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Article

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

by
Nicolas Bejerman
1,2,*,
Ralf Dietzgen
3,* and
Humberto Debat
1,2,*
1
Instituto de Patología Vegetal—Centro de Investigaciones Agropecuarias—Instituto Nacional de Tecnología Agropecuaria (IPAVE—CIAP—INTA), Camino 60 Cuadras Km 5,5, Córdoba X5020ICA, Argentina
2
Unidad de Fitopatología y Modelización Agrícola, Consejo Nacional de Investigaciones Científicas y Técnicas, Camino 60 Cuadras Km 5,5, Córdoba X5020ICA, Argentina
3
Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD 4072, Australia
*
Authors to whom correspondence should be addressed.
Viruses 2023, 15(12), 2402; https://doi.org/10.3390/v15122402
Submission received: 7 November 2023 / Revised: 7 December 2023 / Accepted: 8 December 2023 / Published: 10 December 2023
(This article belongs to the Special Issue The World of Rhabdoviruses)
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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.

1. Introduction

In the current metagenomics era, the rapid discovery of novel viruses has unveiled a rich and diverse evolutionary landscape of replicating entities, that present intricate challenges in their systematic classification [1]. To address this phenomenon, diverse strategies have emerged, culminating in a comprehensive proposal for establishing a megataxonomy of the virus world [2]. However, despite extensive efforts to characterize the viral component of the biosphere, it is evident that only a minuscule fraction, likely encompassing less than one percent of the entire virosphere, has been comprehensively characterized to date [3,4]. Consequently, our understanding of the vast global virome remains limited, with its remarkable diversity and its interactions with various host organisms [5,6,7,8]. To fill this knowledge gap, researchers have used the mining of publicly available transcriptome datasets obtained through High-Throughput Sequencing (HTS) as an efficient and inexpensive strategy [6,9,10,11]. This data-driven approach to virus discovery has become increasingly valuable, given the wealth of freely available datasets within the Sequence Read Archive (SRA) maintained by the National Center for Biotechnology Information (NCBI), which is continually expanding at an extraordinary rate. These data represents a substantial, albeit still somewhat limited and potentially biased, portion of the organisms inhabiting our world, thus making the NCBI-SRA database a cost-effective and efficient resource for the identification of novel viruses [12]. Serratus [6] has become an invaluable and exciting tool that facilitates comprehensive data mining, thus accelerating virus sequence discovery at a pace never witnessed before. In terms of virus taxonomy, a consensus statement has emphasized the importance of incorporating viruses known solely based on metagenomic data into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV) [13]. This recognition underscores the significance of metagenomic approaches in expanding our understanding of the global virome and adapting taxonomic frameworks to accommodate the ever-expanding diversity of viruses [14].
The family Rhabdoviridae is composed of members with negative-sense single-stranded RNA genomes that infect a broad range of hosts including plants, amphibians, fish, mammals, reptiles, insects, and other arthropods, and they include many pathogens of significance to public health, agriculture, and fisheries [15,16]. Almost all rhabdovirus genomes are unsegmented, but interestingly, plant-associated rhabdoviruses with bi-segmented genomes and a shared evolutionary history of rhabdoviruses have been included in the family in both genera Dichorhavirus and Varicosavirus [15,16]. Cytorhabdovirus is one of the genera that include plant-infecting viruses (family Rhabdoviridae, subfamily Betarhabdovirinae) [16]. Most cytorhabdoviruses exhibit a genome organization characterized by the presence of six conserved canonical genes encoded in the order 3′– nucleocapsid protein (N) – phosphoprotein (P) – movement protein (P3) – matrix protein (M) – glycoprotein (G) – large polymerase (L) –5′, and up to four additional accessory genes with unknown functions, leading to diverse genome organizations [17]. With some exceptions, the presence and synteny of the canonical genes are strictly conserved, nevertheless, some cytorhabdoviruses lack the G gene [9]. The viral genes are separated by conserved gene junction sequences, and the whole coding region is flanked by 3′ leader and 5′ trailer sequences that possess partially complementary ends, which could form a panhandle structure during viral replication [15].
In this study, through mining of publicly available sequence data, we identified 93 novel cytorhabdoviruses including five viruses with an unprecedented tri-segmented genome, which represent the first tri-segmented genomes among rhabdoviruses. Our findings will significantly advance the taxonomical classification of cytorhabdoviruses, allowing us to split this genus into three genera and shed new light on the evolutionary landscape of this group of plant rhabdoviruses.

2. Material and Methods

2.1. Identification of Cytorhabdovirus-Like Sequences from Public Plant RNA-Seq Datasets

We analyzed the Serratus database using the Serratus Explorer tool v1 [6] and queried the predicted RNA-dependent RNA polymerase protein (RdRP) of cytorhabdoviruses available at the NCBI-refseq database. The SRA libraries that matched the query sequences (alignment identity > 45%; score > 10) were further explored in detail.

2.2. Sequence Assembly and Virus Identification

Virus discovery was implemented as described elsewhere [10,11]. In brief, the raw nucleotide sequence reads from each SRA experiment that matched the query sequences in the Serratus platform were downloaded from their associated NCBI BioProjects. The datasets were pre-processed by trimming and filtering with the Trimmomatic v0.40 tool as implemented in http://www.usadellab.org/cms/?page=trimmomatic (accessed on 6 October 2023) with standard parameters. The resulting reads were assembled de novo with rnaSPAdes using standard parameters on the Galaxy server (https://usegalaxy.org/, accessed on 6 October 2023). The transcripts obtained from de novo transcriptome assembly were subjected to bulk local BLASTX searches (E-value < 1 × 10−5) against cytorhabdovirus refseq protein sequences available at https://www.ncbi.nlm.nih.gov/protein?term=txid11305[Organism], accessed on 6 October 2023. The resulting viral sequence hits of each dataset were explored in detail. Tentative virus-like contigs were curated (extended and/or confirmed) by iterative mapping of each SRA library’s filtered reads. This strategy was used to extract a subset of reads related to the query contig, used the retrieved reads from each mapping to extend the contig and then repeat the process iteratively using as query the extended sequence [10]. The extended and polished transcripts were reassembled using Geneious v8.1.9 (Biomatters Ltd., Auckland, New Zealand) alignment tool with high sensitivity parameters.

2.3. Bioinformatics Tools and Analyses

2.3.1. Sequence Analyses

ORFs were predicted with ORFfinder (minimal ORF length 120 nt, genetic code 1, https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 6 October 2023), functional domains and architecture of translated gene products were determined using InterPro (https://www.ebi.ac.uk/interpro/search/sequence-search, accessed on 6 October 2023) and the NCBI Conserved domain database—CDD v3.20 (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 6 October 2023) with E-value = 0.1. Further, HHPred and HHBlits as implemented in https://toolkit.tuebingen.mpg.de/#/tools/, accessed on 6 October 2023, were used to complement annotation of divergent predicted proteins by hidden Markov models. Transmembrane domains were predicted using the TMHMM version 2.0 tool (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 6 October 2023) and signal peptides were predicted using the SignalP version 6.0 tool (https://services.healthtech.dtu.dk/services/SignalP-6.0/, accessed on 6 October 2023). The presence of gene junction sequences flanking ORFs was also included as a criterion to determine the potential coding sequences. The predicted proteins were then subjected to NCBI-BLASTP searches against the non-redundant protein sequences (nr) database.

2.3.2. Pairwise Sequence Identity

Percentage amino acid (aa) sequence identities of the predicted L protein of all viruses identified in this study, as well as those available in the NCBI database were calculated using SDTv1.2 [18] based on MAFFT 7.505 (https://mafft.cbrc.jp/alignment/software, accessed on 6 October 2023), alignments with standard parameters. Virus names and abbreviations of cytorhabdoviruses already reported are shown in Supplementary Table S1.

2.3.3. Phylogenetic Analysis

Phylogenetic analysis based on the predicted L and N proteins of all plant cytorhabdoviruses, listed in Table S1, was conducted using MAFFT 7.505 with multiple aa sequence alignments using FFT-NS-i. The aligned aa sequences were used as the input in MEGA11 software [19] to generate phylogenetic trees by the maximum-likelihood method (best-fit model = WAG + G + F). Local support values were computed using bootstraps with 1000 replicates. L and N proteins of selected varicosaviruses and alphanucleorhabdoviruses were used as outgroups.

3. Results

3.1. Summary of Discovered Viral Sequences

In this study, through identification, assembly, and curation of raw NCBI-SRA reads of publicly available transcriptomic data we obtained the coding complete viral genomic sequences of 93 novel viruses with genetic and evolutionary links to cytorhabdoviruses. The phylogenetic relationships of the now significantly expanded number of known cytorhabdoviruses provide support for splitting the genus Cytorhabdovirus to establish three genera (Figure 1) that represent distinct evolutionary lineages, which we propose to name Alphacytorhabdovirus (Table 1), Betacytorhabdovirus (Table 2) and Gammacytorhabdovirus (Table 3). Strikingly, five unprecedented viruses with a tri-segmented genome were also identified and their full-length viral genomic sequences were assembled (Table 4), including the corrected full-length coding genome segments of the previously reported Picris cytorhabdovirus 1 (PiCRV1) [20], which had one RNA segment missing, as well as its RNA2 partially annotated.

3.2. Genus Alphacytorhabdovirus

The full-length coding regions of 38 novel putative alphacytorhabdoviruses were assembled in this study, including three variants of the same virus associated with different plant hosts, and two host variants of two other viruses (Table 1). The newly identified viruses were associated with 39 plant host species and a wetland metagenome study (Table 1). Most of the apparent host plants are herbaceous dicots (27/39), while 6 hosts are monocots and another 6 are woody dicots (Table 1).
The genomic organization of the 38 novel alphacytorhabdoviruses was quite similar, with few exceptions, with six distinct genomic organizations observed (Table 1, Figure 2B). Two virus genomes have no additional accessory genes and have the genome organization 3′-N-P–P3-M-G-L-5′ (Table 1, Figure 2B), while 14 viruses had an overlapping ORF within the P-encoding ORF, named P′, one virus had an accessory ORF between the G and L genes displaying a 3′-N-P–P3-M-G-P6-L-5′ genomic organization and 20 viruses had both those accessory ORFs (Table 1, Figure 2B). Another virus also had two accessory ORFs, one located between the P3 and the M genes, and the other between the G and L genes, displaying a 3′-N-P–P3-P4-M-G-P7-L-5′ genomic organization (Table 1, Figure 2B). Another newly identified virus also had two accessory ORFs, one between the G and L genes, and the other following the L gene, showing a 3′-N-P–P3-M-G-P6-L-P8-5′ genomic organization (Table 1, Figure 2B). P4 and P8 proteins yielded no hits when BlastP searches were carried out, and no conserved domains were identified in these proteins. On the other hand, transmembrane domains were identified in each P′ protein, as well as in each protein encoded by the accessory ORF located between the G and L genes.
The consensus gene junction sequences of the novel alphacytorhabdoviruses identified in our study were highly similar to those of previously reported phylogenetically related cytorhabdoviruses (Table 5).
Pairwise aa sequence identity values between each of the L proteins of the 38 novel viruses and those from known alphacytorhabdoviruses varied significantly, ranging from 36.16% to 85.65% (Table S2), while sequence identity for variants of the same virus ranged from 89.01% to 96.28% (Table S2). On the other hand, the highest L protein aa sequence identity with those cytorhabdoviruses proposed to be classified as betacytorhaboviruses and gammacytorhabdoviruses was 33.84% (Table S2).
A phylogenetic analysis based on the L protein aa sequence showed that the 38 novel viruses grouped with 33 known cytorhabdoviruses in a distinct major cluster (Figure 1). Within this cluster of 71 viruses, several clades could be distinguished (Figure 2A). One major clade and other minor ones were composed of viruses that do not have accessory ORFs between the G and L genes (Figure 2), while another clade grouped together viruses with an accessory ORF between the G and L genes (Figure 2). Other clusters grouped together viruses with distinct genomic organizations (Figure 2). A similar topology was observed in the phylogenetic tree based on the N protein aa sequences (Figure S1).

3.3. Genus Betacytorhabdovirus

The full-length coding regions of 39 novel putative betacytorhabdoviruses were assembled in this study (Table 2), including two distinct variants of the same virus. Based on the database information, the identified viruses were associated with 36 plant host species and two peat soil metagenomes (Table 2). Interestingly, 18/36 hosts are woody dicots, while 13/36 hosts are herbaceous dicots, and the other five hosts are monocots (Table 2).
The genomic organization of the 39 novel betacytorhabdoviruses was quite diverse, with 12 distinct genomic organizations observed (Table 2, Figure 3B). Several (12/39) viruses lack additional accessory genes and have the conserved basic genome organization 3′-N-P–P3-M-G-L-5′, but 11 of those genomes have a significantly shorter G gene (Table 2, Figure 3B). Other viruses (16/39) had an accessory ORF between the G and L genes displaying a 3′-N-P–P3-M-G-P6-L-5′ genomic organization. One virus had an accessory ORF, located after the L gene, thus displaying a 3′-N-P–P3-M-G-L-P7-5′ genomic organization, and one virus had an accessory ORF between the P3 and M genes showing a 3′-N-P–P3-P4-M-G-L-5′ genome organization (Table 2, Figure 3B). One virus had two accessory ORFs, one between the G and L genes, and the other after the L gene showing a 3′-N-P–P3-M-G-P6-L-P8-5′ genome organization (Table 2, Figure 3B). Yet another virus had two accessory ORFs in the same position; however, this virus lacked a discernable P3 gene, thus displaying a 3′-N-P–M-G-P5-L-P7-5′ genome organization (Table 2, Figure 3B). Four viruses had three accessory ORFs each in their genome, located either between the G and L genes displaying a 3′-N-P–P3-M-G-P6-P7-P8-L-5′ genome organization, or two accessory ORFs between the G and L genes and another between the N and P genes showing a 3′-N-X-P–P3-M-G-P7-P8-L-5′ genome organization or two accessory ORFs located between the P3 and the M genes, and another one after the L gene, displaying a 3′-N-P–P3-P4-M-G-L-P7-5′ genomic organization (Table 2, Figure 3B). On the other hand, one virus appeared to only have four genes in the order 3′-N-P-P3-L-5′, while the genome of two other viruses had five genes in the order 3′-N-P–P3-M-L-5′ but lacking the G gene (Table 2, Figure 3B). The P4 protein encoded by the virus named Passiflora betacytorhabdovirus 1 showed no hits when BlastP searches were carried out, and no known conserved domains were identified, whereas the P4 protein encoded by Sesamum virus 1 had no hits against the database, but a transmembrane domain and a Signal peptide were predicted. No hits against the database, nor conserved domains were found in those proteins encoded by the accessory ORF located after the L gene, or by the ORFs located after the accessory ORF which encodes the P6 protein in those viruses that have more than one accessory ORF between the G and L genes. Transmembrane domains were identified in the P6 protein which is encoded by an accessory ORF located between the G and L genes in those viruses, named P5 in Kobresia betacytorhabdovirus 1 and P7 in Justicia betacytorhabdovirus 1 and Passiflora betacytorhabdovirus 1.
The consensus gene junction sequences among the novel betacytorhabdoviruses identified in this study and those already known, showed some variability, mainly in the length of the intergenic spacer (Table 5).
Pairwise aa sequence identity values between each of the L proteins of the 39 novel viruses and those from known betacytorhabdoviruses varied significantly, ranging between 27% and 80.06% (Table S2), while the L protein identity for variants of the same virus ranged between 93.47% and 99.29% (Table S2). The highest L protein aa sequence identity with those cytorhabdoviruses proposed to be classified as betacytorhaboviruses and gammacytorhabdoviruses was 33.84% (Table S2).
Phylogenetic analysis based on L protein aa sequences showed that the 39 novel viruses grouped with 20 known cytorhabdoviruses in a distinctive major group that we named betacytorhabdoviruses (Figure 1). Within this distinct group of 59 viruses, several evolutionary clades could be distinguished (Figure 3A). One clade grouped together all viruses that have a short G gene and share a similar genomic organization with no additional accessory ORFs in their genomes except for Yerba mate virus A, which clustered basal to this clade (Figure 3). Another clade grouped together viruses with two accessory genes located between the P and M genes, exemplified by Sesamum betacytorhabdovirus 1 (Figure 3). Several other clades that grouped together viruses with a similar genomic organization were also observed (Figure 3). However, other clusters grouped together viruses with diverse genomic organizations (Figure 3). A similar topology was observed in the phylogenetic tree based on the N protein aa sequences (Figure S1).

