Chicken Fibroblasts Research Articles

Evaluation of chicken embryo extract and egg yolk extract as alternatives to basic cell culture medium supplement

BackgroundFetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS).MethodsSpecific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum.ResultsFibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively.ConclusionThis investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted.

Transdifferentiation of fibroblasts into muscle cells to constitute cultured meat with tunable intramuscular fat deposition

Current studies on cultured meat mainly focus on the muscle tissue reconstruction in vitro, but lack the formation of intramuscular fat, which is a crucial factor in determining taste, texture, and nutritional contents. Therefore, incorporating fat into cultured meat is of superior value. In this study, we employed the myogenic/lipogenic transdifferentiation of chicken fibroblasts in 3D to produce muscle mass and deposit fat into the same cells without the co-culture or mixture of different cells or fat substances. The immortalized chicken embryonic fibroblasts were implanted into the hydrogel scaffold, and the cell proliferation and myogenic transdifferentiation were conducted in 3D to produce the whole-cut meat mimics. Compared to 2D, cells grown in 3D matrix showed elevated myogenesis and collagen production. We further induced fat deposition in the transdifferentiated muscle cells and the triglyceride content could be manipulated to match and exceed the levels of chicken meat. The gene expression analysis indicated that both lineage-specific and multifunctional signalings could contribute to the generation of muscle/fat matrix. Overall, we were able to precisely modulate muscle, fat, and extracellular matrix contents according to balanced or specialized meat preferences. These findings provide new avenues for customized cultured meat production with desired intramuscular fat contents that can be tailored to meet the diverse demands of consumers.

Transdifferentiation of fibroblasts into muscle cells to constitute cultured meat with tunable intramuscular fat deposition.

Current studies on cultured meat mainly focus on the muscle tissue reconstruction in vitro, but lack the formation of intramuscular fat, which is a crucial factor in determining taste, texture, and nutritional contents. Therefore, incorporating fat into cultured meat is of superior value. In this study, we employed the myogenic/lipogenic transdifferentiation of chicken fibroblasts in 3D to produce muscle mass and deposit fat into the same cells without the co-culture or mixture of different cells or fat substances. The immortalized chicken embryonic fibroblasts were implanted into the hydrogel scaffold, and the cell proliferation and myogenic transdifferentiation were conducted in 3D to produce the whole-cut meat mimics. Compared to 2D, cells grown in 3D matrix showed elevated myogenesis and collagen production. We further induced fat deposition in the transdifferentiated muscle cells and the triglyceride content could be manipulated to match and exceed the levels of chicken meat. The gene expression analysis indicated that both lineage-specific and multifunctional signalings could contribute to the generation of muscle/fat matrix. Overall, we were able to precisely modulate muscle, fat, and extracellular matrix contents according to balanced or specialized meat preferences. These findings provide new avenues for customized cultured meat production with desired intramuscular fat contents that can be tailored to meet the diverse demands of consumers.

Establishment of Big Bone chicken fibroblast cell bank and study of its biological characteristics

A fibroblast bank of Big Bone chicken was established using tissue adherent culture method. This cell bank included 29 embryo samples, had stocks of 147 cryogenically-preserved vials each containing 2~3Å~106 cells, and met all the cell line quality control standards established by the American Type Culture Collection (ATCC). The cells cultured in vitro showed the typical morphology of fibroblasts. The growth curve assumed the “S” shape, and the cell population doubling time (PDT) was about 30h. Cell viability was 98.1% before cryopreservation and 96.6% after recovery. All tests for microbial contamination were negative. The isoenzyme pattern was of species specificity. The frequency of diploid cells was 91%. The transfection efficiency of three fluorescent protein genes fluctuated between 10.6% and 26.5%. These results showed that the cells cultured in vitro grew well and had stable genetic properties. This research thus does not only preserve the poultry genetic resources of Big Bone chicken at the cell level, but also opens new ways for preserving important genetic resources of endangered animals in the form of somatic cells.

