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Biting midges (Culicoides) are vectors of many pathogens of medical and veterinary importance, but their viromes are poorly characterized compared to certain other hematophagous arthropods, e.g., mosquitoes and ticks. The goal of this study was to use metagenomics to identify viruses in Culicoides from Mexico. A total of 457 adult midges were collected in Chihuahua, northern Mexico, in 2020 and 2021, and all were identified as female Culicoides reevesi. The midges were sorted into five pools and homogenized. An aliquot of each homogenate was subjected to polyethylene glycol precipitation to enrich for virions, then total RNA was extracted and analyzed by unbiased high-throughput sequencing. We identified six novel viruses that are characteristic of viruses from five families (Nodaviridae, Partitiviridae, Solemoviridae, Tombusviridae, and Totiviridae) and one novel virus that is too divergent from all classified viruses to be assigned to an established family. The newly discovered viruses are phylogenetically distinct from their closest known relatives, and their minimal infection rates in female C. reevesi range from 0.22 to 1.09. No previously known viruses were detected, presumably because viral metagenomics had never before been used to study Culicoides from the Western Hemisphere. To conclude, we discovered multiple novel viruses in C. reevesi from Mexico, expanding our knowledge of arthropod viral diversity and evolution.

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Mosquitoes can transmit several pathogenic viruses to humans, but their natural viral community is also composed of a myriad of other viruses such as insect-specific viruses (ISVs) and those that infect symbiotic microorganisms. Besides a growing number of studies investigating the mosquito virome, the majority are focused on few urban species, and relatively little is known about the virome of sylvatic mosquitoes, particularly in high biodiverse biomes such as the Brazilian biomes. Here, we characterized the RNA virome of 10 sylvatic mosquito species from Atlantic forest remains at a sylvatic-urban interface in Northeast Brazil employing a metatranscriptomic approach. A total of 16 viral families were detected. The phylogenetic reconstructions of 14 viral families revealed that the majority of the sequences are putative ISVs. The phylogenetic positioning and, in most cases, the association with a high RNA-dependent RNA polymerase amino acid divergence from other known viruses suggests that the viruses characterized here represent at least 34 new viral species. Therefore, the sylvatic mosquito viral community is predominantly composed of highly divergent viruses highlighting the limited knowledge we still have about the natural virome of mosquitoes in general. Moreover, we found that none of the viruses recovered were shared between the species investigated, and only one showed high identity to a virus detected in a mosquito sampled in Peru, South America. These findings add further in-depth understanding about the interactions and coevolution between mosquitoes and viruses in natural environments. IMPORTANCE: Mosquitoes are medically important insects as they transmit pathogenic viruses to humans and animals during blood feeding. However, their natural microbiota is also composed of a diverse set of viruses that cause no harm to the insect and other hosts, such as insect-specific viruses. In this study, we characterized the RNA virome of sylvatic mosquitoes from Northeast Brazil using unbiased metatranscriptomic sequencing and in-depth bioinformatic approaches. Our analysis revealed that these mosquitoes species harbor a diverse set of highly divergent viruses, and the majority comprises new viral species. Our findings revealed many new virus lineages characterized for the first time broadening our understanding about the natural interaction between mosquitoes and viruses. Finally, it also provided several complete genomes that warrant further assessment for mosquito and vertebrate host pathogenicity and their potential interference with pathogenic arboviruses.

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Hibiscus is not native to Colombia but well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of its leaves, was collected for virome analysis using meta-transcriptomic high-throughput sequencing technology. Bioinformatic analysis identified 12.5% of the total reads in the Ribo-Zero cDNA library which mapped to viral genomes. BLAST searches revealed the presence of carlavirus, potexvirus, and of known members of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus in the sample; confirmed by RT-PCR with virus-specific primers followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the mixed infection. Both RNA dependent RNA polymerase and coat protein gene sequences of the potex and carla viruses shared less than 72% nucleotide and 80% amino acid identities with any alphaflexi- and betaflexi-virus sequences available in GenBank, identifying three novel carlavirus and one potexvirus species in the Hibiscus rosa-sinensis plant. The detection of physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus are also new reports from Colombia. Overall, the meta-transcriptome analysis identified the complex virome associated with the black spot symptoms on hibiscus leaves and demonstrated the diversity of virus genera tolerated in the mixed infection of a single H. rosa-sinensis plant.