3.4. Genus Gammacytorhabdovirus

The full-length coding regions of 16 novel putative gammacytorhabdoviruses were assembled in this study (Table 3), bringing the number of potential members of this proposed genus to 18, by the inclusion of two previously reported cytorhabdoviruses (Figure 4A). The newly identified viruses were tentatively associated with 15 plant host species and the fungus Hymenoscyphus fraxineus (Table 3). Most of the host plants (12/15) were herbaceous dicots, while two were orchids, and one was a dicot tree (Table 3).
The genomic organization of the 16 gammacytorhabdoviruses is quite similar with only a few exceptions. A common feature of all newly identified gammacytorhabdoviruses is the absence of the G gene. Twelve viruses had five genes in the order 3′-N-P–P3-M-L-5′; while two viruses had four genes in the order 3′-N-P–P3-L-5′ and lacked the M gene. Interestingly, the two viruses associated with Fraxinus display a genomic organization 3′-N-P–P3-M-P5-L-5′, with six genes including a small ORF located between the M and L genes (Table 3, Figure 4B). The predicted P5 protein showed no hits when BlastP searches were carried out, but one transmembrane domain was identified.
The consensus gene junction sequences of the novel gammacytorhabdoviruses identified in this study were highly similar and resembled those of the two previously reported phylogenetically related cytorhabdoviruses (Table 5).
Pairwise aa sequence identity values between each L protein of the 18 proposed gammacytorhabdoviruses varied significantly, ranging between 49% and 84% (Table S2). However, the highest sequence identity with those cytorhabdoviruses proposed to be classified as alphacytorhaboviruses and betacytorhabdoviruses was 33.4% (Table S2).
The phylogenetic analysis based on the L protein aa sequence showed that the 16 novel viruses grouped with two known cytorhabdoviruses in a distinct group (Figure 1). Within this distinct group of 18 viruses, most of the clusters grouped together viruses with the same genome organization and/or type of hosts, such as both Fraxinus-associated viruses, the cluster that grouped the carrot, celery, and Trachyspermum-associated viruses, the cluster composed of the Heliosperma and Silene-associated viruses, or the cluster composed of the Argyranthemum and Lonas-associated viruses (Figure 4). Interestingly, the orchid-associated viruses (Cypripedium, Epipactis and Gymnadenia) did not share a similar genomic organization and did not cluster together (Figure 4). A similar topology was observed in the phylogenetic tree based on the N protein aa sequences (Figure S1).

3.5. Tri-Segmented Rhabdoviruses

Unexpectedly, the full-length coding regions of four novel viruses that consisted of three genome segments were also assembled (Table 4). The best hits of the L, N, P2, P3, and P4 proteins encoded by those four tri-segmented viruses were the cognate proteins encoded by Picris cytorhabdovirus 1 (PiCRV1) [20]. Two genome segments of this virus had previously been assembled, annotated, and deposited in GenBank (Accession # OL472127 and OL472128), but the assembled PiCRV1 N protein gene was significantly shorter than the N gene assembled for the four novel tri-segmented viruses. Consequently, we re-analyzed the SRA deposited by [20] and we were able to extend the sequence of the N gene, but also to assemble a previously unrecognized third segment. Thus, five tri-segmented rhabdo-like viruses, subsequently referred to as trirhaviruses, were identified from the SRA data analysis. We propose to rename the Picris-associated virus as Picris trirhavirus 1.
RNA1 of all the tri-segmented viruses had one gene that encodes the L protein (Table 4, Figure 5A). RNA2 of four of these viruses had four genes in the order 3′-N-P2-P3-P4-5′ while one virus has five genes in its RNA2 in the order 3′-N-P2-P3-P4-P5-5′ (Table 4, Figure 5A). RNA3 of all tri-segmented viruses had 4 genes, where the first three encoded proteins, named as P6, P7 and P8, are homologous and syntenic to each other. The protein encoded at the 5′ end of segment 3 in the Chrysantheum and Medicago tri-segmented viruses is homologous to the P5 protein identified in the Alnus tri-segmented virus genome, while the proteins encoded in this position in the Erysimum, Picris and Alnus tri-segmented viruses are not homologous neither to P5 nor to each other, thus named as P9, P10 and P11, respectively (Table 4, Figure 5A).
One interesting feature discovered when the RNA segment ends of the Alnus, Erysmum and Picris tri-segmented viruses were analyzed, is that the 30 to 40 nucleotides located at the end of the 5′trailer of each one of the segments are 99% to 100% identical (Figure 5B). BlastP searches of each encoded protein showed that the L protein sequence of all tri-segmented rhabdoviruses was more similar to the L protein of cytorhabdoviruses than to the L protein of any other rhabdovirus. On the other hand, for every tri-segmented virus, the N protein best hits were the N proteins encoded by varicosaviruses or nucleorhabdoviruses. No similarity hits were found for P2, P3, P4, P6, P7, P8, P9, P10 or P11 in databases even with relaxed parameters. Strikingly, the P5 proteins showed hits against the putative silencing suppressor protein encoded by emaraviruses (family Fimoviridae) (Table 4). A signal P was predicted in each P2 and P5 proteins, while transmembrane domains were predicted in each P4 and P8 proteins. However, no conserved domains were predicted in any of the other viral proteins.
The consensus gene junction sequences of the tri-segmented rhabdoviruses are highly similar and like those previously reported for cytorhabdoviruses proposed to be classified as alphacytorhabdoviruses (Table 5).
Pairwise aa sequence identity values between each of the L proteins of the five tri-segmented viruses did not vary significantly, ranging between 55% and 66% (Table S2). On the other hand, the highest sequence identity with those cytorhabdoviruses proposed to be classified as alphacytorhaboviruses, betacytorhabdoviruses or gammacytorhabdoviruses was only 32% (Table S2). The highest sequence identity of trirhaviruses with the alpha-, beta- and gammanucleorhabdoviruses was 28.5%, and with varicosaviruses and gymnorhaviruses, the highest sequence identity was 28.6% and 27.5%, respectively.
The phylogenetic analysis based on deduced L protein aa sequences placed all tri-segmented rhabdoviruses into a distinct clade which is basal to all cytorhabdoviruses (Figure 1). The Alnus and Chrysantheum tri-segmented viruses grouped together (Figure 1), and these viruses are also the most similar in pairwise sequence identity values of their L proteins, but their RNA2 genomic organization is different (Figure 5A). The second cluster included the Erysmum, Medicago and Picris tri-segmented viruses (Figure 1), which have a similar genomic organization (Figure 5A). On the other hand, the phylogenetic tree based on deduced N protein aa sequences placed all tri-segmented rhabdoviruses into a distinct clade which is basal to all plant rhabdoviruses (Figure S1).

4. Discussion

4.1. Discovery of Novel Cytorhabdo-Like Viruses Expands Their Diversity and Evolutionary History

In the last few years, several novel cytorhabdoviruses that do not induce visible disease symptoms have been reported in HTS studies [91,92,93,94,95,96,97,98,99,100,101]. Moreover, many novel cytorhabdoviruses were identified when metatranscriptomic data publicly available at the Transcriptome Shotgun Assembly (TSA) sequence databases was mined [9]. On the other hand, the NCBI-SRA database, where many cytorhabdo-like virus sequences are likely hidden, remains significantly underexplored. This is because, traditionally, viruses were not expected to be present in sequence libraries of non-symptomatic plants. Nevertheless, the development of the Serratus tool [6] has greatly facilitated the exploration of the SRA database, which otherwise would be tedious and time-consuming, allowing us to carry out the most extensive search to date for cytorhabdovirus-like sequences. This substantial in silico directed search resulted in the identification and assembly of the full coding regions of 93 novel putative cytorhabdovirus members, representing a 1.7-fold increase in the known cytorhabdoviruses. The phylogenetic relationships, as well as the genomic features of the now expanded number of known cytorhabdoviruses, provide strong support for splitting the genus Cytorhabdovirus to establish three genera that we propose to name as Alphacytorhabdovirus, Betacytorhabdovirus and Gammacytorhabdovirus. However, the major highlight of our data mining efforts was the first-ever identification of rhabdoviruses with a tri-segmented genome. Thus, our findings clearly highlight the significance of data-driven virus discovery to increase our understanding of the genomic diversity, evolutionary trajectory, and singularity of the rhabdoviruses.

4.2. Proposed New Genus Alphacytorhabdovirus

The full-length coding regions of 38 novel alphacytorhabdoviruses were assembled in this study. Most of the associated host plants were herbaceous dicots (69% of the assigned hosts), in line with previous findings as 90% of the previously identified alphacytorhabdoviruses were also associated with herbaceous dicots. Thus, these viruses likely have a host adaptation trajectory leading to preferentially infecting herbaceous dicots during their evolution. The assigned hosts of six of the newly identified alphacytorhabdoviruses were monocots, and represent the first alphacytorhabdoviruses associated with monocots hosts. No apparent concordant evolutionary history with their plant hosts was observed for the monocot-infecting viruses, like what was previously reported for invertebrate and vertebrate rhabdoviruses [102]. Furthermore, one newly identified virus was associated with a wetland metagenome study, but even after extensive assessment of the corresponding libraries we were not able to clearly assign a host to this virus.
All but one alphacytorhabdovirus identified so far had at least the six basic plant rhabdovirus genes N, P, P3, M, G and L reported for cytorhabdoviruses [17]. The exception was one virus associated with the host Pogostemon, known as “patchouly chlorosis-associated cytorhabdovirus”, which was found to have a truncated G gene. It was speculated that this truncation may be linked to the fact that patchouli plants are primarily propagated vegetatively and may not require a functional G protein [94]. One distinctive feature of alphacytorhabdoviruses is the presence of an overlapping ORF within the P-encoding ORF, named P’ in most of their proposed members (65/71). At least one transmembrane domain was identified in each P′ protein predicted in the genomes of the alphacytorhabdoviruses assembled in this study. This is consistent with what has been previously reported for cytorhabdoviruses, where at least one transmembrane domain was identified in every P′ protein [9]. Hence, it could be speculated that this protein serves a membrane-associated function. Nevertheless, additional research should be directed toward the functional characterization of this intriguing protein. Moreover, 42/71 alphacytorhabdoviruses have an accessory ORF between the G and L genes. The encoded small protein contains transmembrane domains, and it was speculated that it may have membrane-associated functions similar to viroporins of vertebrate rhabdoviruses [9]. Other accessory ORFs were also detected in only two alphacytorhabdoviruses identified in this study. One of them, named P4, was located between the P3 and M genes in one virus and another one, dubbed P8, was found between the L gene and the 5′ trailer. No significant hits were found for P4 or P8 when BlastP searches were carried out, and no conserved domains were identified in these proteins. Another small accessory ORF, named P7, was previously reported to be located between the P6 and L genes of strawberry virus 1 [103]. Neither prediction of functional domains nor BLASTP searches against nonredundant GenBank database returned any significant hits [103]. Thus, further studies should be focused on the functional characterization of the P4, P7 and P8 proteins to gain knowledge about their potential roles.
The consensus gene junction sequences among the alphacytorhabdoviruses are highly similar, likely indicating a common evolutionary history for this group of viruses. The nt sequence identity between the genomes of alphacytorhabdoviruses varied significantly ranging from 36% to 86%. This suggests that there may be still an unknown amount of “virus dark matter” within some clusters of the alphacytorhabdoviruses space worth exploring, which may contain some yet-to-be-discovered alphacytorhabdoviruses. Moreover, the highest sequence identity with those viruses not classified as potential alphacytorhabdoviruses is very low, which is common among plant rhabdoviruses, which are characterized by a high level of diversity in both genome sequence and organization [15]. On the other hand, when we analyzed the diversity between variants of viruses which are likely members of the same species, the sequence identity ranged from 89% to 96%.
Among all plant rhabdoviruses studied so far, there is a strong correlation between phylogenetic relationships and vector types [17]. Many members grouped within the alphacytorhabdoviruses have been shown to be aphid-transmitted [17,99], except for patchouly chlorosis-associated cytorhabdovirus, which was speculated to be vertically transmitted [94]. We, therefore, predict that the novel alphacytorhabdoviruses described here are likely aphid-transmitted. In support of this, the Triticum-associated virus was also found in a sequencing library of the aphid Sitobium avenae, thus providing some evidence for aphids as potential vectors of the newly identified alphacytorhabdoviruses.
The observed phylogenetic relationships suggest a common evolutionary history for alphacytorhabdoviruses, with four major clades observed. All viruses in the clade including the well-studied lettuce necrotic yellows virus do not have an accessory ORF between the G and L genes; thus, these viruses may represent the ancestral clade within the alphacytorhabdoviruses. In another clade, all members but two had an accessory ORF between the G and L genes; therefore, these viruses may have evolved from an ancestor that already had that ORF, which is absent in the patchouly chlorosis-associated cytorhabdovirus and primula alphacytohabdovirus 2 genomes, while strawberry virus 1 acquired another accessory ORF during its evolution. In another clade, there are two major clusters, one that includes viruses that likely evolved from the ancestral ancestor, while the other cluster showed a more complex evolutionary history including members with distinct numbers of genes within their genomes. The fourth clade also showed a more complex evolutionary history because it included members with distinct genomic organizations, where many viruses acquired accessory ORFs during their evolution, mostly in the position between the G and L genes.
We propose to classify this group of evolutionary-related viruses into a novel genus within the family Rhabdoviridae, subfamily Betarhabdovirinae for which we suggest the name “Alphacytorhabdovirus”. Based on the phylogenetic insights and the observed genetic distance of the newly identified viruses we tentatively propose 86% aa sequence identity of the L protein as the threshold for species demarcation in this newly proposed genus which will include 72 members, for which the complete coding-sequence is available.