Transcriptome-wide N6-methyladenosine modification and microRNA jointly regulate the infection of avian leukosis virus subgroup J in vitro

N6-methyladenosine (m6A) methylation in transcripts has been suggested to influence tumorigenesis in liver tumors caused by the avian leukosis virus subgroup J (ALV-J). However, m6A modifications during ALV-J infection in vitro remain unclear. Herein, we performed m6A and RNA sequencing in ALV-J-infected chicken fibroblasts (DF-1). A total of 51 differentially expressed genes containing differentially methylated peaks were identified, which were markedly enriched in microRNAs (miRNAs) in cancer cells as well as apoptosis, mitophagy and autophagy, RNA degradation, and Hippo and MAPK signaling pathways. Correlation analysis indicated that YTHDC1 (m6A-reader gene) plays a key role in m6A modulation during ALV-J infection. The env gene of ALV-J harbored the strongest peak, and untranslated regions and long terminal repeats also contained peaks of different degrees. To the best of our knowledge, this is the first thorough analysis of m6A patterns in ALV-J-infected DF-1 cells. Combined with miRNA profiles, this study provides a useful basis for future research into the key pathways of ALV-J infection associated with m6A alteration.

PMAIP1 promotes J subgroup avian leukosis virus replication by regulating mitochondrial function

Avian leukosis virus Subgroup J (ALV-J) exhibits high morbidity and pathogenicity, affecting approximately 20% of poultry farms. It induces neoplastic diseases and immunosuppression. Phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), a proapoptotic mitochondrial protein in the B-cell lymphoma-2 (Bcl-2) family, plays a role in apoptosis in cancer cells. However, the connection between the PMAIP1 gene and ALV-J pathogenicity remains unexplored. This study investigates the potential impact of the PMAIP1 gene on ALV-J replication and its regulatory mechanisms. Initially, we examined PMAIP1 expression using quantitative real-time PCR (qRT-PCR) in vitro and in vivo. Furthermore, we manipulated PMAIP1 expression in chicken fibroblast cells (DF-1) and assessed its effects on ALV-J infection through qRT-PCR, immunofluorescence assay (IFA), and western blotting (WB). Our findings reveal a significant down-regulation of PMAIP1 in the spleen, lung, and kidney, coupled with an up-regulation in the bursa and liver of ALV-J infected chickens compared to uninfected ones. Additionally, DF-1 cells infected with ALV-J displayed a notable up-regulation of PMAIP1 at 6, 12, 24, 48, 74, and 108 h. Over-expression of PMAIP1 enhanced ALV-J replication, interferon expression, and proinflammatory factors. Conversely, interference led to contrasting results. Furthermore, we observed that PMAIP1 promotes virus replication by modulating mitochondrial function. In conclusion, the PMAIP1 gene facilitates virus replication by regulating mitochondrial function, thereby enriching our understanding of mitochondria-related genes and their involvement in ALV-J infection, offering valuable insights for avian leukosis disease resistance strategies.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species

BackgroundThe gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes.MethodsIn this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed.ResultsThis novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines.ConclusionsAn AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite–host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.Graphical

Transcriptomic analysis of the hypoxia-inducible factor 1α impact on the gene expression profile of chicken fibroblasts under hypoxia

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional regulator that mediates cellular adaptive responses to hypoxia. Hypoxia-inducible factor 1α (HIF-1α) is involved in the development of ascites syndrome (AS) in broiler chickens. Therefore, studying the effect of HIF-1α on the cellular transcriptome under hypoxic conditions will help to better understand the mechanism of HIF-1α in the development of AS in broilers. In this study, we analyzed the gene expression profile of the chicken fibroblast cell line (DF-1) under hypoxic conditions by RNA-seq. Additionally, we constructed the HIF-1α knockdown DF-1 cell line by using the RNAi method and analyzed the gene expression profile under hypoxic conditions. The results showed that exposure to hypoxia for 48 h had a significant impact on the expression of genes in the DF-1 cell line, which related to cell proliferation, stress response, and apoptosis. In addition, after HIF-1α knockdown more differential expression genes appeared than in wild-type cells, and the expression of most hypoxia-related genes was either down-regulated or remained unchanged. Pathway analysis results showed that differentially expressed genes were mainly enriched in pathways related to cell proliferation, apoptosis, and oxidative phosphorylation. Our study obtained transcriptomic data from chicken fibroblasts at different hypoxic times and identified the potential regulatory network associated with HIF-1α. This data provides valuable support for understanding the transcriptional regulatory mechanism of HIF-1α in the development of AS in broilers.

