Chicken Embryonic Fibroblast Cells Research Articles

A Novel Recombinant Modified Vaccinia Ankara Virus expressing Interleukin-13 Receptor α2 Antigen for Potential Cancer Immunotherapy.

Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cell-mediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors in vivo, indicating that host immune responses against IL-13Rα2 need further augmentation. The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing IL-13RΑ2 (rMVA-IL13RΑ2) virus and study in vitro infectivity and efficacy against IL-13Rα2 positive cell lines. We constructed a recombinant MVA expressing IL-13Rα2 and a green fluorescent protein (GFP) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2. Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13α2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP+ fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13-PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells. rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.

Whole transcriptome profiling reveals a lncMDP1 that regulates myogenesis by adsorbing miR-301a-5p targeting CHAC1

Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in different developmental stages of chicken primary myoblasts (CPMs) using RNA sequencing (RNA-seq) technology. The dual luciferase reporter system was performed using chicken embryonic fibroblast cells (DF-1), and functional studies quantitative real-time polymerase chain reaction (qPCR), cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), flow cytometry cycle, RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence, and western blotting assay. Our research demonstrated that miR-301a-5p had a targeted binding ability to lncMDP1 and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). The results revealed that lncMDP1 regulated the proliferation and differentiation of myoblasts via regulating the miR-301a-5p/CHAC1 axis, and CHAC1 promotes muscle regeneration. This study fulfilled the molecular regulatory network of skeletal muscle development and providing an important theoretical reference for the future improvement of chicken meat performance and meat quality.

Single Amino Acid Substitution in the Matrix Protein of Rabies Virus Is Associated with Neurovirulence in Mice.

Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

Different Infectivity of Swine Enteric Coronaviruses in Cells of Various Species.

Swine enteric coronaviruses (SECoVs), including porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), have caused high mortality in piglets and, therefore, pose serious threats to the pork industry. Coronaviruses exhibit a trend of interspecies transmission, and understanding the host range of SECoVs is crucial for improving our ability to predict and control future epidemics. Here, the replication of PDCoV, TGEV, and PEDV in cells from different host species was compared by measuring viral genomic RNA transcription and protein synthesis. We demonstrated that PDCoV had a higher efficiency in infecting human lung adenocarcinoma cells (A549), Madin-Darby bovine kidney cells (MDBK), Madin-Darby canine kidney cells (MDCK), and chicken embryonic fibroblast cells (DF-1) than PEDV and TGEV. Moreover, trypsin can enhance the infectivity of PDCoV to MDCK cells that are nonsusceptible to TGEV. Additionally, structural analyses of the receptor ectodomain indicate that PDCoV S1 engages Aminopeptidase N (APN) via domain II, which is highly conserved among animal species of different vertebrates. Our findings provide a basis for understanding the interspecies transmission potential of these three porcine coronaviruses.

Differential analysis of IBV-infected primary chicken embryonic fibroblasts and immortalized DF-1.

Infectious bronchitis virus (IBV) is responsible for high morbidity and mortality as well as substantial economic losses worldwide. Transcriptome sequencing of IBV-infected chicken embryonic fibroblast and DF-1 cells revealed that the virus elicits antiviral immunity in cells after viral infection, but IBV cannot activate DF-1 cells to produce sufficient amounts of viral structures to assemble into complete virions, which may be caused by the interactions between cytokines. The study of IBV cellular adaptations is important for vaccine development and investigation of the pathogenesis of IBV.

Clostridium perfringens sialidase interaction with Neu5Ac α-Gal sialic acid receptors by in-silico observation and its impact on monolayers cellular behavior structure.

This study aims to evaluate the effect of Clostridium perfringens sialidase treatment on monolayer cell behavior using computational screening and an in vitro approach to demonstrate interaction between enzyme-based drugs and ligands in host cells. The in silico study was carried out by molecular docking analysis used to predict the interactions between atoms that occur, followed by genetic characterization of sialidase from a wild isolate. Sialidase, which has undergone further production and purification processes exposed to chicken embryonic fibroblast cell culture, and observations-based structural morphology of cells compared between treated cells and normal cells without treatment. Based on an in silico study, C. perfringens sialidase has an excellent binding affinity with Neu5Acα (2.3) Gal ligand receptor with Gibbs energy value (∆G)-7.35 kcal/mol and Ki value of 4.11 µM. Wild C. perfringens isolates in this study have 99.1%-100% similarity to the plc gene, NanH, and NanI genes, while NanJ shows 93.18% similarity compared to the reference isolate from GenBank. Sialidase at 750 and 150 mU may impact the viability, cell count, and cell behavior structure of fibroblast cells by significantly increasing the empty area and perimeter of chicken embryo fibroblast (CEF) cells, while at 30 mU sialidase shows no significant difference compared with mock control. Sialidase-derived C. perfringens has the capacity to compete with viral molecules for attachment to host sialic acid based on in silico analysis. However, sialidase treatment has an impact on monolayer cell fibroblasts given exposure to high doses.

