Chicken Cells Research Articles

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.

Virus-inhibitory activity of the antigen complex of opportunistic pathogenic bacteria against SARS-CoV-2

Introduction. The antigen complex of opportunistic pathogenic bacteria (ACOPB) has a protective effect against avian influenza viruses, herpes virus type 2, and other viruses that cause acute respiratory viral infections. In the context of the COVID-19 pandemic, an important task is to find out whether ACOPB has a protective effect against SARS-CoV-2.
 The purpose of the study was to evaluate in vitro the ACOPB virus-inhibitory activity against the Dubrovka laboratory strain of SARS-CoV-2.
 Materials and methods. The study was performed using Vero cell line CCL-81, human peripheral blood mononuclear cells (PBMCs), mouse monoclonal anti-idiotypic antibodies structurally mimicking biological effects of human interferons (IFNs), the Dubrovka laboratory strain of SARS-CoV-2. The infectivity of the virus was assessed by two methods: by virus titration using cell cultures and the limiting dilution method when the results are assessed by a cytopathic effect; the second method was a plaque assay. The in vitro virus inhibition test was performed using the cell culture susceptible to SARS-CoV-2; the mixture containing a specific dose of the virus and a two-fold dilution of ACOPB was transferred to the cell culture after the ACOPB medication had interacted with the virus at 4C for 2 hours. The ACOPB virus-inhibitory activity against SARS-CoV-2 was assessed by the functional activity of / and IFN receptors (RIFN) in human PBMCs induced in vitro by ACOPB and the ACOPB mixture with the specific dose of SARS-CoV-2. The RIFN expression level was measured by the indirect membrane immunofluorescence test.
 Results. Hemagglutination assay using chicken, mouse, guinea pig, and human red blood cells was performed for detection of the SARS-CoV-2 inhibitory protein. The lysate of Vero CCL-81 cells infected with SARS-CoV-2 Dubrovka demonstrated the highest hemagglutination activity with guinea pig red blood cells and low titers of hemagglutination in the virus-containing fluid. The virus inhibition test in the Vero CCL-81 cell culture demonstrated that ACOPB inhibited 10 doses of SARS-CoV-2 Dubrovka with the titer 1 : 32, providing 100% protection of the cell culture for 8 days (the monitoring period). ACOPB induced / and RIFN expression on membranes of human PBMCs in in vitro cultures and decreased RIFN / and expression after its interaction with SARS-CoV-2 Dubrovka.
 Conclusion. The experimental studies including the virus inhibition test in the cell culture susceptible to SARS-CoV-2 Dubrovka and the indirect membrane immunofluorescence assay using monoclonal anti-idiotypic antibodies mimicking IFN-like properties demonstrated that ACOPB had both an immunomodulatory and a virus-inhibitory effect.

Emergence of biased hypermutation in a heterologous additional transcription unit in recombinant lentogenic Newcastle disease virus.

Recombinant Newcastle disease virus (rNDV) strains engineered to express foreign genes from an additional transcription unit (ATU) are considered as candidate live-attenuated vector vaccines for human and veterinary use. Early during the COVID-19 pandemic we and others generated COVID-19 vaccine candidates based on rNDV expressing a partial or complete SARS-CoV-2 spike (S) protein. In our studies, a number of the rNDV constructs did not show high S expression levels in cell culture or seroconversion in immunized hamsters. Sanger sequencing showed the presence of frequent A-to-G transitions characteristic of adenosine deaminase acting on RNA (ADAR). Subsequent whole genome rNDV sequencing revealed that this biased hypermutation was exclusively localized in the ATU expressing the spike gene, and was related to deamination of adenosines in the negative strand viral genome RNA. The biased hypermutation was found both after virus rescue in chicken cell line DF-1 followed by passaging in embryonated chicken eggs, and after direct virus rescue and subsequent passaging in Vero E6 cells. Levels of biased hypermutation were higher in constructs containing codon-optimized as compared to native S gene sequences, suggesting potential association with increased GC content. These data show that deep sequencing of candidate recombinant vector vaccine constructs in different phases of development is of crucial importance in the development of NDV-based vaccines.