4.3. Proposed New Genus Betacytorhabdovirus

The full-length coding regions of 39 novel betacytorhabdoviruses were assembled in this study. Interestingly, half of the associated hosts were woody dicots, while 35% (7/20) of the previously identified cytorhabdoviruses of this group are also associated with woody dicots. Thus, many betacytorhabdoviruses likely infect woody dicots, which may be a distinctive feature of this group of viruses. Most of the monocot-infecting betacytorhabdoviruses grouped together suggesting a shared co-divergence for these viruses. Two newly identified betacytorhabdoviruses which clustered with monocot-infecting viruses were associated with a peat soil metagenome study. Therefore, it is tempting to speculate that monocots could be associated with these viruses.
The genomic organization of the betacytorhabdoviruses is quite diverse, with 16 distinct genomic organizations discernable among its 59 putative members. Almost a quarter (14/59) of betacytorhabdoviruses lacked additional accessory genes and had at least the six basic genes N, P, P3, M, G and L reported for cytorhabdoviruses [17]. Nevertheless, 12 betacytorhabdoviruses had a shorter G gene. Four viruses lacked the G gene altogether, while one also lacked the M gene. The G protein was found to be essential for virus acquisition by arthropod vectors [15], but it is not essential for replication and systemic movement [104]. Some isolates of the betacytorhabdovirus citrus-associated rhabdovirus were recently shown to have a defective G gene, thus it was speculated that the lack of a functional G gene could provide an evolutionary advantage in fruit trees that are propagated artificially by asexual modes, such as cutting and grafting [105]. Moreover, the recently identified Rudbeckia virus 1, which was identified in Rudbeckia seeds, lacked the G gene [96]. Thus, it was predicted to be vertically transmitted by seeds without the help of a vector which may have favored the loss of the G gene during its evolution [96]. Hence, one might be inclined to speculate that viruses lacking the G gene or having a shorter G gene could potentially undergo vertical transmission. Furthermore, infections with viruses that lack the M gene have been reported to be asymptomatic [106], which is additional evidence supporting vertical transmission of the virus. Moreover, it has been shown, using a nucleorhabdovirus as a model, that cooperative M-G interactions are needed for some of the functions that involve the M protein [107]. Thus, perhaps in those viruses that lack the G gene, the M gene could become dispensable and may be prone to be lost during evolution like in the Cypripedium-associated virus, which lacks both genes. Further studies should experimentally assess these conjectures. Moreover, many betacytorhabdoviruses (27/59) have an accessory ORF between the G and L genes. This small protein has transmembrane domains, and it was speculated that it may have membrane-associated functions similar to viroporins in vertebrate rhabdoviruses [9]. Several other accessory ORFs were also identified in betacytorhabdoviruses reported in this study suggesting a complex evolutionary history where many members acquired additional ORFs during adaptation to their hosts. Four betacytorhabdoviruses had an accessory ORF between the L gene and the 5′trailer. Ten betacytorhabdoviruses had two ORFs between the P and M genes, while three others had four ORFs between these genes [17]. One of these ORFs encodes the putative cell-to-cell movement protein, which is named P3 in all but the Yerba mate virus A, where this protein is named P4 [91]. The other accessory proteins are named P4, P5 and P6. The P4 protein encoded by the newly identified Sesamum virus 1, and the one encoded by the known Bemisia tabaci associated virus, Cucurbit cytorhabdovirus 1, Yerba mate chlorosis associated virus, soybean blotchy mosaic virus, papaya virus E and Aristolochia-associated cytorhabdovirus; as well as the P5 protein encoded by barley yellow striate mosaic virus (BYSMV) and maize yellow striate virus, are small proteins (70–80aa) with a predicted transmembrane domain, suggesting a membrane association function. Indeed, the BYSMV P5 was shown to be targeted to the endoplasmic reticulum and it was suggested that the features of this protein are reminiscent of the small hydrophobic proteins of tupaia rhabdovirus [108]. Two viruses had two additional accessory ORFs between the viroporin-like protein gene and the L gene, while another virus had one accessory ORF in that position. An overlapping ORF within the one encoding the viroporin-like protein was found in a cytorhabdovirus associated with the Linden tree Tillia cordata [95]. In the Justicia-associated virus, an accessory ORF was found between the N and P genes. An accessory ORF located in this position has been described for some alphanucleorhabdoviruses [17], but Justicia-associated virus appears to be the first cytorhabdovirus with an ORF in this position. For the above accessory ORFs, except for the viroporin-like proteins and the P4/P5 proteins, BlastP results were orphans, no known signals, or domains present, and no clues towards their putative (conserved?) function were found. Thus, further studies should be focused on the functional characterization of these proteins to gain essential knowledge regarding the proteome of the accessory ORFs of betacytorhabdoviruses.
The consensus gene junction sequences of the novel and previously reported betacytorhabdoviruses showed some variability, but there appears to be a correlation with the phylogenetic relationships thus supporting a common evolutionary history for these viruses.
When we analyzed the diversity between variants of viruses that likely belong to the same species, nt sequence identity ranged from 93.5% to 99%. On the other hand, the pairwise aa sequence identity among betacytorhabdoviruses L protein showed a great variation ranging between 27% and 80% which suggests that there may be many more betacytorhabdoviruses yet to be discovered. Moreover, the sequence identity with those viruses not classified as potential betacytorhabdoviruses is very low (<33.9%), which is a common feature among plant rhabdoviruses, that are characterized by a high level of diversity in both genome sequence and organization [15]. Furthermore, this high sequence diversity coupled with the distinct genomic architecture displayed by betacytorhabdoviruses, and the complex evolutionary history as shown in the phylogenetic analyses may set the foundation to further split this proposed genus in the future once additional members can be identified.
Among all plant rhabdoviruses studied so far, there is a strong correlation between phylogenetic relationships and vector types [17]. Some betacytorhabdoviruses have been shown to be transmitted by planthoppers, others by leafhoppers and others by whiteflies [17]. We, therefore, predict that the potential vectors of the novel betacytorhabdoviruses may be whiteflies, planthoppers, leafhoppers and likely non-aphid arthropods, like psyllids. Those betacytorhabdoviruses that lack the G gene or with a shorter G gene are likely vertically transmitted.
The phylogenetic analysis of betacytorhabdoviruses revealed several major clades suggesting a complex evolutionary history. One clade grouped together all viruses with a short G gene and without accessory genes, except for Yerba mate virus A. These viruses, with one exception, are associated with woody dicots; therefore, it is tempting to speculate that the ancestor virus was adapted to infect woody plants and that Yerba mate virus A acquired an additional ORF during its host adaptation. Another clade includes most of the viruses with two genes between the P and M genes but no accessory ORFs in other positions in their genomes likely indicating that they share the same ancestor. Certain clusters encompassed viruses that share common genomic organization, while other clusters featured viruses with unique genomic structures. An example of this diversity can be seen in the cluster that includes Cypripedium- and Mango-infecting viruses. This highlights the intricate evolutionary history of most betacytorhabdoviruses, with many of them acquiring additional genes during their evolution. Notably, these gene acquisitions were primarily concentrated between the P and M genes or between the G and L genes.
Based on the phylogenetic insights and the observed genetic distances of the newly identified viruses we tentatively propose an aa sequence identity of 82% in the L protein as the threshold for species demarcation in this newly proposed genus which will include 59 members, for which the complete coding-sequence is available.

4.4. Proposed New Genus Gammacytorhabdovirus

The full-length coding regions of 16 novel gammacytorhabdoviruses were assembled in this study, and most of the associated host plants were herbaceous dicots. Three viruses were linked to orchids, while two were associated with the woody tree Fraxinus but, interestingly, one of them was identified in a library of the fungal pathogen (Hymenoscyphus fraxineus) sampled from this woody tree.
The common feature of all 18 gammacytorhabdoviruses identified so far (16 in this study and two in [9]) is the lack of a G gene in their genome. The G gene was shown, using as a model an infectious clone of the Sonchus yellow net virus, to be not essential for replication and systemic movement [104]. Those two viruses associated with Fraxinus, have an additional ORF between the M and L genes, which we named P5. Interestingly, transmembrane domains were predicted for P5 suggesting a membrane-associated function for this protein that has a similar size to cytorhabdovirus viroporin-like proteins, which also have transmembrane domains [9]. Nevertheless, no distant hits with viroporin-like proteins were found when we used HHblits on the predicted P5 protein. One previously identified gammacytorhabdovirus, associated with the orchid Gymandenia, lacks not only the G gene but also the P3 gene. Thus, how GymDenV1 moves from cell to cell remains to be unraveled, but no cell-to-cell movement protein has either been identified in the fungi-transmitted varicosaviruses [9]. Strikingly, two gammacytorhabdoviruses identified in this study, one associated with the orchid Epipactis and the other with the parasitic plant Rhopalocnemis, do not have M and G genes. Previous studies have assumed that the nucleocapsid core (NC) proteins N, P and L are essential for virus replication and transcription and that the M protein is required for condensation of the core during virion assembly [15]. M protein appears to be required for the long-distance movement of the virus within the plant [104], and an infectious clone of a plant rhabdovirus lacking the M gene displayed reduced infectivity, a vasculature-confined tissue tropism and no visible symptoms [106]. Moreover, it has been shown, using a nucleorhabdovirus as a model, that cooperative M-G interactions are needed for some of the functions that involve the M protein [107]. Thus, it is tempting to speculate that in those viruses that lack the G gene, the M gene could be dispensable, and may have been lost during the evolution of the Epipactis- and Rhopalocnemis-associated viruses.
It has been suggested that the fungi-transmitted varicosaviruses, which do not encode a G protein [10], may have originated through trans-kingdom horizontal gene transfer events between fungi and plants, adapting specifically to a plant-based lifestyle [5]. The absence of the G gene, coupled with the detection of one of the recently identified gammacytorhabdoviruses in a fungal library, raises the possibility that these viruses might be transmitted by a fungal vector rather than by arthropods, as is commonly observed in viruses classified as alpha- and betacytorhabdoviruses. This serves as another distinguishing characteristic of the gammacytorhabdoviruses. Thus, further studies should focus on the potential vector and the mode of transmission of gammacytorhabdoviruses.
Another distinctive feature of gammacytorhabdoviruses is that the intergenic spacer of their gene junctions starts with an A instead of a typical G, like all other plant rhabdoviruses [9,10] suggesting a unique evolutionary history of these viruses.
The nt sequence identity among gammacytorhabdoviruses showed a high variation ranging between 49% and 84%. Moreover, the pairwise aa sequence identity with the L protein of those viruses not classified as potential gammacytorhabdoviruses is very low (<33.5%), suggesting unknown gammacytorhabdovirus diversity is yet to be discovered.
Interestingly, the three orchid-associated viruses (Cypripedium, Gymnadenia, and Epipactis) have different genomic organization, where one virus lacks the G gene, another does not encode the G and M proteins, while the third does not have the P3 and G genes. Moreover, they are not grouped together in the phylogenetic tree, thus they likely did not share a common evolutionary history. On the other hand, most of the viruses infecting herbaceous dicot hosts, as well as those associated with woody trees, clustered together according to the host family, suggesting a shared host-virus co-divergence in those clades.
We propose to classify this group of evolutionary-related viruses sharing the lack of the G gene in their genomes as a distinctive feature, into a novel genus within the family Rhabdoviridae, subfamily Betarhabdovirinae for which we suggest the name “Gammacytorhabdovirus”. Based on the phylogenetic insights and the observed genetic distance of the newly identified viruses we tentatively propose an aa sequence identity of 85% in the L protein as the threshold for species demarcation in this newly proposed genus which will include 18 members, for which the complete coding-sequences are available.

