Novel insights into tomato leaf curl New Delhi virus introduction and evolution in Southeastern France using an advanced long-read sequencing workflow.

The Mediterranean population of tomato leaf curl New Delhi virus (ToLCNDV-ES) is characterized by a high genetic uniformity, distinguishing it from its Asian counterparts. ToLCNDV-ES is thought to have a monophyletic origin, likely resulting from a single recombination event, prior to its spread throughout the Mediterranean region. Following its first detection in southeastern France in 2020, ToLCNDV-ES re-emerged in France in 2022. Our analysis based on advanced long-read sequencing, circular DNA profiling, and phylogeny indicates both local persistence of French ToLCNDV-ES and multiple independent introduction events. Signatures of positive selection were identified in French ToLCNDV-ES populations, whereas no clear evidence of recombination was found. Bayesian time-structured phylogenetic analyses suggest that introductions in France occurred between 2018 and 2021 from the major ToLCNDV-ES clade, while several Italian ToLCNDV-ES isolates diverged prior to the virus introduction in the Mediterranean basin. Overall, this study demonstrates the value of an optimized long-read sequencing approach for resolving circular DNA virus diversity, and sheds light on the complex evolutionary history of ToLCNDV-ES in the Mediterranean Basin, particularly in southeastern France.