Chicken Embryonic Fibroblasts Research Articles

TMT-based quantitative proteomic analysis of IBDV-infected CEF cells reveals a favorable role of chicken IRF10 in IBDV replication via suppressing type-I interferon expression

Infectious bursal disease (IBD) is an acute, highly contagious disease caused by infectious bursal disease virus (IBDV), causing huge economic losses to the poultry industry worldwide. Currently, the emerging variant strains of IBDV and new recombinants in the field are circulating in many countries and poses severe threats to the development of poultry industry. Elucidation of the pathogenesis of IBDV infection will be of great help to the development of vaccines for control of IBDV infection. In this study, liquid chromatography tandem-mass spectrometry (LC–MS/MS) combined with tandem mass tag (TMT) labeling was performed to determine the expressions of nucleus proteins in IBDV-infected chicken embryonic fibroblast (CEF) cells 24 h post-infection (hpi). Our data show that a total of 236 nucleus proteins were differentially expressed in IBDV-infected cells vs mock-infected controls, and that among those proteins, 171 were significantly upregulated while 65 downregulated. Bioinformatics analysis reveals that the differentially expressed proteins (DEPs) were mainly involved in immune response, DNA replication, mismatch repair, and RIG-I-like receptor (RLR) signaling. Consistently, the expression of ten selected upregulated genes (IRF10, IRF7, IRF1, STAT1, ATF3, GTF3A, CSRP3, RARB, BASP1, and NF-κB1) markedly increased as examined by quantitative real‐time PCR (qRT-PCR). Furthermore, the expression of IRF10 was upregulated both in the cytoplasm and nucleus of DF-1 cells as examined by Western Blot. Moreover, knockdown of IRF10 remarkably inhibited IBDV replication via promoting IFN-I response, and overexpression of IRF10 significantly suppressed type I interferon and ISGs expression in both mock and IBDV-infected cells, suggesting that IRF10 serve as a negative regulator for host antiviral response. These results provide clues to further investigation into host–IBDV interactions and the underlying mechanisms of IBDV infection.

CHIR99021 and Brdu Are Critical in Chicken iPSC Reprogramming via Small-Molecule Screening.

Background/Objectives: Induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells into cells with most of the ESC (embryonic stem cell) characteristics show promise toward solving ethical problems currently facing stem cell research and eventually yield clinical grade pluripotent stem cells for therapies and regenerative medicine. In recent years, an increasing body of research suggests that the chemical induction of pluripotency (CIP) method can yield iPSCs in vitro, yet its application in avian species remains unreported. Methods: Herein, we successfully obtained stably growing chicken embryonic fibroblasts (CEFs) using the tissue block adherence method and employed 12 small-molecule compounds to induce chicken iPSC formation. Results: The final optimized iPSC induction system was bFGF (10 ng/mL), CHIR99021 (3 μM), RepSox (5 μM), DZNep (0.05 μM), BrdU (10 μM), BMP4 (10 ng/mL), vitamin C (50 μg/mL), EPZ-5676 (5 μM), and VPA (0.1 mM). Optimization of the induction system revealed that the highest number of clones was induced with 8 × 104 cells per well and at 1.5 times the original concentration. Upon characterization, these clones exhibited iPSC characteristics, leading to the development of a stable compound combination for iPSC generation in chickens. Concurrently, employing a deletion strategy to investigate the functionality of small-molecule compounds during induction, we identified CHIR99021 and BrdU as critical factors for inducing chicken iPSC formation. Conclusions: In conclusion, this study provides a reference method for utilizing small-molecule combinations in avian species to reprogram cells and establish a network of cell fate determination mechanisms.

Rapid adaptive evolution of avian leukosis virus subgroup J in response to biotechnologically induced host resistance.

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.

Optimization of process parameters for specific pathogen-free chicken embryonic fibroblast cultivation for yellow fever vaccine production

The yellow fever (YF) vaccine is usually produced with egg-based methods, which has limitations, including potential adverse effects and low production yields. Alternatively, producing the vaccine using Vero cells or HEK 293 cells can overcome some of these issues, but these methods are significantly more expensive. In the current study, the YF vaccine candidate 17DD virus was produced in primary chicken embryo fibroblast (CEF) cells. The primary CEF cells isolation from eggs was optimized through a two-step process. In the first step, the important parameters that contribute to the development of the egg embryo, such as egg position, relative humidity (RH), and incubation time are optimized. In second step, primary CEF release parameters namely; trypsin volume and incubation temperature are optimized. Both steps were optimized using statistical methods. Further, the seeding cell density of isolated CEF was also optimized. It was observed that 5 x 104 cells/cm2 gave the highest virus titer of 3.89 million PFU/ml. The 17DD yields achieved in primary CEFs are much higher than egg-based production and it is an economically viable method.

