Location: Endemic Poultry Viral Diseases Research
Project Number: 6040-32000-074-026-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2020
End Date: Sep 30, 2021
Objective:
The object is to define the transcriptome and proteome of host and virus RNA transcripts and proteins in feather follicle epithelial cells using Gallid herpesvirus 2 and mutant viruses containing deletion in the conserved herpesvirus protein kinase gene.
Approach:
The approach is to generate viruses containing a series of GaHV-2 mutants with alterations in the genes involved in horizontal transmission. Chickens will be infected with these mutants and wild type viruses, and a late times postinoculation, feather follicle epithelial cells will be harvested. Some cells will be collected for RNA extraction, while others will be harvested for protein analysis. We will use guanidinium isothiocyanate technology to isolate the RNA. RNA integrity will be assessed using an RNA 6000 nanochip on a Bioanalyszer 2100. Poly A + RNA will be isolated using a Dynabeads mRNA purification kit. rRNA depleted total RNA and PolyA selected RNA will be sequenced using two platforms: Nanopore and Illumina. For direct RNA -sequencing using nanopore, RNA preps will be spike with a synthetic poly A + RNA standard and sequenced on a MinION MkIb with R9.4 flow cells for a runtime of 18 hrs. Base-calling will be performed using Albacore 1.2.1. For Illumina sequencing, TruSeq stranded RNA libraries will be prepared from the two different types of RNA preps using random hexamers. Following bar-coding, paired-end sequencing will be performed using HiSeq 4000. Frozen protein samples will be reduced and alkylated. Following precipitation, the samples will be subjected to sequential Endoproteinase lysine-C (Lys-C)/trypsin digestion, and their concentrations determined using a BCA assay. The proteins will be labeled using an isobaric TMT10plex kit and phophoenriched. Samples will be fractionated using liquid chromatography with a 120-minute gradient and analyzed using mass spectrometry.
