A systematic review on current approaches in bat virus discovered between 2018-2022.

Zoonotic viruses are widely seen as the primary threat for future pandemics. Bats are the most diverse group of mammals, with more than 1400 species distributed across most habitats on Earth. So far, 31 known virus families were associated with bats, although the understanding of most viruses were insufficient. Continuous efforts to discover, understand and monitor these bats viruses, is thereby an area of public health interest. This systematic review was designed to catalogue publications reporting novel bat virus discoveries within PubMed, SCOPUS, and Web of Science databases, within a 5-year period from 2018-2022. Various experimental parameters, including sampling locations, methodology, bat species diversity, similarity to known viruses, species demarcation of new viruses, and genomic sequencing strategies, were extracted from 41 publications and analyzed. In total, 72 novel viruses from 19 virus families were identified between 2018 and 2022, particularly from Genomoviridae (DNA viruses) and Coronaviridae (RNA viruses). That said, only a limited number of bat families featured extensively despite noticeable shift towards next generation sequencing methods and metagenomics pipeline for virus identification across different sampling methods. This review aims to provide a comprehensive analysis of the global efforts made over the past five years to identify and characterize emerging viruses in bat species, and to provide a detailed overview of the current technologies and methodologies used in these studies.

Highly divergent and diverse viral community infecting sylvatic mosquitoes from Northeast Brazil.

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.

Complete genome sequence of a novel amalgavirus in sponge gourd, Luffa cylindrica.

A novel monopartite dsRNA virus, tentatively named "sponge gourd amalgavirus 1" (SGAV1), was discovered by high-throughput sequencing in sponge gourd (Luffa cylindrica) displaying mosaic symptoms in Jiashan County, Zhejiang Province, China. The genome of SGAV1 is 3,447 nucleotides in length and contains partially overlapping open reading frames (ORFs) encoding a putative replication factory matrix-like protein and a fusion protein, respectively. The fusion protein of SGAV1 shares 57.07% identity with the homologous protein of salvia miltiorrhiza amalgavirus 1 (accession no. DAZ91057.1). Phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) protein suggests that SGAV1 belongs to the genus Amalgavirus of the family Amalgaviridae. Moreover, analysis of SGAV1-derived small interfering RNAs indicated that SGAV1 was actively replicating in the host plant. Semi-quantitative RT-PCR showed higher levels of SGAV1 expression in leaves than in flowers and fruits. This is the first report of a novel amalgavirus found in sponge gourd in China.

Discovery of Novel Viruses in Culicoides Biting Midges in Chihuahua, Mexico.

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.

Entourage: all-in-one sequence analysis software for genome assembly, virus detection, virus discovery, and intrasample variation profiling.

BACKGROUND: Pan-virus detection, and virome investigation in general, can be challenging, mainly due to the lack of universally conserved genetic elements in viruses. Metagenomic next-generation sequencing can offer a promising solution to this problem by providing an unbiased overview of the microbial community, enabling detection of any viruses without prior target selection. However, a major challenge in utilising metagenomic next-generation sequencing for virome investigation is that data analysis can be highly complex, involving numerous data processing steps. RESULTS: Here, we present Entourage to address this challenge. Entourage enables short-read sequence assembly, viral sequence search with or without reference virus targets using contig-based approaches, and intrasample sequence variation quantification. Several workflows are implemented in Entourage to facilitate end-to-end virus sequence detection analysis through a single command line, from read cleaning, sequence assembly, to virus sequence searching. The results generated are comprehensive, allowing for thorough quality control, reliability assessment, and interpretation. We illustrate Entourage's utility as a streamlined workflow for virus detection by employing it to comprehensively search for target virus sequences and beyond in raw sequence read data generated from HeLa cell culture samples spiked with viruses. Furthermore, we showcase its flexibility and performance on a real-world dataset by analysing a preassembled Tara Oceans dataset. Overall, our results show that Entourage performs well even with low virus sequencing depth in single digits, and it can be used to discover novel viruses effectively. Additionally, by using sequence data generated from a patient with chronic SARS-CoV-2 infection, we demonstrate Entourage's capability to quantify virus intrasample genetic variations, and generate publication-quality figures illustrating the results. CONCLUSIONS: Entourage is an all-in-one, versatile, and streamlined bioinformatics software for virome investigation, developed with a focus on ease of use. Entourage is available at https://codeberg.org/CENMIG/Entourage under the MIT license.

