Expanding the known nucleorhabdovirus world: the final chapter in a trilogy exploring the hidden diversity of plant-associated rhabdoviruses

Nucleorhabdoviruses, classified into four distinct genera within the family Rhabdoviridae (subfamily Betarhabdovirinae ), are plant-infecting viruses, characterized by nucleus-associated, enveloped, bacilliform virions. Nucleorhabdoviruses possess an unsegmented, single-stranded, negative-sense RNA genome (ca. 12-15.2 kb) that encodes six to eight proteins. Here, by exploring large publicly available metatranscriptomics datasets, we report the identification and genomic characterization of 47 novel viruses with genetic and evolutionary hallmarks of nucleorhabdoviruses. These viruses are associated with 45 distinct host plant species and were previously hidden in public database. Our findings significantly broaden the known host range of nucleorhabdoviruses, including the first nucleorhabdovirus associated with ferns and the first gammanucleorhabdoviruses linked to dicot hosts. Genetic divergence and evolutionary analyses suggest that all these novel viruses likely represent members of novel species. Phylogenetic reconstruction indicates that ten novel viruses are related to alphanucleorhabdoviruses, 25 to betanucleorhabdoviruses, eight to deltanucleorhabdoviruses and four to gammanucleorhabdoviruses. This study constitutes the final chapter in a trilogy chronicling a data mining expedition into the cryptic diversity of plant-associated rhabdoviruses, a journey that began with varicosaviruses and continued with cytorhabdoviruses. These new findings yield the most comprehensive phylogeny of nucleorhabdoviruses to date, significantly expanding our genomic understanding and illuminating the phylogenetic relationships and evolutionary dynamics of this virus group. Further, this work underscores the value of large-scale sequence data mining in advancing our understanding of the hidden world of plant rhabdoviruses.