Hybrid Whole-Genome Sequencing for Genetic Stability Assessment of Infectious Laryngotracheitis Virus Vaccine Strains

BACKGROUND: Genetic stability of live-attenuated infectious laryngotracheitis virus (ILTV) vaccines is essential for consistent efficacy and safety; however, marker-based assessments targeting partial genes are often insufficient given the virus's large, structurally complex genome. The ILTV genome contains long internal inverted repeats (IRs) that can give rise to genomic isomers, complicating short-read assembly and accurate resolution of genome structure. METHODS: To overcome these limitations, we used a hybrid whole-genome sequencing (WGS) strategy, combining Oxford Nanopore Technologies (ONT) long reads to improve assembly contiguity with Illumina short reads for high-accuracy polishing at the single-nucleotide level. Using this approach, we generated complete de novo genome assemblies for the commercial Serva and Salsbury #146 vaccine strains. RESULTS: The assemblies showed high sequence concordance with targeted regions validated by Sanger sequencing. Whole-genome analysis further enabled detection and independent validation of a structural inversion in the unique short (US) region of the Salsbury strain, consistent with herpesvirus genome isomerization. To enable phylogenetic inference despite structural variability, we performed a pangenome-based analysis to define a conserved core-genome dataset that robustly resolved vaccine-associated lineages, separating Serva- and Salsbury-derived strains. CONCLUSIONS: Collectively, these findings show that a hybrid WGS workflow can generate high-confidence genome assemblies for the specific commercial ILTV vaccine vials analyzed and can support QC-relevant detection of major structural variations. Because this study is cross-sectional (two strains; single lot/vial per strain), it cannot distinguish potential biological lot-to-lot variation from methodological differences, and a comprehensive genetic stability evaluation will require applying this workflow across defined passage levels and/or multiple production lots.