Hecatomb: an integrated software platform for viral metagenomics.

BACKGROUND: Modern sequencing technologies offer extraordinary opportunities for virus discovery and virome analysis. Annotation of viral sequences from metagenomic data requires a complex series of steps to ensure accurate annotation of individual reads and assembled contigs. In addition, varying study designs will require project-specific statistical analyses. FINDINGS: Here we introduce Hecatomb, a bioinformatic platform coordinating commonly used tasks required for virome analysis. Hecatomb means "a great sacrifice." In this setting, Hecatomb is "sacrificing" false-positive viral annotations using extensive quality control and tiered-database searches. Hecatomb processes metagenomic data obtained from both short- and long-read sequencing technologies, providing annotations to individual sequences and assembled contigs. Results are provided in commonly used data formats useful for downstream analysis. Here we demonstrate the functionality of Hecatomb through the reanalysis of a primate enteric and a novel coral reef virome. CONCLUSION: Hecatomb provides an integrated platform to manage many commonly used steps for virome characterization, including rigorous quality control, host removal, and both read- and contig-based analysis. Each step is managed using the Snakemake workflow manager with dependency management using Conda. Hecatomb outputs several tables properly formatted for immediate use within popular data analysis and visualization tools, enabling effective data interpretation for a variety of study designs. Hecatomb is hosted on GitHub (github.com/shandley/hecatomb) and is available for installation from Bioconda and PyPI.

Detection, sequencing, and tissue distribution of piscine orthoreovirus 2-like virus in diseased coho salmon in Alaska.

We performed a diagnostic disease investigation on a cohort of coho salmon (Oncorhynchus kisutch) fingerlings in Alaska exhibiting anorexia, gaping mouths, anemia, and increased mortality. Histologic examination revealed mild-to-severe myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis, moderate splenic histiocytosis, and mild renal histiocytosis. Piscine orthoreoviruses 1 and 3 were not detected by molecular methods, and no other viruses could be cultured on 3 common diagnostic fish cell lines. De novo assembly produced a viral genome of 10 linear segments with >80% homology to piscine orthoreovirus 2 (PRV2) encoding all 11 PRV2 proteins. An in situ hybridization probe using RNAscope was developed against 697 viral nucleotides identified by sequencing, which revealed viral genome in heart, spleen, gill, kidney, liver, blood, and the lamina propria of the intestines. Our findings are supportive of a novel piscine orthoreovirus most closely related to PRV2 associated with morbidity and mortality of coho salmon in the northeastern Pacific.

A parasite odyssey: An RNA virus concealed in Toxoplasma gondii.