Chicken CH25H inhibits ALV-J replication by promoting cellular autophagy.

Autophagy plays an important role in host antiviral defense. The avian leukosis virus subgroup J (ALV-J) has been shown to inhibit autophagy while promoting viral replication. The underlying autophagic mechanisms, however, are unknown. Cholesterol 25-hydroxylase (CH25H) is a conserved interferon-stimulated gene, which converts cholesterol to a soluble antiviral factor, 25-hydroxycholesterol (25HC). In this study, we further investigated the autophagic mechanism of CH25H resistance to ALV-J in chicken embryonic fibroblast cell lines (DF1). Our results found that overexpression of CH25H and treatment with 25HC promoted the autophagic markers microtubule-associated protein 1 light chain 3 II (LC3II) and autophagy-related gene 5(ATG5), while decreased autophagy substrate p62/SQSTM1 (p62) expression in ALV-J infection DF-1 cells. Induction of cellular autophagy also reduces the levels of ALV-J gp85 and p27. ALV-J infection, on the other hand, suppresses autophagic marker protein LC3II expression. These findings suggest that CH25H-induced autophagy is a host defense mechanism that aids in ALV-J replication inhibition. In particular, CH25H interacts with CHMP4B and inhibits ALV-J infection in DF-1 cells by promoting autophagy, revealing a novel mechanism by which CH25H inhibits ALV-J infection. Although the underlying mechanisms are not completely understood, CH25H and 25HC are the first to show inhibiting ALV-J infection via autophagy.

Lnc90386 Sponges miR-33-5p to Mediate Mycoplasma gallisepticum-Induced Inflammation and Apoptosis in Chickens via the JNK Pathway.

Mycoplasma gallisepticum (MG) is one of the most important pathogens, that causes chronic respiratory disease (CRD) in chickens. Long non-coding RNAs (lncRNAs) are emerging as new regulators for many diseases and some lncRNAs can function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we found that miR-33-5p was significantly up-regulated both in MG-infected chicken embryonic lungs and chicken embryo fibroblast cells (DF-1), and Lnc90386 negatively correlated with miR-33-5p. miR-33-5p, as a new regulator for MG infection, repressed apoptosis, inflammatory factors in DF-1 cells by targeting JNK1. Further analyses showed that Lnc90386 sponged miR-33-5p to weaken its inhibitory effect on JNK1, forming the ceRNA regulatory network. Furthermore, knockdown of Lnc90386 significantly inhibited apoptosis and inflammatory factors, and promoted DF-1 cells proliferation. However, co-treatment with miR-33-5p inhibitor and Lnc90386 siRNA showed that knockdown of Lnc90386 could partially eliminate the inhibiting effect of miR-33-5p inhibitor on inflammation, cell apoptosis and proliferation. In conclusion, Lnc90386 sponges miR-33-5p to defend against MG infection by inhibiting the JNK signaling pathway.

A Role for the Chicken Interferon-Stimulated Gene CMPK2 in the Host Response Against Virus Infection.

