Multiplex NGS Panel for whole-genome recovery and molecular epidemiology of canine morbillivirus

Canine morbillivirus, also known as canine distemper virus (CDV), is a multi-host pathogen infecting both carnivorous and non-carnivorous species worldwide. However, the limited availability of complete CDV genomes hinders detailed phylogenetic and epidemiological research. Here, we developed a multiplex PCR-based genomic panel optimized for Illumina next-generation sequencing to obtain full-length CDV genomes from clinical samples directly. Designed from 173 global sequences, it was validated using a commercial vaccine strain and 15 field isolates from five Latin American countries. The method consistently achieved over 97% genome coverage, with average depths exceeding 1200×, even in samples with high Ct values. Whole-genome phylogenetic analysis of the obtained sequences resolved three lineages with higher resolution than H gene trees, revealing previously undetected sub-lineage structure. This approach also detected intra-host variants, offering insights into the natural diversity of CDV. This cost-effective, scalable method allows high-quality genome recovery without virus isolation, broadening genomic surveillance and enhancing the molecular epidemiology of CDV.