Bisphenol A aggravate selenium deficiency-induced apoptosis via miR-215-3p/Dio1 to activate ROS/PI3K/AKT pathway in chicken arterial.

Both bisphenol A (BPA) and selenium (Se) deficiency can affect the expression of microRNAs (miRNAs), which can specifically regulate its target mRNA and induce apoptosis, and play a significant role in cardiovascular injury diseases. To explore the mechanism of apoptosis induced by BPA and Se deficiency in chicken arterial endothelial tissue and the role of miRNAs in this process, the model of BPA exposure/Se deficiency in chicken and PAEC cells have been employed. The targeting relationship between miR-215-3p and iodothyronine deiodinase 1 (Dio1) in PAEC was verified by double luciferase gene report. The level of miR-215-3p was detected by qRT-PCR. The oxidative stress level of arterial endothelial cells was detected by oxidative stress kit and DCFH-DA probe method. The PI3K/AKT pathway, mitochondrial dynamics, and apoptosis-related genes were detected by qRT-PCR and western blot. The mitochondrial ATP level and nitric oxide synthases (NOSs) level were detected with the kit. TUNEL, acridine orange/ethidium bromide,and flow cytometry were used to detect the level of apoptosis. The results showed that BPA exposure and Se deficiency led to overexpression of miR-215-3p, aggravated oxidative stress, inhibited activation of PI3K/AKT pathway, promoted mitochondrial division, increased expression of apoptosis related genes, and finally led to apoptosis of chicken arterial endothelial cells. We also established knockdown/overexpression models of miR-215-3p and Dio1 in vitro, and found that overexpression of miR-215-3p and knockout of Dio1 can induce apoptosis. Interestingly, miR-215-3p-Inhibitor and N-acetyl- l-cysteine (NAC) partially prevented apoptosis caused by BPA exposure and Se deficiency, and LY294002 aggravated apoptosis. These results suggest that BPA exposure aggravates the apoptosis of Se deficient arterial endothelial cells in chickens by regulating the ROS/PI3K/AKT pathway activated by miR-215-3p/Dio1. The miR-215-3p/Dio1 axis provides a new way to understand the toxic mechanism of BPA exposure and Se deficiency, and reveals a new regulatory model of apoptosis damage in vascular diseases.

Effects of chronic stress and intestinal inflammation on commercial poultry health and performance: A review

The gastrointestinal tract provides the biological environment for nutrient digestion and absorption. Its physical and chemical barriers are crucial to protect from invading pathogens and toxic substances. On this basis, the intactness of the gastrointestinal tract, with its multiple functions and impacts, is one of the key prerequisites for human and animal health. Undoubtedly, the functions of a healthy gut system also largely benefit the welfare and performance of animals in farming systems such as poultry industries. Broiler chickens grow rapidly, as a result of rigorous genetic programs, due to the high absorption capacity of intestinal epithelia for nutrients, the quick transport of nutrients to the muscle, and their efficient conversion into energy and biomass. Due to oxygen metabolism or enteric commensal bacteria, intestinal epithelial cells create reactive oxygen and nitrogen species physiologically. However, increased generation of these oxidants goes along with the formation of free radicals resulting in oxidative stress causing lipid peroxidation and dramatic molecular changes in the structure and function of the cell and mitochondrial membranes. These effects contribute to chronic oxidative stress and inflammation of the gastrointestinal tract and generally affect all chicken organs, tissues, and cells. Hence, all forms of chronic stress, regardless of the origin, negatively impact the chicken's overall performance, health, and welfare. This review article highlights some enteric inflammation models and biomarkers to evaluate gut integrity in chickens and discusses the repercussions that chronic stress and intestinal inflammation have on the health and performance of commercial poultry

The Effects of Freezing Media on the Characteristics of Male and Female Chicken Primordial Germ Cell Lines.