Virus infection can lead to the production of interferon, which activates the JAK/STAT pathway and induces the expression of multiple downstream interferon-stimulated genes (ISGs) to achieve their antiviral function. Cytidine/uridine monophosphate kinase 2 (CMPK2) gene has been identified as an ISG in human and fish, and is also known as a rate-limiting enzyme in mitochondria to maintain intracellular UTP/CTP levels, which is necessary for de novo mitochondrial DNA synthesis. By mining previous microarray data, it was found that both Avian Influenza Virus (AIV) and Newcastle Disease Virus (NDV) infection can lead to the significant upregulation of chicken CMPK2 gene. However, little is known about the function of CMPK2 gene in chickens. In the present study, the open reading frame (ORF) of chicken CMPK2 (chCMPK2) was cloned from DF-1, a chicken embryo fibroblasts cell line, and subjected to further analysis. Sequence analysis showed that chCMPK2 shared high similarity in amino acid with CMPK2 sequences from all the other species, especially reptiles. A thymidylate kinase (TMK) domain was identified in the C-terminus of chCMPK2, which is highly conserved among all species. In vitro, AIV infection induced significant increases in chCMPK2 expression in DF-1, HD11, and the chicken embryonic fibroblasts (CEF), while obvious increase only detected in DF-1 cells and CEF cells after NDV infection. In vivo, the expression levels of chCMPK2 were up-regulated in several tissues from AIV infected chickens, especially the brain, spleen, bursa, kidney, intestine, heart and thymus, and notable increase of chCMPK2 was detected in the bursa, kidney, duodenum, lung, heart, and thymus during NDV infection. Here, using MDA5 and IFN-β knockdown cells, we demonstrated that as a novel ISG, chCMPK2 could be regulated by the MDA5/IFN-β pathway. The high expression level of exogenous chCMPK2 displayed inhibitory effects on AIV and NDV as well as reduced viral RNA in infected cells. We further demonstrated that Asp135, a key site on the TMK catalytic domain, was identified as critical for the antiviral activities of chCMPK2. Taken together, these data demonstrated that chCMPK2 is involved in the chicken immune system and may play important roles in host anti-viral responses.

Antiviral Effect of Lithium Chloride on Replication of Marek's Disease Virus in Chicken Embryonic Fibroblasts.

To investigate the antiviral effect of lithium chloride (LiCl) on the replication of Marek’s disease virus (MDV) in chicken embryonic fibroblast (CEF) cells, real-time PCR, Western blotting, plaque counting, and indirect immunofluorescence experiments were performed at different time points of LiCl treated CEF cells with virus infection. The results demonstrated that LiCl could affect multiple steps of virus replication and inhibit viral gene expression and protein synthesis in a dose- and time-dependent manner. Moreover, LiCl could directly affect viral infectivity as well. In addition, LiCl significantly affected the gene expression of IFN-β related genes in virus-infected cells. These results indicate that LiCl significantly inhibits MDV replication and proliferation in CEF cells and it has the potential to be used as an antiviral agent against MDV.

Transcriptional regulation of CYP19A1 expression in chickens: ESR1, ESR2 and NR5A2 form a functional network

Aromatase, encoded by CYP19A1, is responsible for the conversion of androgen to estrogen, which plays a vital role in the development and function of the ovary and functions in many other physiological processes in both sexes. Instead of being expressed in ovarian granulosa cells, as in mammals, CYP19A1 is expressed in chickens in the theca cells of ovarian follicles, and the mechanism of CYP19A1 expression regulation remains unknown. Here, using immunofluorescence and western blotting assay, we first confirmed that CYP19A1 and FOXL2 (Forkheadbox L2) were coexpressed in pre-granulosa cells of female chicken embryonic gonads, while FOXL2 did not affect aromatase expression at embryonic stages. Second, our research showed that CYP19A1, ESR1 (estrogen receptor alpha), ESR2 (estrogen receptor beta) and NR5A2 (liver receptor homologue-1) were coexpressed in the theca cell layers of chicken small yellow follicles. There was cross-talk between CYP19A1 and candidate transcription factors (ESR1, ESR2 and NR5A2), which was identified by generating a reliable theca cell culture model. Using luciferase assays in theca cells and chicken embryonic fibroblast (DF-1) cells, the results suggested that ESR1 and NR5A2 had potential effects on CYP19A1 promoter activity in chickens. Overexpression of ESR1, ESR2 and NR5A2 in chicken embryonic fibroblast (DF-1) cells upregulated the protein expression of CYP19A1, mutually restricted each other and formed a potential regulatory network to coordinate the expression of CYP19A1. To conclude, our results indicated that FOXL2 cannot regulate the expression of CYP19A1 at chicken embryonic stages and after sexual maturity, ESR1, ESR2 and NR5A2 form a functional network to affect the expression of CYP19A1. These results laid a foundation for further research on the transcriptional regulation of chicken aromatase.

