Location: Endemic Poultry Viral Diseases ResearchTitle: Genotyping of infectious laryngotracheitis virus using MinION sequencing.
|GARCIA, MARICARMEN - University Of Georgia|
|RIBLET, SYLVA - University Of Georgia|
|VOLKENING, JEREMY - Base2bio|
Submitted to: Herpesvirus International Workshop
Publication Type: Proceedings
Publication Acceptance Date: 7/20/2019
Publication Date: 7/20/2019
Citation: Spatz, S.J., Garcia, M., Riblet, S., Ross, T.A., Volkening, J., Taylor, T.L., Kim, T.N., Afonso, C.L. 2019. Genotyping of infectious laryngotracheitis virus using MinION sequencing. Proceedings of the Herpesvirus International Workshop, Knoxville, Tennessee, July 20-24, 2019.
Technical Abstract: Infectious laryngotracheitis (ILT) is an acute highly contagious upper respiratory infection of poultry caused by Gallid alphaherpesvirus 1. Recent genotyping studies of field strains from Australia and Korea suggest that circulating virulent field viruses are natural recombinants of vaccine strains. The identification of circulating viruses that are either distantly or closely related to vaccine strains and their characterization is currently a subject of great interest. Genotyping of infectious laryngotracheitis virus have traditional been done using allelic variations of three viral alleles gB, gM and UL47/gG using (PCR-RFLP) analysis. This has grouped US ILTV strains into nine genotypes (I-IX). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORFA/ORFB) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III- (TCO), IV - (CEO), V – (virulent CEO-like), VI – (virulent US) and VII/VIII/IX – (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real time amplicon sequencing using the single-allele assay and MinION sequencing.