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

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.

Complete genome sequence of patchouli chlorosis-associated cytorhabdovirus, a new cytorhabdovirus infecting patchouli plants in Brazil.

A cytorhabdovirus, tentatively named "patchouli chlorosis-associated cytorhabdovirus" (PCaCV), was identified in a patchouli plant, using high-throughput sequencing, and its genome sequence was confirmed by Sanger sequencing. The PCaCV genome consists of 12,913 nucleotides and contains six open reading frames in the order 3'-N-P'-P-P3-M-(G)-L-5'. The glycoprotein gene was found to contain stop codons in the coding frame; hence, this gene is considered defective. PCaCV is most closely related to tomato yellow mottle-associated virus, sharing 61.1% nucleotide sequence identity in the complete genome and 73.9% amino acid sequence identity in the L protein. These data suggest that PCaCV should be considered a new member of the genus Cytorhabdovirus, and the binomial species name "Cytorhabdovirus patchoulii" is proposed.

Genome Characterization and Pathogenicity of Two New Hyptis pectinata Viruses Transmitted by Distinct Insect Vectors.

Two newly described viruses belonging to distinct families, Rhabdoviridae and Geminiviridae, were discovered co-infecting Hyptis pectinata from a tropical dry forest of Ecuador. The negative-sense RNA genome of the rhabdovirus, tentatively named Hyptis latent virus (HpLV), comprises 13,765 nucleotides with seven open reading frames separated by the conserved intergenic region 3'-AAUUAUUUUGAU-5'. Sequence analyses showed identities as high as 56% for the polymerase and 38% for the nucleocapsid to members of the genus Cytorhabdovirus. Efficient transmission of HpLV was mediated by the pea aphid (Acyrthosiphon pisum) in a persistent replicative manner. The single-stranded DNA genome of the virus tentatively named Hyptis golden mosaic virus (HpGMV) shared homology with members of the genus Begomovirus with bipartite genomes. The DNA-A component consists of 2,716 nucleotides (nt), whereas the DNA-B component contains 2,666 nt. Pairwise alignments using the complete genomic sequence of DNA-A of HpGMV and closest relatives showed identities below the cutoff (<91% shared nt) established by the ICTV as species demarcation, indicating that HpGMV should be classified in a distinct begomovirus species. Transmission experiments confirmed that the whitefly Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) is a vector of HpGMV.

Illuminating the Plant Rhabdovirus Landscape through Metatranscriptomics Data.

Rhabdoviruses infect a large number of plant species and cause significant crop diseases. They have a negative-sense, single-stranded unsegmented or bisegmented RNA genome. The number of plant-associated rhabdovirid sequences has grown in the last few years in concert with the extensive use of high-throughput sequencing platforms. Here, we report the discovery of 27 novel rhabdovirus genomes associated with 25 different host plant species and one insect, which were hidden in public databases. These viral sequences were identified through homology searches in more than 3000 plant and insect transcriptomes from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) using known plant rhabdovirus sequences as the query. The identification, assembly and curation of raw SRA reads resulted in sixteen viral genome sequences with full-length coding regions and ten partial genomes. Highlights of the obtained sequences include viruses with unique and novel genome organizations among known plant rhabdoviruses. Phylogenetic analysis showed that thirteen of the novel viruses were related to cytorhabdoviruses, one to alphanucleorhabdoviruses, five to betanucleorhabdoviruses, one to dichorhaviruses and seven to varicosaviruses. These findings resulted in the most complete phylogeny of plant rhabdoviruses to date and shed new light on the phylogenetic relationships and evolutionary landscape of this group of plant viruses. Furthermore, this study provided additional evidence for the complexity and diversity of plant rhabdovirus genomes and demonstrated that analyzing SRA public data provides an invaluable tool to accelerate virus discovery, gain evolutionary insights and refine virus taxonomy.

Joá yellow blotch-associated virus, a new alphanucleorhabdovirus from a wild solanaceous plant in Brazil.

