p < 0.01). IFA revealed that rLP78 adhered to DF-1 cells, and this adherence was prevented by mouse anti-rLP78 serum. Furthermore, rLP78 was found to bind to the DF-1 cells membrane proteins in a dose-dependent manner by enzyme-linked immunosorbent assay (ELISA). Screening of DF-1 cells membrane proteins by western blotting showed that proteins with molecular weight of 35-40 kDa and 55-70 kDa bound to rLP78. Moreover, rLP78 was identified to be a fibronectin/plasminogen binding protein. The sensitivity and specificity of rLP78-based iELISA were 85.7 and 94.1%, respectively. The maximum dilution of positive serum (HI titer, 1:128) detected via rLP78-based iELISA was 1:6,400, whereas that detected using a commercial ELISA kit was 1:12,800-1:25,600. Both rLP78-based iELISA and the commercial ELISA kit detected seroconversion after 7 days of challenge and immunization. No cross-reactivity with positive sera against other avian pathogens was observed in rLP78-based iELISA. Collectively, these results indicate that LP78 is a fibronectin/plasminogen-binding adhesion protein of M. synoviae and a potential diagnostic antigen. The present study will facilitate a better understanding of the pathogenesis of M. synoviae and the development of new diagnostic.

Transcriptome Analysis Reveals the Potential Role of Long Noncoding RNAs in Regulating Fowl Adenovirus Serotype 4-Induced Apoptosis in Leghorn Male Hepatocellular Cells.

Hepatitis-hydropericardium syndrome (HHS) is caused by fowl adenovirus serotype 4 (FAdV-4) and has resulted in considerable economic losses to the poultry industry globally. FAdV-4 elicits apoptosis in host cells. Long noncoding RNAs (lncRNAs) have emerged as important regulatory RNAs with profound effects on various biological processes, including apoptosis. However, it remains unknown whether lncRNAs participate in FAdV-4-induced apoptosis. In this study, RNA sequencing was applied to determine the transcription of cellular lncRNA in leghorn male hepatocellular (LMH) cells infected with FAdV-4. Cellular RNA transcription analysis demonstrated that FAdV-4 infection elicited 1798 significantly differentially expressed (DE) lncRNAs in infected LMH cells at 24 h post-infection (hpi) compared to mock control infection. In addition, 2873 DE mRNAs were also found. Target prediction and analyses revealed that 775 DE lncRNAs whose 671 target mRNAs were among the DE mRNAs were involved in several signaling pathways, including the AMPK signaling pathway, p53 signaling pathway and insulin signaling pathway. From these 775 DE lncRNAs, we identified 71 DE lncRNAs related to apoptosis based on their target gene functions. Subsequently, lncRNA 54128 was selected from the 71 identified DE lncRNAs, and its role in FAdV-4-induced apoptosis was verified. LncRNA 54128 interference significantly suppressed the rate of apoptosis, which was accompanied by reduced BMP4 transcription levels. To the best of our knowledge, this is the first study to analyze host lncRNA transcription during FAdV-4 infection. Our findings provide a better understanding of host responses to FAdV-4 infection and provide new directions for understanding the potential association between lncRNAs and FAdV-4 pathogenesis.

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.

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.

An Optimization Routing Algorithm Based on Segment Routing in Software-Defined Networks.

Software-defined networks (SDNs) are improving the controllability and flexibility of networks as an innovative network architecture paradigm. Segment routing (SR) exploits an end-to-end logical path and is composed of a sequence of segments as an effective routing strategy. Each segment is represented by a middle point. The combination of SR and SDN can meet the differentiated business needs of users and can quickly deploy applications. In this paper, we propose two routing algorithms based on SR in SDN. The algorithms aim to save the cost of the path, alleviate the congestion of networks, and formulate the selection strategy by comprehensively evaluating the value of paths. The simulation results show that compared with existing algorithms, the two proposed algorithms can effectively reduce the consumption of paths and better balance the load of the network. Furthermore, the proposed algorithms take into account the preferences of users, actualize differentiated business networks, and achieve a larger comprehensive evaluation value of the path compared with other algorithms.

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.

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).

Mouse trophoblastic cells exhibit a dominant invasiveness phenotype over cancer cells.

Invasion process occurs both in mammalian embryo implantation during development and malignant cancer cell metastasis. We investigated the interactions between trophoblasts and metastatic cancer cells and found the phenomenon that mouse trophoblastic cells invaded the monolayer of malignant cancer cells in vitro and appeared the general trait of invasiveness to more than 30 types of malignant cancer cell lines which were derived from different histological origins and with different invasive or metastatic potential. We further investigated the cellular and molecular changes in the process of mouse trophoblastic cells invading human ovarian cancer HO-8910 cells. The results show that the invasion of trophoblastic cells lead HO-8910 cells near mouse embryo to apoptosis, and expression of cell-cycle-related protein cyclinD1 and Ki-67 mRNA were steadily remained both in mouse blastocysts and human ovarian cancer HO-8910 cells, which in part explain the proliferation activities of these cells. Our study also shows that expression of some proteins including MMP-9, FAK and Integrinαvß3 was changeable in trophoblastic cells and HO-8910 cells in the process of blastocyst invasion, which suggested temporal expression of these molecules may involved in the invasive behavior of trophoblasts cells to cancer cells.