Gga-miR-142-3p negatively regulates Mycoplasma gallisepticum (HS strain)-induced inflammatory cytokine production via the NF-κB and MAPK signaling by targeting TAB2

Mycoplasma gallisepticum (MG), a notorious avian pathogen, leads to considerable economic losses in the poultry industry. MG infection is characterized by severe, uncontrollable inflammation and host DNA damage. Micro ribonucleic acids (miRNAs) have emerged as important regulators in microbial pathogenesis. However, the role of miRNAs in MG infection is poorly characterized. In this study, we validated the functional roles of gga-miR-142-3p. The relative expression of gga-miR-142-3p in the lungs of the MG-infected chicken embryos and the MG-infected chicken embryonic fibroblast cell line (DF-1) was determined by reverse transcription quantitative real-time PCR analysis. Bioinformatics database was used to analysis the target gene of gga-miR-142-3p. The luciferase reporter assay as well as gene expression analysis were conducted to validate the target gene. To further explore the biological functions of gga-miR-142-3p upon MG infection, the cell proliferation was quantified using Cell Counting Kit-8 (CCK-8). Meanwhile, cell cycle analysis and apoptosis were measured using a flow cytometer. gga-miR-142-3p was significantly upregulated in both MG-infected chicken-embryo lungs and the DF-1 cells. gga-miR-142-3p over expression significantly downregulated the expression of pro-inflammatory cytokines, including interleukin-1β, interleukin-6 and tumor necrosis factor alpha after MG infection. Meanwhile, gga-miR-142-3p enhanced the host defense against MG infection by facilitating cell proliferation, promoting cell progression and inhibiting cell apoptosis. Interestingly, TAB2 knockdown groups show similar results, whereas, TAB2 over-expression groups and gga-miR-142-3p inhibitor groups had thoroughly opposite results. The expression of p-p65 in nuclear factor kappa B (NF-κB) and p-p38 in the mitogen-activated protein kinase (MAPK) pathway was decreased when gga-miR-142-3p was over-expressed. Upon MG infection, upregulation of gga-miR-142-3p alleviates inflammation by negatively regulating the signaling pathways of NF-κB and MAPKs by targeting TAB2 and facilitates cell proliferation by inhibiting cell apoptosis and promoting cell cycle progression to defend against MG infection.

Comprehensive profiling analysis of the N6-methyladenosine-modified circular RNA transcriptome in cultured cells infected with Marek\u2019s disease virus

Marek’s disease virus (MDV) induces severe immunosuppression and lymphomagenesis in the chicken, its natural host, and results in a condition that investigated the pathogenesis of MDV and have begun to focus on the expression profiling of circular RNAs (circRNAs). However, little is known about how the expression of circRNAs is referred to as Marek’s disease. Previous reports have is regulated during MDV replication. Here, we carried out a comprehensive profiling analysis of N6-methyladenosine (m6A) modification on the circRNA transcriptome in infected and uninfected chicken embryonic fibroblast (CEF) cells. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) revealed that m6A modification was highly conserved in circRNAs. Comparing to the uninfected group, the number of peaks and conserved motifs were not significantly different in cells that were infected with MDV, although reduced abundance of circRNA m6A modifications. However, gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses revealed that the insulin signaling pathway was associated with the regulation of m6A modified circRNAs in MDV infection. This is the first report to describe alterations in the transcriptome-wide profiling of m6A modified circRNAs in MDV-infected CEF cells.

Interplay of the ubiquitin proteasome system and the innate immune response is essential for the replication of infectious bronchitis virus.

Infectious bronchitis virus (IBV) is the only coronavirus known to infect poultry. The replication and pathogenesis of IBV are poorly understood, mainly because of the unavailability of a robust cell culture system. Here, we report that an active ubiquitin proteasome system (UPS) is necessary for efficient replication of IBV in Vero cells. Synthesis of IBV-specific RNA as well as viral protein is hampered in the presence of chemical inhibitors specific for the UPS. Like other coronaviruses, IBV encodes a papain-like protease (PLpro) that exhibits in vitro deubiquitinase activity in addition to proteolytically processing the replicase polyprotein. Our results show that the IBV PLpro enzyme inhibits the synthesis of interferon beta (IFNβ) in infected chicken embryonic fibroblast (DF-1) cells and that this activity is enhanced in the presence of melanoma differentiation-associated protein 5 (MDA5) and TANK binding kinase 1 (TBK1). IBV PLpro, when overexpressed in DF-1 cells, deubiquitinates MDA5 and TBK1. Both of these proteins, along with other adapter molecules such as MAVS, IKKε, and IRF3, form a signaling cascade for the synthesis of IFNβ. Ubiquitination of MDA5 and TBK1 is essential for their activation, and their deubiquitination by IBV PLpro renders them unable to participate in antiviral signaling. This study shows for the first time that there is cross-talk between the UPS and the innate immune response during IBV infection and that the deubiquitinase activity of IBV PLpro is involved in its activity as an IFN antagonist. This insight will be useful for designing better antivirals targeting the catalytic activity of the IBV PLpro enzyme.

