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Abstract: As the world manages the impact of a global pandemic caused by COVID-19, the discovery of new antiviral agents has become way more relevant and urgent. Viruses are submicroscopic infectious agents that replicate inside the living cells of different organisms. These viruses use nucleic acids (both DNA and RNA) for further replication and maturity inside the cells. Some of the viruses responsible for various human and plant diseases belong to the classes of Picornaviridae, Retroviridae, Orthomyxoviri-dae, Flaviviridae, Pneumoviridae, Virgaviridae, and Hepadnaviridae, and their treatment options are lim-ited or non-existent. The consistent reemergence and resistance development in the viral strains demand the discovery and development of new antiviral drugs possessing better efficacy. Bio-active compounds isolated from fungi can be the source of new compounds with enhanced potency and new mechanisms of action. Fungi are known to produce a diverse lot of secondary metabolites due to their existence in harsh and testing climates which are often inhabitable for many organisms. Because of these unique environ-ments, fungi produce a variety of secondary metabolites of different chemical classes like alkaloids, qui-nones, furanone, pyrones, benzopyranoids, xanthones, terpenes, steroids, peptides, and many acyclic com-pounds. Fungal metabolites are known to display a wide range of bioactive attributes, i.e., anticancer, antibacterial, antifungal, and anti-Alzheimer's, along with antiviral properties. In this review article, we report over 300 antiviral compounds from fungal sources during the period of 2009 to 2019. The source of these compounds is marine and endophytic fungi and they are arranged based on their antiviral action against different viral families. These compounds offer promise for their use and development as future antiviral drugs.

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.

Hibiscus latent Fort Pierce virus (HLFPV) is a member of the genus Tobamovirus (family Virgaviridae) and a common virus infecting members of the family Malvaceae. This research presents the first report of HLFPV detection in hibiscus plants with viral symptoms and the use of high throughput sequencing (HTS) to detect this virus in a water source in Ecuador. The near complete genome (99.41%) of HLFPV was assembled.

Tomato brown rugose fruit virus (ToBRFV; genus Tobamovirus, family Virgaviridae) was fi rst observed in 2014 on tomatoes in Israel, and otbreaks have recently occurred in China, Mexico, the USA and several EPPO countries. The virus is characterized as highly infectious and persistant. Propagative materials including transplants and seeds, as well as fresh fruits are considered high risk pathways for the introduction of ToBRFV into new areas. Also, ToBRFV is a virus easily spread mechanically by plant-to-plant contact, contaminated tools, shoes, clothing, hands and equipment during performing various plant care measures. Control of ToBRFV is a very diffi cult and it is important to prevent the introduction of the virus by using virus-free propagative materials. Once the virus is introduced, control measures are very limited and mainly rely on the elimination of infected plants and the application of strict hygiene measures, as resistant cultivars are not currently available. Due to its high damage potential for the tomato production, ToBRFV poses a considerable phytosanitary risk for European countries and was added to the EPPO A2 List in 2020.

Plant viruses have been reported to be common in the gut of human adults, presumably as result of food ingestion. In this work, we report that plant viruses can also be found frequently in the gut and oropharynx of children during their first year of life, even when they are exclusively breast-fed. Fecal and oropharynx samples were collected monthly, from birth to 1 year of age, from three apparently healthy children in a semi-rural community and analyzed by next generation sequencing. In 100% of the fecal samples and 65% of the oropharynx samples at least one plant virus was identified. Tobamoviruses in the Virgaviridae family were by far the most frequently detected, with tropical soda apple mosaic virus, pepper mild mottle virus, and opuntia tobamovirus 2 being the most common species. Seventeen complete virus genomes could be assembled, and phylogenetic analyses showed a large diversity of virus strains circulating in the population. These results suggest that children are continuously exposed to an extensive and highly diverse collection of tobamoviruses. Whether the common presence of plant viruses at an early age influences the infant’s immune system, either directly or through interaction with other members of the microbiota, remains to be investigated.

