Novel Tri-Segmented Rhabdoviruses: A Data Mining Expedition Unveils the Cryptic Diversity of Cytorhabdoviruses

Abstract

Cytorhabdoviruses (genus Cytorhabdovirus, family Rhabdoviridae) are plant-infecting viruses with enveloped, bacilliform virions. Established members of the genus Cytorhabdovirus have unsegmented single-stranded negative-sense RNA genomes (ca. 10-16 kb) which encode four to ten proteins. Here, by exploring large publicly available metatranscriptomics datasets, we report the identification and genomic characterization of 93 novel viruses with genetic and evolutionary cues of cytorhabdoviruses. Strikingly, five unprecedented viruses with tri-segmented genomes were also identified. This finding represents the first tri-segmented viruses in the family Rhabdoviridae, and they should be classified in a novel genus within this family for which we suggest the name "Trirhavirus". Interestingly, the nucleocapsid and polymerase were the only typical rhabdoviral proteins encoded by those tri-segmented viruses, whereas in three of them, a protein similar to the emaravirus (family Fimoviridae) silencing suppressor was found, while the other predicted proteins had no matches in any sequence databases. Genetic distance and evolutionary insights suggest that all these novel viruses may represent members of novel species. Phylogenetic analyses, of both novel and previously classified plant rhabdoviruses, provide compelling support for the division of the genus Cytorhabdovirus into three distinct genera. This proposed reclassification not only enhances our understanding of the evolutionary dynamics within this group of plant rhabdoviruses but also illuminates the remarkable genomic diversity they encompass. This study not only represents a significant expansion of the genomics of cytorhabdoviruses that will enable future research on the evolutionary peculiarity of this genus but also shows the plasticity in the rhabdovirus genome organization with the discovery of tri-segmented members with a unique evolutionary trajectory.

Keywords: cytorhabdoviruses; genetic diversity; metatranscriptomics; tri-segmented viruses; virus discovery; virus taxonomy.

Figure 1

Maximum-likelihood phylogenetic tree based on amino acid sequence alignments of the complete L gene of all tri-segmented rhabdoviruses and cytorhabdoviruses reported so far and in this study constructed with the WAG + G + F model. The scale bar indicates the number of substitutions per site. Bootstrap values following 1000 replicates are given at the nodes, but only the values above 50% are shown. The viruses identified in this study are noted with green, red, violet, and blue rectangles according to proposed genus membership. Alphanucleorhabdoviruses, gymnorhaviruses and varicosaviruses were used as outgroups.

Figure 2

(A): An inset of the maximum-likelihood phylogenetic tree shown in Figure 1 was cropped to show those viruses included in the proposed genus Alphacytorhabdovirus. The viruses identified in this study are noted with green squares. (B): genomic organization of the viral sequences used in the phylogeny.

Figure 3

(A): An inset of the maximum-likelihood phylogenetic tree shown in Figure 1 was cropped to show those viruses included in the proposed genus Betacytorhabdovirus. The viruses identified in this study are noted with red squares. (B): genomic organization of the viral sequences used in the phylogeny.

Figure 4

(A): An inset of the maximum-likelihood phylogenetic tree shown in Figure 1 was cropped to show those viruses included in the proposed genus Gammacytorhabdovirus. The viruses identified in this study are noted with violet squares. (B): genomic organization of the viral sequences used in the phylogeny.

Figure 5

(A): genomic organization of all tri-segmented rhabdoviruses identified in this study (B): Alignment of the 5′ trailer sequence ends of the three RNA segments of Alnus-, Erysimum- and Picris-associated viruses. The predicted coding sequences are shown in arrowed rectangles. Colors indicate protein homologies.