Interferon Signaling in Chickens Plays a Crucial Role in Inhibiting Influenza Replication in DF1 Cells

Influenza A viruses (IAV) pose a constant threat to human and poultry health. Of particular interest are the infections caused by highly pathogenic avian influenza (HPAI) viruses, such as H5N1, which cause significant production issues. In response to influenza infection, cells activate immune mechanisms that lead to increased interferon (IFN) production. To investigate how alterations in the interferon signaling pathway affect the cellular response to infection in the chicken, we used CRISPR/Cas9 to generate a chicken cell line that lacks a functional the type I interferon receptor (IFNAR1). We then assessed viral infections with the WSN strain of influenza. Cells lacking a functional IFNAR1 receptor showed reduced expression of the interferon stimulated genes (ISG) such as Protein Kinase R (PKR) and Myxovirus resistance (Mx) and were more susceptible to viral infection with WSN. We further investigated the role or IFNAR1 on low pathogenicity avian influenza (LPAI) strains (H7N9) and a HPAI strain (H5N1). Intriguingly, Ifnar−/− cells appeared more resistant than WT cells when infected with HPAI virus, potentially indicating a different interaction between H5N1 and the IFN signaling pathway. Our findings support that ChIFNAR1 is a key component of the chicken IFN signaling pathway and these data add contributions to the field of host-avian pathogen interaction and innate immunity in chickens.

Reconstruction of a generic genome-scale metabolic network for chicken: investigating network connectivity and finding potential biomarkers

Chicken is the first sequenced avian that has a crucial role in human life for its meat and egg production. Because of various metabolic disorders, study the metabolism of chicken cell is important. Herein, the first genome-scale metabolic model of a chicken cell named iES1300, consists of 2427 reactions, 2569 metabolites, and 1300 genes, was reconstructed manually based on databases. Interactions of metabolic genes for growth were examined for E. coli , S. cerevisiae , human, and chicken metabolic models. The results indicated robustness to genetic manipulation for iES1300 similar to the results for human. iES1300 was integrated with transcriptomics data using algorithms and Principal Component Analysis was applied to compare context-specific models of the normal, tumor, lean and fat cell lines. It was found that the normal model has notable metabolic flexibility in the utilization of various metabolic pathways, especially in metabolic pathways of the carbohydrate metabolism, compared to the others. It was also concluded that the fat and tumor models have similar growth metabolisms and the lean chicken model has a more active lipid and carbohydrate metabolism.

Stability across the Whole Nuclear Genome in the Presence and Absence of DNA Mismatch Repair

We describe the contribution of DNA mismatch repair (MMR) to the stability of the eukaryotic nuclear genome as determined by whole-genome sequencing. To date, wild-type nuclear genome mutation rates are known for over 40 eukaryotic species, while measurements in mismatch repair-defective organisms are fewer in number and are concentrated on Saccharomyces cerevisiae and human tumors. Well-studied organisms include Drosophila melanogaster and Mus musculus, while less genetically tractable species include great apes and long-lived trees. A variety of techniques have been developed to gather mutation rates, either per generation or per cell division. Generational rates are described through whole-organism mutation accumulation experiments and through offspring–parent sequencing, or they have been identified by descent. Rates per somatic cell division have been estimated from cell line mutation accumulation experiments, from systemic variant allele frequencies, and from widely spaced samples with known cell divisions per unit of tissue growth. The latter methods are also used to estimate generational mutation rates for large organisms that lack dedicated germlines, such as trees and hyphal fungi. Mechanistic studies involving genetic manipulation of MMR genes prior to mutation rate determination are thus far confined to yeast, Arabidopsis thaliana, Caenorhabditis elegans, and one chicken cell line. A great deal of work in wild-type organisms has begun to establish a sound baseline, but far more work is needed to uncover the variety of MMR across eukaryotes. Nonetheless, the few MMR studies reported to date indicate that MMR contributes 100-fold or more to genome stability, and they have uncovered insights that would have been impossible to obtain using reporter gene assays.

