ConCreT, a 2D convolutional neural network for taxonomic classification applied to viruses in the phylum Cressdnaviricota.

Taxonomic assignments allow scientists to communicate better with each other. In virology, taxonomy is continually improving towards a more precise and comprehensive framework. With the huge numbers of new viruses being described in metagenomic studies, automated taxonomy tools are urgently needed. A number of such tools have been proposed, and those applying machine learning (ML), mainly in the deep learning branch, stand out with accurate results. Still, there is a demand for tools that are less computationally intensive and that can classify viruses down to the ranks of genus and species. Cressdnaviruses are good subjects for testing such tools, due to their small, circular genomes and the existence of several families and genera with a highly imbalanced number of species. We developed a 2D convolutional neural network for virus taxonomy and tested it for classification of viruses from the phylum Cressdnaviricota. We obtained >98 % accuracy in the final pipeline tested, which we named ConCreT (Convolutional Neural Network for Cressdnavirus Taxonomy). The mixture of augmentation for more imbalanced groups with no augmentation for more balanced ones achieved the best score in the final test.

Virus taxonomy and the role of the International Committee on Taxonomy of Viruses (ICTV).

The taxonomy of viruses is developed and overseen by the International Committee on Taxonomy of Viruses (ICTV), which scrutinizes, approves and ratifies taxonomic proposals, and maintains a list of virus taxa with approved names (https://ictv.global). The ICTV has approximately 180 members who vote by simple majority. Taxon-specific Study Groups established by the ICTV have a combined membership of over 600 scientists from the wider virology community; they provide comprehensive expertise across the range of known viruses and are major contributors to the creation and evaluation of taxonomic proposals. Proposals can be submitted by anyone and will be considered by the ICTV irrespective of Study Group support. Thus, virus taxonomy is developed from within the virology community and realized by a democratic decision-making process. The ICTV upholds the distinction between a virus or replicating genetic element as a physical entity and the taxon category to which it is assigned. This is reflected by the nomenclature of the virus species taxon, which is now mandated by the ICTV to be in a binomial format (genus + species epithet) and is typographically distinct from the names of viruses. Classification of viruses below the rank of species (such as, genotypes or strains) is not within the remit of the ICTV. This article, authored by the ICTV Executive Committee, explains the principles of virus taxonomy and the organization, function, processes and resources of the ICTV, with the aim of encouraging greater understanding and interaction among the wider virology community.

Four principles to establish a universal virus taxonomy.

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.

A Capsid Protein Fragment of a Fusagra-like Virus Found in Carica papaya Latex Interacts with the 50S Ribosomal Protein L17.

Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321-670 and 961-1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus-host interactions.

Begomovirus populations in single plants are complex and may include both well-adapted and poorly-adapted viruses.

Begomoviruses (single-stranded DNA plant viruses transmitted by whiteflies) are economically important pathogens causing epidemics worldwide. Tomato-infecting begomoviruses emerged in Brazil in the 1990's following the introduction of Bemisia tabaci Middle East-Asia Minor 1. It is believed that these viruses evolved from indigenous viruses infecting non-cultivated hosts. However, tomato-infecting viruses are rarely found in non-cultivated hosts, and vice-versa. It is possible that viral populations in a given host are composed primarily of viruses which are well adapted to this host, but also include a small proportion of poorly adapted viruses. Following transfer to a new host, the composition of the viral population would shift rapidly, with the viruses which are better adapted to the new host becoming predominant. To test this hypothesis, we collected tomato and Sida plants growing next to each other at two locations in 2014 and 2018. Total DNA was extracted from tomato and Sida samples from each location and year and used as a template for high-throughput sequencing. Reads were mapped following a highly stringent set of criteria. For the 2014 samples, >98% of the Sida reads mapped to Sida micrantha mosaic virus (SiMMV), but 0.1% of the reads mapped to tomato severe rugose virus (ToSRV). Conversely, >99% of the tomato reads mapped to ToSRV, with 0.18% mapping to SiMMV. For the 2018 samples, 41% of the Sida reads mapped to three Sida-adapted viruses and 0.1% of the reads mapped to ToSRV, while 99.9% of the tomato reads mapped to ToSRV. These results are consistent with the hypothesis that viral populations in a single plant are composed primarily of the virus that is better adapted to the host but also include a small proportion of viruses that are poorly adapted.