Down-regulated gga-miR-223 inhibits proliferation and induces apoptosis of MG-infected DF-1 cells by targeting FOXO3

Mycoplasma gallisepticum (MG) is a major poultry pathogen that can induce Chronic Respiratory Disease (CRD) in chickens, causing serious economic losses in the poultry industry worldwide. Increasing evidence suggests that microRNAs (miRNAs) act as a vital role in resisting microbial pathogenesis and maintaining cellular mechanism. Our previous miRNAs sequencing data showed that gga-miR-223 expression level significantly decreased in MG-infected chicken lungs. The aim of this study was to reveal the role of gga-miR-223 in MG-induced CRD progression. We found that gga-miR-223 was remarkably down regulated and forkhead box O3 (FOXO3) was up-regulated in both MG-infected chicken embryos lungs and the chicken embryonic fibroblast cell line (DF-1) by qPCR. FOXO3 was verified as the target gene of gga-miR-223 through bioinformatics analysis and dual-luciferase reporter assay. Further studies showed that overexpressed gga-miR-223 could promote cell proliferation, cell cycle, and inhibit cell apoptosis by notably promoting the expression of cell cycle marker genes cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 6 (CDK6) and Cyclin D1 (CCND1) and inhibiting the expression of apoptosis markers Bcl-2-like 11(BIM), FAS ligand (FASLG) and TNF-related apoptosis-inducing ligand (TRAIL). As expected, FOXO3 knockdown group got similar results. Overexpression of gga-miR-223 observably promoted cell multiplication, cell cycle progression, and inhibited apoptosis of MG-infected DF-1 cells, while inhibited gga-miR-223 had the opposite effect. Taken together, upon MG-infection, downregulated gga-miR-223 could decrease proliferation, cycle progression, and increase apoptosis through directly targeting FOXO3 to exert an aggravating MG-infectious effect.

Cloning, characterization, and transcriptional activity of β-actin promoter of African catfish (Clarias gariepinus).

Selection of suitable promoters is crucial for the efficient expression of exogenous genes in transgenic animals. Although one of the most effective promoters, the β-actin promoter, has been widely studied in fish species, it still remains unknown in the economical important African catfish (Clarias gariepinus). In this study, the β-actin promoter of African catfish (cgβ-actinP) was cloned and characterized. In addition, recombinant plasmid pcgβ-actinP-EGFP with enhanced green fluorescent protein (GFP) gene as the reporter gene was constructed to verify the transcriptional activity. We obtained a cgβ-actinP fragment length of 1405bp, consisting 104bp of the 5' proximal promoter, 96bp of the first exon, and 1205bp of the first intron. Similar to those of other fish species, cgβ-actinP contains three key transcription regulatory elements (CAAT box, CArG motif, and TATA box). GFP-specific fluorescent signals were detected in chicken embryonic fibroblasts cells (DF-1 cells) transfected with pcgβ-actinP-EGFP, which was approximately 1.11 times of the positive control. In addition, GFP was effectively expressed in zebrafish larvae microinjected with linearized cgβ-actinP-EGFP, with expression rate reaching approximately 49.84%. Our data indicate that cgβ-actinP could be a potential candidate promoter in the practice of constructing "all fish" transgenic fish.