Recently, in vitro gene preservation has gained ground thanks to its lower cost and higher stability compared to in vivo techniques. One of the methods that can preserve female-specific W chromosome-linked genes is primordial germ cell (PGC) freezing. PGCs can be isolated from Hamburger-Hamilton stage 14-16 embryos via blood sampling. In our experiment, we used two newly established Black Transylvanian naked neck chicken cell lines and four cell lines from our gene bank. We compared two different freezing media (FAM1 and FAM2) in this study. The cell number and viability of the PGCs were measured before freezing (BF) and after thawing on Day 0, Day 1, and Day 7 of cultivation. We analyzed the germ cell-specific chicken vasa homologue (CVH) expression profile in PGCs using RT-qPCR. We found that on Day 0, immediately after thawing, the cell number in cell lines frozen with the FAM2 medium was significantly higher than in the FAM1-treated ones. On Day 1 and Day 7, the cell number and viability were also higher in most cell lines frozen with FAM2, but the difference was insignificant. The freezing also affected the chicken vasa homologue gene expression in male lines treated with both freezing media.

Temperature-induced reactivation of Marek's disease virus-transformed T cells ex vivo.

Marek's disease virus (MDV) establishes latency in chicken T lymphocytes that can lead to T cell transformation and cancer. Transformed Marek's disease chicken cell lines (MDCCs) can be expanded ex vivo and provide a valuable model to study latency, transformation, and reactivation. Here, we developed MDCCs from chickens infected with MDV that fluoresce during lytic replication and reactivation. Sodium butyrate treatment increased fluorescent protein expression as evidenced by fluorescent microscopy, flow cytometry, and western blotting; however, it caused significant apoptosis and necrosis. Treatment of MDCCs by decreasing the temperature resulted in robust MDV reactivation without significant induction of apoptosis and necrosis. Furthermore, MDV reactivation was significantly affected by the time in culture that can affect downstream reactivation analyses. In all, our data show that fluorescent protein expression during reactivation is a robust tool to examine viral replication in live cells ex vivo, and temperature treatment is an efficient technique to induce reactivation without punitive effects on cell viability seen with chemical treatment.

Sedeveria pink ruby Extract-Mediated Synthesis of Gold and Silver Nanoparticles and Their Bioactivity against Livestock Pathogens and in Different Cell Lines.

Biological synthesis of metal nanoparticles has a significant impact in developing sustainable technologies for human, animal, and environmental safety. In this study, we synthesized gold and silver nanoparticles (NPs) using Sedeveria pink ruby (SP) extract and characterized them using UV-visible spectrophotometry, FESEM-EDX, HR-TEM, XRD, and FT-IR spectroscopy. Furthermore, antimicrobial and antioxidant activities and cytotoxicity of the synthesized NPs were evaluated. UV-visible absorption spectra showed λmax at 531 and 410 nm, corresponding to the presence of SP gold NPs (SP-AuNPs) and SP silver NPs (SP-AgNPs). Most NPs were spherical and a few were triangular rods, measuring 5-30 and 10-40 nm, respectively. EDX elemental composition analysis revealed that SP-AuNPs and SP-AgNPs accounted for >60% and 30% of NPs, respectively. Additionally, some organic moieties were present, likely derived from various metabolites in the natural plant extract, which acted as stabilizing and reducing agents. Next, the antimicrobial activity of the NPs against pathogenic microbes was tested. SP-AgNPs showed potent antibacterial activity against Escherichia coli and Yersinia pseudotuberculosis. Moreover, at moderate and low concentrations, both NPs exhibited weak cytotoxicity in chicken fibroblasts (DF-1) and macrophages (HD11) as well as human intestinal cancer cells (HT-29). Meanwhile, at high concentrations, the NPs exhibited strong cytotoxicity in both chicken and human cell lines. Therefore, the synthesized SP-AuNPs and SP-AgNPs may act as promising materials to treat poultry diseases.

