Location: Endemic Poultry Viral Diseases ResearchTitle: Recombineering of the infectious laryngotracheitis genome using synthetic genomics assembly
|GARCIA, MARICARMEN - University Of Georgia|
|FUCHS, WALTER - Friedrich-Loeffler-institut|
|RIBLET, SYLVA - University Of Georgia|
|LOCOMAN, CARLOS - Melbourne University|
|VOLKENING, JEREMY - Base2bio|
Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 7/21/2018
Publication Date: 7/21/2018
Citation: Spatz, S.J., Garcia, M., Fuchs, W., Ross, T.A., Riblet, S., Kim, T.N., Locoman, C., Volkening, J. 2018. Recombineering of the infectious laryngotracheitis genome using synthetic genomics assembly. Proceedings of the 43rd International Herpesvirus Workshop, Vancouver, Canada, July 21-25, 2018.
Technical Abstract: The study of gene function of the herpesvirus, infectious laryngotracheitis virus (ILTV) has been difficult due to the lack of an infectious clone. To overcome this, large ILTV DNA fragments (>40 kb) were cloned into the cosmid pSuperCos to generate a series of overlapping cosmids. These cosmids and a yeast centromere plasmid (ycp) containing sequences that span the TRS/UL junction, were used in transfection experiments to reconstitute virus. To demonstrate the usefulness of this system, we generated three mutant viruses containing packaging signal deletions and insertions. Interestingly, the D-type genome of ILTV is unlike most other alphaherpesviruses and contains two Pac-2 sites at the 5’ end of the UL region. In transfection experiments, wild type virus was reconstituted using the cosmid clones and the ycp clone (BC114) containing one Pac-1 site and two Pac-2 sites. However, in transfection experiments with the cosmid clones and a mutated ycp clone (KLO26) containing only one Pac-2 site and one Pac-1 site, viable viruses were also generated. Similarly, viable “marker” viruses were reconstituted using the ycp clone (modKLO) containing the gene encoding green fluorescent protein (gfp) inserted into the redundant Pac-2 site. Reconstituted cell-free viruses (vBC, vKLO, and vModKLO) from transfected LMH cells were able to infect chicken kidney (CK) cells and exhibited growth kinetics similar to the parental wild type virus (USDA challenge strain). To determine whether the reconstituted viruses retained their virulent phenotypes, specific pathogen free chickens were inoculated with the viruses and succumbed to clinical disease to similar degrees as birds inoculated with wild type virus. Overall this study demonstrated that viable virus could be reconstituted from a collection of recombinant clones and the redundant Pac-2 site is nonessential for infectivity both in vitro and in vivo.