We are entering a 'Platinum Age of Virus Discovery', an era marked by exponential growth in the discovery of virus biodiversity, and driven by advances in metagenomics and computational analysis. In the ecosystem of a human (or any animal) there are more species of viruses than simply those directly infecting the animal cells. Viruses can infect all organisms constituting the microbiome, including bacteria, fungi, and unicellular parasites. Thus the complexity of possible interactions between host, microbe, and viruses is unfathomable. To understand this interaction network we must employ computationally assisted virology as a means of analyzing and interpreting the millions of available samples to make inferences about the ways in which viruses may intersect human health. From a computational viral screen of human neuronal datasets, we identified a novel narnavirus Apocryptovirus odysseus (Ao) which likely infects the neurotropic parasite Toxoplasma gondii. Previously, several parasitic protozoan viruses (PPVs) have been mechanistically established as triggers of host innate responses, and here we present in silico evidence that Ao is a plausible pro-inflammatory factor in human and mouse cells infected by T. gondii. T. gondii infects billions of people worldwide, yet the prognosis of toxoplasmosis disease is highly variable, and PPVs like Ao could function as a hitherto undescribed hypervirulence factor. In a broader screen of over 7.6 million samples, we explored phylogenetically proximal viruses to Ao and discovered nineteen Apocryptovirus species, all found in libraries annotated as vertebrate transcriptome or metatranscriptomes. While samples containing this genus of narnaviruses are derived from sheep, goat, bat, rabbit, chicken, and pigeon samples, the presence of virus is strongly predictive of parasitic Apicomplexa nucleic acid co-occurrence, supporting the fact that Apocryptovirus is a genus of parasite-infecting viruses. This is a computational proof-of-concept study in which we rapidly analyze millions of datasets from which we distilled a mechanistically, ecologically, and phylogenetically refined hypothesis. We predict that this highly diverged Ao RNA virus is biologically a T. gondii infection, and that Ao, and other viruses like it, will modulate this disease which afflicts billions worldwide.

Discovery and characterization of novel DNA viruses in Apis mellifera: expanding the honey bee virome through metagenomic analysis.

To date, many viruses have been discovered to infect honey bees. In this study, we used high-throughput sequencing to expand the known virome of the honey bee, Apis mellifera, by identifying several novel DNA viruses. While the majority of previously identified bee viruses are RNA, our study reveals nine new genomes from the Parvoviridae family, tentatively named Bee densoviruses 1 to 9. In addition, we characterized a large DNA virus, Apis mellifera filamentous-like virus (AmFLV), which shares limited protein identities with the known Apis mellifera filamentous virus. The complete sequence of AmFLV, obtained by a combination of laboratory techniques and bioinformatics, spans 152,678 bp. Linear dsDNA genome encodes for 112 proteins, of which 49 are annotated. Another large virus we discovered is Apis mellifera nudivirus, which belongs to a group of Alphanudivirus. The virus has a length of 129,467 bp and a circular dsDNA genome, and has 106 protein encoding genes. The virus contains most of the core genes of the family Nudiviridae. This research demonstrates the effectiveness of viral binning in identifying viruses in honey bee virology, showcasing its initial application in this field.IMPORTANCEHoney bees contribute significantly to food security by providing pollination services. Understanding the virome of honey bees is crucial for the health and conservation of bee populations and also for the stability of the ecosystems and economies for which they are indispensable. This study unveils previously unknown DNA viruses in the honey bee virome, expanding our knowledge of potential threats to bee health. The use of the viral binning approach we employed in this study offers a promising method to uncovering and understanding the vast viral diversity in these essential pollinators.

A tale of caution: How endogenous viral elements affect virus discovery in transcriptomic data.

Large-scale metagenomic and -transcriptomic studies have revolutionized our understanding of viral diversity and abundance. In contrast, endogenous viral elements (EVEs), remnants of viral sequences integrated into host genomes, have received limited attention in the context of virus discovery, especially in RNA-Seq data. EVEs resemble their original viruses, a challenge that makes distinguishing between active infections and integrated remnants difficult, affecting virus classification and biases downstream analyses. Here, we systematically assess the effects of EVEs on a prototypical virus discovery pipeline, evaluate their impact on data integrity and classification accuracy, and provide some recommendations for better practices. We examined EVEs and exogenous viral sequences linked to Orthomyxoviridae, a diverse family of negative-sense segmented RNA viruses, in 13 genomic and 538 transcriptomic datasets of Culicinae mosquitoes. Our analysis revealed a substantial number of viral sequences in transcriptomic datasets. However, a significant portion appeared not to be exogenous viruses but transcripts derived from EVEs. Distinguishing between transcribed EVEs and exogenous virus sequences was especially difficult in samples with low viral abundance. For example, three transcribed EVEs showed full-length segments, devoid of frameshift and nonsense mutations, exhibiting sufficient mean read depths that qualify them as exogenous virus hits. Mapping reads on a host genome containing EVEs before assembly somewhat alleviated the EVE burden, but it led to a drastic reduction of viral hits and reduced quality of assemblies, especially in regions of the viral genome relatively similar to EVEs. Our study highlights that our knowledge of the genetic diversity of viruses can be altered by the underestimated presence of EVEs in transcriptomic datasets, leading to false positives and altered or missing sequence information. Thus, recognizing and addressing the influence of EVEs in virus discovery pipelines will be key in enhancing our ability to capture the full spectrum of viral diversity.