The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens

Avian influenza viruses (AIV) of the H7N7 subtype are enzootic in the wild bird reservoir in Europe, cause infections in poultry, and have sporadically infected humans. The non-structural protein PB1-F2 is encoded in a second open frame in the polymerase segment PB1 and its sequence varies with the host of origin. While mammalian isolates predominantly carry truncated forms, avian isolates typically express full-length PB1-F2. PB1-F2 is a virulence factor of influenza viruses in mammals. It modulates the host immune response, causing immunopathology and increases pro-inflammatory responses. The role of full-length PB1-F2 in IAV pathogenesis as well as its impact on virus adaptation and virulence in poultry remains enigmatic. Here, we characterised recombinant high pathogenicity AIV (HPAIV) H7N7 expressing or lacking PB1-F2 in vitro and in vivo in chickens. In vitro, full-length PB1-F2 modulated viability of infected chicken fibroblasts by limiting apoptosis. In chickens, PB1-F2 promoted gastrointestinal tropism, as demonstrated by enhanced viral replication in the gut and increased cloacal shedding. PB1-F2’s effects on cellular immunity however were marginal. Overall, chickens infected with full-length PB1-F2 virus survived for shorter periods, indicating that PB1-F2 is also a virulence factor in bird-adapted viruses.

Diet Supplementation with Prinsepiae Nux Extract in Broiler Chickens: Its Effect on Growth Performance and Expression of Antioxidant, Pro-Inflammatory, and Heat Shock Protein Genes.

Heat stress significantly undermines the poultry industry by escalating rates of morbidity and mortality and impairing growth performance. Our recent findings indicate that Prinsepiae Nux extract (PNE) effectively stimulates the Nrf2 signaling pathway, a vital element in cellular antioxidant stress responses. This study further explores the prospective benefits of supplementing PNE into poultry feed to enhance broiler growth in heat-stressed conditions. An Nrf2-luciferase reporter assay was developed in a chicken fibroblast cell line, demonstrating that PNE induces Nrf2 activity in a concentration-dependent manner. Real-time RT-PCR results showed that PNE intensifies the expression of Nrf2-responsive targets such as Ho1 and Nqo1 in chicken fibroblasts. A total of 160 one-day-old Arbor Acres broiler chicks were randomly assigned into four groups, each receiving a basal diet supplemented with either 0% (control), 0.1% PNE, 1% PNE, or commercial electrolyte for 35 days. Broilers were raised in an environment where the ambient temperature exceeded 30 °C for approximately seven hours each day, fluctuating between 26 and 34 °C, which is known to induce mild heat stress. The findings reveal that a 1% PNE supplement led to a significant decrease in the feed conversion ratio (FCR) compared to the control group. Moreover, chickens supplemented with 1% PNE exhibited a substantial increase in hepatic mRNA expression of antioxidant genes, such as Nqo1, Gclc, Sod2, Cat, and heat shock protein-related genes including Hsp90 and Hsf1, and a decrease in pro-inflammatory cytokine genes Il-6 and Il-1β. Consequently, PNE holds potential as a feed supplement to strengthen the antioxidant defenses of broilers and build heat stress resilience in the poultry industry.