In this study, we explored the viral diversity and community structures of the ticks collected from dogs and detected possible tick-borne viruses (TBVs) using metagenomic analysis. Additionally, full-length sequences and the structural, phylogenetic and molecular features of the possible TBVs were characterized using bioinformatics tools. The sequence run produced 12,254,268 reads and 6,667,259 unique reads from the tick pools. Further analysis of the viral reads revealed that 92.73% were similar to ssRNA viruses, and 90.5% of the total viral sequences belonged to Non-classified viral families. Of the 29 classified viral families, most virome sequences were homologous with vertebrate viruses from Circoviridae, plant viruses from Virgaviridae, phages from Microviridae, insect viruses from Baculoviridae, and giant viruses from Mimiviridae. In the process of assembly of reads, large contigs representing four virus families were identified, including Phenuiviridae, Chuviridae, Parvoviridae and Non-classified families. Some sequences of Non-classified family shared a highly divergent amino acid sequence identities from existing virus sequences. The importance of these newly identified virus contigs to public and veterinary health needs additional researches. Because of the lack of the acquired viral reads from these families, we only demonstrated the complete sequences of Circoviridae in detail. The complete canine circovirus (CaCV)-NC21 genome detected from police dogs was 2,063 bp long with a GC content of 53.6%. Our results suggested that tick viromes collected from dogs contained diverse sequences with a broad range of animal, insect, plant, and phage viruses, which may reflect the ecological characteristics of the dogs and their ticks. Furthermore, our study revealed the existence of the partial contigs belonging to the four virus families in ticks. More researches are needed to verify their importance to public and veterinary health. As well, our detection of CaCv-NC21 demonstrated that ticks can harbor and potentially transmit canine circoviruses.

Locusts and grasshoppers are one of the most dangerous agricultural pests. Environmentally benign microbial pesticides are increasingly desirable for controlling locust outbreaks in fragile ecosystems. Here we use metagenomic sequencing to profile the rich viral communities in 34 grasshopper species and report 322 viruses, including 202 novel species. Most of the identified viruses are related to other insect viruses and some are targeted by antiviral RNAi pathway, indicating they infect grasshoppers. Some plant/fungi/vertebrate associated viruses are also abundant in our samples. Our analysis of relationships between host and virus phylogenies suggests that the composition of viromes is closely allied with host evolution, and there is significant phylogenetic relatedness between grasshoppers and viruses from Lispiviridae, Partitiviridae, Orthomyxoviridae, Virgaviridae and Flaviviridae. Overall, this study is a thorough exploration of viruses in grasshoppers and provide an essential evolutionary and ecological context for host-virus interaction in Acridoidea.Author SummaryLocusts are the most destructive migratory pest in the world and continue to cause massive damages that endanger food security and threaten millions of people in 21st century. While chemical pesticides are still heavily relied on, biological pesticides developed from natural pathogens offer a reliable, less harmful alternative for controlling locust outbreaks in fragile ecosystems. Unfortunately, little is known about natural pathogens infecting this pest. In this study, we profile the viral communities in 34 grasshopper species include some major swarming species. While we identified as many as 202 novel viral species associated with grasshoppers, some of them are of potential to be developed as biocontrol agents. Our analysis of relatedness of phylogenies of grasshoppers and associated viruses helps to shed light on the eco-evolutionary interactions between insects and viruses. This work provides a valuable dataset of both academic and applied interest.

Plants and fungi are closely associated through parasitic or symbiotic relationships in which bidirectional exchanges of cellular contents occur. Recently, a plant virus was shown to be transmitted from a plant to a fungus, but it is unknown whether fungal viruses can also cross host barriers and spread to plants. In this study, we investigated the infectivity of Cryphonectria hypovirus 1 (CHV1, family Hypoviridae), a capsidless, positive-sense (+), single-stranded RNA (ssRNA) fungal virus in a model plant, Nicotiana tabacum. CHV1 replicated in mechanically inoculated leaves but did not spread systemically, but coinoculation with an unrelated plant (+)ssRNA virus, tobacco mosaic virus (TMV, family Virgaviridae), or other plant RNA viruses, enabled CHV1 to systemically infect the plant. Likewise, CHV1 systemically infected transgenic plants expressing the TMV movement protein, and coinfection with TMV further enhanced CHV1 accumulation in these plants. Conversely, CHV1 infection increased TMV accumulation when TMV was introduced into a plant pathogenic fungus, Fusarium graminearum. In the in planta F. graminearum inoculation experiment, we demonstrated that TMV infection of either the plant or the fungus enabled the horizontal transfer of CHV1 from the fungus to the plant, whereas CHV1 infection enhanced fungal acquisition of TMV. Our results demonstrate two-way facilitative interactions between the plant and fungal viruses that promote cross-kingdom virus infections and suggest the presence of plant–fungal-mediated routes for dissemination of fungal and plant viruses in nature.