We identified a novel plant rhabdovirus infecting native joá (Solanum aculeatissimum) plants in Brazil. Infected plants showed yellow blotches on the leaves, and typical enveloped bacilliform rhabdovirus particles associated with the nucleus were seen in thin sections by electron microscopy. The virus could be graft-transmitted to healthy joá and tomato plants but was not mechanically transmissible. RT-PCR using degenerate plant rhabdovirus L gene primers yielded an amplicon from extracted total RNA, the sequence of which was similar to those of alphanucleorhabdoviruses. Based on close sequence matches, especially with the type member potato yellow dwarf virus (PYDV), we adopted a degenerate-primer-walking strategy towards both genome ends. The complete genome of joá yellow blotch-associated virus (JYBaV) is comprised of 12,965 nucleotides, is less than 75% identical to that of its closest relative PYDV, and clusters with PYDV and other alphanucleorhabdoviruses in L protein phylogenetic trees, suggesting that it should be taxonomically classified in a new species in the genus Alphanucleorhabdovirus, family Rhabdoviridae. The genome organization of JYBaV is typical of the 'PYDV-like' subgroup of alphanucleorhabdoviruses, with seven genes (N-X-P-Y-M-G-L) separated by conserved intergenic regions and flanked by partly complementary 3' leader and 5' trailer regions.

Natural Defect of a Plant Rhabdovirus Glycoprotein Gene: A Case Study of Virus-Plant Coevolution.

Seven isolates of a putative cytorhabdovirus (family Rhabdoviridae, order Mononegavirales) designated as citrus-associated rhabdovirus (CiaRV) were identified in citrus, passion fruit, and paper bush from the same geographical area in China. CiaRV, bean-associated cytorhabdovirus (Brazil), and papaya virus E (Ecuador) should be taxonomically classified in the species Papaya cytorhabdovirus. Due to natural mutations, the glycoprotein (G) and P4 genes were impaired in citrus-infecting isolates of CiaRV, resulting in an atypical rhabdovirus genome organization of 3' leader-N-P-P3-M-L-5' trailer. The P3 protein of CiaRV shared a common origin with begomoviral movement proteins (family Geminiviridae). Secondary structure analysis and trans-complementation of movement-deficient tomato mosaic virus and potato virus X mutants by CiaRV P3 supported its function in viral cell-to-cell trafficking. The wide geographical dispersal of CiaRV and related viruses suggests an efficient transmission mechanism, as well as an underlying risk to global agriculture. Both the natural phenomenon and experimental analyses demonstrated presence of the "degraded" type of CiaRV in citrus, in parallel to "undegraded" types in other host plant species. This case study shows a plant virus losing the function of an important but nonessential gene, likely due to host shift and adaption, which deepened our understanding of course of natural viral diversification.

Transmission of the Bean-Associated Cytorhabdovirus by the Whitefly Bemisia tabaci MEAM1.

The knowledge of genomic data of new plant viruses is increasing exponentially; however, some aspects of their biology, such as vectors and host range, remain mostly unknown. This information is crucial for the understanding of virus-plant interactions, control strategies, and mechanisms to prevent outbreaks. Typically, rhabdoviruses infect monocot and dicot plants and are vectored in nature by hemipteran sap-sucking insects, including aphids, leafhoppers, and planthoppers. However, several strains of a potentially whitefly-transmitted virus, papaya cytorhabdovirus, were recently described: (i) bean-associated cytorhabdovirus (BaCV) in Brazil, (ii) papaya virus E (PpVE) in Ecuador, and (iii) citrus-associated rhabdovirus (CiaRV) in China. Here, we examine the potential of the Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) to transmit BaCV, its morphological and cytopathological characteristics, and assess the incidence of BaCV across bean producing areas in Brazil. Our results show that BaCV is efficiently transmitted, in experimental conditions, by B. tabaci MEAM1 to bean cultivars, and with lower efficiency to cowpea and soybean. Moreover, we detected BaCV RNA in viruliferous whiteflies but we were unable to visualize viral particles or viroplasm in the whitefly tissues. BaCV could not be singly isolated for pathogenicity tests, identification of the induced symptoms, and the transmission assay. BaCV was detected in five out of the seven states in Brazil included in our study, suggesting that it is widely distributed throughout bean producing areas in the country. This is the first report of a whitefly-transmitted rhabdovirus.

Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico.