Chicken miR-148a-3p regulates immune responses against AIV by targeting the MAPK signalling pathway and IFN-γ

MicroRNAs are involved in the immune systems of host animals and play essential roles in several immune-related pathways. In the current study, we investigated the systemic biological function of the chicken miRNA gga-miR-148a-3p on immune responses in chicken lines resistant and susceptible to HPAIV-H5N1. We found that gga-miR-148a expression in the lung tissue of H5N1-resistant chickens was significantly downregulated during HPAIV-H5N1 infection. Overexpression of gga-miR-148a and a reporter construct with wild type or mutant IFN-γ, MAPK11, and TGF-β2 3′ untranslated region (3′ UTR)-luciferase in chicken fibroblasts showed that gga-miR-148a acted as a direct translational repressor of IFN-γ, MAPK11, and TGF-β2 by targeting their 3′ UTRs. Furthermore, miR-148a directly and negatively influenced the expression of signalling molecules related to the MAPK signalling pathway, including MAPK11, TGF-β2, and Jun, and regulated antiviral responses through interferon-stimulated genes and MHC class I and class II genes by targeting IFN-γ. Downstream of the MAPK signalling pathway, several proinflammatory cytokines such as IL-1β, IFN-γ, IL-6, TNF-α, IFN-β, and interferon-stimulated genes were downregulated by the overexpression of gga-miR-148a. Our data suggest that gga-miR-148a-3p is an important regulator of the MAPK signalling pathway and antiviral response. These findings improve our understanding of the biological functions of gga-miR-148a-3p, the mechanisms underlying the MAPK signalling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens as well as the role of gga-miR-148a-3p in improving the overall performance of chicken immune responses for breeding disease-resistant chickens.

Fibroblast test cells of embryo of Super Java Chicken as an indicator to test toxicity and malignancy

Fibroblast is one component of connective tissue cells in polygonal or stellate with cytoplasmic processes or projections. In vitro, tissue culture is an excellent medium used in biomedical research. The goal of this study was to analyze Toxicity and Malignancy in Embryo of Super Java Chicken as Potential Candidates of Strong Poultry in Indonesia. This study used Preparation of fibroblast primary cell culture of Java super chicken embryos, Pathogenicity test using fibroblast cells from Super Java chicken, Toxicity test using fibroblast cells from Super Java Chicken. Primary fibroblast cell culture of Java super chicken embryos was prepared from 10-day-old brood chicken eggs free of pathogens. Cells were prepared in 96-well microplates with minimal essential medium (MEM) containing 10 % fetal calf serum (FCS) 100 IU/ug penicillin/streptomycinVirus isolates were diluted in stages from 10-2 to 10-5 and inoculated into Java Super Chicken Fibroblast cell culture. The result showed that the negative control of the samples had a faster proliferative power than the fibroblast cell culture of Java super chicken, which was treated with concentrations of 10-2; 10-3; 10-4; 10-5. Moreover, before being inoculated with the virus, the confluent fibroblast cells of Java super chicken looked oval and regular. The day after infection, syncytia (large multinucleated cells) began to form on a small scale and became more pronounced on the second and third post-infection days. CPE was found in the 10-2,10-3 and 10-4 virus dilutions, and CPE was not found in the 10-5 dilution.

Mycoplasma synoviae lipid-associated membrane proteins identification and expression changes when exposed to chicken cells.

Mycoplasma synoviae is a significant cause of respiratory disease and synovitis among chickens, and has an adverse economic impact on broiler breeding efforts. The present study was designed to develop a systematic understanding of the role that M. synoviae lipid-associated membrane proteins (LAMPs) may play in the virulence of this pathogen. Bioinformatics tools were used to identify 146 predicted membrane proteins and lipoproteins in the M. synoviae proteome. Then, Triton X-114 was used to extract LAMPs that were subsequently identified via LC-MS/MS. This approach enabled the detection of potential LAMPs, and the top 200 most abundant proteins detected using this strategy were subject to further analysis. M. synoviae cells (100 MOI) were exposed to chicken fibroblasts (DF-1) and macrophages (HD-11) in a 1:1 mixed culture. Analysis of LAMP transcripts identified 72 up-regulated LAMP genes which were analyzed in depth by bioinformatics. GO analysis revealed these genes to be enriched in the nucleotide binding, sulfur amino acid transmembrane transporter activity, tRNA binding, rRNA modification, and transition metal ion transport pathways. Moreover, KEGG enrichment analysis suggested that these genes were enriched in the biosynthesis of secondary metabolites, carbon metabolism, glycolysis/gluconeogenesis, and nitrogen metabolism pathways.