The cryo‐EM structure of the CENP‐A nucleosome in complex with ggKNL2

Centromere protein A (CENP‐A) nucleosomes containing the centromere‐specific histone H3 variant CENP‐A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP‐A deposition via the CENP‐A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP‐C‐like motif, and our previous study suggested that ggKNL2 directly binds to the CENP‐A nucleosome to recruit HJURP/CENP‐A to the centromere. However, the molecular basis for CENP‐A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo‐EM structure of the chicken CENP‐A nucleosome in complex with a ggKNL2 fragment containing the CENP‐C‐like motif. Chicken KNL2 distinguishes between CENP‐A and histone H3 in the nucleosome using the CENP‐C‐like motif and its downstream region. Both the C‐terminal tail and the RG‐loop of CENP‐A are simultaneously recognized as CENP‐A characteristics. The CENP‐A nucleosome–ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression.

Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations.

During the 2020 to 2022 epizootic of highly pathogenic avian influenza virus (HPAI), several infections of mammalian species were reported in Europe. In the Netherlands, HPAI H5N1 virus infections were detected in three wild red foxes (Vulpes vulpes) that were submitted with neurological symptoms between December of 2021 and February of 2022. A histopathological analysis demonstrated that the virus was mainly present in the brain, with limited or no detection in the respiratory tract or other organs. Limited or no virus shedding was observed in throat and rectal swabs. A phylogenetic analysis showed that the three fox viruses were not closely related, but they were related to HPAI H5N1 clade 2.3.4.4b viruses that are found in wild birds. This suggests that the virus was not transmitted between the foxes. A genetic analysis demonstrated the presence of the mammalian adaptation E627K in the polymerase basic two (PB2) protein of the two fox viruses. In both foxes, the avian (PB2-627E) and the mammalian (PB2-627K) variants were present as a mixture in the virus population, which suggests that the mutation emerged in these specific animals. The two variant viruses were isolated, and virus replication and passaging experiments were performed. These experiments showed that the mutation PB2-627K increases the replication of the virus in mammalian cell lines, compared to the chicken cell line, and at the lower temperatures of the mammalian upper respiratory tract. This study showed that the HPAI H5N1 virus is capable of adaptation to mammals; however, more adaptive mutations are required to allow for efficient transmission between mammals. Therefore, surveillance in mammals should be expanded to closely monitor the emergence of zoonotic mutations for pandemic preparedness. IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses caused high mortality among wild birds from 2021 to 2022 in the Netherlands. Recently, three wild foxes were found to be infected with HPAI H5N1 viruses, likely due to the foxes feeding on infected birds. Although HPAI is a respiratory virus, in these foxes, the viruses were mostly detected in the brain. Two viruses isolated from the foxes contained a mutation that is associated with adaptation to mammals. We show that the mutant virus replicates better in mammalian cells than in avian cells and at the lower body temperature of mammals. More mutations are required before viruses can transmit between mammals or can be transmitted to humans. However, infections in mammalian species should be closely monitored to swiftly detect mutations that may increase the zoonotic potential of HPAI H5N1 viruses, as these may threaten public health.

Chicken or Porcine Aminopeptidase N Mediates Cellular Entry of Pseudoviruses Carrying Spike Glycoprotein from the Avian Deltacoronaviruses HKU11, HKU13, and HKU17.

