An alternative genomic target for proper phylogenetic classification of canine distemper virus (Morbillivirus canis)

The high variability of canine distemper virus (CDV) highlights the need for adequate genomic analyses to accurately classify viral variants, with whole-genome sequencing (WGS) being the most recommended strategy for this purpose. However, for practical/economic reasons, CDV variants have often been classified by genetic analysis of the hemagglutinin (H) gene, whose suitability for proper lineage identification has been discussed. Herein, we propose an alternative genomic target for genetic classification of CDV in scenarios in which WGS is not feasible. Initially, we designed high coverage primers to amplify an internal region of the gene previously suggested as a potential target for CDV classification (P/V/C gene). The primers were based on 113 WGS of different CDV lineages available (GenBank) and target a 568 bp amplicon. The putative amplicon was then evaluated in silico for its ability to reproduce the genetic classification by WGS. After this analysis, we optimized the RT-PCR and evaluated its performance by testing different biological samples previously identified as CDV positive. Amplicons generated from these samples were sequenced and phylogenetically analyzed. Our in silico analysis confirmed that the classification based on this amplicon fully reproduces the classification from the CDV WGS. Following this validation, we demonstrated that our primers were able to amplify CDV genetic material from different clinical samples. Finally, the amplicons were easily sequenced by the Sanger method, allowing proper CDV classification. In conclusion, we propose an alternative genomic target for proper phylogenetic classification of CDV, which may be an alternative when WGS is not possible.