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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.

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A novel virga-like virus, provisionally named Rubber tree latent virus 2 (RTLV2), was identified from rubber tree (Hevea brasiliensis). It is a close relative of the previously reported Rubber tree latent virus 1 (RTLV1). The complete genomes of RTLV1 and RTLV2 were sequenced and comparatively analyzed in terms of genome organization, putative gene products and phylogenetic relationship. Both RTLV1 and RTLV2 have positive-sense single-stranded RNA genomes that encode seven open reading frames (ORFs), forming a similar genomic layout. In phylogenetic analyses based on replicase and coat protein amino acid sequences, RTLV1 and RTLV2 were clustered with unclassified virga-like viruses. They are distinct from currently recognized plant virus families. RTLV1 and RTLV2 can be distinguished from members of Virgaviridae by the presence of a putative coat protein duplex and a poly(A) tail at the 3'-terminus. The authenticity of RTLV1 and RTLV2 as infectious viruses was confirmed through field investigations and transmissibility assays. In conclusion, RTLV1 and RTLV2 represent a novel plant virus group that does not readily fit into current virus families.

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A previously uncharacterized torradovirus species infecting potatoes was detected by high-throughput sequencing from field samples from Peru and in customs intercepts in potato tubers that originated from South America in the United States of America and the Netherlands. This new potato torradovirus showed high nucleotide sequence identity to an unidentified isometric virus (SB26/29), which was associated with a disease named potato rugose stunting in southern Peru characterized over two decades ago. Thus, this virus is tentatively named potato rugose stunting virus (PotRSV). The genome of PotRSV isolates sequenced in this study were composed of two polyadenylated RNA segments. RNA1 ranges from 7,086 to 7,089 nt and RNA2 from 5,228 to 5,230 nt. RNA1 encodes a polyprotein containing the replication block (helicase-protease-polymerase), whereas RNA2 encodes a polyprotein cleaved into a movement protein and the three capsid proteins (CPs). Pairwise comparison among PotRSV isolates revealed amino acid identity values greater than 86% in the protease-polymerase (Pro-Pol) region and greater than 82% for the combined CPs. The closest torradovirus species, squash chlorotic leaf spot virus, shares amino acid identities of ∼58 and ∼41% in the Pro-Pol and the combined CPs, respectively. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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Babaco (Vasconcellea × heilbornii) is a subtropical species in the Caricaceae family. The plant is native to Ecuador and represents an important crop for hundreds of families. The objective of this study was to characterize, at the genomic level, two new babaco viruses identified by high-throughput sequencing. The viruses, an ilarvirus and a nucleorhabdovirus, were found in a symptomatic babaco plant from a commercial nursery in the Azuay province of Ecuador. The tripartite genome of the new ilarvirus, provisionally named babaco ilarvirus 1 (BabIV-1), is related to subgroup 3 ilarviruses, including apple mosaic virus, apple necrotic mosaic virus, and prunus necrotic ringspot virus as the closest relatives. The genome of the nucleorhabdovirus, provisionally named babaco nucleorhabdovirus 1 (BabRV-1), showed the closest relation with joa yellow blotch-associated virus and potato yellow dwarf nucleorhabdovirus. Molecular-based detection methods found BabIV-1 and BabRV-1 in 21% and 36%, respectively, of plants surveyed in a commercial babaco nursery, highlighting the importance of enforcing virus testing and nursery certification programs for babaco.