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 cellmediated 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-IL13Rα2 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.

Mechano-activated connexin hemichannels and glutathione transport protect lens fiber cells against oxidative insults

Long-lived lens fiber cells require a robust cellular protective function against oxidative insults to maintain their hemostasis and viability; however, the underlying mechanism is largely obscure. In this study, we unveiled a new mechanism that protects lens fiber cells against oxidative stress-induced cell death. We found that mechano-activated connexin (Cx) hemichannels (HCs) mediate the transport of glutathione (GSH) into chick embryonic fibroblasts (CEF) and primary lens fiber cells, resulting in a decrease in the accumulation of intracellular reactive oxygen species induced by both H2O2 and ultraviolet B, providing protection to lens fiber cells against cell apoptosis and necrosis. Furthermore, HCs formed by both homomeric Cx50 or Cx46 and heteromeric Cx50/Cx46 were mechanosensitive and could transport GSH into CEF cells. Notably, mechano-activated Cx50 HCs exhibited a greater capacity to transport GSH than Cx46 HCs. Consistently, the deficiency of Cx50 in single lens fiber cells led to a higher level of oxidative stress. Additionally, outer cortical short lens fiber cells expressing full length Cxs demonstrated greater resistance to oxidative injury compared to central core long lens fibers. Taken together, our results suggest that the activation of Cx HCs by interstitial fluid flow in cultured epithelial cells and isolated fiber cells shows that HCs can serve as a pathway for moving GSH across the cell membrane to offer protection against oxidative stress.

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.

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.

Forsythoside A protects against Zearalenone-induced cell damage in chicken embryonic fibroblasts via mitigation of endoplasmic reticulum stress.

Zearalenone (ZEA) is a non-steroidal estrogenic mycotoxin that exerts its toxic effects through various damage mechanisms such as oxidative stress, endoplasmic reticulum stress (ERS), mitochondrial damage, cell cycle arrest, and apoptosis. At present, there are few studies on drugs that can rescue ZEA-induced chicken embryonic fibroblasts damage. Forsythoside A (FA) is one of effective ingredients of traditional Chinese medicine that plays a role in various biological functions, but its antitoxin research has not been investigated so far. In this study, in vitro experiments were carried out. Chicken embryo fibroblast (DF-1) cells was used as the research object to select the appropriate treatment concentration of ZEA and examined reactive oxygen species (ROS), mitochondrial membrane potential, ERS and apoptosis to investigate the effects and mechanisms of FA in alleviating ZEA-induced cytotoxicity in DF-1 cells. Our results showed that ZEA induced ERS and activated the unfolded protein response (UPR) leading to apoptosis, an apoptotic pathway characterized by overproduction of Lactate dehydrogenase (LDH), Caspase-3, and ROS and loss of mitochondrial membrane potential. We also demonstrated that FA help to prevent ERS and attenuated ZEA-induced apoptosis in DF-1 cells by reducing the level of ROS, downregulating GRP78, PERK, ATF4, ATF6, JNK, IRE1, ASK1, CHOP, BAX expression, and up-regulating Bcl-2 expression. Our results provide a basis for an in-depth study of the mechanism of toxic effects of ZEA on chicken cells and the means of detoxification, which has implications for the treatment of relevant avian diseases.

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.

Isolation and culture of chickenembryonic synovial fibroblasts.

Cells obtained from chicken embryos are often preferred for in vitro studies. These cells, which easily adapt to rapid and continuous growth in the appropriate cell culture environment, are thought to be one of the effective methods in the investigation of leg diseases that are frequently observed in poultry. Leg diseases, especially affecting the joints in chickens, cause locomotor problems and adversely affect animal welfare. In addition, they cause significant health problems and increase mortality. It is known that synovial fibroblasts play an important role in joint diseases. In this study, chicken embryonic synovial fibroblasts were isolated from tissue explants taken from the tibio-metatarsal joint region of brown layer chicken embryos. Characterization of cells was evaluated by immunocytochemistry and hemacolor staining. chicken embryonic synovial fibroblasts showed a strong positive reaction to the vimentin antibody. As a result of hemacolor staining, it was noted that the cell morphology was spindle-shaped. The absence of macrophages in chicken embryonic synovial fibroblast culture was confirmed by the carbon powder uptake. In this present study, we aim to present a useful cell culture protocol such as primary culture, passage, and characterization suitable for chicken embryonic synovial fibroblast to be used in the new scientific research.

A Novel, Efficient Method to Isolate Chicken Primordial Germ Cells from Embryonic Blood Using Cell Culture Inserts.