Characterization of the genomic sequence of a circo-like virus and of three chaphamaparvoviruses detected in mute swan (Cygnus olor).

We report the complete genomes of four ssDNA viruses: a circular replication-associated protein-encoding single-stranded DNA virus belonging to a clade previously detected only in mammals, and three chaphamaparvoviruses, which were detected by viromic surveillance of mute swan (Cygnus olor) fecal samples from the United Kingdom.

Meta-Transcriptomic Analysis Uncovers the Presence of Four Novel Viruses and Multiple Known Virus Genera in a Single Hibiscus rosa-sinensis Plant in Colombia.

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.

A brief tale of two pioneering moments: Europe's first discovery of Tick-Borne Encephalitis (TBE) virus beyond the Soviet Union and the largest alimentary TBE outbreak in history.

The emergence of tick-borne encephalitis (TBE) in Europe marked several significant milestones. The discovery of TBE in Czechoslovakia in 1948, with Gallia and Krejcí simultaneously isolating the TBE virus (TBEV) from human samples for the first time in Europe outside the Soviet Union, was pivotal. Subsequent TBEV isolation from ticks suggested the viral transmission via this vector. In 1951, the outbreak in Roznava in Slovakia (Czechoslovakia) revealed an unexpected mode of transmission, unpasteurized milk from a local dairy, challenging existing understanding. Investigations exposed illicit practices of mixing cow's milk with goat's milk for economic gains. Laboratory research confirmed the outbreak was caused by TBEV, which was substantiated by serological analyses. This was the first and largest documented alimentary TBE outbreak in history. In this review, we delve into both published sources and unpublished archival data, offering a comprehensive understanding of these historic accomplishments and shedding light on these pivotal moments.

Virome profiling of fig wasps (Ceratosolen spp.) reveals virus diversity spanning four realms.

We investigated the virome of agaonid fig wasps (Ceratosolen spp.) inside syconia ("fruits") of various Ficus trees fed upon by frugivores such as pteropodid bats in Sub-Saharan Africa. This virome includes representatives of viral families spanning four realms and includes near-complete genome sequences of three novel viruses and fragments of five additional potentially novel viruses evolutionarily associated with insects, fungi, plants, and vertebrates. Our study provides evidence that frugivorous animals are exposed to a plethora of viruses by coincidental consumption of fig wasps, which are obligate pollinators of figs worldwide.

Characterisation of putative novel tick viruses and zoonotic risk prediction.

Tick-associated viruses remain a substantial zoonotic risk worldwide, so knowledge of the diversity of tick viruses has potential health consequences. Despite their importance, large amounts of sequences in public data sets from tick meta-genomic and -transcriptomic projects remain unannotated, sequence data that could contain undocumented viruses. Through data mining and bioinformatic analysis of more than 37,800 public meta-genomic and -transcriptomic data sets, we found 83 unannotated contigs exhibiting high identity with known tick viruses. These putative viral contigs were classified into three RNA viral families (Alphatetraviridae, Orthomyxoviridae and Chuviridae) and one DNA viral family (Asfarviridae). After manual checking of quality and dissimilarity towards other sequences in the data set, these 83 contigs were reduced to five contigs in the Alphatetraviridae from four putative viruses, four in the Orthomyxoviridae from two putative viruses and one in the Chuviridae which clustered with known tick-associated viruses, forming a separate clade within the viral families. We further attempted to assess which previously known tick viruses likely represent zoonotic risks and thus deserve further investigation. We ranked the human infection potential of 133 known tick-associated viruses using a genome composition-based machine learning model. We found five high-risk tick-associated viruses (Langat virus, Lonestar tick chuvirus 1, Grotenhout virus, Taggert virus and Johnston Atoll virus) that have not been known to infect human and two viral families (Nairoviridae and Phenuiviridae) that contain a large proportion of potential zoonotic tick-associated viruses. This adds to the knowledge of tick virus diversity and highlights the importance of surveillance of newly emerging tick-associated diseases.