From Contagium vivum fluidum to Riboviria: A Tobacco Mosaic Virus-Centric History of Virus Taxonomy.

Viruses were discovered as agents of disease in the late 19th century, but it was not until the 1930s that the nature of these agents was elucidated. Nevertheless, as soon as viral diseases started to be recognized and cataloged, there were attempts to classify and name viruses. Although these early attempts failed to be adopted by the nascent virology community, they are evidence of the human compulsion to try to organize the natural world into well-defined categories. Different classification schemes were proposed during the 20th century, but again none were widely embraced by virologists. In 1966, with the creation of the International Committee on Nomenclature of Viruses (eventually renamed as the International Committee on Taxonomy of Viruses), a more organized effort led to an official taxonomy in which viruses were classified into families and genera. At present, a much better understanding of the evolutionary relationships among viruses has led to the establishment of a 15-rank taxonomy based primarily on these evolutionary relationships. This review of virus taxonomy will be centered on the tobacco mosaic virus (TMV), the agent of the disease studied by Dmitry Ivanovsky and the first virus to be recognized as such, which was often historically at the center of major advancements in virology during the 20th century.

How To Be a Successful Monopartite Begomovirus in a Bipartite-Dominated World: Emergence and Spread of Tomato Mottle Leaf Curl Virus in Brazil.

Begomoviruses are members of the family Geminiviridae, a large and diverse group of plant viruses characterized by a small circular single-stranded DNA genome encapsidated in twinned quasi-icosahedral virions. Cultivated tomato (Solanum lycopersicum L.) is particularly susceptible and is infected by >100 bipartite and monopartite begomoviruses worldwide. In Brazil, 25 tomato-infecting begomoviruses have been described, most of which are bipartite. Tomato mottle leaf curl virus (ToMoLCV) is one of the most important of these and was first described in the late 1990s but has not been fully characterized. Here, we show that ToMoLCV is a monopartite begomovirus with a genomic DNA similar in size and genome organization to those of DNA-A components of New World (NW) begomoviruses. Tomato plants agroinoculated with the cloned ToMoLCV genomic DNA developed typical tomato mottle leaf curl disease symptoms, thereby fulfilling Koch's postulates and confirming the monopartite nature of the ToMoLCV genome. We further show that ToMoLCV is transmitted by whiteflies, but not mechanically. Phylogenetic analyses placed ToMoLCV in a distinct and strongly supported clade with other begomoviruses from northeastern Brazil, designated the ToMoLCV lineage. Genetic analyses of the complete sequences of 87 ToMoLCV isolates revealed substantial genetic diversity, including five strain groups and seven subpopulations, consistent with a long evolutionary history. Phylogeographic models generated with partial or complete sequences predicted that the ToMoLCV emerged in northeastern Brazil >700 years ago, diversifying locally and then spreading widely in the country. Thus, ToMoLCV emerged well before the introduction of MEAM1 whiteflies, suggesting that the evolution of NW monopartite begomoviruses was facilitated by local whitefly populations and the highly susceptible tomato host. IMPORTANCE Worldwide, diseases of tomato caused by whitefly-transmitted geminiviruses (begomoviruses) cause substantial economic losses and a reliance on insecticides for management. Here, we describe the molecular and biological properties of tomato mottle leaf curl virus (ToMoLCV) from Brazil and establish that it is a NW monopartite begomovirus indigenous to northeastern Brazil. This answered a long-standing question regarding the genome of this virus, and it is part of an emerging group of these viruses in Latin America. This appears to be driven by widespread planting of the highly susceptible tomato and by local and exotic whiteflies. Our extensive phylogenetic studies placed ToMoLCV in a distinct strongly supported clade with other begomoviruses from northeastern Brazil and revealed new insights into the origin of Brazilian begomoviruses. The novel phylogeographic analysis indicated that ToMoLCV has had a long evolutionary history, emerging in northeastern Brazil >700 years ago. Finally, the tools used here (agroinoculation system and ToMoLCV-specific PCR test) and information on the biology of the virus (host range and whitefly transmission) will be useful in developing and implementing integrated pest management (IPM) programs targeting ToMoLCV.