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit-specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit-specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

Sequencing, genome analysis and prevalence of a cytorhabdovirus discovered in Carica papaya.

The complete genome of a new rhabdovirus infecting papaya (Carica papaya L.) in Ecuador, named papaya virus E, was sequenced and characterized. The negative-sense single-stranded RNA genome consists of 13,469 nucleotides with six canonical open reading frames (ORFs) and two accessory short ORFs predicted between ORFs corresponding to P3 (movement protein) and M (matrix protein). Phylogenetic analyses using amino acid sequences from the nucleocapsid, glycoprotein and polymerase, grouped the virus with members of the genus Cytorhabdovirus, with rice stripe mosaic virus, yerba mate chlorosis-associated virus and Colocasia bobone disease-associated virus as closest relatives. The 3' leader and 5' trailer sequences were 144 and 167 nt long, respectively, containing partially complementary motifs. The motif 3'-AUUCUUUUUG-5', conserved across rhabdoviruses, was identified in all but one intergenic regions; whereas the motif 3'-ACAAAAACACA-5' was found in three intergenic junctions. This is the first complete genome sequence of a cytorhabdovirus infecting papaya. The virus was prevalent in commercial plantings of Los Ríos, the most important papaya producing province of Ecuador. Recently, the genome sequence of bean-associated cytorhabdovirus was reported. The genome is 97% identical to that of papaya virus E, indicating that both should be considered strains of the same virus.

Novel bird's-foot trefoil RNA viruses provide insights into a clade of legume-associated enamoviruses and rhabdoviruses.

Here, we report the identification and characterization of two novel viruses associated with bird's-foot trefoil. Virus sequences related to those of enamoviruses (ssRNA (+); Luteoviridae; Enamovirus) and nucleorhabdoviruses (ssRNA (-); Rhabdoviridae; Nucleorhabdovirus) were detected in Lotus corniculatus transcriptome data. The genome of the tentatively named "bird's-foot trefoil-associated virus 1" (BFTV-1) is a 13,626-nt-long negative-sense ssRNA. BFTV-1 encodes six predicted gene products in the antigenome orientation in the canonical order 3'-N-P-P3-M-G-L-5'. The genome of the proposed "bird's-foot trefoil-associated virus 2" (BFTV-2) is 5,736 nt long with a typical 5΄-PO-P1-2-IGS-P3-P5-3' enamovirus genome structure. Phylogenetic analysis indicated that BFTV-1 is closely related to datura yellow vein nucleorhabdovirus and that BFTV-2 clusters into a monophyletic lineage of legume-associated enamoviruses. This subclade of highly related and co-divergent legume-associated viruses provides insights into the evolutionary history of the enamoviruses.

Two Rhabdoviridae: Dillard's Draw virus, a putative new virus, and Merida virus from Culex tarsalis (Diptera: Culicidae) in New Mexico, USA.

The U.S. Air Force conducts vector and arbovirus surveillance worldwide. We report on two Rhabdoviridae detected at Holloman Air Force Base in Otero County, New Mexico including the nearly complete 11-kb genome of Merida virus, which was not previously known from the USA, and a novel virus tentatively named Dillard's Draw virus. Merida virus was previously only known from Mexico. Dillard's Draw virus represents a novel arbovirus most closely related to an avian pathogen from Australia and shares some genetic similarity to Durham virus from the eastern United States.

Unveiling the complete genome sequence of clerodendrum chlorotic spot virus, a putative dichorhavirus infecting ornamental plants.

The genus Dichorhavirus includes plant-infecting rhabdoviruses with bisegmented genomes that are horizontally transmitted by false spider mites of the genus Brevipalpus. The complete genome sequences of three isolates of the putative dichorhavirus clerodendrum chlorotic spot virus were determined using next-generation sequencing (Illumina) and traditional RT-PCR. Their genome organization, sequence similarity and phylogenetic relationship to other viruses, and transmissibility by Brevipalpus yothersi mites support the assignment of these viruses to a new species of dichorhavirus, as suggested previously. New data are discussed stressing the reliability of the current rules for species demarcation and taxonomic status criteria within the genus Dichorhavirus.