4.5. Tri-Segmented Rhabdoviruses

All rhabdoviruses identified to date have unsegmented genomes, except for the dichorhaviruses and most varicosaviruses which have bi-segmented genomes [10,15]. Unexpectedly, five novel viruses with tri-segmented genomes were identified in this study, including the corrected full-length coding genome segments of the previously reported PiCRV1 [20].
RNA1 of all these tri-segmented viruses had only one gene that encodes the L protein, which is similar to the bi-segmented rhabdoviruses where the L protein is the only gene product present in the varicosaviruses RNA1, and in the dichorhaviruses RNA2 [10,17]. RNA2 of four of the viruses has four genes, while the Alnus tri-segmented virus has five genes. Five genes are present in RNA1 of dichorhaviruses [17], while three to five genes are present in RNA2 of varicosaviruses [10], with the N gene being the only orthologous gene between them. RNA3 of all tri-segmented viruses has four genes, where the first three encoded proteins are homologous. The protein encoded at the end of this segment in the Chrysanthemum and Medicago tri-segmented viruses is homologous to P5 on RNA2 of the Alnus tri-segmented virus genome, while the proteins located in this position in the Erysimum, Picris and Alnus tri-segmented viruses are unique. This genomic organization is unique among rhabdoviruses [15,16] and represents the first known tri-segmented rhabdovirus genomes. Other segmented negative-sense RNA viruses (NSR), belonging to the order Bunyavirales, have one or two genes on each RNA segment. Thus, the genomic organization of the tri-segmented rhabdoviruses identified in this study is likely distinctive among NSR viruses.
The ends of the 5′ trailer region of all genome segments are conserved in the tri-segmented viruses identified in our study. A similar feature is observed in the other segmented rhabdoviruses and NSR viruses, which may be linked to RNA-dependent RNA polymerase-mediated recognition for replication [15].
BlastP searches of the L protein encoded on RNA1 of all identified tri-segmented viruses showed that this protein is most closely related to the L protein encoded by cytorhabdoviruses, while the best hits for the N protein were the N proteins coded by varicosaviruses or nucleorhabdoviruses. This suggests that these two proteins, which are located on different RNA segments, have distinct evolutionary histories. On the other hand, no hits were found for P2, P3, P4, P6, P7, P8, P9, P10 or P11. Strikingly, P5 showed hits against the putative RNA silencing suppressor protein encoded by emaraviruses (family Fimoviridae), plant viruses with segmented, linear, single-stranded, negative-sense genomes [109] in the order Bunyavirales [110], while rhabdoviruses are classified in the order Mononegavirales [16]. Viral RNA silencing suppressors are required for systemic infection of the plant host and the presence of these proteins suggests that the tri-segmented viruses detected here are plant-associated [5].
A signal peptide was predicted in each P5 protein, which may be associated with its RNA silencing suppressor function, and in each P2 protein. Interestingly, a signal peptide is present in the movement protein (MP) encoded by emaraviruses [110], but none were identified in the MP encoded by plant rhabdoviruses [9], and no distant hits with any MP were found when using HHblits on the P2. Transmembrane domains were predicted in each P4 and P8 proteins suggesting a membrane-associated function for these proteins. P4 size is similar to that reported for cytorhabdovirus viroporin-like proteins, which also have transmembrane domains [9] while P8 size is similar to that reported for the glycoprotein encoded by plant rhabdoviruses [9], but no distant hit with any viroporin-like protein or glycoprotein was found using HHblits. No conserved domains were found in the other coded proteins. Thus, further studies should be focused on the functional characterization of the P2, P3, P4, P6, P7, P8, P9, P10 and P11 proteins to gain fundamental insights about the proteome of the tri-segmented viruses beyond the N, L and P5 proteins.
The novel tri-segmented viruses also resemble rhabdoviruses in possessing similar conserved gene junctions that are also highly similar to those present in the alphacytorhabdoviruses.
The pairwise aa sequence identities between the L proteins of all the tri-segmented viruses were not low at all, ranging between 55% and 66%, which may suggest that tri-segmented rhabdoviruses are evolutionarily younger than unsegmented ones.
The phylogenetic analysis based on deduced L protein aa sequences placed all tri-segmented viruses into a distinct clade within the plant rhabdoviruses that is grouped with the cytorhabdoviruses rather than with varicosaviruses or nucleorhabdoviruses, whereas the phylogenetic tree based on the N protein placed the tri-segmented viruses in a clade which is basal to all plant rhabdoviruses. The complex evolutionary history of this divergent group of viruses suggests that they share a unique evolutionary history among rhabdoviruses. It is tempting to speculate that the RNA segment encoding the L protein evolved from a cytorhabdovirus ancestor, while the RNA segment encoding the N protein may have evolved from a rhabdovirus ancestor of all tri-segmented viruses, except for the Alnus-associated virus. The presence of an emaravirus-related protein in its RNA2 segment, as well as in the RNA3 segment of the Chrysanthemum- and Medicago-associated tri-segmented viruses leads us to speculate that these segments may have emerged from the recombination of a negative-sense rhabdovirus ancestor and an emaravirus. On the other hand, the RNA3 segment of the viruses from Alnus, Erysimum and Picris may have evolved from a segmented negative-sense rhabdovirus ancestor.
Taken together, these tri-segmented viruses may be taxonomically classified in a novel genus within the family Rhabdoviridae, subfamily Betarhabdovirinae for which we suggest the name “Trirhavirus”. Based on the phylogenetic insights and the observed genetic distance of the newly identified viruses we tentatively propose an aa sequence identity of 80% in the L protein as threshold for species demarcation in this proposed genus.

4.6. Strengths and Limitations of Sequence Discovery through Data Mining

As demonstrated previously by Bejerman and colleagues [10] and in this study with the Picris-associated virus, the independent validation through re-analyzes of the NCBI-SRA raw data of viruses assembled with unexpected genomes is important to enhance our comprehension and confidence in the genomic architecture of RNA viruses assembled via HTS data. However, the inability to revisit the original biological material for replication of results and verification of the assembled viral genome sequences is a significant weakness of the data mining approach in virus discovery. Moreover, potential issues such as contamination, low sequencing quality, spill-over, and other technical artifacts pose a risk of yielding false-positive detections, chimeric assemblies, or difficulties in accurately assigning host organisms. Therefore, researchers should be cautious when scrutinizing publicly available SRA data for virus detection. To bolster and complement such results, the acquisition of new RNAseq datasets from the predicted plant hosts is strongly recommended. Furthermore, the absence of a directed strategy for verifying genomic segment termini, such as the use of Rapid Amplification of cDNA Ends (RACE), presents challenges in determining bona fide RNA virus ends, especially considering the conserved functional and structural cues observed in rhabdoviruses [15]. Despite these limitations, certain aspects of our virus discovery strategy can help mitigate some of these challenges and provide additional evidence for identification. For example, when the same putative virus is consistently identified in multiple independent libraries originating from the same plant host, when there is substantial coverage of virus-related reads when multiple RNA segments of the virus are detected within a single library, or when different viral strains are identified in plants that are closely related in terms of their evolutionary history. Nonetheless, it is essential to acknowledge that associations and detections provided in this work and other data-driven studies should be viewed as preliminary and should be complemented through subsequent studies.

5. Conclusions

In conclusion, this study underlines the significance of analyzing SRA public data as a valuable tool, not only for expediting the discovery of novel viruses but also for gaining insights into their evolutionary history and enhancing virus classification. Through this approach, we conducted a search for hidden cytorhabdovirus-like sequences, which significantly expanded the number of putative cytorhabdoviruses. It also allowed us to unequivocally split this group of viruses into three genera resulting in the most comprehensive cytorhabdoviruses phylogeny to date, highlighting their diversity and complex evolutionary dynamics. The major finding of our study was the first-ever identification of tri-segmented rhabdoviruses, which shows the extensive plasticity inherent to the rhabdovirus genome organization including members with unique and intriguing evolutionary trajectories. Thus, future studies should explore various unresolved aspects of these viruses, such as potential symptoms, vertical transmission, and possible vectors.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v15122402/s1, Figure S1: Maximum-likelihood phylogenetic tree based on amino acid sequence alignments of the complete N protein 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. Table S1: Virus names and abbreviations of cytorhabdovirus sequences used in this study. Table S2: Amino acid identity of the complete L ORF.

Author Contributions

Conceptualization, N.B. and H.D.; data analysis, N.B. and H.D.; writing—original draft preparation, N.B.; writing—review and editing, N.B., R.D. and H.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Nucleotide sequence data reported are available in the Third Party Annotation Section of the DDBJ/ENA/GenBank databases under the accession numbers TPA: BK064247-BK064360.

Acknowledgments

We would like to express genuine appreciation to the producers of the original data used for this work, which are cited in Table 1, Table 2, Table 3 and Table 4. By ensuing open science practices with accessible raw sequence data in open public repositories, they supported contributions based on secondary data analyses.

Conflicts of Interest

The authors declare no conflict of interest.