Members of deltacoronavirus (DCoV) have mostly been identified in diverse avian species as natural reservoirs, though the porcine DCoV (PDCoV) is a major swine enteropathogenic virus with global spread. The important role of aminopeptidase N (APN) orthologues from various mammalian and avian species in PDCoV cellular entry and interspecies transmission has been revealed recently. In this study, comparative analysis indicated that three avian DCoVs, bulbul DCoV HKU11, munia DCoV HKU13, and sparrow DCoV HKU17 (Chinese strain), and PDCoV in the subgenera Buldecovirus are grouped together at whole-genome levels; however, the spike (S) glycoprotein and its S1 subunit of HKU17 are more closely related to night heron DCoV HKU19 in Herdecovirus. Nevertheless, the S1 protein of HKU11, HKU13, or HKU17 bound to or interacted with chicken APN (chAPN) or porcine APN (pAPN) by flow cytometry analysis of cell surface expression of APN and by coimmunoprecipitation in APN-overexpressing cells. Expression of chAPN or pAPN allowed entry of pseudotyped lentiviruses with the S proteins from HKU11, HKU13 and HKU17 into nonsusceptible cells and natural avian and porcine cells, which could be inhibited by the antibody against APN or anti-PDCoV-S1. APN knockdown by siRNA or knockout by CRISPR/Cas9 in chicken or swine cell lines significantly or almost completely blocked infection of these pseudoviruses. Hence, we demonstrate that HKU11, HKU13, and HKU17 with divergent S genes likely engage chAPN or pAPN to enter the cells, suggesting a potential interspecies transmission from wild birds to poultry and from birds to mammals by certain avian DCoVs. IMPORTANCE The receptor usage of avian deltacoronaviruses (DCoVs) has not been investigated thus far, though porcine deltacoronavirus (PDCoV) has been shown to utilize aminopeptidase N (APN) as a cell receptor. We report here that chicken or porcine APN also mediates cellular entry by three avian DCoV (HKU11, HKU13, and HKU17) spike pseudoviruses, and the S1 subunit of three avian DCoVs binds to APN in vitro and in the surface of avian and porcine cells. The results fill the gaps in knowledge about the avian DCoV receptor and elucidate important insights for the monitoring and prevention of potential interspecies transmission of certain avian DCoVs. In view of the diversity of DCoVs, whether this coronavirus genus will cause novel virus to emerge in other mammals from birds, are worthy of further surveillance and investigation.

Calsequestrin in Purkinje cells of mammalian cerebellum

Cerebellum is devoted to motor coordination and cognitive functions. Endoplasmic reticulum is the largest intracellular calcium store involved in all neuronal functions. Intralumenal calcium binding proteins play a pivotal role in calcium storage and contribute to both calcium release and uptake. Calsequestrin, a key calcium binding protein of sarco-endoplasmic reticulum in skeletal and cardiac muscles, was identified in chicken and fish cerebellum Purkinje cells, but its expression in mammals and human counterpart has not been studied in depth. Aim of the present paper was to investigate expression and localization of Calsequestrin in mammalian cerebellum. Calsequestrin was found to be expressed at low level in cerebellum, but specifically concentrated in Calbindin D28- and zebrin- immunopositive-Purkinje cells. Two additional fundamental calcium store markers, sarco-endoplasmic calcium pump isoform 2, SERCA2, and Inositol-trisphosphate receptor isoform 1, IP3R1, were found to be co-expressed in the region, with some localization peculiarities. In conclusion, a new marker was identified for Purkinje cells in adult mammals, including humans. Such a marker might help in staminal neuronal cells specification and in dissection of still unknown neurodegeneration and physio-pathological effects of dysregulated calcium homeostasis.

Genome editing to create avian influenza resistance in chickens. Proceeding of The First International Avian Influenza Summit, University of Arkansas- October 16-17, 2023”

Avian influenza (AI) significantly threatens global poultry production and public health. Efforts to combat this viral disease have been predominantly focused on vaccination strategies, with limited success in controlling its spread. This study explores a novel approach utilizing genome editing techniques to enhance avian influenza resistance in chickens. Specifically, we employ CRISPR-Cas9 technology to target the ANP32 genes, endogenous proteins co-opted by influenza for replication in chicken cells. Through the precise modification of these genes, we aimed to elucidate their role in conferring resistance to low and high pathogenic avian influenza strains. We employed genome editing methodologies to introduce specific modifications in the chicken genome of reproductive cells. By introducing targeted genetic alterations in the germ cells, we created chickens that transmit their resistance trait to subsequent generations of chickens. The outcomes of this research have the potential to change avian influenza management strategies, offering a new paradigm for disease resistance in poultry populations. This approach holds promise for sustainable and cost-effective AI management strategies, reducing reliance on continuous vaccination campaigns. Moreover, the knowledge gained from our study may have broader implications for understanding host-influenza interactions in multiple species and inform future strategies for combating emerging infectious diseases at the human-animal interface.

