Location: Endemic Poultry Viral Diseases ResearchTitle: Investigation of the virus genome concatemers of Gallid alphaherpesvirus 2 during in vitro replication by MinION sequencing
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/17/2019
Publication Date: 7/20/2019
Citation: Kim, T.N., Spatz, S.J. 2019. Investigation of the virus genome concatemers of Gallid alphaherpesvirus 2 during in vitro replication by MinION sequencing [abstract]. 44th Annual Herpesvirus Workshop, Knoxville, Tennessee, July 20-24, 2019.
Technical Abstract: Marek’s disease (MD), caused by Gallid alphaherpesvirus 2 also known as Marek’s disease virus (MDV), is a highly contagious lymphoproliferative disease of chickens. MDV has a class E genome with unique long (UL) and short (US) regions, and each unique segment is flanked by inverted repeats, i.e. terminal repeat long (TRL), internal repeat long (IRL), internal repeat short (IRS) and terminal repeat short (TRS). Like herpes simplex virus, the replication intermediates of MDV are concatemers that isomerize into four distinct isomers. To investigate the concatemerization of the MDV genome during replication, a Bacterial Artificial Chromosome (BAC) clone (Md5B40) of MDV strain Md5 was modified to contain an additional FseI restriction endonuclease cleavage site at the 3’ end of UL region by two-step Red recombineering. With the additional FseI site in Md5B40-BAC genome, one copy of Md5B40-BAC genome can be fragmented by FseI treatment as 109.4 kb (UL) and 74.5 kb fragments (IRL-IRS-US-TRS-TRL). MDV was reconstituted from modified Md5B40-BAC clones and the reconstituted Md5B40-Fse has comparable virus growth to parental virus. The fragmented Md5B40-Fse virus genomes were analyzed by MinION sequencing. The details of long-reads sequence analysis and the replication intermediate forms of MDV genome during in vitro passage will be discussed.