Unraveling the Nrf2-ARE Signaling Pathway in the DF-1 Chicken Fibroblast Cell Line: Insights into T-2 Toxin-Induced Oxidative Stress Regulation.

The T-2 toxin (T2) poses a major threat to the health and productivity of animals. The present study aimed to investigate the regulatory mechanism of Nrf2 derived from broilers against T2-induced oxidative damage. DF-1 cells, including those with normal characteristics, as well as those overexpressing or with a knockout of specific components, were exposed to a 24 h treatment of 50 nM T2. The primary objective was to evaluate the indicators associated with oxidative stress and the expression of downstream antioxidant factors regulated by the Nrf2-ARE signaling pathway, at both the mRNA and protein levels. The findings of this study demonstrated a noteworthy relationship between the up-regulation of the Nrf2 protein and a considerable reduction in the oxidative stress levels within DF-1 cells (p < 0.05). Furthermore, this up-regulation was associated with a notable increase in the mRNA and protein levels of antioxidant factors downstream of the Nrf2-ARE signaling pathway (p < 0.05). Conversely, the down-regulation of the Nrf2 protein was linked to a marked elevation in oxidative stress levels in DF-1 cells (p < 0.05). Additionally, this down-regulation resulted in a significant decrease in both the mRNA and protein expression of antioxidant factors (p < 0.05). This experiment lays a theoretical foundation for investigating the detrimental impacts of T2 on broiler chickens. It also establishes a research framework for employing the Nrf2 protein in broiler chicken production and breeding. Moreover, it introduces novel insights for the prospective management of oxidative stress-related ailments in the livestock and poultry industry.

Bacillus coagulant HYI (BC-HYI) Alleviates LPS-Elicited Oxidative Stress by Engaging the Nrf2/HO-1 Signaling Pathway and Regulates Gut Macrobiotics in Laying Chickens

In the current study, Bacillus coagulants had a role in combating oxidative stress by inhibiting the growth of intestinal pathogens. However, there are few studies on reducing the mechanisms of oxidative stress. Therefore, this study aimed to explore the effects and underlying mechanisms of B. coagulant HYI (BC-HYI) treatment on growth and intestinal functions in laying chickens under LPS-induced oxidative stress. The in vivo experimental group included five groups of laying chicks: normal control, LPS group, B6 group, B7 group and B8 group. The test consisted of six repetitions in each group, with six animals in each repetition. In the in vitro experiment, an LPS-induced oxidative stress model of chicken fibroblast DF-1 cells was established, and the DF-1 cells were divided into control group, LPS-treated group, B5 group, B6 group and B7 group. On the one hand, we found that BC-HYI can inhibit pathological changes in some intestinal tissues. On the other hand, BC-HYI supplementation has a dual effect on the gut microbiota, promoting the proliferation of beneficial microbes such as Barbarella, Lactobacillus, and Antibacterial while maintaining symbiotic balance. The abundance of Barbarella, Bactericide, and Cloistral was significantly different between the LPS group and the BC-HYI group (p &lt; 0.01). Moreover, compared with the LPS group, BC-HYI significantly decreased reactive oxygen species levels and prevented cell apoptosis (p &lt; 0.01). It used to prevent oxidative stress by activating the Nrf2-ARE/HO-1 signaling pathway, enhancing the scavenging of free radicals, and reducing oxidative damage. BC-HYI alleviated oxidative stress in laying chickens by modulating the gut microbiota and activating the Nrf2-ARE/HO-1 signaling pathway. In summary, laying chickens and cell experiments indicate that BC-HYI supplementation can improve the enzyme function of antioxidants, regulate intestinal barrier function and activate the Nrf2-ARE/HO-1 signaling pathway to regulate intestinal barrier function.