Molecular analysis of chicken interferon-alpha inducible protein 6 gene and transcriptional regulation.

Interferon-alpha inducible protein 6 (IFI6) is an interferon-stimulated gene (ISG), belonging to the FAM14 family of proteins and is localized in the mitochondrial membrane, where it plays a role in apoptosis. Transcriptional regulation of this gene is poorly understood in the context of inflammation by intracellular nucleic acid-sensing receptors and pathological conditions caused by viral infection. In this study, chicken IFI6 (chIFI6) was identified and studied for its molecular features and transcriptional regulation in chicken cells and tissues, i.e., lungs, spleens, and tracheas from highly pathogenic avian influenza virus (HPAIV)-infected chickens. The chIFI6-coding sequences contained 1638 nucleotides encoding 107 amino acids in three exons, whereas the duck IFI6-coding sequences contained 495 nucleotides encoding 107 amino acids. IFI6 proteins from chickens, ducks, and quail contain an IF6/IF27-like superfamily domain. Expression of chIFI6 was higher in HPAIV-infected White Leghorn chicken lungs, spleens, and tracheas than in mock-infected controls. TLR3 signals regulate the transcription of chIFI6 in chicken DF-1 cells via the NF-κB and JNK signaling pathways, indicating that multiple signaling pathways differentially contribute to the transcription of chIFI6. Further research is needed to unravel the molecular mechanisms underlying IFI6 transcription, as well as the involvement of chIFI6 in the pathogenesis of HPAIV in chickens.

Chicken Serum과 Horse Serum이 닭 근육위성세포의 증식과 분화에 미치는 영향

For efficient production of cultured meat, chicken breast muscle cells and chicken leg muscle cells were proliferated and differentiated using medium with various serums, and proliferation and differentiation ability were analyzed. Chicken breast muscle cells and chicken leg muscle cells were cultured with serum-free medium (NC), medium with 20% FBS (C), medium with 10% chicken serum (CS) and 5% horse serum (HS) (T1), medium with 10% CS (T2), medium with 5% HS (T3). Using the immunofluorescence staining, Pax7 and nuclei were stained in chicken breast muscle cells and chicken leg muscle cells to confirm that they were muscle satellite cells. As a result of cell counting, both cells cultured in T1 medium proliferated the most. Chicken breast muscle cells and chicken leg muscle cells were subcultured from passage 1 to passage 6 using T1 medium. In both cells, viability and number of live cells decreased with increasing passage, but chicken leg muscle cells had higher viability and number of live cells than chicken breast muscle cells in all passages. Cells were differentiated using differentiation medium containing 10% chicken serum and 5% horse serum for each passage. It was confirmed that the differentiation ability of chicken leg muscle cells was more maintained as passage increased than chicken breast muscle cells. Therefore, it is considered that the results of chicken cells will become the basic data for the development of cultured meat.

The Chick Embryo Xenograft Model for Malignant Pleural Mesothelioma: A Cost and Time Efficient 3Rs Model for Drug Target Evaluation.

Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess drug targets using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, potentially offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies.