Primordial germ cells (PGCs) play a crucial role in preserving poultry genetic resources and conducting transgenic research. A system for the rapid isolation of PGCs from single chicken embryonic blood was established in this paper. We found that PGCs can migrate to the lower layer of chicken embryonic fibroblasts (CEFs) through pores smaller than their diameter, while blood cells cannot, when co-cultured with CEFs of passages two to three. Based on the characteristics of PGCs, we developed a new PGC isolation method (cell culture insert/CEF adhesion method) that utilizes a 3 μm cell culture insert and CEFs of passages two to three. Using this method, approximately 700 PGCs can be isolated from the blood of a single chicken embryo at Hamburger and Hamilton (H&H) stage 17 of development. The separation rate achieved was 87.5%, with a separation purity of 95%. The separation rate of this method was 41.4% higher than the common Percoll density gradient centrifugation method and 33.6% higher than lysis with ACK buffer. PGCs isolated from embryonic blood could proliferate 37-fold within 2 weeks when cultured in a feeder-free culture system. They also continued to express the SSEA-1 and DAZL proteins and retained the ability to migrate in vivo. Overall, PGCs separated using cell culture inserts/CEF adhesion method retain their stem cell characteristics and migration ability. PGCs also exhibit good proliferation efficiency, making them suitable for subsequent transgenic experiments or genetic resource preservation.

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.

Efficacy of a recombinant turkey herpesvirus (H9) vaccine against H9N2 avian influenza virus in chickens with maternal-derived antibodies.

Although vaccines have been widely used for many years, they have failed to control H9N2 avian influenza virus (AIV) in the field in China. The high level of maternal-derived antibodies (MDAs) against H9N2 virus contributes to the H9N2 influenza vaccine failure in poultry. The study aimed to generate a new vaccine to overcome MDAs interference in H9N2 vaccination in chickens. We used turkey herpesvirus (HVT) as a vaccine vector to express H9 hemagglutinin (HA) proteins. The recombinant HVT expressing H9 HA proteins (rHVT-H9) was successfully generated and characterized in primary chicken embryonic fibroblasts (CEFs). Western blot and indirect immunofluorescence assay (IFA) showed that the rHVT-H9 consistently expressed HA proteins. In addition, the rHVT-H9 had similar growth kinetics to the parent HVT. Preliminary animal experiments showed that compared to the conventional inactivated whole virus (IWV) vaccine, the rHVT-H9 stimulated robust humoral immunity in chickens with passively transferred antibodies (PTAs) that were used to mimic MDAs. Transmission experiments showed that the rHVT-H9 induced both humoral and cellular immunity in chickens with PTAs. Furthermore, we used mathematical models to quantify the vaccine's efficacy in preventing the transmission of H9N2 AIV. The results showed that the rHVT-H9 reduced the virus shedding period and decreased the reproduction ratio (R) value in chickens with PTAs after homologous challenge. However, the vaccination in this trial did not yet bring R < 1. In summary, we generated a new rHVT-H9 vaccine, which stimulated strong humoral and cellular immunity, reducing virus shedding and transmission of H9N2 AIV even in the presence of PTAs in chickens.

Small RNA interfering effects in chicken embryonic fibroblasts using two sites 8664/p201 and 2122/pll3.7 of chicken s1p1/edg 1 gene

Introduction: Currently, short interfering RNA (siRNA) is an experimental tool in molecular biology to study gene function in cell culture and in vivo in model of organisms (mouse and chicken embryos, etc.) Method: Lentivirus production technique by human embryonic kidney 293T cell lines (HEK293T) with two (2) sites 8664/P201 and 2122/Pll.3.7 of the sphingosine 1 phosphate receptor gene and endothelial differentiation 1 gene (S1P1/ Chicken EDG1) was used to show the effects of small interfering RNA in chicken embryonic fibroblasts during embryogenesis. After preparation of plasmids 8664/P201 and 2122/Pll3.7, lentivirus packaging, transfection with 2xHBS, virus titration and chicken egg preparation, microinjection of lentivirus was performed in the embryos chicken. Results: The results obtained after the microinjection of lentivirus into chicken embryos from the second day to the fourth day of incubation showed the effects of small interfering RNA in the embryonic development of chicken materialized by bleeding in the yolk sac of embryos therefore a decrease in gene function. Conclusion: Previous studies have already proven that expression of small interfering RNA decreases in embryos of certain vertebrates such as mammals.

Small RNA interfering effects in chicken embryonic fibroblasts using two sites 8664/p201 and 2122/pll3.7 of chicken s1p1/edg 1 gene

Small RNA interfering effects in chicken embryonic fibroblasts using two sites 8664/p201 and 2122/pll3.7 of chicken s1p1/edg 1 gene