A new bipartite begomovirus naturally infecting Pyrenacantha sp. in Mozambique.

A begomovirus was isolated from a Pyrenacantha sp. plant with yellow mosaic symptoms collected in a maize production field in Mozambique. The complete DNA-A and DNA-B components have a genomic organization typical of Old World, bipartite begomoviruses. Based on the current ICTV species demarcation criteria for the genus Begomovirus, the virus isolate, named Pyrenacantha yellow mosaic virus (PyYMV), is a member of a new species, for which the name "Begomovirus pyrenacanthae" is proposed. Alignment of their common regions (CR) indicated a 35-nt insertion in the DNA-A CR. The nt sequence identity between the CRs is only 83% but increases to 96% when the 35-nt insertion is removed from the alignment. This is the first report of a begomovirus naturally infecting Pyrenacantha spp.

Establishment of five new genera in the family Geminiviridae: Citlodavirus, Maldovirus, Mulcrilevirus, Opunvirus, and Topilevirus.

Geminiviruses are plant-infecting, circular single-stranded DNA viruses that have a geminate virion morphology. These viruses infect both cultivated and non-cultivated monocotyledonous and dicotyledonous plants and have a wide geographical distribution. Nine genera had been established within the family Geminiviridae (Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Eragrovirus, Grablovirus, Mastrevirus, Topocuvirus, and Turncurtovirus) as of 2020. In the last decade, metagenomics approaches have facilitated the discovery and identification of many novel viruses, among them numerous highly divergent geminiviruses. Here, we report the establishment of five new genera in the family Geminiviridae (Citlodavirus, Maldovirus, Mulcrilevirus, Opunvirus, and Topilevirus) to formally classify twelve new, divergent geminiviruses.

Guidance for creating individual and batch latinized binomial virus species names.

The International Committee on Taxonomy of Viruses recently adopted, and is gradually implementing, a binomial naming format for virus species. Although full Latinization of these names remains optional, a standardized nomenclature based on Latinized binomials has the advantage of comparability with all other biological taxonomies. As a language without living native speakers, Latin is more culturally neutral than many contemporary languages, and words built from Latin roots are already widely used in the language of science across the world. Conversion of established species names to Latinized binomials or creation of Latinized binomials de novo may seem daunting, but the rules for name creation are straightforward and can be implemented in a formulaic manner. Here, we describe approaches, strategies and steps for creating Latinized binomials for virus species without prior knowledge of Latin. We also discuss a novel approach to the automated generation of large batches of novel genus and species names. Importantly, conversion to a binomial format does not affect virus names, many of which are created from local languages.

ICTV Virus Taxonomy Profile: Geminiviridae 2021.

The family Geminiviridae includes viruses with mono- or bipartite single-stranded, circular DNA genomes of 2.5-5.2 kb. They cause economically important diseases in most tropical and subtropical regions of the world. Geminiviruses infect dicot and monocot plants and are transmitted by insect vectors. DNA satellites are associated with some geminiviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Geminiviridae which is available at ictv.global/report/geminiviridae.

Small but mighty: Functional landscape of the versatile geminivirus-encoded C4 protein.