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Ophioviruses (genus Ophiovirus, family Aspiviridae) are plant-infecting viruses with non-enveloped, filamentous, naked nucleocapsid virions. Members of the genus Ophiovirus have a segmented single-stranded negative-sense RNA genome (ca. 11.3-12.5 kb), encompassing three or four linear segments. In total, these segments encode four to seven proteins in the sense and antisense orientation, both in the viral and complementary strands. The genus Ophiovirus includes seven species with viruses infecting both monocots and dicots, mostly trees, shrubs and some ornamentals. From a genomic perspective, as of today, there are complete genomes available for only four species. Here, by exploring large publicly available metatranscriptomics datasets, we report the identification and molecular characterization of 33 novel viruses with genetic and evolutionary cues of ophioviruses. Genetic distance and evolutionary insights suggest that all the detected viruses could correspond to members of novel species, which expand the current diversity of ophioviruses ca. 4.5-fold. The detected viruses increase the tentative host range of ophioviruses for the first time to mosses, liverwort and ferns. In addition, the viruses were linked to several Asteraceae, Orchidaceae and Poaceae crops/ornamental plants. Phylogenetic analyses showed a novel clade of mosses, liverworts and fern ophioviruses, characterized by long branches, suggesting that there is still plenty of unsampled hidden diversity within the genus. This study represents a significant expansion of the genomics of ophioviruses, opening the door to future works on the molecular and evolutionary peculiarity of this virus genus.

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The family Tymoviridae comprises positive-sense RNA viruses, which mainly infect plants. Recently, a few Tymoviridae-like viruses have been found in mosquitoes, which feed on vertebrate sources. We describe a novel Tymoviridae-like virus, putatively named, Guachaca virus (GUAV), isolated from Culex pipiens and Culex quinquefasciatus species of mosquitoes and collected in the rural area of Santa Marta, Colombia. After a cytopathic effect was observed in C6/36 cells, RNA was extracted and processed through the NetoVIR next-generation sequencing protocol, and data were analyzed through the VirMAP pipeline. Molecular and phenotypic characterization of the GUAV was achieved using a 5'/3' RACE, transmission electron microscopy, amplification in vertebrate cells, and phylogenetic analysis. A cytopathic effect was observed in C6/36 cells three days post-infection. The GUAV genome was successfully assembled, and its polyadenylated 3' end was corroborated. GUAV shared only 54.9% amino acid identity with its closest relative, Ek Balam virus, and was grouped with the latter and other unclassified insect-associated tymoviruses in a phylogenetic analysis. GUAV is a new member of a family previously described as comprising plant-infecting viruses, which seem to infect and replicate in mosquitoes. The sugar- and blood-feeding behavior of the Culex spp., implies a sustained contact with plants and vertebrates and justifies further studies to unravel the ecological scenario for transmission.

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Morbilliviruses are negative-sense single-stranded monosegmented RNA viruses in the family Paramyxoviridae (order Mononegavirales). Morbilliviruses infect diverse mammals including humans, dogs, cats, small ruminants, seals, and cetaceans, which serve as natural hosts. Here, I report the identification and characterization of novel viruses detected in public RNAseq datasets of South American long-haired and olive field mice. The divergent viruses dubbed Ratón oliváceo morbillivirus (RoMV) detected in renal samples from mice collected from Chile and Argentina are characterized by an unusually large genome including long intergenic regions and the presence of an accessory protein between the F and H genes redounding in a genome architecture consisting in 3'-N-P/V/C-M-F-hp-H-L-5'. Structural and functional annotation, genetic distance, and evolutionary insights suggest that RoMV is a member of a novel species within genus Morbillivirus tentatively named as South American mouse morbillivirus. Phylogenetic analysis suggests that this mouse morbillivirus is closely related to and clusters into a monophyletic group of novel rodent-borne morbilliviruses. This subclade of divergent viruses expands the host range, redefines the genomic organization and provides insights on the evolutionary history of genus Morbillivirus.

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The number of viral-associated neoplasms reported in wildlife has increased over the last decades, likely because of growing research efforts and a potentially greater burden of carcinogenic pathogens. Herein, we describe a primary gastric T-cell lymphoma in one free-ranging giant armadillo (Priodontes maximus) from Brazilian Pantanal infected by a novel gammaherpesvirus, proposed as Cingulatid gammaherpesvirus 1 (CiHV-1). By chromogenic in situ hybridisation against Epstein-Barr virus some neoplastic cells were labeled. Subsequently, a molecular screening was carried out to detect the occurrence of this pathogen in other giant armadillos in the same region. Overall, this novel virus was detected in 14.3% (3/21) of the tested giant armadillos. We suggest this herpesvirus, the first in Xenarthra, as a plausible aetiology of the neoplasm. The implications of CiHV-1 for this species are uncertain; while no outbreaks of disease have been recorded, the present study raises concerns. Further research is warranted to assess the real significance of CiHV-1 and its potential oncogenic role in this species.