Diversity and connectedness of brine shrimp viruses in global hypersaline ecosystems.

Brine shrimp (Artemia) has existed on Earth for 400 million years and has major ecological importance in hypersaline ecosystems. As a crucial live food in aquaculture, brine shrimp cysts have become one of the most important aquatic products traded worldwide. However, our understanding of the biodiversity, prevalence and global connectedness of viruses in brine shrimp is still very limited. A total of 143 batches of brine shrimp (belonging to seven species) cysts were collected from six continents including 21 countries and more than 100 geographic locations worldwide during 1977-2019. In total, 55 novel RNA viruses were identified, which could be assigned to 18 different viral families and related clades. Eleven viruses were dsRNA viruses, 16 were +ssRNA viruses, and 28 were-ssRNA viruses. Phylogenetic analyses of the RNA-directed RNA polymerase (RdRp) showed that brine shrimp viruses were often grouped with viruses isolated from other invertebrates and fungi. Remarkably, most brine shrimp viruses were related to those from different hosts that might feed on brine shrimp or share the same ecological niche. A notable case was the novel brine shrimp noda-like virus 3, which shared 79.25% (RdRp) and 63.88% (capsid proteins) amino acid identity with covert mortality nodavirus (CMNV) that may cause losses in aquaculture. In addition, both virome composition and phylogenetic analyses revealed global connectedness in certain brine shrimp viruses, particularly among Asia and Northern America. This highlights the incredible species diversity of viruses in these ancient species and provides essential data for the prevalence of RNA viruses in the global aquaculture industry. More broadly, these findings provide novel insights into the previously unrecognized RNA virosphere in hypersaline ecosystems worldwide and demonstrate that human activity might have driven the global connectedness of brine shrimp viruses.

Optimized Recovery of Viral DNA and RNA from Blood Plasma for Viral Metagenomics.

Metagenomics is vastly improving our ability to discover new viruses, as well as their possible associations with disease. However, metagenomics has also changed our understanding of viruses in general. This is because we can find viruses in healthy hosts in the absence of disease, which changes the perspective of viruses as mere pathogens and offers a new perspective in which viruses function as important components of ecosystems. In concrete, human blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. These viruses are human anelloviruses and, to a lower extent, human pegiviruses. Viral metagenomics' major challenge is the correct isolation of the viral nucleic acids from a specific sample. For the protocol to be successful, all steps must be carefully chosen, in particular those that optimize the recovery of viral nucleic acids. Here, we present a procedure that allows the recovery of both DNA and RNA viruses from plasma samples.

Application of Nanopore-Based Sequencing to Identify Virus Infections in Woody Plants.

Plant viruses threaten the yield and quality of crops. Efficient and affordable pathogen diagnosis is crucial to regulate the trade of plant materials and for disease management and control. Sequencing technology based on Illumina platform is a powerful tool for the identification of plant viruses, but it requires long and expensive protocols, cumbersome equipment, and significant cost per library. Nanopore sequencing technology, developed by Oxford Nanopore Technologies (ONT), is a recent sequencing system very easy to use, suitable for onsite-field detection, and associated with low costs. Coupled with its portability, nanopore technology has great application prospects in the field of quick detection of plant viruses. In this protocol, we expose in detail the application of cDNA-PCR nanopore-based sequencing for the detection of plant viruses.

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.

Identification and characterization of two closely related virga-like viruses latently infecting rubber trees (Hevea brasiliensis).

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.