The fast-paced evolution of viruses enables them to quickly adapt to the organisms they infect by constantly exploring the potential functional landscape of the proteins encoded in their genomes. Geminiviruses, DNA viruses infecting plants and causing devastating crop diseases worldwide, produce a limited number of multifunctional proteins that mediate the manipulation of the cellular environment to the virus' advantage. Among the proteins produced by the members of this family, C4, the smallest one described to date, is emerging as a powerful viral effector with unexpected versatility. C4 is the only geminiviral protein consistently subjected to positive selection and displays a number of dynamic subcellular localizations, interacting partners, and functions, which can vary between viral species. In this review, we aim to summarize our current knowledge on this remarkable viral protein, encompassing the different aspects of its multilayered diversity, and discuss what it can teach us about geminivirus evolution, invasion requirements, and virulence strategies.

Taxonomy update for the family Alphasatellitidae: new subfamily, genera, and species.

Alphasatellites (family Alphasatellitidae) are circular, single-stranded DNA molecules (~1-1.4 kb) that encode a replication-associated protein and have commonly been associated with some members of the families Geminiviridae, Nanoviridae, and Metaxyviridae (recently established). Here, we provide a taxonomy update for the family Alphasatellitidae following the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021. The taxonomic update includes the establishment of the new subfamily Petromoalphasatellitinae. This new subfamily includes three new genera as well as the genus Babusatellite, which previously belonged to the subfamily Nanoalphasatellitinae. Additionally, three new genera and 14 new species have been established in the subfamily Geminialphasatellitinae, as well as five new species in the subfamily Nanoalphasatellitinae.

Complete genome sequence of a recombinant isolate of yambean mosaic virus from Canavalia ensiformis.

The complete genome sequence of a Brazilian isolate of yambean mosaic virus (YBMV) is presented. High-throughput sequencing (Illumina HiSeq) and Sanger sequencing revealed the complete genome sequence of the YBMV-BRA-6 isolate, found in Canavalia ensiformis. The de novo contigs were assembled into a 9612 nucleotides (nt) long scaffold, excluding the 3'-terminal poly(A) tail, covering the complete genome. The genomic RNA contains an open reading frame (ORF) typical of members of the genus Potyvirus, family Potyviridae, encoding a large putative polyprotein of 3078 amino acids (aa) and a small overlapping PIPO ORF. Pairwise comparisons showed that the YBMV-BRA-6 isolate sequence shares 88.1% nt identity for the complete genome and 90.6% aa identity for the polyprotein with the YBMV-SR isolate. Phylogenetic analysis grouped both isolates together and close to bean common mosaic virus (BCMV). The polyprotein cleavage sites were predicted and a recombination event is described.

Revealing the Complexity of Sweepovirus-Deltasatellite-Plant Host Interactions: Expanded Natural and Experimental Helper Virus Range and Effect Dependence on Virus-Host Combination.

Sweepoviruses are begomoviruses (genus Begomovirus, family Geminiviridae) with ssDNA genomes infecting sweet potato and other species of the family Convolvulaceae. Deltasatellites (genus Deltasatellite, family Tolecusatellitidae) are small-size non-coding DNA satellites associated with begomoviruses. In this study, the genetic diversity of deltasatellites associated with sweepoviruses infecting Ipomoea indica plants was analyzed by further sampling the populations where the deltasatellite sweet potato leaf curl deltasatellite 1 (SPLCD1) was initially found, expanding the search to other geographical areas in southern continental Spain and the Canary Islands. The sweepoviruses present in the samples coinfected with deltasatellites were also fully characterized by sequencing in order to define the range of viruses that could act as helper viruses in nature. Additionally, experiments were performed to assess the ability of a number of geminivirids (the monopartite tomato leaf deformation virus and the bipartite NW begomovirus Sida golden yellow vein virus, the bipartite OW begomovirus tomato leaf curl New Delhi virus, and the curtovirus beet curly top virus) to transreplicate SPLCD1 in their natural plant hosts or the experimental host Nicotiana benthamiana. The results show that SPLCD1 can be transreplicated by all the geminivirids assayed in N. benthamiana and by tomato leaf curl New Delhi virus in zucchini. The presence of SPLCD1 did not affect the symptomatology caused by the helper viruses, and its effect on viral DNA accumulation depended on the helper virus-host plant combination.