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Paramyxoviruses have a broad host range and geographic distribution, including human pathogens transmitted by bats, such as Nipah and Hendra viruses. In this study, we combined high-throughput sequencing and molecular approaches to investigate the presence of paramyxoviruses in neotropical bats (Microchiroptera suborder) in Brazil. We discovered and characterized three novel paramyxoviruses in the kidney tissues of apparently healthy common vampire bats (D. rotundus) and Seba's short-tailed bats (C. perspicillata), which we tentatively named Kanhgág virus (KANV), Boe virus (BOEV), and Guató virus (GUATV). In this study, we classified these viruses as putative species into the Macrojêvirus genus, a newly proposed genus of the Orthoparamyxovirinae subfamily. Using RT-PCR, we detected these viruses in 20.9% (9 out of 43) of bats tested, and viral RNA was detected exclusively in kidney tissues. Attempts to isolate infectious virus were successful for KANV and GUATV. Our results expand the viral diversity, host range, and geographical distribution of the paramyxoviruses.

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The use of high-throughput sequencing (HTS) for virus diagnostics, as well as the importance of this technology as a valuable tool for discovery of novel viruses has been extensively investigated. In this review, we consider the application of HTS approaches to uncover novel plant viruses with a focus on the negative-sense, single-stranded RNA virosphere. Plant viruses with negative-sense and ambisense RNA (NSR) genomes belong to several taxonomic families, including Rhabdoviridae, Aspiviridae, Fimoviridae, Tospoviridae, and Phenuiviridae. They include both emergent pathogens that infect a wide range of plant species, and potential endophytes which appear not to induce any visible symptoms. As a consequence of biased sampling based on a narrow focus on crops with disease symptoms, the number of NSR plant viruses identified so far represents only a fraction of this type of viruses present in the virosphere. Detection and molecular characterization of NSR viruses has often been challenging, but the widespread implementation of HTS has facilitated not only the identification but also the characterization of the genomic sequences of at least 70 NSR plant viruses in the last 7 years. Moreover, continuing advances in HTS technologies and bioinformatic pipelines, concomitant with a significant cost reduction has led to its use as a routine method of choice, supporting the foundations of a diverse array of novel applications such as quarantine analysis of traded plant materials and genetic resources, virus detection in insect vectors, analysis of virus communities in individual plants, and assessment of virus evolution through ecogenomics, among others. The insights from these advancements are shedding new light on the extensive diversity of NSR plant viruses and their complex evolution, and provide an essential framework for improved taxonomic classification of plant NSR viruses as part of the realm Riboviria. Thus, HTS-based methods for virus discovery, our 'new eyes,' are unraveling in real time the richness and magnitude of the plant RNA virosphere.

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The nearly complete genome sequences of two Cucumis melo endornavirus (CmEV) strains were obtained using deep sequencing while investigating fecal samples for the presence of gastroenteritis viruses. The Brazilian CmEV BRA/TO-23 (aa positions 116-5027) and BRA/TO-74 (aa positions 26-5057) strains were nearly identical to the reference CmEV CL-01 (USA) and SJ1 (South Korea) strains, showing 97% and 98% of nucleotide and amino acid identity, respectively. Endornaviruses are not known to be associated with human disease and their presence may simply reflect recent dietary consumption. Metagenomic analyses offered an opportunity to identify for the first time in Brazil a newly described endornavirus species.