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Viruses 15 02402 g001
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 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.
Viruses 15 02402 g001
Viruses 15 02402 g002
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 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.
Viruses 15 02402 g002
Viruses 15 02402 g003
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 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.
Viruses 15 02402 g003
Viruses 15 02402 g004
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 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.
Viruses 15 02402 g004
Viruses 15 02402 g005
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.
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.
Viruses 15 02402 g005
Table 1. Summary of novel alphacytorhabdoviruses identified from plant RNA-seq data available on NCBI.
Table 1. Summary of novel alphacytorhabdoviruses identified from plant RNA-seq data available on NCBI.
Plant HostTaxa/
Family		Virus Name/
Abbreviation		Bioproject ID/
Data Citation		Length (nt)/CoverageAccession NumberProtein IDLength (aa)Highest Scoring Virus-Protein/E-Value/Query Coverage %/Identity % (Blast P)
Greater burdock
(Arctium lappa)		Dicot/AsteraceaeArctium alphacytorhabdovirus 1/
ArcACRV1		PRJNA598011/
[21]		12768/310.2XBK064262N450WhIV5-N/0.0/91/63.07
P300SaV1-P/1e-100/100/50.67
P′115no hits
P3225TrARV1-P3/6e-93/97/59.91
M182CnV2-M/2e-54/98/47.49
G552TrARV1-G/0.0/98/55.09
L2097WhIV5-L/0.0/99/64.42
Silvery wormwood
(Artemisia argyi)		Dicot/AsteraceaeArtemisia alphacytorhabdovirus 1/
ArtACRV1		PRJNA397671/
[22]		12978//55.94XBK064263N454LNYV-N/0.0/88/60.30
P315LNYV-P/9e-91/93/50.68
P3307LNYV-P3/2e-119/96/54.52
M184LNYV-M/2e-48/96/45.76
G552LNYV-G/0.0/99/51.18
L2074LNYV-L/0.0/99/68.25
Common wormwood
(Artemisia montana)		Dicot/AsteraceaeArtemisia alphacytorhabdovirus 2/
ArtACRV2		PRJDB8414/
Kyoto University, Japan, unpublished		14344/82.73XBK064264N479RVCV-N/1e-175/98/51.95
P343RCVC-P/1e-84/92/44.55
P′88RVCV-P′/2e-10/64/49.12
P3241RVCV-P3/2e-74/78/56.08
M182RVCV-M/1e-49/83/50.66
G569RVCV-G/0.0/92/55.51
P664RVCV-P6/3e-11/90/44.83
L2086RVCV-L/0.0/99/64.72
Sievers wormwood
(Artemisia sieversiana)		Dicot/AsteraceaeArtemisia alphacytorhabdovirus 3/
ArtACRV3		PRJNA834888/
[23]		14339/57.85XBK064265N476RVCV-N/2e-171/98/50.62
P342RCVC-P/6e-82/93/43.69
P′103RVCV-P′/3e-13/66/54.93
P3240RVCV-P3/3e-75/82/52.40
M185RVCV-M/1e-52/82/51.97
G571RVCV-G/0.0/91/56.76
P664RVCV-P6/7e-14/100/48.44
L2089RVCV-L/0.0/99/63.43
Desert broom
(Baccharis sarothroides)		Dicot/AsteraceaeBaccharis alphacytorhabdovirus 1/
BacACRV1		PRJNA716650/
Romero, M., UNAM, Mexico, unpublished		13581/25.63XBK064266N470CCyV1-N/1e-153/87/52.68
P297CCyV1-P/2e-56/98/40.74
P′85no hits
P3337StrV2-P3/4e-120/93/56.78
M168CCyV1-M/5e-34/95/38.65
G550CCyV1-G/1e-160/92/43.14
L2074CCyV1-L/0.0/99/59.24
Large bittercress (Cardamine amara)Dicot/BrassicaceaeCardamine alphacytorhabdovirus 1/
CarACRV1		PRJDB4989/
[24]		13209/83.24XBK064267N457PaCRV1-N/0.0/99/72.35
P316PaCRV1-P/5e-134/63.26
P′109PaCRV1-P′/1e-21/48.11
P3224PaCRV1-P3/8e-122/98/75.57
M164PaCRV1-M/1e-89/100/73.78
G569PaCRV1-G/0.0/95/75.64
P670PaCRV1-P6/5e-26/97/63.24
L2092PaCRV1-L/0.0/100/78.30
Greater celandine
(Chelidonium majus)		Dicot/PapaveraceaeChelidonium alphacytorhabdovirus 1/
CheACRV1		PRJNA376854/
Zhao, L., Jinlin, China, unpublished		12148/68.31XBK064268N415TpVA-N/0.0/100/70.19
P325TpVA-P/2e-146/100/63.38
P′71no hits
P3200TpVA-P3/8e-117/98/80.71
M169TpVA-M/2e-73/92/69.43
G552TpVA-G/0.0/98/71.72
P666GlLV1-P6/2e-14/100/57.58
L2072TpVA-L/0.0/100/80.41
Indian chrysanthemum
(Chrysanthemum indicum)		Dicot/AsteraceaeChrysanthemum alphacytorhabdovirus 1/
ChrACRV1		PRJNA361213/
[25]		12715/71.27XBK064269N448WhIV5-N/0.0/99/60.22
P301SaV1-P/4e-100/99/51
P′140no hits
P3225TrARV1-P3/1e-91/97/57.92
M19CnV2-M/6e-50/91/45.60
G549SaV1-G/0.0/100/53.42
L2097WhIV5-L/0.0/99/63.89
Bear corn
(Conopholis americana)		Dicot/OrobanchaceaeConopholis alphacytorhabdovirus 1/
ConACRV1		PRJEB21674/
1000 Plant (1KP) Transcriptomes Initiative, Unpublished		13083/178.6XBK064270N467CCyV1-N/1e-157/94/49.1
P299CCyV1-P/4e-70/100/42.35
P′87no hits
P3328StrV2-P3/3e-119/83/60.58
M166BCRV2-M/7e-32/93/38.06
G546CCyV1-G/8e-151/99/40.26
L2076CCyV1-L/0.0/98/58.75
Cardoon
(Cynara cardunculus)		Dicot/AsteraceaeCynara alphacytorhabdovirus 1/
CynACRV1		PRJNA590905/
[26]		13726/33.22XBK064271N472TCRV1/0.0/100/80.08
P311TCRV1-P/3e-153/100/69.97
P′132TCRV1-P′/1e-23/75/53.54
P3350TCRV1-P3/0.0/99/80
M179TCRV1-M/3e-100/100/78.77
G562TCRV1-G/0.0/98/70.40
L2144TCRV1-L/0.0/98/83.25
Fischer´s spurge
(Euphorbia fischeriana)		Dicot/EuphorbiaceaeEuphorbia alphacytorhabdovirus 1/
EupACRV1		PRJNA693977/
[27]		13713/109.3XBK064272N451PeVA-N/0.0/95/68.41
P330PeVA-P/2e-100/99/53.62
P3223PeVA-P3/1e-84/100/55.61
P4130no hits
M184PeVA-M/5e-57/96/48.88
G559PeVA-G/0.0/95/68.35
P741no hits
L2089PeVA-L/0.0/99/69.3
Tikoua fig
(Ficus tikoua)		Dicot/MoraceaeFicus alphacytorhabdovirus 1/
FicACRV1		PRJNA432314/
Bai, Y., Guiyang University, China,
unpublished		13839/18.65XBK064274N458SCV-N/1e-140/97/48.80
P316SCV-P/3e-52/88/36.51
P′84no hits
P3228SCV-P3/2e-75/100/50.43
M182SCV-M/7e-43/82/48.67
G563SCV-G/0.0/94/50.46
P669ADV-P6/3e-09/98/42.03
L2097SCV-L/0.0/99/60.22
Garlic
(Allium sativum)		Monocot/AmaryllidaceaeGarlic alphacytorhabdovirus 1/
GarACRV1		PRJNA772184/
Liu, T., IBFC, China, unpublished		13400/76.43XBK064275N468LNYV-N/3e-160/93/53.17
P298TpVB-P/4e-53/94/34.80
P3329StrV2-P3/5e-126/98/58.28
M174LNYV-M/3e-35/97/40.94
G554TpVB-G/5e-175/98/44.97
L2075LYMV-L/0.0/99/58.43
Herb bennet
(Geum urbanum)		Dicot/RosaceaeGeum alphacytorhabdovirus 1/
GeuACRV1		PRJEB23354/
[28]		12756/12.97XBK064276N459StrV2-N/0.0/98/73.57
P295StrV2-P/7e-128/100/60.34
P′98StrV2-P′/3e-22/98/55.67
P3319StrV2-P3/1e-171/100/74.22
M177BCRV2-M/7e-78/92/68.29
G546BCRV2-G/0.0/96/69.57
L2093BCRV2-L/0.0/99/72.89
English ivy
(Hedera helix)		Dicot/AraliaceaeHedera alphacytorhabdovirus 1/
HedACRV1		PRJEB21674/
1000 Plant (1KP) Transcriptomes Initiative, Unpublished		12588/11.88XBK064277N459StrV2-N/0.0/97/71.27
P295StrV2-P/3e-128/100/61.02
P′98StrV2-P′/2e-22/98/53.61
P3319StrV2-P3/2e-171/100/74.30
M172BCRV2-M/2e-78/99/65.50
G546BCRV2-G/0.0/99/68.19
L2100BCRV2-L/0.0/98/72.95
Plum-leaved holly
(Ilex asprella)		Dicot/AquifoliaceaeIlex alphacytorhabdovirus 1/
IleACRV1		PRJNA736810/
[29]		14540/166.3XBK064278N452AcCV-N/0.0/99/56.87
P333AcCV-P/1e-67/96/39.76
P′93no hits
P3370AcCV-P3/1e-130/99/52.49
M184AcCV-1e-33/99/37.30
G553AcCV-G/0.0/96/49.06
P654no hits
L2137AcCV-L/0.0/98/60.77
P8134no hits
Lucerne
(Medicago sativa)		Dicot/FabaceaeMedicago alphacytorhabdovirus 1/
MedACRV1		PRJNA644634/
[30]		13586/9.75XBK064279N431StrV1-N/0.0/97/60.48
P360StrV1-1e-73/98/39.12
P′64StrV1-P′/2e-13/100/54.69
P3224StrV1-P3/7e-81/99/52.47
M191StrV1-M/5e-43/83/44.65
G547StrV1-G/0.0/99/60.44
P680StrV1-P6/6e-11/76/45.9
L2085StrV1-L/0.0/99/68.21
Horse mint
(Mentha longifolia)		Dicot/LamiaceaeMentha alphacytorhabdovirus 1/
MenACRV1		PRJNA779119/
Wang, B., Shaoguan University, unpublished		12387/56.72XBK064280N422TpVA-N/9e-163/100/54.14
P316TpVA-/7e-45/96/32.18
P′101no hits
P3195TpVA-P3/6e-68/97/55.38
M163GlLV1-M/6e38/95/45.16
G551TpVA-G0.0/97/55.84
P667GlLV1-P6/4e-07/100/50.75
L2072TpVA-L/0.0/99/62.64
Indian mulberry
(Morinda officinalis)		Dicot/RubiaceaeMorinda alphacytorhabdovirus 1_Mor/MorACRV1_MorPRJNA717096/
[31]		13023/25.34XBK064281N463CCyV1-N/0.0/99/56.87
P300CCyV1-P/7e-68/98/42.71
P′81no hits
P3346BCRV2-P3/1e-115/76/59.7
M172CCyV1-M/7e-29/92/37.58
G548CCyV1-G/6e-171/93/45.14
L2075CCyV1-L/0.0/99/59.16
Chinese holly
(Ilex cornuta)		Dicot/AquifoliaceaeMorinda alphacytorhabdovirus 1_Ile/MorACRV1_IlePRJNA399054/
[32]		12876/20.54XBK064282N463CCyV1-N/0.0/99/56.29
P300CCyV1-P/1e-66/98/43.39
P′85no hits
P3346BCRV2-P3/2e-116/76/60.08
M184CCyV1-M/2e-27/88/36.31
G549CCyV1-G/7e-167/98/43.57
L2075CCyV1-L/0.0/99/59.16
Oak
(Quercus robur)		Dicot/FagaceaeOak alphacytorhabdovirus 1/
OakACRV1		PRJNA322128/
[33]		12817/21.81XBK064283N453LYMV-N/2e-109/93/40.47
P304LYMV-P/1e-20/88/29.58
P′104no hits
P3376AscSyV1-P3/2e-46/66/35.97
M176CCyV1-M/3e-06/59/28.57
G543TrARV1-G/3e-7/98/30.16
L2078BCRV2-L/0.0/99/45.57
Holy basil
(Ocimum tenuiflorum)		Dicot/LamiaceaeOcimum alphacytorhabdovirus 1/
OciACRV1		PRJNA251328/
[34]		12478/24.87XBK064284N425KePCyV-N/0.0/99/76.65
P327KePCyV-P/1e-116/99/54.41
P′93no hits
P3205KePCyV-P3/1e-105/99/70.53
M155KePCyV-M/5e-58/98/56.21
G563KePCyV-G/0.0/96/70.46
P660StrV1-P6/2e-08/83/50
L2078KePCyV-L/0.0/99/77.36
Scented pelargonium
(Pelargonium X hybrid)		Dicot/GeraniaceaePelargonium alphacytorhabdovirus 1/
PelACRV1		PRJNA883637/
Saint-Marcoux, D., Lyon University, France, unpublished		12332/91.55XBK064285N413TpVA-N/1e-164/98/56.23
P328TpVA-P/2e-53/97/36.25
P′84no hits
P3203TpVA-P3/2e-91/96/68.02
M170TpVA.M/5e-37/90/44.81
G552TpVA-G/0.0/95/60.71
P658no hits
L2073TpVA-L/0.0/99/63.7
Moso bamboo
(Phyllostachys edulis)		Monocot/PoaceaePhyllostachys alphacytorhabdovirus 1/
PhyACRV1		PRJNA350353/
[35]		12947/94.47XBK064286N455LNYV-N/2e-138/97/46.55
P296LNYV-P/2e-44/93/37.28
P′83no hits
P3328StrV2-P3/7e-140/96/59.81
M193LNYV-M/6e-28/90/34.27
G544StrV2-G/8e-144/90/40.93
L2070BCRV2-L/0.0/99/57.21
Peltate green dragon
(Pinellia peltata)		Monocot/AraceaePinellia alphacytorhabdovirus 1/
PinACRV1		PRJNA623739/
[36]		13438/126.5XBK064287N479AscSyV1-N/4e-131/90/44.24
P305DV1-P/8e-47/98/36.75
P′87no hits
P3373AscSyV1-P3/9e-105/89/47.51
M170TCRV1-M/1e-05/88/31.21
G568WhIV4/2e-134/95/36.73
P661no hits
L2106WhIV4/0.0/99/48.13
Patchouli
(Pogostemom cablin)		Dicot/LamiaceaePogostemom alphacytorhabdovirus 1_Pog/
PogACRV1_Pog		PRJNA660501/
[37]		13171/250.8XBK064288N462CCyV1-N/0.0/99/55.17
P300CCyV1-P/1e-69/96/40.79
P′81no hits
P3347StrV2-P3/3e-116/77/61.94
M179CCyV1-M/3e-38/92/36.14
G557CCyV1-G/1e-160/93/40.92
L2070CCyV1-L/0.0/58.03
Black pepper
(Piper nigrum)		Dicot/PiperaceaePogostemom alphacytorhabdovirus 1_ Pip/