Distribution and genetic variability of alfalfa dwarf virus, a cytorhabdovirus associated with alfalfa dwarf disease in Argentina.

In 2010, a novel cytorhabdovirus named alfalfa dwarf virus (ADV) was detected for the first time in lucerne crops in Argentina showing dwarfism, in mixed infections with several other viruses. ADV appears to be endemic to Argentina and has not been reported elsewhere. In this study, we have investigated the genetic variability of ADV based on the complete nucleoprotein (N) gene of 13 isolates from different lucerne-growing regions in Argentina. Phylogenetic and sequence identity analyses showed that all ADV isolates are closely related and have not diverged more than 1% in the N gene despite geographical separation. These data provide further evidence that ADV is new to science and emerged and spread very recently. A total of 43 single-nucleotide polymorphisms were identified between the ADV isolates studied. Analysis of N gene ORF sequence revealed a mutational bias, with more transitions than transversions. In all cases, the ratio of non-synonymous/synonymous nucleotide changes was < 1, indicating that ADV N gene is under predominantly purifying selection.

The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile.

We have determined the complete genome sequence of a new rhabdovirus, tentatively named Caligus rogercresseyi rhabdovirus Ch01 (CrRV-Ch01), which was found in the parasite Caligus rogercresseyi, present on farmed Atlantic salmon (Salmo salar) in Chile. The genome encodes the five canonical rhabdovirus proteins in addition to an unknown protein, in the order N-P-M-U (unknown)-G-L. Phylogenetic analysis showed that the virus clusters with two rhabdoviruses (Lepeophtheirus salmonis rhabdovirus No9 and Lepeophtheirus salmonis rhabdovirus No127) obtained from another parasitic caligid, Lepeophtheirus salmonis, present on farmed Atlantic salmon on the west coast of Norway.

Identification and Characterization of Citrus Chlorotic Spot Virus, a New Dichorhavirus Associated with Citrus Leprosis-Like Symptoms.

Local chlorotic spots resembling early lesions characteristic of citrus leprosis (CL) were observed in leaves of two sweet orange (Citrus sinensis L.) trees in Teresina, State of Piauí, Brazil, in early 2017. However, despite the similarities, these spots were generally larger than those of a typical CL and showed rare or no necrosis symptoms. In symptomatic tissues, transmission electron microscopy revealed the presence of viroplasms in the nuclei of the infected parenchymal cells and rod-shaped particles with an average size of approximately 40 × 100 nm, resembling those typically observed during infection by dichorhaviruses. A bipartite genome of the putative novel virus, tentatively named citrus chlorotic spot virus (CiCSV) (RNA1 = 6,518 nucleotides [nt] and RNA2 = 5,987 nt), revealed the highest nucleotide sequence identity values with the dichorhaviruses coffee ringspot virus strain Lavras (73.8%), citrus leprosis virus N strain Ibi1 (58.6%), and orchid fleck virus strain So (56.9%). In addition to citrus, CiCSV was also found in local chlorotic lesions on leaves of the ornamental plant beach hibiscus (Talipariti tiliaceum (L.) Fryxell). Morphological characterization of mites recovered from the infected plants revealed at least two different types of Brevipalpus. One of them corresponds to Brevipalpus yothersi. The other is slightly different from B. yothersi mites but comprises traits that possibly place it as another species. A mix of the two mite types collected on beach hibiscus successfully transmitted CiCSV to arabidopsis plants but additional work is required to verify whether both types of flat mite may act as viral vectors. The current study reveals a newly described dichorhavirus associated with a citrus disease in the northeastern region of Brazil.

Complete genome sequence of maize yellow striate virus, a new cytorhabdovirus infecting maize and wheat crops in Argentina.