PA-X protein of H9N2 subtype avian influenza virus suppresses the innate immunity of chicken bone marrow-derived dendritic cells

H9N2 subtype avian influenza (AI) is an infectious disease associated with immunosuppression in poultry. Here, the regulation function of PA-X protein was determined on the host innate immune response of H9N2-infected chicken bone marrow-derived DCs (chBM-DCs). Based on 2 mutated viruses expressing PA-X protein (rTX) or deficient PA-X protein (rTX-FS), and the established culture system of chBM-DCs, results showed PA-X protein inhibited viral replication in chBM-DCs but not in non-immune chicken cells (DF-1). Moreover, PA-X protein downregulated the expression of phenotypic markers (CD40, CD86, and MHCII) and proinflammatory cytokine (IL-12 and IL-1β) of chBM-DCs. The mixed lymphocyte reaction between chBM-DCs and chicken T cells showed PA-X protein significantly decreased H9N2-infected chBM-DCs to induce T cell proliferation, implying a suppression of the DC-induced downstream T cell response. Taken together, these findings indicated that PA-X protein is a key viral protein to help H9N2 subtype AIVs escape the innate immunity of chBM-DCs.

Antimicrobial and cytotoxicity properties of biosynthesized gold and silver nanoparticles using D. brittonii aqueous extract

Recently, the production of nanoparticles using biological resources has gained considerable attention due to their application for animal and human well-being. In this study, we used a green synthesis to fabricate gold and silver nanoparticles by reducing HAuCl 4 and AgNO 3 into AuNPs and AgNPs, respectively, using Dudleya brittonii (DB) extract. The physio-chemical properties of the synthesized nanoparticles were analyzed using a UV–vis spectrophotometer, FESEM, EDX, HR-TEM, AFM and FT-IR. Furthermore, the antimicrobial and cytotoxicity activities of DB-AuNPs and DB-AgNPs against livestock pathogenic bacteria and different cell lines, as well as anti-oxidant activity, were investigated. DB synthesized AuNPs and AgNPs were mostly spherical with a few triangular rods and sizes ranging of 5–25 nm and 10–40 nm, respectively. The in vitro antibacterial and antifungal studies demonstrated the DB-AuNPs and DB-AgNPs have good antibacterial activity against E. coli and other livestock pathogens, including Y. pseudotuberculosis and S. typhi . Cell studies revealed that the higher concentrations of both DB-AuNPs and DB-AgNPs (1 µg/ml to 1 mg/ml) showed potent cytotoxicity in chicken cells after 24 hrs, whereas the middle and lower concentrations of DB-AuNPs and DB-AgNPs did not show cytotoxicity in selected cell lines after 24 hrs. In addition, the DB synthesized AuNPs and AgNPs exhibited good free scavenging activity in a dose-dependent manner. Therefore, the biosynthesized nanoparticles can be utilized by the livestock industry to develop an effective source against livestock microbial infections.

Transcriptome alterations in chicken HD11 cells with steady knockdown and overexpression of RIPK2 gene

Receptor interacting protein kinase 2 (RIPK2) is involved in a variety of signaling pathway to produce a series of inflammatory cytokines in response to a diverse of bacterial, viral and protozoal pathogens. However, the underlying regulating of RIPK2 remain unknown. Transcriptome alterations in chicken HD11 cells following RIPK2 overexpression or silencing by shRNA were analyzed by next-generation sequencing. Both overexpression and knockdown of the RIPK2 gene caused wide-spread changes in gene expression in chicken HD11 cells. Differentially expressed genes (DEGs) caused by altered RIPK2 gene expression were associated with multiple biological processes linked with biological regulation, response to stimulus, cell communication, and signal transduction etc. KEGG analysis revealed that many of the DEGs were enriched in VEGF signaling pathway, ECM-receptor interaction, Focal adhesion, TGF-beta signaling pathway etc. Moreover, we show that initiation genes, TGFB1 and TGFB3, in the TGF-beta signaling pathway are biological targets regulated by RIPK2 in chicken HD11 cells. This is the first transcriptome-wide study in which RIPK2-regulated genes in chicken cells have been screened. Our findings elucidate the molecular events associated with RIPK2 in chicken HD11 cells.

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.