PogACRV1_Pip		PRJNA580359/
[38]		13063/22.35XBK064289N462CCyV1-N/0.0/99/54.96
P300CCyV1-P/3e-67/99/40.79
P′81no hits
P3347StrV2-P3/3e-116/77/62.31
M179CCyV1-M/5e-39/87/39.74
G557CCyV1-G/7e-164/91/42.88
L2070CCyV1-L/0.0/58.75
Tropical soda apple
(Solanum viarum)		Dicot/SolanaceaePogostemom alphacytorhabdovirus 1_Sol/
PogACRV1_Sol		PRJNA666394/
[39]		13138/26.34XBK064290N462CCyV1-N/3e-179/99/54.84
P300CCyV1-P/5e-63/99/39.79
P′116no hits
P3351StrV2-P3/4e-115/76/61.57
M179CCyV1-M/4e-40/87/40.38
G558CCyV1-G/2e-157/95/40.98
L2070CCyV1-L/0.0/58.31
Patchouli
(Pogostemom cablin)		Dicot/LamiaceaePogostemom alphacytorhabdovirus 2/
PogACRV2		PRJNA660501/
[37]		13209/218.9XBK064291N421StrV1-N/0.0/98/78.71
P359StrV1-P/8e-164/100/66.12
P′64StrV1-P′/9e-25/100/73.44
P3224StrV1-P3/4e-134/100/81.7
M179StrV1-M/2e-82/99/65.36
G549StrV1-G/0.0/100/74.05
P669StrV1-P6/2e-31/100/72.46
L2083StrV1-L/0.0/99/82.4
Patchouli
(Pogostemom cablin)		Dicot/LamiaceaePogostemom alphacytorhabdovirus 3_Pog/
PogACRV3_Pog		PRJNA511937/
[40]		13252/202.3XBK064292N449BmV1/0.0/99/71.05
P293BmV1-P/1e-127/100/64.85
P′86BmV1-P′/5e-20/100/52.33
P3353BmV1-P3/0.0/96/72.14
M165BmV1-M/1e-53/93/51.3
G544BmV1-G/0.0/95/71.43
P671no hits
L2110BmV1-L/0.0/99/72.52
Crepe myrtle
(Lagerstroemia indica)		Dicot/LythraceaePogostemom alphacytorhabdovirus 3_Lag/
PogACRV3_Lag		PRJNA32094/
[41]		13149/11.36XBK064293N449BmV1/0.0/99/72.20
P294BmV1-P/1e-128/100/63.61
P′86BmV1-P′/3e-23/100/55.81
P3353BmV1-P3/0.0/96/72.14
M180BmV1-M/4e-55/83/55.63
G544BmV1-G/0.0/95/70.10
P671no hits
L2108BmV1-L/0.0/99/72.04
Candelabra primrose
(Primula chungensis)		Dicot/PrimulaceaePrimula alphacytorhabdovrus1/
PriACRV1		PRJNA616180/
Wang, X., BI, Kunming, China, unpublished		12953/237.5XBK064294N450LYMV-N/0.0/99/72.1
P307LYMV-P/2e-127/98/61.26
P′103no hits
P3311LYMV-P3/2e-161/100/70.74
M174LYMV-M/2e-68/98/56.4
G549LYMV-G/0.0/98/60.19
L2066LYMV-L/0.0/100/73.91
Glory primrose
(Primula oreodoxa)		Dicot/PrimulaceaePrimula alphacytorhabdovirus 2/
PriACRV2		PRJNA544868/
[42]		12146/21.43XBK064295N414TpVA-N/1e-160/98/54.57
P327GlLV1-P/2e-51/99/33.63
P′82no hits
P3201TpVA-P3/4e-86/98/64.5
M167GlLV1-M/5e-42/91/43.79
G559GlLV1-G/0.0/94/58.87
L2072TpVA-L0.0/99/64.58
Beach rose
(Rosa rugosa)		Dicot/RosaceaeRose
alphacytorhabdovirus 1/
RosACRV1		PRJNA498442/
[43]		12601/230.8XBK064296N425TpVA-N/7e-152/98/51.78
P313TpVA-P/3e-56/95/37.38
P′80no hits
P3167GlLV1-P3/4e-67/97/57.83
M172TpVA-M/5e-27/100/34.48
G593GlLV1-G/0.0/94/50.45
P667GlLV1-P6/1e-04/100/41.79
L2068TpVA-L/0.0/99/64.85
Korean bramble
(Rubus coreanus)		Dicot/RosaceaeRubus
alphacytorhabdovirus 1/
RubACRV1		PRJNA401210/
[44]		14682/33.28XBK064297N474DV1-N/91/3e-108/40.14
P297DV1-P/2e-38/98/33.22
P′93DV1-P′/0.029/88/32.56
P3366BmV1-P3/2e-106/93/45.45
M186DV1-M/5e-10/81/28.1
G573WhIV4-G/6e-154/93/41.2
P664no hits
L2109WhIV4-L/0.0/99/47.44
Barbed skullcap
(Scutellaria barbata)		Dicot/LamiaceaeScutellaria
alphacytorhabdovirus 1/
ScuACRV1		PRJNA653305/
[45]		13187/56.32XBK064298N447BmV1-N/0.0/99/68.6
P295BmV1-P/4e-121/100/61.36
P′86BmV1-P′/3e-24/100/53.49
P3350BmV1-P3/0.0/98/72.46
M165BmV1-M/9e-52/93/50.65
G547BmV1-G/0.0/95/70.86
P675no hits
L2103BmV1-0.0/99/71.44
Piggyback plant
(Tolmiea menziesii)		Dicot/SaxifragaceaeTolmiea alphacytorhabdovirus 1/
TolACRV1		PRJNA507776/
[46]		12746/19.26XBK064299N460StrV2-N/0.0/97/67.11
P295BCRV2-P/4e-130/100/62.03
P′102StrV2-P′/3e-19/96/43.88
P3333BCRV2-P3/7e-166/94/72.01
M182BCRV2-M/3e-78/92/67.86
G545BCRV2-G/0.0/96/73.14
L2091BCRV2-L/0.0/99/77.05
Wheat
(Triticum aestivum)		Monocot/PoaceaeTriticum alphacytorhabdovirus 1/
TriACRV1		PRJNA577739/
Li, Y., Hebei, China, unpublished		13955/87.91XBK064300N474AscSyV1-N/1e-134/93/44.02
P315DV1-P/2e-47/97/36.81
P′87DV1-P′/0.021/97/34.12
P3345AscSyV1-P3/5e-109/90/48.96
M190BmV1-M/3e-07/98/27.15
G561DV1-G/6e-130/86/39.64
P655no hits
L2106WhIV4-L/0.0/98/48.4
Long-leaved bladderwort
(Utricularia longifolia)		Dicot/LentibulariaceaeUtricularia alphacytorhabdovirus 1/
UtrACRV1		PRJNA354080/
Tang, C., Nanjing University, China, unpublished		13017/25.91XBK064301N454PaCRV1-N/4e-157/98/49.89
P324PaCRV1-P/6e-57/100/35.17
P3217PaCRV1-P3/2e73/98/52.47
M203PaCRV1-M/1e-46/80/41.1
G571PaCRV1-G/0.0/98/48.23
P663no hits
L2089PaCRV1-L/0.0/98/59.1
Wetland metagenome-Wetland metagenome associated alphacytorhabdovirus 1/
WMaACRV1		PRJNA338276/
[47]		12726/43.4XBK064302N445PeVA-N/4e-174/89/57.39
P301PeVA-P/1e-71/99/43.93
P′99no hits
P3219PeVA-P3/1e-60/98/44.39
M172PeVA-M/2e-45/99/43.93
G551PeVA-G/0.0/95/53.86
P652no hits
L2093PeVA-L/0.0/99/62.45
Malabar cardamon
(Wurfbainia villosa)		Monocot/ZingiberaceaeWurfbainia alphacytorhabdovirus 1/
WurACRV1		PRJNA471573/
Wang, H., Guangzhou, China, unpublished		13348/59.28XBK064303N465BmV1-N/0.0/98/60.87
P297BmV1-P/6e-81/99/46.49
P′93BmV1-P′/1e-09/89/43.37
P3356BmV1-P3/8e-143/97/56.32
M187BmV1-M/1e-19/81/32.68
G546BmV1-G/0.0/96/57.47
P685no hits
L2116BmV1-L/0.0/99/56.5
Maize
(Zea mays)		Monocot/PoaceaeZea alphacytorhabdovirus 1/
ZeaACRV1		PRJNA543910/
Wang, J., Anhui, China, unpublished		14358/38.58XBK064304N477RVCV-N/0.0/97/53.45
P329RVCV-P/3e-90/96/46.5
P′93RVCV-P′/1e-05/50/48.94
P3242RVCV-P36e-78/82/54.5
M181RVCV-M/6e-48/96/48.28
G573RVCV-G/0.0/95/55.21
P664RVCV-P6/5e-11/100/42.19
L2085RVCV-L/0.0/99/65.42
Table 2. Summary of novel betacytorhabdoviruses identified in plant RNA-seq data available on NCBI.
Table 2. Summary of novel betacytorhabdoviruses identified in plant RNA-seq data available on NCBI.
Plant HostTaxa/
Family		Virus Name/
Abbreviation		Bioproject ID/
Data Citation		Length (nt)/CoverageAccession NumberProtein IDLength (aa)Highest Scoring Virus-Protein/E-Value/Query Coverage %/Identity % (Blast P)
Rock wormwood
(Artemisia rupestris)		Dicot/AsteraceaeArtemisia betacytorhabdovirus 1/
ArtBCRV1		PRJNA730219/
[48]		13426/527.8XBK064305N484NCMV-N/2e-41/94/28.1
P340no hits
P3201RudV1-P3/3e-09/62/26.72
M202no hits
G524PpVE-G/7e-24/89/22.67
P684no hits
L2076RudV1-L/0.0/99/42.91
Zip begonia
(Begonia conchifolia)		Dicot/BegoniaceaeBegonia betacytorhabdovirus 1/
BegBCRV1		PRJEB26711/
[49]		13838/71.85XBK064306N447TiCRV1-N/5e-52/62/34.21
P299TiCRV1-P/2e-19/97/28.04
P3181no hits
M206TiCRV1-M/7e-10/83/23.12
G572TiCRV1-G/2e-70/85/27.93
P668no hits
L2161TiCRV1-L/0.0/99/41.72
White birch
(Betula pendula)		Dicot/BetulaceaeBetula betacytorhabdovirus 1/
BetBCRV1		PRJEB29260/
[50]		14744/26.93XBK064307N449RaCV-N/8e-69/90/34.24
P485no hits
P3195RaCV-P4/6e-19/91/23.46
M196RaCV-M/1e-04/83/23.78
G556RaCV-G/3e-59/92/27.08
P6138no hits
L2242RaCV-L/0.0/92/40.01
Himalayan birch
(Betula utilis)		Dicot/BetulaceaeBetula betacytorhabdovirus 2/
BetBCRV2		PRJNA638802/
Kumar, N., CSIR, India, unpublished		15147/15.49XBK064308N443RaCV-N/2e-78/92/36.01
P470no hits
P3196RaCV-P4/3e-22/89/28.41
M193RaCV-M/4e-08/86/25.68
G551RaCV-G/2e-53/87/27.44
P6138no hits
L2246RaCV-L/0.0/94/39.58
Buffalo grass
(Bouteloa dactyloides)		Monocot/PoaceaeBouteloa betacytorhabdovirus 1/
BouBCRV1		PRJNA297834/
[51]		14127/373.6XBK064309N452RudV1-N/2e-65/95/32.07
P381RVR-P/1e-07/17/33.33
P3195RudV1-P3/9e-36/89/39.13
M199RudV1-M/5e-28/84/34.32
G508NCMV-G/5e-21/94/23.43
P672no hits
P7255no hits
P8190no hits
L2070RudV1-L/0.0/99/49.04
Hardy garden mum (Chrysanthemum morifolium)Dicot/AsteraceaeChrysanthemum betacytorhabdovirus 1/
ChrBCRV1		PRJNA397042/
[52]		13309/99.94XBK064310N450MaCyV-N/1e-45/92/30.07
P333RVR-P/0.007/26/29.55
P3198TaEV1-P3/3e-11/67/29.93
M206no hits
G511PpVe-G/7e-22/69/24.66
P686no hits
L2075RudV1-0.0/99/42.21
Siberian hazelnut
(Corylus heterophylla)		Dicot/BetulaceaeCorylus betacytorhabdovirus 1/
CorBCRV1		PRJNA899668/
Sun, J, Lianoning, China, unpublished		15228/21.34XBK064311N461RaCV/7e-64/93/31.96
P479no hits
P3197YmVA-P4/4e-14/71/28.97
M201RaCV-M/4e-04/87/21.59
G555PpVE-G/3e-73/94/27.58
P6141no hits
L2257RaCV-L/0.0/89/40.21
Buffalo gourd
(Cucurbita foetidissima)		Dicot/CucurbitaceaeCucurbita betacytorhabdovirus 1/
CucBCRV1		PRJNA473174/
Sun, University of California, USA, unpublished		12969/336.9XBK064312N450MaCyV-N/1e-47/94/29.88
P302no hits
P3195RudV1-P3/3e-08/59/31.03
M209RudV1-M/2e-06/71/27.81
G514RSMV-G/5e-22/93/23.35
P679no hits
L2079RudV1-L/0-0/99/41.7
Slipper orchid (Cypripedium flavum)Monocot/OrchidaceaeCypripedium betacytorhabdovirus 1/
CypBCRV1		PRJNA479379/
[53]		9958/85.93XBK064313N430MYSV-N/1e-38/56/36
P280no hits
P3198no hits
L2114RaCV-L/0.0/91/33.81
Keladan
(Dryobalanops oblongifolia)		Dicot/DipterocarpaceaeDryobalanops betacytorhabdovirus 1/
DryBCRV1		PRJDB8182/
[54]		14393/134.5XBK064314N495YmVA-N/1e-95/98/35.8
P598YmVA-P/6e-07/23/32
P3234YmVa-P4/1e-29/68/38.04
M273no hits
G261no hits
L2259YmVA-L/0.0/98/42.34
Durian
(Durio zibethinus)		Dicot/MalvaceaeDurio betacytorhabdovirus 1/
DurBCRV1		PRJNA400310/
[55]		12791/52.47XBK064315N434NCMV-N/6e-52/97/33.03
P318no hits
P3193PMuMaV-P3/8e-13/68/28.79
M174no hits
G539RSMV-G/6e-34/84/24.74
P662no hits
L2057MYSV-L/0.0/98/45.16
Littleleaf honey locust
(Gleditsia microphylla)		Dicot/FabaceaeGleditsia betacytorhabdovirus 1/
GleBCRV1		PRJNA848854/
[56]		13339/29.81XBK064316N483YmVA-N/9e86/99/33.61
P454no hits
P3240YmVA-P4/6e-37/69/37.35
M251no hits
G162no hits
L2240YmVA-L/0.0/99/40.15
Chinese licorice
(Glycyrrhiza inflata)		Dicot/FabaceaeGlycyrrhiza betacytorhabdovirus 1/
GlyBCRV1		PRJNA574093/
[57]		14755/67.34XBK064317N493YmVA-N/8e-95/96/37.89
P500YmVA-P/3e-25/19/57.14
P3238YmVA-P4/2e-39/76/37.57
M280no hits
G169no hits
L2264YmVA-L/0.0/98/42.05
Pennywort
(Hepatica nobilis)		Dicot/RanunculaceaeHepatica betacytorhabdovirus 1/
HepBCRV1		PRJDB6630/
Nodai Genome Research Center, Japan, unpublished		10440/20.81XBK064318N432RVR-N/2e-77/92/35.78
P384CBDaV-P/6e-07/52/24.63
P3183RVR-P3/9e-13/70/27.91
M162no hits
L2074RVR-L/0.0/99/48.03
Kentia palm
(Howea forsteriana)		Monocot/ArecaceaeHowea betacytorhabdovirus 1/
HowBCRV1		PRJNA244607/
[58]		13727/58.86XBK064319N447TiCRV1-N/7e-46/53/36.86
P301TiCRV1-P/6e-10/95/24.76
P3173TiCRV1-P3/5e-11/81/25.53
M211TiCRV1-M/1e-10/91/24.37
G557TiCRV1-G/1e-58/92/26.15
P669no hits
