Avianflu could decimate Australian blackswans uq_news
]. Briefly, 17-day-old chicken and 21-day-old Pekin duck embryonated eggs were purchased from Darling Downs Hatchery . Primary aortic endothelial cells were cultured from the aortic arches of chicken and duck embryos as described previously using EGM-2MV medium with 10% FBS [305, 307].
All avian cells were cultured at 40 °C 5% CO2 unless otherwise stated. The black swans used for cell isolation were sourced from Currumbin Wildlife Animal Hospital , Queensland, Australia, and Tauranga Harbor, Bay of Plenty, New Zealand . The animals were either euthanized due to a terminal illness or as part of a government-approved cull. Tibiotarsi and femurs were obtained from all birds. The bones were then partially opened in the middle to expose the bone marrow and then submerged in pre-chilled Dulbecco’s modified Eagle medium in T175 flasks. Flasks were then transferred to a personal containment level 2 laboratory for further processing within 4 to 3 h post-mortem. Bone marrow was subsequently removed and resuspended in pre-chilled DMEM. A 15-ml suspension of DMEM containing bone marrow was then layered after filtering through a 40-μm sterile filter over 15ml of Lymphoprep® in a 50-ml SepMate® tube. The layer of cells at the DMEM-Lymphoprep interface was separated according to the SepMate manufacturer’s instructions. The separated cell layer was then resuspended in 1ml EGM-2MV media and enumerated. The cell concentration was adjusted to approximately 1 × 10cells per ml before transferring to a Petri dish with 10ml EGM-2MV media. The Petri dish was incubated at 40°C with 5% CO2. The media was replaced every other day until cells became confluent. Bone marrow-derived primary cells of passage 4 were cultured to confluency and incubated with 1.75μg/ml Alexa Fluor™ 488 Acetylated low-density lipoprotein for 4 h. The cells were detached by washing twice with PBS and incubating with 0.05% Trypsin-EDTA . The cells were mixed in 2% FBS in PBS and subsequently sorted with the Moflo Astrios ® High-Speed cell sorter under sterile conditions. Ac-LDL-stained cells were sorted based on prior knowledge of high and low ac-LDL intake [303] using BD FACSDiva™ . The sorted cells were counted and transferred to gelatine-coated plates and grown in EGM-2MV . Cells at post-sorted passages were known as black swan primary endothelial cells . The purity of bsEPCs was confirmed as described by Additional fileThe experiment was designed to include two groups viz. challenge and control, from each of the species, i.e., black swan, chicken and duck. Each group consisted of three biological replicates of endothelial cells.A/Chicken/Vietnam/0008/2004 virus was amplified in MDCK cells. All experiments using HPAIVs were performed under physical containment level 3 settings at the Australian Centre for Disease Preparedness . Primary endothelial cells were grown to at least 60% confluency and were inoculated with 5 × 10PFU/mL of VN04 for 60 min at 40°C under physical containment 3 settings. The inoculum was removed, and the cells were washed once with PBS, and EGM-2MV media without FBS was added. The cells were incubated for an additional 5 h.According to the manufacturer’s instructions, cellular RNA was extracted from cultured cells using the RNeasy Plus Kit . Subsequently, 3M of sodium acetate and 100% ethanol were added for RNA precipitation.Paired-end, 150bp long, RNAseq was performed by Macrogen approximately 40 million reads per sample. Library preparation was performed using the SMARTer® ultra-low kit . All the libraries were barcoded and subsequently sequenced using the Novaseq® 6000 with Novaseq® 6000 S4 Reagent Kit as per the manufacturer’s instructions using within a single lane in a single cell to avoid potential technical variations. RNAseq reads were separated into original samples based on the corresponding bar code.RNAseq-based differential gene expression analysis was performed after assessing the quality of the raw reads using the FastQC tool before and after adaptor and quality trimming with TrimGalore version 0.6.2 . RNAseq reads were then quantified at the transcript level using Salmon v1.5.2. First, the indexes were created for the black swan reference transcriptome and the Ensembl version 104 chicken and duck transcriptomes with following parameters -l -g -r input_r1 -f input_r2 --validateMappings -o ). The transcript abundance data of each species were imported through the R package “tximport” by transforming data from transcripts to gene-level quantification. The expression data from each species were independently analyzed for differential gene expression. Once RNAseq read libraries were normalized for sequencing depth and RNA composition, differential gene expression analysis was performed using DESeq2 version 1.30.1 [
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