Location: Endemic Poultry Viral Diseases Research
Project Number: 6040-32000-083-018-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: May 1, 2023
End Date: Apr 30, 2024
Objective:
To define the spatial transcriptome of host and virus RNA transcripts within individual cells in the skin of chicken infected with gallid ahphaherpesvirus-2.
Approach:
Chickens will be infected with wildtype and transmission mutants of Marek’s disease virus, and at late times postinoculation, skin biopsies will be collected and frozen. Four tissue samples will be fixed onto cell capture slides that contain barcoded capture oligonucleotides. Once fixed, the tissue sample will be stained with either H&E or IF staining and microscopically imaged. Then, the tissue will be permeabilized to release the mRNA so it can be captured with the 5,000 barcoded capture oligonucleotides that bind to the RNA in the tissue. Finally, the captured mRNA will be converted into cDNA, and sequencing libraries will be prepared. Each barcoded oligonucleotide will capture the transcripts from 1-10 cells. The Visium spatial gene expression platform will link both host and viral transcripts to a specific location on the slide, allowing a visual depiction of the transcriptional landscape.