N-linked glycosylation at site 158 of the HA protein of H5N6 highly pathogenic avian influenza virus is important for viral biological properties and host immune responses

Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

Knockout of IRF7 Highlights its Modulator Function of Host Response Against Avian Influenza Virus and the Involvement of MAPK and TOR Signaling Pathways in Chicken

Interferon regulatory factor 7 (IRF7) is known as the master transcription factor of the type I interferon response in mammalian species along with IRF3. Yet birds only have IRF7, while they are missing IRF3, with a smaller repertoire of immune-related genes, which leads to a distinctive immune response in chickens compared to in mammals. In order to understand the functional role of IRF7 in the regulation of the antiviral response against avian influenza virus in chickens, we generated IRF7-/- chicken embryonic fibroblast (DF-1) cell lines and respective controls (IRF7wt) by utilizing the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. IRF7 knockout resulted in increased viral titers of low pathogenic avian influenza viruses. Further RNA-sequencing performed on H6N2-infected IRF7-/- and IRF7wt cell lines revealed that the deletion of IRF7 resulted in the significant down-regulation of antiviral effectors and the differential expression of genes in the MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) signaling pathways. Dynamic gene expression profiling of the host response between the wildtype and IRF7 knockout revealed potential signaling pathways involving AP1 (activator protein 1), NF-κB (nuclear factor kappa B) and inflammatory cytokines that may complement chicken IRF7. Our findings in this study provide novel insights that have not been reported previously, and lay a solid foundation for enhancing our understanding of the host antiviral response against the avian influenza virus in chickens.

Linc-GALMD1 Regulates Viral Gene Expression in the Chicken.

A rapidly increasing number of reports on dysregulated long intergenic non-coding RNA (lincRNA) expression across numerous types of cancers indicates that aberrant lincRNA expression may be a major contributor to tumorigenesis. Marek’s disease (MD) is a T cell lymphoma of chickens induced by Marek’s disease virus (MDV). Although we have investigated the roles of lincRNAs in bursa tissue of MDV-infected chickens in previous studies, the molecular mechanisms of lincRNA functions in T cells remain poorly understood. In the present study, Linc-GALMD1 was identified from CD4+ T cells and MSB1 cells, and its expression was significantly downregulated in MD-resistant line of birds in response to MDV challenge. Furthermore, loss-of-function experiments indicated that linc-GALMD1 significantly affected the expression of 290 genes in trans. Through integrated analysis of differentially expressed genes (DEGs) induced by MDV and linc-GALMD1, we found that IGLL1 gene expression levels had a positive correlation with the degree of MD infection and could potentially serve as an indicator for clinical diagnosis of MD. Moreover, an interaction between MDV and linc-GALMD1 was also observed. Accordingly, chicken embryonic fibroblast cells were inoculated with MDV with and without the linc-GALMD1 knockdown, and the data showed that linc-GALMD1 could repress MDV gene expression during the course of MDV infection. These findings uncovered a role of linc-GALMD1 as a viral gene regulator and suggested a function of linc-GALMD1 contributing to tumor suppression by coordinating expression of MDV genes and tumor-related genes and regulating immune responses to MDV infection.

Coupling metabolomics analysis and DOE optimization strategy towards enhanced IBDV production by chicken embryo fibroblast DF-1 cells

Infectious bursal disease (IBD) caused by IBD virus (IBDV) is highly contagious viral and vaccination in chicken embryo has been an effective mean to prevent acute infection. However, the current production of IBDV vaccine faces serious batch instability and external contamination. The chicken embryonic fibroblast cell line DF-1 is widely used for the proliferation of avian viruses and vaccine production. Thus, optimizing the production of IBDV by DF-1 cells has an important application value. Combining metabolomics analysis and a Design of Experiments (DOE) statistical strategy, this study successfully optimized the process of IBDV production by DF-1 cells. Differential analysis and time series analysis of metabolite data in both IBDV-infected and uninfected DF-1 cells were performed by multivariate statistical analysis. The results showed that the intracellular metabolite intensities of glycolysis, the pentose phosphate pathway, the nucleoside synthesis pathway, lipid metabolism, and glutathione metabolism were upregulated, and the TCA cycle underwent a slight downregulation after IBDV infection of DF-1 cells. Based on the metabolome results and DOE statistical optimization method, the additive components suitable for IBDV proliferation were determined. The IBDV titer increased by 20.7 times upon exogenous addition of cysteine, methionine, lysine and nucleosides in the control medium, which is consistent with the predicted result (20.0 times) by a multivariate quadratic equation. This study provides a strategy for the efficient production of IBDV vaccines and could potentially be utilized to improve the production of other viral vaccines and biologics.