The avian retroviral receptor Tva mediates the uptake of transcobalamin bound vitamin B12 (cobalamin)

The Avian sarcoma and leukosis viruses (ASLVs) are important chicken pathogens. Some of the virus subgroups, including ASLV-A and K, utilize the Tva receptor for cell entrance. Though Tva was identified three decades ago, its physiological function remains unknown. Previously, we have noted an intriguing resemblance and orthology between the chicken gene coding for Tva and the human gene coding for CD320, a receptor involved in cellular uptake of transcobalamin (TC) in complex with vitamin B12/cobalamin (Cbl). Here we show that both the transmembrane and the glycosylphosphatidylinositol (GPI)-anchored form of Tva in the chicken cell line DF-1 promotes the uptake of Cbl with help of expressed and purified chicken TC. The uptake of TC-Cbl complex was monitored using an isotope- or fluorophore-labeled Cbl. We show that (i) TC-Cbl is internalized in chicken cells; and (ii) the uptake is lower in the Tva-knockout cells and higher in Tva-overexpressing cells when compared with wild type chicken cells. The relation between physiological function of Tva and its role in infection was elaborated by showing that infection with ASLV subgroups (targeting Tva) impairs the uptake of TC-Cbl, while this is not the case for cells infected with ASLV-B (not recognized by Tva). In addition, exposure of the cells to a high concentration of TC-Cbl alleviates the infection with Tva-dependent ASLV. IMPORTANCE: We demonstrate that the ASLV receptor Tva participates in the physiological uptake of TC-Cbl, because the viral infection suppresses the uptake of Cbl and vice versa. Our results pave the road for future studies addressing the issues: (i) whether a virus infection can be inhibited by TC-Cbl complexes in vivo; and (ii) whether any human virus employs the human TC-Cbl receptor CD320. In broader terms, our study sheds light on the intricate interplay between physiological roles of cellular receptors and their involvement in virus infection.

Chicken Second Branchial Arch Progenitor Cells Contribute to Heart Musculature in vitro and in vivo

In the past, the heart muscle was thought to originate from a single source of myocardial progenitor cells. More recently, however, an additional source of myocardial progenitors has been revealed to be the second heart field, and chicken embryos were important for establishing this concept. However, there have been few studies in chicken on how this field contributes to heart muscles in vitro. We have developed an ex vivo experimental system from chicken embryos between stages HH17–20 to investigate how mesodermal progenitors in the second branchial arch (BA2) differentiate into cardiac muscles. Using this method, we presented evidence that the progenitor cells within the BA2 arch differentiated into beating cardiomyocytes in vitro. The beating explant cells were positive for cardiac actin, Nkx2.5, and ventricular myosin heavy chain. In addition, we performed a time course for the expression of second heart field markers (Isl1 and Nkx2.5) in the BA2 from stage HH16 to stage HH21 using in situ hybridization. Accordingly, using EGFP-based cell labeling techniques and quail-chicken cell injection, we demonstrated that mesodermal cells from the BA2 contributed to the outflow tract and ventricular myocardium in vivo. Thus, our findings highlight the cardiogenic potential of chicken BA2 mesodermal cells in vitro and in vivo.

Establishment of a genetically engineered chicken DF-1 cell line for efficient amplification of influenza viruses in the absence of trypsin

Background The initial step of influenza infection is binding of the virus to specific sialic acid receptors expressed by host cells. This is followed by cell entry via endocytosis. Cleavage of the influenza virus hemagglutinin (HA) protein is critical for infection; this is performed by host cell proteases during viral replication. In cell culture systems, HA is cleaved by trypsin added to the culture medium. The vast majority of established cell lines are mammalian. Results In the present study, we generated genetically engineered chicken DF-1 cell lines overexpressing transmembrane protease, serine 2 (TMPRSS2, which cleaves HA), ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3GAL1, which plays a role in synthesis of α-2,3 linked sialic acids to which avian-adapted viruses bind preferentially), or both. We found that overexpression of TMPRSS2 supports the virus life cycle by cleaving HA. Furthermore, we found that overexpression of ST3GAL1 increased the viral titer. Finally, we showed that overexpression of both TMPRSS2 and ST3GAL1 increased the final viral titer due to enhanced support of viral replication and prolonged viability of the cells. In addition, overexpression of these genes of interest had no effect on cell proliferation and viability. Conclusions Taken together, the results indicate that these engineered cells could be used as a cell-based system to propagate influenza virus efficiently in the absence of trypsin. Further studies on influenza virus interactions with chicken cell host factors could be studied without the effect of trypsin on cells.