Chicken miR-26a-5p modulates MDA5 during highly pathogenic avian influenza virus infection

MicroRNAs play crucial roles in immune-related pathways in host animals. In this study, we aimed to investigate the systemic biological function of gga-miR-26a-5p, a chicken miRNA, in the immune responses to HPAIV H5N1 infection in the Vietnamese Ri chicken line. Our results showed a significant downregulation in gga-miR-26a expression in the lung tissue of Ri chickens during HPAIV H5N1 infection. Overexpression of gga-miR-26a and the reporter construct, either containing the wildtype or mutant melanoma differentiation-associated protein 5 (MDA5) 3′ untranslated region (3′ UTR)-luciferase, into a chicken fibroblast cell line, revealed that gga-miR-26a can act as a direct translational repressor of MDA5 by targeting the 3′ UTRs. Additionally, miR-26a negatively regulated the expression of the signaling molecules related to the MDA5 signaling pathway, including MDA5, mitochondrial antiviral-signaling (MAVS), interferon regulatory factor 7 (IRF7), p38 mitogen-activated protein kinases, and nuclear factor-kappa B (NF-κB). Moreover, downstream of the IRF7 and NF-κB signaling pathway, the proinflammatory cytokines such as IL-1β, IFN-γ, IFN-α, IFN-β, and the interferon-stimulated gene (Mx1) were, likewise, downregulated by the overexpression of gga-miR-26a. These findings suggest that gga-miR-26a-5p serves as an important regulator in the MDA5 signaling pathway and antiviral response. Overall, our results contribute to an improved understanding of the biological functions of gga-miR-26a-5p, alongside the mechanisms underlying the MDA5 signaling pathway, and the antiviral response to HPAIV-H5N1 infection in chickens.

Modification of surface glycan by expression of beta-1,4-N-acetyl-galactosaminyltransferase (B4GALNT2) confers resistance to multiple viruses infection in chicken fibroblast cell.

Infectious viruses in poultry, such as avian influenza virus (AIV) and Newcastle disease virus (NDV), are one of the most major threats to the poultry industry, resulting in enormous economic losses. AIVs and NDVs preferentially recognize α-2,3-linked sialic acid to bind to target cells. The human beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) modifies α-2,3-linked sialic acid-containing glycan by transferring N-acetylgalactosamine to the sub-terminal galactose of the glycan, thus playing a pivotal role in preventing viruses from binding to cell surfaces. However, chickens lack a homolog of the B4GALNT2 gene. Here, we precisely tagged the human B4GALNT2 gene downstream of the chicken GAPDH so that the engineered cells constitutively express the human B4GALNT2. We performed a lectin binding assay to analyze the modification of α-2,3-linked sialic acid-containing glycan by human B4GALNT2. Additionally, we infected the cells with AIV and NDV and compared cell survivability, viral gene transcription, and viral titer using the WST-1 assay, RT-qPCR and TCID50 assay, respectively. We validated human B4GALNT2 successfully modified α-2,3-linked sialic acid-containing glycan in chicken DF-1 cells. Following viral infection, we showed that human B4GALNT2 reduced infection of two AIV subtypes and NDV at 12-, 24-, and 36-hours post-infection. Moreover, cells expressing human B4GALNT2 showed significantly higher cell survivability compared to wild-type DF-1 cells, and viral gene expression was significantly reduced in the cells expressing human B4GALNT2. Collectively, these results suggest that artificially expressing human B4GALNT2 in chicken is a promising strategy to acquire broad resistance against infectious viruses with a preference for α-2,3-linked sialic acids such as AIV and NDV.

Isolation and characterization of a novel goose ovaritis-associated cluster 3 Tembusu virus