L2145TiCRV1-L/0.0/99/40.22
Sweet potato
(Ipomoea batatas)		Dicot/ConvolvulaceaeIpomoea betacytorhabdovirus 1/
IpoBCRV1		PRJNA626066/
Read, ARC, SouthAfrica, unpublished		12811/9.76XBK064320N448NCMV-N/2e-55/93/32.62
P327RVR-P/0.001/25/26.76
P3196RudV1-P3/1e-06/56/30
M210RudV1-M/0.015/70/24.68
G533RSMV-G/1e-22/86/22.76
P6101no hits
L2071TaEV1-1/0.0/99/41.46
Malabar nut
(Justicia adhatoda)		Dicot/AcanthaceaeJusticia betacytorhabdovirus 1/
JusBCRV1		PRJNA842169/
[59]		15957/148.3XBK064321N463RaCV-N/3e-89/99/37.26
X180no hits
P408no hits
P3198RaCV-P4/4e-31/95/31.94
M199RaCV-M/1e-08/83/23.7
G574RaCV-G/8e-96/89/32.44
P7150RaCV-P7/1e-06/82/29.27
P8140no hits
L2236RaCV/0.0/96/45.34
Royle´s sedge
(Kobresia royleana)		Monocot/CyperaceaeKobresia betacytorhabdovirus 1/
KobBCRV1		PRJNA588660/
Qu, G., Lhasa, China, unpublished		14255/34.61XBK064322N542RSMV-N/7e-91/97/36.17
P550RSMV-P/8e-07/17/34.38
M175RSMV-M/0.003/88/26.28
G545RSMV-G/1e-124/94/39.24
P589no hits
L2098RsMV-L/0.0/99/52.39
P7106no hits
Plate-seed conebush
(Leucadendron platyspermum)		Dicot/ProteaceaeLeucadendron betacytorhabdovirus 1/
LeuBCRV1		PRJEB45774/
[60]		12698/181.2XBK064323N445NCMV-N/1e-115/93/43.97
P408NCMV-P/2e-32/72/31.76
P3192NCMV-P3/3e-09/76/26.35
M188TaEV1-M/8e-08/86/26.83
G533RSMV-G/9e-34/94/26.07
P663no hits
L2081RudV1-L/0.0/99/42.01
Black goji
(Lycium ruthenicum)		Dicot/SolanaceaeLycium betacytorhabdovirus 1/
LycBCRV1		PRJNA505629/
[61]		14855/75.64XBK064324N504YmVA-N/2e-78/92/35.16
P515YmVA-P/1e-20/22/45.76
P3239YmVA-P4/2e-37/95/34.06
M286YmVA-M/0.003/67/23.35
G208no hits
L2260YmVA-L/0.0/98/41.21
Mango
(Mangifera indica)		Dicot/AnacardiaceaeMango betacytorhabdovirus 1/
ManBCRV1		PRJNA487154/
[62]		13826/421.1XBK064325N477YmVA-N/8e-35/84/26.25
P359no hits
P3167no hits
M195no hits
G577RVR-G/7e-20/78/22.41
P695no hits
L2148RaCV-L/0.0/94/35.18
White mulberry
(Morus alba)		Dicot/MoraceaeMorus betacytorhabdovirus 1/
MorBCRV1		PRJNA597172/
[63]		15904/125.4XBK064326N502YmVA-N/8e-99/91/36.15
P617YmVA-P/5e-32/74/29.3
P3231YmVA-P4/5e-39/84/37.44
M263no hits
G215no hits
L2260YmVA-L/0.0/98/43.14
Nitre bush
(Nitraria tangutorum)		Dicot/NitrariaceaeNitraria betacytorhabdovirus 1/
NitBCRV1		PRJNA686177
[64]		15520/48.23XBK064327N433RaCV-N/2e-121/99/42.96
P544RaCV-P/2e-14/51/27.97
P3189RaCV-P4/1e-38/95/37.57
M187RaCV-M/2e-21/95/30.56
G583RaCV-G/6e-98/86/34.9
P6153RaCV-P7/3e-09/59/32.97
L2246RaCV-L/0.0/99/44.89
Hall´s panicgrass
(Panicum hallii)		Monocot/PoaceaePanicum betacytorhabdovirus 1/
PanBCRV1		PRJNA306692/
[65]		12136/60.31XBK064328N418CBDaV-N/0.0/99/72.9
P280CBDaV-P/2e-128/100/66.07
P3195CBDaV-P3/3e-104/94/75.14
M172CBDaV-M/1e-83/98/68.05
G505CBDaV-G/0.0/100/66.14
L2068CBDaV-L/0.0/100/75.87
Blue passionflower
(Passiflora caerulea)		Dicot/PassifloraceaePassiflora betacytorhabdovirus 1/
PasBCRV1		PRJEB21674/
1000 Plant (1KP) Transcriptomes Initiative, Unpublished		13471/32.88XBK064329N500TiCRV1-N/8e-130/92/44.49
P317TiCRV1-P/6e-35/89/32.77
P3188TiCRV1-P3/1e-22/80/29.14
P469no hits
M209TiCRV1-M/5e-48/85/47.19
G541TiCRV1-G/4e-160/90/44.85
P772no hits
L2138TiCRV1-L/0.0/99/62.66
Peat soil-Peat soil associated betacytorhabdovirus 1/
PSaBCRV1		PRJNA412438/
[66]		12663/58.67XBK064330N436MYSV-N/5e-96/99/38.79
P315NCMV-P/2e-26/86/31.62
P3186BYSMV-P3/1e-21/78/36.99
M169MYSV-M/7e-09/94/24.07
G505MaCyV-G/2e-75/95/31.43
P656no hits
L2083MaCyV-L/0.0/99/53.75
Peat soil-Peat soil associated betacytorhabdovirus 2/
PSaBCRV2		PRJNA570134/
JGI, USA, unpublished		14865/74.65XBK064331N457RudV1-N/2e-65/88/33.41
P397BYSMV-P/4e-09/26/35.51
P3194RudV1-P3/9e-29/78/36.84
M199RudV1-M/2e-21/88/31.64
G516PpVe-G/8e-35/82/25.61
P666no hits
P7276no hits
P8182no hits
L2066RudV1-L/0.0/99/50.63
Pentaphragma spicatumDicot/PentaphragmataceaePentaphragma betacytorhabdovirus 1/
PenBCRV1		PRJNA636634/
[67]		12983/164.1XBK064332N446TiCRV1-N/2e-50/57/34.91
P288TiCRV1-P/8e-17/87/27.21
P3178no hits
M197TiCRV1-M/0.008/84/25.44
G550TiCRV1-G/3e-76/91/30.18
P663no hits
L2160TiCRV1-L/0.0/99/41.12
Amur cork tree
(Phellodendron amurense)		Dicot/RutaceaePhellodendron betacytorhabdovirus 1/
PheBCRV1		PRJNA817294/
[68]		14292/177.4XBK064333N488YmVA-N/1e-85/97/34.43
P541YmVA-P/3e-40/61/34.47
P3241YmVA-P4/7e-30/78/32.28
M294no hits
G251no hits
L2258YmVA-L/0.0/98/41.42
Desert poplar
(Populus pruinosa)		Dicot/SalicaceaePopulus betacytorhabdovirus 1/
PopBCRV1		PRJNA354971/
Yu, L., Lanzhou, China, unpublished		15094/59.43XBK064334N432RaCV-N/8e-118/99/42.73
P569RaCV-P/6e-10/50/22.37
P3188RaCV-P4/1e-33/90/34.71
M201RaCV-M/1e-17/85/26.59
G586RaCV-G/2e-102/92/32.84
P6150RaCV-P7/2e-10/78/31.15
L2246RaCV-L/0.0/99/45.75
Kudzu
(Pueraria montana)		Dicot/FabaceaePueraria betacytorhabdovirus 1/
PueBCRV1		PRJNA515956/
[69]		13614/145.6XBK064335N481YmVA-N/1e-69/97/33.74
P338YmVA-P/8e-11/75/41.35/
P3230YmVA-P4/3e-20/58/36.57
M255no hits
G166no hits
L2254YmVA-L/0.0/98/39.88
Sesame
(Sesamum indicum)		Dicot/PedaliaceaeSesamum
betacytorhabdovirus 1_Ses/
SesBCRV1_Ses		PRJNA644139/
[70]		13565/178.3XBK064336N439CuCV1-N/5e-72/95/34.95
P340YmCaV-P/9e-15/58/30.10
P3183SbBMV-P3/2e-28/73/41.18
P476no hits
M224CuCV1-M/1e-23/75/30.59
G575YmCaV-G/2e-100/84/35.74
L2113CuCV1-L/0.0/99/48.07
Madagascar periwinkle (Catharanthus roseus)Dicot/ApocynaceaeSesamum
betacytorhabdovirus 1_Cat/
SesBCRV1_Cat		PRJNA246273/
[71]		13497/58.97XBK064337N440CuCV1-N/1e-71/95/35.33
P340YmCaV-P/9e-15/58/30.10
P3183SbBMV-P3/9e-28/73/41.18
P476no hits
M224CuCV1-M/7e-24/75/30.59
G575YmCaV-G/3e-100/84/35.95
L2113CuCV1-L/0.0/99/48.02
Schiedea pentandraDicot/CaryophyllaceaeSchiedea betacytorhabdovirus 1/
SchBCRV1		PRJNA491458
[72]		12964/214.6XBK064338N439MYSV-N/3e-55/93/33.01
P379NCMV-P/6e-95/98/42.89
P3206RVR-P3/8e-10/62/30.47
M182no hits
G524RSMV-G/1e-32/95/24.86
L2062RudV1-L/0.0/99/43.63
P7114no hits
Japanese pagoda tree
(Sophora japonica)		Dicot/FabaceaeSophora betacytorhabdovirus 1/
SopBCRV1		PRJNA797104/
[73]		13767/149.6XBK064339N501YmVA-N/3e-94/91/36.54
P493YmVA-P/2e-37/72/31.27
P3241YmVA-P4/1e-39/93/35.29
M283YmVA-M/0.001/61/24.57
G137no hits
L2255YmVA-L/0.0/97/41.79
Red clover
(Trifolium pratense)		Dicot/FabaceaeTrifolium betacytorhabdovirus 1/
TriBCRV1		PRJNA561285/
[74]		13511/285.1XBK064340N429BYSMV-N/4e-46/90/34.43
P372CBDaV-P/6e-06/30/26.55
P3218PMuMaV-P3/5e-19/66/31.65
M176AntAmV1-M/4e-04/66/25.42
G529RSMV-G/4e-37/91/25.2
P672no hits
L2069BYSMV-L/0.0/99/45.15
P8175no hits
Broad bean
(Vicia faba)		Dicot/FabaceaeVicia betacytorhabdovirus 1/
VicBCRV1		PRJNA591424/
[75]		12101/269.5XBK064341N434MaCyV-N/3e-60/95/31.13
P441RVR-P/0.035/16/30.99
P3186RVR-P3/8e-22/75/34.04
M164no hits
L2099CBDaV-L/0.0/98/44.27
Japanese prickly ash
(Zanthoxilum ailanthoides)		Dicot/RutaceaeZanthoxilum betacytorhabdovirus 1/
ZanBCRV1		PRJNA656412/
[76]		16669/78.34XBK064342N488YmVA-N/1e-88/92/33.98
P627YmVA-P/2e-25/22/47.18
P3243YmVA-P4/9e-26/70/33.53
M276no hits
G278no hits
L2278YmVA-L/0.0/98/41.48
Japanese prickly ash
(Zanthoxilum ailanthoides)		Dicot/RutaceaeZanthoxilum betacytorhabdovirus 2/
ZanBCRV2		PRJNA656412/
[76]		15584/94.97XBK064343N492YmVA-N/8e-88/91/35.01
P579YmVA-P/6e-37/61/33.33
P3242YmVA-P4/8e-27/68/37.35
M270no hits
G281no hits
L2280YmVA-L/0.0/98/40.79
Japanese prickly ash
(Zanthoxilum ailanthoides)		Dicot/RutaceaeZanthoxilum betacytorhabdovirus 3/
ZanBCRV3		PRJNA656412/
[76]		16283/50.29XBK064344N492YmVA-N/3e-88/98/34.2
P578YmVA-P/6e-27/17/57.84
P3242YmVA-P4/2e-24/80/32.82
M272no hits
G283no hits
L2282YmVA-L/0.0/99/41.24
Table 3. Summary of novel gammacytorhabdoviruses identified from plant RNA-seq data available on NCBI.
Table 3. Summary of novel gammacytorhabdoviruses identified from plant RNA-seq data available on NCBI.
Plant HostTaxa/
Family		Virus Name/
Abbreviation		Bioproject ID/
Data Citation		Length (nt)/CoverageAccession NumberProtein IDLength (aa)Highest Scoring Virus-Protein/E-Value/Query Coverage %/Identity % (Blast P)
Teide marguerite (Argyranthemum tenerifae)Dicot/AsteraceaeArgyranthemum gammacytorhabdovirus 1/
ArgGCRV1		PRJNA491458/
[72]		10801/20.11XBK064345N450GymDenV1-N/1e-98/94/41.31
P297no hits
P3231TrAV1-P3/7e-28/96/29.91
M176GymDenV1-M/1e-24/88/33.97
L2068GymDenV1-L/0.0/99/55.15
carrot (Daucus carota)Dicot/ApiaceaeDaucus gammacytorhabdovirus 1/
DauGCRV1		PRJNA745346/
Chakrabarti, S., CSIR-IICB, unpublished		11730/9.31XBK064346N459TrAV1-N/2e-133/95/47.42
P328TrAV1-P/2e-36/99/30.65
P3230TrAV1-P3/4e-52/95/39.73
M201GymDenV1-M/2e-23/91/33.33
L2069TrAV1-L/0.0/99/64.34
celery (Apium graveolens)Dicot/ApiaceaeApium gammacytorhabdovirus1/
ApiGCRV1		PRJNA543957/
[77]		12008/165.3XBK064347N455TrAV1-N/4e-173/94/57.83
P325TrAV1-P/2e-81/87/47.44
P3233TrAV1-P3/4e-81/94/53-95
M197TrAV1-M/4e-61/94/51.87
L2069TrAV1-L/0.0/100/72.5
Chinese goldthread
(Coptis chinensis)		Dicot/RanunculaceaeCoptis gammacytorhabdovirus 1/
CopGCRV1		PRJNA361017/
[78]		11214/17.41XBK064348N437GynDenV1-N/5e-118/99/42.6
P286TrAV1-P/2e-30/97/28.52
P3227TrAV1-P3/1e-47/93/35.81
M187GymDenV1-M/2e-40/93/41.95
L2069TrAV1-L/0.0/99/61.74
Bigseed alfalfa dodder (Cuscuta indecora)Dicot/ConvolvulaceaeCuscuta gammacytorhabdovirus 1/
CusGCRV1		PRJNA543296/
[79]		10772/35.14XBK064349N429TrAV1-N/7e-108/96/43.68
P301GymDenV1-P/3e-21/92/29.87
P3220TrAV1-P3/3e-16/97/25.23
M196GymDenV1-M/6e-13/80/32.1
L2054GymDenV1-L/0.0/99/50.17
Nevada dodder
(Cuscuta nevadensis)		Dicot/ConvolvulaceaeCuscuta gammacytorhabdovirus 2/
CusGCRV2		PRJNA561399/
Frangione, E., Canada, unpublished		10700/33.29XBK064350N429TrAV1-N/1e-106/96/42.49
P302GymDenV1-P/9e-20/83/27.97
P3220TrAV1-P3/2e-17/95/27.78
M188GymDenV1-M/6e-17/85/34.15
L2054GymDenV1-L/0.0/99/50.85
Slipper orchid (Cypripedium flavum)Monocot/OrchidaceaeCypripedium gammacytorhabdovirus 1/
CypGCRV1		PRJNA479379/
[53]		10872/30.26XBK064351N437GymDenV1-N/2e-117/96/45.5
P283GymDenV1-P/2e-46/96/35.1
P3228TrAV1-P3/4e-36/94/34.86
M213GymDenV1-M/4e-38/83/37.64
L2069GymDenV1-L/0.0/99/60.34
Violet helleborine
(Epipactis purpurata)		Monocot/OrchidaceaeEpipactis gammacytorhabdovirus 1/
EpiGCRV1		PRJNA450088/
[80]		11001/38.65XBK064352N452GymDenV1-N/2e-102/86/42.36