Modulation of Heat-Shock Proteins Mediates Chicken Cell Survival against Thermal Stress

Heat stress is one of the most challenging environmental stresses affecting domestic animal production, particularly commercial poultry, subsequently causing severe yearly economic losses. Heat stress, a major source of oxidative stress, stimulates mitochondrial oxidative stress and cell dysfunction, leading to cell damage and apoptosis. Cell survival under stress conditions needs urgent response mechanisms and the consequent effective reinitiation of cell functions following stress mitigation. Exposure of cells to heat-stress conditions induces molecules that are ready for mediating cell death and survival signals, and for supporting the cell’s tolerance and/or recovery from damage. Heat-shock proteins (HSPs) confer cell protection against heat stress via different mechanisms, including developing thermotolerance, modulating apoptotic and antiapoptotic signaling pathways, and regulating cellular redox conditions. These functions mainly depend on the capacity of HSPs to work as molecular chaperones and to inhibit the aggregation of non-native and misfolded proteins. This review sheds light on the key factors in heat-shock responses for protection against cell damage induced by heat stress in chicken.

Immunomodulatory effects of thymol and cinnamaldehyde in chicken cell lines

Thymol and cinnamaldehyde are phytogenic feed additives that have been developed to improve gut health, immunity and growth performance in poultry and swine. This study evaluated the immune modulating effects of a thymol and cinnamaldehyde blend (TCB) in the intestinal system of poultry in vitro, using two chicken cell lines, LMH (liver cell line) which has been used to mimic epithelial cell responses, and HD-11 (monocyte/macrophage-like). Cells with high viability (>95%) from established cell lines were cultured in the presence of TCB at concentrations ranging from 1 ng/ml to 100 ng/ml. The viability, transepithelial electrical resistance (TEER) and phagocytic capacity of co-cultured LMH cells, with or without stimulation with lipopolysaccharide (LPS), was subsequently evaluated. The expression of cytokines, chemokines and pattern recognition receptors by HD-11 monocytes/macrophages was measured by RT-PCR and by proteomic analysis. TCB was well tolerated by both cell lines (cell viability >90% after co-culture with TCB at 100 ng/ml for 48 h with or without LPS). Epithelial integrity of LMH cells (as assessed by TEER) was increased by TCB (10 ng/ml) after 4 h incubation, versus untreated controls, and phagocytic capacity of HD-11 cells was increased, in a dose-dependent manner (P<0.05). In HD-11 cells, TCB (10 ng/ml) downregulated the relative expression of pro-inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8 and the transcription factor cyclooxygenase-2 and upregulated expression of anti-inflammatory IL-10, versus untreated controls (P<0.05). In summary, under the tested conditions, TCB enhanced the epithelial barrier integrity of poultry hepatocytes, increased phagocytic activity and production of anti-inflammatory cytokines by monocytes and macrophages. These results indicated how supplementing TCB in poultry diets can increase bird performance, by increasing in vivo cell membrane integrity (especially important in the gut) and assisting in immune responses, which can liberate energy for growth.

HMEJ-mediated efficient site-specific gene integration in chicken cells

Background The production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species. Results Here we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system in chicken DF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in in chicken PGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 in chicken PGCs through the insertion of a fluorescent protein in the frame at the 3′ end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chicken eggs, suggested its good promise for the mass production of protein with pharmaceutical importance using the chicken oviduct system. Conclusions Taken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration in chicken PGCs, which holds great potential for the biopharmaceutical engineering of chicken cells.

Evaluation of a Recombinant Newcastle Disease Virus Expressing Human IL12 against Human Breast Cancer

The Newcastle disease virus (NDV) strain AF2240 is an avian avulavirus that has been demonstrated to possess oncolytic activity against cancer cells. However, to illicit a greater anti-cancer immune response, it is believed that the incorporation of immunostimulatory genes such as IL12 into a recombinant NDV backbone will enhance its oncolytic effect. In this study, a newly developed recombinant NDV that expresses IL12 (rAF-IL12) was tested for its safety, stability and cytotoxicity. The stability of rAF-IL12 was maintained when passaged in specific pathogen free (SPF) chicken eggs from passage 1 to passage 10; with an HA titer of 29. Based on the results obtained from the MTT cytotoxic assay, rAF-IL12 was determined to be safe as it only induced cytotoxic effects against normal chicken cell lines and human breast cancer cells while sparing normal cells. Significant tumor growth inhibition (52%) was observed in the rAF-IL12-treated mice. The in vivo safety profile of rAF-IL12 was confirmed through histological observation and viral load titer assay. The concentration and presence of the expressed IL12 was quantified and verified via ELISA assay. In summary, rAF-IL12 was proven to be safe, selectively replicating in chicken and cancer cells and was able to maintain its stability throughout several passages; thus enhancing its potential as an anti-breast cancer vaccine.