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A metagenomics approach was used to detect novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico. A total of 1359 mosquitoes of 7 species and 5 genera (Aedes, Anopheles, Culex, Mansonia and Psorophora) were sorted into 37 pools, homogenized and inoculated onto monolayers of Aedes albopictus (C6/36) cells. A second blind passage was performed and then total RNA was extracted and analysed by RNA-seq. Two novel viruses, designated Uxmal virus and Mayapan virus, were identified. Uxmal virus was isolated from three pools of Aedes (Ochlerotatus) taeniorhynchus and phylogenetic data indicate that it should be classified within the recently proposed taxon Negevirus. Mayapan virus was recovered from two pools of Psorophora ferox and is most closely related to unclassified Nodaviridae-like viruses. Two recognized viruses were also detected: Culex flavivirus (family Flaviviridae) and Houston virus (family Mesoniviridae), with one and two isolates being recovered, respectively. The in vitro host ranges of all four viruses were determined by assessing their replicative abilities in cell lines of avian, human, monkey, hamster, murine, lepidopteran and mosquito (Aedes, Anopheles and Culex) origin, revealing that all viruses possess vertebrate replication-incompetent phenotypes. In conclusion, we report the isolation of both novel and recognized RNA viruses from mosquitoes collected in Mexico, and add to the growing plethora of viruses discovered recently through the use of metagenomics.

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We report here the complete genome sequence of a bipartite virus, herein denoted WLPRV/human/BRA/TO-34/201, from a sample collected in 2015 from a two-year-old child in Brazil presenting acute gastroenteritis. The virus has 98-99% identity (segments 2 and 1, respectively) with the Wuhan large pig roundworm virus (unclassified RNA virus) that was recently discovered in the stomachs of pigs from China. This is the first report of a Wuhan large pig roundworm virus detected in human specimens, and the second genome described worldwide. However, the generation of more sequence data and further functional studies are required to fully understand the ecology, epidemiology, and evolution of this new unclassified virus.

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The golden orb-weaver spider Nephila clavipes, known for its sexual size dimorphism, is abundant and widespread in the New World. The first annotated genome of orb-weaver spiders, exploring N. clavipes, has recently been reported. The study, focused primarily on the diversity of silk specific genes, shed light into the complex evolutionary history of spiders. Furthermore, a robust transcriptome analysis provided a massive resource for N. clavipes RNA survey. Here, I present evidence of viral sequences corresponding to the first 10 extant virus species associated to N. clavipes and indeed, nephilids. The putatively new species are linked to ssRNA positive-strand viruses, such as Picornavirales, and to ssRNA negative-strand and dsRNA viruses. In addition, I detected sequence data of new strains of two recently reported arthropod viruses, which complemented and extended the corresponding sequence references. The identified viruses appear to be complete, potentially functional, and presenting the typical architecture and consistent viral domains. The intrinsic nature of the detected sequences and their absence in the recently generated genome assembly, suggest that they correspond to bona fide RNA virus sequences. The available RNA data allowed for the first time to address a tissue/organ specific analysis of virus loads/presence in spiders, suggesting a complex spatial and differential distribution of the tentative viruses, encompassing the spider brain and also silk and venom glands. Until recently, the virus landscape associated to spiders remained elusive. The discovered viruses described here provide only a fragmented glimpse of the potential magnitude of the Aranea virosphere. Future studies should focus not only on complementing and expanding these findings, but also on addressing the potential ecological role of these viruses, which might influence the biology of these outstanding arthropod species.

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In this study, we describe a novel putative Enamovirus member, Grapevine enamovirus-1 (GEV-1), discovered by high-throughput sequencing (HTS). A limited survey using HTS of 17 grapevines (Vitis spp.) from the south, southeast, and northeast regions of Brazil led to the detection of GEV-1 exclusively on southern plants, infecting four grapevine cultivars (Cabernet Sauvignon, Semillon, CG 90450, and Cabernet franc) with a remarkable identity of around 99% at the nucleotide level. This novel virus was only detected in multiple-virus infected plants exhibiting viral-like symptoms. GEV-1 was also detected on a cv. Malvasia Longa by RT-PCR. We performed graft-transmissibility assays on GEV-1. The organization, products, and cis-acting regulatory elements of GEV-1 genome are also discussed here. The near complete genome sequence of GEV-1 was obtained during the course of this study, lacking only part of the 3' untranslated terminal region. This is the first report of a virus in the family Luteoviridae infecting grapevines. Based on its genomic properties and phylogenetic analyses, GEV-1 should be classified as a new member of the genus Enamovirus.

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