A rhabdovirus infecting maize and wheat crops in Argentina was molecularly characterized. Through next-generation sequencing (NGS) of symptomatic leaf samples, the complete genome was obtained of two isolates of maize yellow striate virus (MYSV), a putative new rhabdovirus, differing by only 0.4% at the nucleotide level. The MYSV genome consists of 12,654 nucleotides for maize and wheat virus isolates, and shares 71% nucleotide sequence identity with the complete genome of barley yellow striate mosaic virus (BYSMV, NC028244). Ten open reading frames (ORFs) were predicted in the MYSV genome from the antigenomic strand and were compared with their BYSMV counterparts. The highest amino acid sequence identity of the MYSV and BYSMV proteins was 80% between the L proteins, and the lowest was 37% between the proteins 4. Phylogenetic analysis suggested that the MYSV isolates are new members of the genus Cytorhabdovirus, family Rhabdoviridae. Yellow striate, affecting maize and wheat crops in Argentina, is an emergent disease that presents a potential economic risk for these widely distributed crops.

Molecular characterization of yerba mate chlorosis-associated virus, a putative cytorhabdovirus infecting yerba mate (Ilex paraguariensis).

We present the molecular characterization of a new virus infecting yerba mate (Ilex paraguariensis St. Hil.) in Argentina. Deep sequencing of diseased yerba mate plants showing chlorotic linear patterns, chlorotic rings, and vein yellowing resulted in the identification of a new virus resembling plant rhabdoviruses in sequence and genome structure. We have determined the complete genome sequence of this virus, which is 12,876 nt long. Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3'-N-P-P3-P4-M-G-L-5'. Phylogenetic analysis suggested that the described virus is a new member of the genus Cytorhabdovirus, which was supported by the observation of rhabdovirus-like particles within the cytoplasm of infected yerba mate cells. The virus has been tentatively named "yerba mate chlorosis-associated virus" (YmCaV). The availability of the YmCaV genome sequence will contribute to assessing the genetic variability of this virus and determining its role in this yerba mate disease.

Almendravirus: A Proposed New Genus of Rhabdoviruses Isolated from Mosquitoes in Tropical Regions of the Americas.

The Rhabdoviridae is a diverse family of negative-sense single-stranded RNA viruses, many of which infect vertebrate hosts and are transmitted by hematophagous arthropods. Others appear to be arthropod specific, circulating only within arthropod populations. Herein, we report the isolation and characterization of three novel viruses from mosquitoes collected from the Americas. Coot Bay virus was isolated from Anopheles quadrimaculatus mosquitoes collected in the Everglades National Park, Florida; Rio Chico virus was isolated from Anopheles triannulatus mosquitoes collected in Panama; and Balsa virus was isolated from two pools of Culex erraticus mosquitoes collected in Colombia. Sequence analysis indicated that the viruses share a similar genome organization to Arboretum virus and Puerto Almendras virus that had previously been isolated from mosquitoes collected in Peru. Each genome features the five canonical rhabdovirus structural protein genes as well as a gene encoding a class 1A viroporin-like protein (U1) located between the G and L genes (3'-N-P-M-G-U1-L-5'). Phylogenetic analysis of complete L protein sequences indicated that all five viruses cluster in a unique clade that is relatively deeply rooted in the ancestry of animal rhabdoviruses. The failure of all viruses in this clade to grow in newborn mice or vertebrate cells in culture suggests that they may be poorly adapted to replication in vertebrates.

Merida virus, a putative novel rhabdovirus discovered in Culex and Ochlerotatus spp. mosquitoes in the Yucatan Peninsula of Mexico.

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.

Detection and survey of coffee ringspot virus in Brazil.

Coffee ringspot virus (CoRSV) a member of the proposed genus "Dichorhavirus", was surveyed on commercial and research farms spanning an area responsible for the majority of Coffea arabica production in Brazil. Virus-infected plants were found at one hundred percent of locations (n = 45) sampled. All cultivars, regardless of cherry color, were found to serve as hosts, suggesting that there is limited resistance in commercially employed germplasm. Reverse transcription PCR analysis revealed that the virus is contained within symptomatic lesions, with little systemic spread throughout leaves. Phylogenetic analysis based on the ORF1 (nucleocapsid) gene identified a strong geo-spatial relationship among isolates, which clustered into three clades. Despite low genetic diversity among isolates, variation in symptom expression was observed in the experimental host Chenopodium quinoa. Our analyses support the hypothesis that the spread of CoRSV is constrained by the clonal expansion of thelytokous populations of Brevipalpus phoenicis. The widespread occurrence of this virus suggests that it is much more prevalent than previously thought.