P300GymDenV1-P/4e-21/93/26.51
P3225TrAV1-P3/2e-26/64/34.72
L2064GymDenV1-L/0.0/99/57.83
Common ash
(Fraxinus excelsior)		Dicot/OleaceaeFraxinus gammacytorhabdovirus 1/
FraGCRV1		PRJEB4958/
[81]		11521/50.58XBK064353N443TrAV1-1e-96/94/41.96
P284GymDenV1-P/1e-26/94/28.81
P3224TrAV1-P3/7e-29/97/29.41
M184GymDenV1-M/2e-30/85/38.22
P565no hits
L2068GymDenV1-L0.0/99/55.78
Ash dieback (Hymenoscyphus fraxineus)-Fraxinus gammacytorhabdovirus 2/
FraGCRV2		PRJEB7998/
[82]		11737/44.16XBK064354N439GymDenV1-2e-101/89/40.61
P285GymDenV1-P/8e-39/94/30.51
P3224TrAV1-P3/6e-33/96/34.84
M187GymDenV1-M/7e-31/86/36.65
P555no hits
L2068GymDenV1-L0.0/99/56
Dwarf heliosperma (Heliosperma pusillum)Dicot/CaryophyllaceaeHeliosperma gammacytorhabdovirus 1/
HelGCRV1		PRJNA760819/
[83]		11579/19.27XBK064355N436GymDenV1-N/3e-102/90/41.65
P308GymDenV1-P/2e-30/84/31.9
P3221TrAV1-P3/1e-27/95/31.63
M206GymDenV1-M/5e-29/83/35.67
L2063GymDenV1-L/0.0/99/58.31
Kenaf
(Hibiscus cannabinus)		Dicot/MalvaceaeHibiscus gammacytorhabdovirus 1/
HibGCRV1		PRJNA602109/
[84]		11079/23.52XBK064356N458GymDenV1-N/3e-77/88/35.39
P391GymDenV1-P/6e-08/62/25.99
P3221TrAV1-P3/2e-16/78/26.92
M194GymDenV1-M/9e-12/79/26.45
L2063TrAV1-L/0.0/99/53.86
Golden ageratum
(Lonas annua)		Dicot/AsteraceaeLonas gammacytorhabdovirus 1/
LonGCRV1		PRJNA371565/
[85]		11920/115.3XBK064357N450GymDenV1-N/5e-106/88/44.75
P297GymDenV1-P/1e-20/94/26.56
P3231TrAV1-P3/3e-24/95/27.6
M176GymDenV1-M/5e-24/88/30.77
L2068GymDenV1-L/0.0/99/55.40
Mantano river lupine
(Lupinus mantaroensis)		Dicot/FabaceaeLupinus gammacytorhabdovirus 1/
LupGCRV1		PRJNA318864/
[86]		11196/44.85XBK064358N430TrAV1-N/8e-105/98/40.95
P314GymDenV1-P/9e-20/85/25.91
P3221TrAV1-P3/1e-16/76/30.41
M189GymDenV1-M/1e-08/84/27.16
L2057TrAV1-L/0.0/99/51.47
Stinkhorn clubhead
(Rhopalocnemis phalloides)		Dicot/BalanophoraceaeRhopalocnemis
gammacytorhabdovirus 1/
RhoGCRV1		PRJNA737177/
[87]		11024/32.23XBK064359N469GymDenV1-N/6e-110/86/43.06
P305GymDenV1-P/1e-17/84/26.16
P3231TrAV1-P3/7e-25/92/29.17
L2071GymDenV1-L/0.0/99/55.86
Bladder campion
(Silene vulgaris)		Dicot/CaryophyllaceaeSilene gammacytorhabdovirus 1/
SilGCRV1		PRJNA104951/
[88]		11500/37.54XBK064360N435GymDenV1-N/6e-107/89/43.83
P311GymDenV1-P/3e-35/88/29.14
P3221TrAV1-P3/9e-33/97/31.96
M209GymDenV1-M/5e-31/80/36.09
L2066GymDenV1-L/0.0/99/58.04
Table 4. Summary of trirhaviruses identified from plant RNA-seq data available on NCBI, including the reannotation of Picris cytorhabdovirus 1 sequence.
Table 4. Summary of trirhaviruses identified from plant RNA-seq data available on NCBI, including the reannotation of Picris cytorhabdovirus 1 sequence.
Plant HostTaxa/
Family		Virus Name/
Abbreviation		Bioproject ID/
Data Citation		RNA Segment/
Length (nt)/Coverage		Accession NumberProtein IDLength (aa)Highest Scoring Virus-Protein/E-Value/Query Coverage %/Identity % (Blast P)
Red alder
(Alnus rubra)		Dicot/BetulaceaeAlnus
trirhavirus 1/
AlTRV1		PRJNA691057/
Bell, C., NCGR, USA, unpublished		RNA1 6699/165.78XBK064247L2043PiCRV1-L/0.0/99/55
BK064248N442PiCRV1-N/6e-62/78/34.72
RNA2 5289/302.95X P2341PiCRV1-40kDa/2e-148/99/62.28
P3201PiCRV1-21kDA/5e-21/89/29.61
P472PiCRV1-8kDa/1e-15/100/52.78
P5312PCLSaV-P5/4e-26/53/34.94
BK064249P6260no hits
P7165no hits
RNA3 4586/211.42X P8515no hits
P11289no hits
Hardy garden mum (Chrysanthemum morifolium)Dicot/AsteraceaeChrysanthemum trirhavirus 1/
ChTRV1		PRJNA510496/
Shen R, China, unpublished		RNA1 6332/29.57XBK064250L2047PiCRV1-L/0.0/99/58.11
BK064251N441PiCRV1-N/8e-73/77/37.29
RNA2 4222/105.57X P2348PiCRV1-40kDa/7e-154/98/63.19
P3189PiCRV1-21kDA/1e-31/90/35.84
P472PiCRV1-8kDa/3e-13/100/47.22
BK064252P6265no hits
P7194no hits
RNA3 5133/66.36X P8528no hits
P5354PCLSaV-P5/1e-25/47/37.43
Sierra Nevada wallflower
(Erysimum nevadense)		Dicot/BrassicaceaeErysimum trirhavirus1/
EryTRV1		PRJNA473238/
[89]		RNA1 6524/16.55XBK064253L2039PiCRV1-L/0.0/99/66.22
BK064254N441PiCRV1-N/4e-111/79/45.98
RNA2 3989/22.23X P2346PiCRV1-40kDa/2e-163/99/63.48
P3198PiCRV1-21kDA/1e-36/85/39.18
P494PiCRV1-8kDa/3e-22/76/62.5
BK064255P6316no hits
P7199no hits
RNA3 4307/21.74X P8509no hits
P9143no hits
Lucerne
(Medicago sativa)		Dicot/FabaceaeMedicago
trirhavirus 1/
MeTRV1		PRJNA667169/
[90]
and
PRJNA535257/
JGI, USA, unpublished		RNA1 6495/9.31XBK064256L2040PiCRV1-L/0.0/99/60.28
BK064257N445PiCRV1-N/2e-113/77/48.47
RNA2 3851/25.36X P2343PiCRV1-40kDa/3e-149/97/60.90
P3183PiCRV1-21kDA/2e-26/96/32.78
P472PiCRV1-8kDa/1e-18/100/62.5
BK064258P6274no hits
P7189no hits
RNA3 4565/12.23X P8514no hits
P5303PCLSaV-P5/1e-14/52/33.33
Bristly ox-tongue
(Picris echioides)		Dicot/AsteraceaePicris trirhavirus 1/
PiTRV1		PRJNA772045/
[20]		RNA1 6530/65.27XBK064259L2043PiCRV1-L/0.0/100/100
BK064269N495PiCRV1-N/0.0/72/100
RNA2 4091/87.26X P2345PiCRV1-40kDa/0.0/100/100
P3184PiCRV1-21kDA/5e-134/100/100
P472PiCRV1-8kDa/2e-42/100/100
BK064261P6331no hits
P7199no hits
RNA3 4259/93.17X P8505no hits
P10148no hits
Table 5. Consensus conserved plant rhabdovirus gene junction sequences.
Table 5. Consensus conserved plant rhabdovirus gene junction sequences.
Proposed GenusVirus *3′ End mRNAIntergenic Spacer5′ End mRNA
AlphacytorhabdovirusArcACRV1AAUUAUUUUGAUCUU
ArtACRV1AAUUCUUUUGA(U)nCNN
ArtACRV2AAUUAUUUUGA(U)nCNN
ArtACRV3AAUUAUUUUGA(U)nCNU
BacACRV1AAUUCUUUUGA(U)nCNC
CarACRV1AAUUAUUUUGAUCUU
CheACRV1AAUUAUUUUGAUCUU
ChrACRV1AAAUAUUUUGAUCUU
ConACRV1AAUUCUUUUGAUCNC
CynACRV1AAUU(C/A)UUUUGA(U)nCNN
EupACRV1AAUUAUUUUGAUCUU
FagACRV1AAUUAUUUUGAUCNN
FicACRV1AAUUAUUUUGAUCNN
GarACRV1AAUUCUUUUGN(U)nCNN
GeuACRV1AAUUCUUUUGAUCNC
HedACRV1AAUUCUUUUGNUCNC
IleACRV1AAUUAUUUUGA(U)nCUG
MedACRV1AAUUAUUUUGAUCNN
MenACRV1AAUUAUUUUGAUCUU
MorACRV1AAUUCUUUUGNUCNN
OakACRV1AAUUAUUUUGAUCUU
OciACRV1AAUUAUUUUGAUCUU
PelACRV1AAUUAUUUUGAUCUN
PhyACRV1AAUUCUUUUGAUCUC
PinACRV1AAUUAUUUUGN(U)nCU(U/G)
PogACRV1AAUUCUUUUG(N)nCUC
PogACRV2AAUUAUUUUGAUCNN
PogACRV3AAUUAUUUUGAUCNG
PriACRV1AAUUCUUUUGA(U)nCUN
PriACRV2CAUUAUUUUGAUCUG
RosACRV1AAUUAUUUUGAUCUN
RubACRV1AAUUAUUUUGNUCNN
ScuACRV1AAUUAUUUUG(N)nCNN
TolACRV1AAUUCUUUUGNUCUC
TriACRV1AAUUAUUUUGA(U)nCU(G/U)
UtrACRV1AAUUAUUUUGA(U)nCNN
WMaACRV1AAUUCUUUUGAUCUU
WurACRV1AAUUAUUUUGN(U)nCNN
ZeaACRV1AUUUAUUUUGA(U)nCNN
AcCVAAUUAUUUUGAUCUG
ADVAAUUAUUUUGAUCUU
AscSyV1AAUUAUUUUGNUCNN
BCRV2AAUUCUUUUGNUCNN
BmV1AAUUAUUUUGANCUG
CCyV1AAUUCUUUUG(N)nCUU
ChYDaVAAUUAUUUUGAUCUN
CCRV1AAUUAUUUUGAUCUU
CnV2AAUUAUUUUGAUCUN
DV1AAUUAUUUUGAUCUG
GlLV1AAUUAUUUUGAUCUU
HpLVAAUUAUUUUGAUCNN
KePCyVAAUUAUUUUGAUCUU
LNYVAAUUCUUUUG(N)nCUU
LYMoVAAUUCUUUUG(N)nCUN
NymAV1AUUAAUUUUGAUCUN
PaCRV1AAUUAUUUUGAUCUU
PCaCVAAUUAUUUUGNUCUN
PeVAAAUUAUUUUG(N)nCUN
PNSaVAAUUAUUUUGAUCUN
RVCVAUUUAUUUUGAUCUU
SaV1AUUUAUUUUGAUCNN
SCVAAUUAUUUUGAUCUU
StrV1AAUUAUUUUGAUCUU
StrV2AAUUCUUUUGNUCNN
TCRV1AAUUAUUUUGAUCNN
TpVAAAUUAUUUUGAUCUU
TpVBAAUUCUUUUG(N)nCUN
TrARV1AAUUAUUUUGAUCUU
TYMaVAAUUAUUUUGAUCUU
WhIV4AAUUAUUUUGNUCUU
WhIV5AAUUAUUUUGAUCNN
WhIV6AAUUAUUUUGAUCUN
BetacytorhabdovirusArtBCRV1AUUCUUUUUGUUCUU
BegBCRV1AUAUUUUUUGNCUN
BetBCRV1AUUCUUUUUGG(U)nCUG
BetBCRV2AUUCUUUUUGG(U)nCUG/A
BouBCRV1AUUCUUUUUGCUCUG
ChrBCRV1AUUCUUUUUGUUCUU
CorBCRV1AUUCUUUUUGGUUCUG
CucBCRV1AUUCUUUUUG(N)nCUU
CypBCRV1UUCUUUUUUGACUC
DryBCRV1AUUAUUUUUGGUCCU
DurBCRV1AUUCUUUUUGACUC
GleBCRV1AUUAUUUUUGG(U)nCUN
GlyBCRV1AUUAUUUUUGGUCCU
HepBCRV1AUUAUUUUUGA(U)nCUU
HowBCRV1AUAUUUUUUGACUN
IpoBCRV1AUUCUUUUUGUUCUN
JusBCRV1AUU(A/C)UUUUUGGUUCUN
KobBCRV1AUUCUUUUUGGNCUC
Leu CRV1AUUCUUUUUGACUC
LycBCRV1AUUAUUUUUGGUCCU
ManBCRV1AUUAUUUUUGG(U)nCUN
MorBCRV1AUUAUUUUUGGUCCU
NitBCRV1AUUCUUUUUGGUUCUN
PanBCRV1AUUCUUUUUG(G/A)CUC
PasBCRV1AUAUUUUUUGAUUCUC
PSaBCRV1AUUUAUUUUGACUC
PSaBCRV2AUUAUUUUUGNUCUN
PenBCRV1AUAUUUUUUG(N)nCUU
PheBCRV1AUUAUUUUUGGUUCUC
PopBCRV1AUUCUUUUUGG(U)nCUN
PueBCRV1AUUAUUUUUGGUCCU
SesBCRV1UUCUUUUUUGACUN
SchBCRV1AUUCUUUUUGACUC
SopBCRV1AUUAUUUUUGGUCCU
TriBCRV1AUUCUUUUUGNCUN
VicBCRV1AUUCUUUUUGGCUC
ZanBCRV1AUUAUUUUUGGUCCU
ZanBCRV2AUUAUUUUUGGUCCU
ZanBCRV3AUUAUUUUUGGUCCU
AntAmV1AUUAUUUUUGCUCUU
AriACRVUUAUUUUUUGN(N)nCNN
BeTaV1UUAUUUUUUGACUC
BYSMVAUUAUUUUUGACUC
CBDaVAUUCUUUUUGGCUC
CuCV1AUUAUUUUUGACUC
MaCyVAUUCUUUUUGACUC
MYSVAUUAUUUUUGACUC
NCMVAUUCUUUUUGACUC
PMuMaVAUUAUUUUUG(N)nCUA
PpVEAUUCUUUUUGACCCU
RaCVAUUCUUUUUG(N)nCUN
RVRAUUUAUUUUGACUC
RSMVAUUCUUUUUGCUCUG
RudV1AUUCUUUUUGGUU(N)nCUN
SbBMVUUAUUUUUUGACAC
TaEV1AUUCUUUUUGG(N)nCUN
TiCRV1AUAUUUUUUGA(N)nCUC
YmCaVUUAUUUUUUGACUC
YmVAAUUCUUUUUGGUCCU
GammacytorhabdovirusArgGCRV1AUUCUUUUUAAUCCU
CarGCRV1AUUCUUUUUA(N)nCCU
CelGCRV1AUUCUUUUUA(N)nCNU
CopGCRV1AUUCUUUUUA(N)nCCU
CusGCRV1AUUCUUUUUA(N)nCNN
CusGCRV2AUUCUUUUUA(N)nCCU
CypGCRV1AAUCUUUUUA(N)nCNN
EpiGCRV1AUUCUUUUUAUGUCCU
FraGCRV1AUUCUUUUUA(N)nCNU
FraGCRV2AUUCUUUUUA(N)nCCU
HelGCRV1AUUCUUUUUA(N)nCCU
HibGCRV1AUUCUUUUUA(N)nCNN
LonGCRV1AUUCUUUUUA(N)nCCU
LupGCRV1AUUCUUUUUA(N)nCCU
Rh GCRV1AUUUCUUUUA(N)nCCU
SilGCRV1AUUCUUUUUA(N)nCCU
GymDenV1AAUCUUUUUA(N)nCNN
TrAV1AUUCUUUUUA(N)nCNU
TrirhavirusAlTRV1AAUUCUUUUGN(N)nCUC
ChTRV1AAUUCUUUUGN(N)nCCU
EryTRV1AAUUCUUUUGN(N)nCUC
MeTRV1AAUUCUUUUGN(N)nCU (C/G)
PiTRV1AAUUCUUUUGN(N)nCUN
The consensus gene junction sequences of the viruses identified in this study are highlighted in light grey. * Names and abbreviations of newly identified viruses are listed in Table 1, Table 2, Table 3 and Table 4; while the names and abbreviations of known viruses are listed in Supplementary Table S1.
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MDPI and ACS Style

Bejerman, N.; Dietzgen, R.; Debat, H. Novel Tri-Segmented Rhabdoviruses: A Data Mining Expedition Unveils the Cryptic Diversity of Cytorhabdoviruses. Viruses 2023, 15, 2402. https://doi.org/10.3390/v15122402

AMA Style

Bejerman N, Dietzgen R, Debat H. Novel Tri-Segmented Rhabdoviruses: A Data Mining Expedition Unveils the Cryptic Diversity of Cytorhabdoviruses. Viruses. 2023; 15(12):2402. https://doi.org/10.3390/v15122402

Chicago/Turabian Style

Bejerman, Nicolas, Ralf Dietzgen, and Humberto Debat. 2023. "Novel Tri-Segmented Rhabdoviruses: A Data Mining Expedition Unveils the Cryptic Diversity of Cytorhabdoviruses" Viruses 15, no. 12: 2402. https://doi.org/10.3390/v15122402

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