Experimental evolution of cowpea mild mottle virus reveals recombination-driven reduction in virulence accompanied by increases in diversity and viral fitness.

Major themes in pathogen evolution are emergence, evolution of virulence, host adaptation and the processes that underlie them. RNA viruses are of particular interest due to their rapid evolution. The in vivo molecular evolution of an RNA plant virus was demonstrated here using a necrotic isolate of cowpea mild mottle virus (CPMMV) and a susceptible soybean genotype submitted to serial inoculations. We show that the virus lost the capacity to cause necrosis after six passages through the host plant. When a severe bottleneck was imposed, virulence reduction occurred in the second passage. The change to milder symptoms had fitness benefits for the virus (higher RNA accumulation) and for its vector, the whitefly Bemisia tabaci. Genetic polymorphisms were highest in ORF1 (viral replicase) and were independent of the symptom pattern. Recombination was a major contributor to this diversity - even with the strong genetic bottleneck, recombination events and hot spots were detected within ORF1. Virulence reduction was associated with different sites in ORF1 associated to recombination events in both experiments. Overall, the results demonstrate that the reduction in virulence was a consequence of the emergence of new variants, driven by recombination. Besides providing details of the evolutionary mechanisms behind a reduction in virulence and its effect under viral and vector fitness, we propose that this recombination-driven switch in virulence allows the pathogen to rapidly adapt to a new host and, potentially, switch back.

First complete genome sequence of an isolate of cowpea severe mosaic virus from South America.

In this study, the complete nucleotide sequence of a Brazilian isolate of cowpea severe mosaic virus (CPSMV) is presented for the first time. To date, the CPSMV-DG isolate, from the USA, is the only one with the complete known genome. High-throughput sequencing (Illumina HiSeq) and Sanger sequencing of the total RNA extract from a cowpea plant collected in Teresina city, Brazil, revealed the genome sequence of the CPSMV-Ter1 isolate. RNA-1 and RNA-2 are, respectively, 5921 and 3465 nucleotides (nt) long without the poly(A) tail, and show 77.91% and 76.08% nt sequence identity with CPSMV-DG, considered the type isolate of the species. The open reading frames (ORFs) were determined and the cleavage sites of the polyproteins were predicted. Although the two isolates show a similar genomic organization, there was a low percentage of sequence identity between Ter1 and DG. Furthermore, pairwise comparisons of a partial RNA-1 fragment between CPSMV-Ter1 and 11 CPSMV isolates from Brazil indicated 94.6 to 94.8% nt and 98.9% to 99.4% aa sequence identities.

Intra-host evolution of the ssDNA virus tomato severe rugose virus (ToSRV).

To evaluate and quantify the evolutionary dynamics of the bipartite begomovirus tomato severe rugose virus (ToSRV) in a cultivated and a non-cultivated host, plants of tomato and Nicandra physaloides were biolistically inoculated with an infectious clone and systemically infected leaves were sampled at 30, 75 and 120 days after inoculation. Total DNA was extracted and sequenced in the Illumina HiSeq 2000 platform. The datasets were trimmed with the quality score limit set to 0.01, and the assembly was performed using the infectious clone sequence as reference. SNPs were filtered using a minimum p-value of 0.001 and the sum frequencies were used to calculate the deviation from the original clone sequence. Nucleotide substitution rates were calculated for the two DNA components in both hosts: 1.73 × 10-3 and 3.07 × 10-4 sub/site/year for the DNA-A and DNA-B, respectively, in N. physaloides, and 8.05 × 10-4 and 7.02 × 10-5 sub/site/year the for DNA-A and DNA-B, respectively, in tomato. These values are in the same range of those estimated for viruses with single-stranded RNA genomes and for other begomoviruses. Strikingly, the number of substitutions decreased over time, suggesting the presence of bottlenecks during systemic infection. Determination of Shannon's entropy indicated different patterns of variation in the DNA-A and the DNA-B, suggesting distinct evolutionary forces acting upon each component.