Tembusu virus (TMUV) is a member of the genus Flavivirus in the family Flaviviridae. Currently, TMUV was classified into 4 distinct clusters, with cluster 2 strains widely distributed in duck and goose populations in Asia, causing significant economic losses to the producing industries. In this study, a novel TMUV strain TMUV/goose/CHN/2019/HNU-NX2 (HNU-NX2-2019) was isolated and characterized from geese with ovaritis from Hunan province, China. Phylogenetic analyses of genome and the E gene indicated the present TMUV could be grouped into the newly defined TMUV cluster 3. The genome of HNU-NX2-2019 showed the highest identities of 98.1% to 98.2% to the cluster 3 TMUVs newly identified in 2020 and 2021 from chickens with a severe egg-drop syndrome from Guangdong, Guangxi and Shandong provinces of China, which were all showing a close relation to a mosquito-origin TMUV (KT607936) identified in 2012. Further experiments confirmed HNU-NX2-2019 could grow well in chicken fibroblast cell line DF-1 and in SPF chicken embryos, with titers varied from 107.3 to 108.8 viral genomic copies per mL in the culture solutions. A pilot virus challenge study in 3-day-old chicks demonstrated that this virus could efficiently infect chicks with virus distributed in the brains, small intestines and other visceral organs, with titers varied from 105.4 to 106.7viral genomic copies per gram of the tissues. Furthermore, HNU-NX2-2019 can induce specific antibody in ducklings but with no obvious disease and virus shedding, and on necropsy no TMUV was detected in the tissues in the present study. This is the first report to identify a novel cluster 3 TUMV from goose, and further demonstrated this goose TMUV strain could infect chicken efficiently but not in ducklings under the present experimental conditions, which highlighted intensive attentions may be paid to this novel mosquito-origin cluster 3 TMUV.

Targeted knockout of Mx in the DF-1 chicken fibroblast cell line impairs immune response against Newcastle disease virus

Newcastle disease virus (NDV) is an RNA virus taking poultry as the host, and the Newcastle disease (ND) caused by NDV is one of the diseases with serious damage to the health of poultry. Mx encoding by myxovirus resistance gene, induced by type I interferon (IFN), has a wide range of antiviral and GTPase activities in human, mice, and other species via inhibition virus replication. However, the antiviral ability of chicken Mx is still a controversial issue. To explore the effect of chicken Mx post-NDV infection, Mx-knockout DF-1 cells were constructed via CRISPR/Cas9 gene editing system. The number of copies of NDV was detected by RT-qPCR, and the mRNA expression levels of IRF-7, IFN-α, IFN-β, TNF-α, p21, p27, and Bak in DF-1 cells were analyzed after NDV infection. Compared with control cells, virus titers were much higher in Mx-knockout DF-1 cells post-NDV infection. The deficiency of Mx aggravated the cell pathological features post-NDV infection, and promoted the expression levels of IRF-7, IFN-α, IFN-β, and pro-inflammatory cytokine TNF-α in host cells. In addition, cells with Mx deficiency could alleviate the harm from virus by enhancing the expression of p21, p27, and Bak, which related to cell proliferation apoptosis. In conclusion, Mx played an important role in antivirus invasion. In the absence of Mx, cells could alleviate the harm from virus infection via retarding cell proliferation and enhancing cell apoptosis.

Study on Propagation and Adaptation of EDS-76 Avian Adenovirus in Duck and SPF Primary Embryonic Chicken Cell Culture Comparison to Duck and SPF Embryonated Chicken Eggs.

Egg drop syndrome (EDS) is a major viral infectious poultry disease with severe economic losses in laying hens. The disease is caused by an adenovirus and can be transmitted horizontally and vertically. This study investigated the EDS virus (EDSV) infection in duck embryo fibroblasts (DEF), specific pathogen-free (SPF) embryo fibroblasts, and SPF egg embryos using different methods. The results were compared to the virus culture in duck and SPF chicken eggs. Duck and chicken fibroblast cells were used as the primary cell culture in Dulbecco's Modified Eagle Medium, and the low-pathogenic duck adenovirus was used to infect the ducks and SPF fibroblasts primary cell cultures, as well as the duck and SPF eggs. The titer of the virus was measured by hemagglutination assay, ECID50, plaque-forming unit, and TCID50 methods. The results revealed that EDSV could proliferate in the chorioallantoic membrane of DEF cells and duck eggs, compared to the chorioallantoic membrane of chicken embryo fibroblasts (CEF) and SPF chicken eggs. The findings showed that duck egg embryos and primary DEF cell lines are more appropriate for EDSV replication, compared to CEF and SPF chicken eggs. This suggests that the use of DEF culture for producing avian adenovirus EDS-76 is a suitable alternative for the embryonic egg culture.