Aim. The development of an inventory of phytoviruses affecting vegetable and melon crops in the South of the Russian Far East. Discussion. On the basis of many years of original research, carried out on a regular basis by the Laboratory of Virology (Federal Scientific Centre of East Asia Biodiversity, Far Eastern Branch, Russian Academy of Sciences) since 1962 as well as available data in the literature, information about the symptoms of diseases, circulation, reserve plants and vectors are presented for the alfalfa mosaic virus (Bromoviridae, Alfamovirus); tomato aspermy virus and cucumber mosaic virus (Bromoviridae, Cucumovirus); cauliflower mosaic virus (Caulimoviridae, Caulimovirus); garlic mosaic virus (Potyviridae, Carlavirus); tobacco etch virus, allium yellow dwarf virus, bean yellow mosaic virus, watermelon mosaic virus 2, turnip mosaic virus and bean common mosaic virus (Potyviridae, Potyvirus); radish mosaic virus (Picornavirales, Secoviridae, Comovirus); tobacco ringspot virus (Picornavirales, Secoviridae, Nepo-virus); cucumber green mottle mosaic virus, tobacco mosaic virus and tomato mosaic virus (Virgaviridae, Tobamovirus). Conclusion. The information presented forms the basis for the development of a set of diagnostic test systems for phytovirus diseases of vegetable and melon crops; a necessary element of activities directed to the improvement of food productivity and security of the Russian Federation in the Far East.

Poa semilatent virus (PSLV), Lychnis ringspot virus (LRSV), and Barley stripe mosaic virus (BSMV) are members of the genus Hordeivirus in the family Virgaviridae. However, the biological properties and molecular genetics of PSLV have not been compared with other hordeiviruses. Here, we have constructed an infectious cDNA clone of the PSLV Canadian strain and provided evidence that PSLV differs from BSMV and LRSV. First, unlike the other two hordeiviruses that replicate in chloroplasts, PSLV induces dramatic structural changes in peroxisome during its infection in barley. The αa replication protein also localizes to peroxisomes, suggesting that PSLV replication occurs in peroxisomes. Second, PSLV encodes a γb protein that shares 19 to 23% identity with those of other hordeiviruses, and its activity as a viral suppressor of RNA (VSR) silencing is distinct from those of BSMV and LRSV. Substitution of the BSMV γb protein with that of PSLV or LRSV revealed a negative correlation between VSR activity and symptom severity of the recombinant BSMV derivatives. Intriguingly, the Ser-Lys-Leu (SKL) peroxisome-targeting signals differ among γb proteins of various hordeiviruses, including some BSMV strains. The presence of the C-terminal SKL motif in the γb protein impairs its silencing suppressor activity and influences symptoms. Finally, we developed a PSLV-based virus-induced gene silencing vector that induced strong and effective silencing phenotypes of endogenous genes in barley, wheat, and millet. Our results shed new light on hordeivirus pathogenesis and evolution, and provide an alternative tool for genomics studies of model hosts and economically important monocots.

Sisal is a common name for different plant varieties in the genus Agave (especially Agave sisalana) used for high-quality natural leaf fiber extraction. Despite the economic value of these plants, we still lack information about the diversity of viruses (virome) in non-tequilana species from the genus Agave. In this work, by associating RNA and DNA deep sequencing we were able to identify 25 putative viral species infecting A. sisalana, A. fourcroydes, and Agave hybrid 11648, including one strain of Cowpea Mild Mottle Virus (CPMMV) and 24 elements likely representing new viruses. Phylogenetic analysis indicated they belong to at least six viral families: Alphaflexiviridae, Betaflexiviridae, Botourmiaviridae, Closteroviridae, Partitiviridae, Virgaviridae, and three distinct unclassified groups. We observed higher viral taxa richness in roots when compared to leaves and stems. Furthermore, leaves and stems are very similar diversity-wise, with a lower number of taxa and dominance of a single viral species. Finally, approximately 50% of the identified viruses were found in all Agave organs investigated, which suggests that they likely produce a systemic infection. This is the first metatranscriptomics study focused on viral identification in species from the genus Agave. Despite having analyzed symptomless individuals, we identified several viruses supposedly infecting Agave species, including organ-specific and systemic species. Surprisingly, some of these putative viruses are probably infecting microorganisms composing the plant microbiota. Altogether, our results reinforce the importance of unbiased strategies for the identification and monitoring of viruses in plant species, including those with asymptomatic phenotypes.

Tomato mottle mosaic virus (ToMMV) is a noteworthy virus which belongs to the Virgaviridae family and causes serious economic losses in tomato. Here, we isolated and cloned the full-length genome of a ToMMV Chinese isolate (ToMMV-LN) from a naturally infected tomato (Solanum lycopersicum L.). Sequence analysis showed that ToMMV-LN contains 6399 nucleotides (nts) and is most closely related to a ToMMV Mexican isolate with a sequence identity of 99.48%. Next, an infectious cDNA clone of ToMMV was constructed by a homologous recombination approach. Both the model host N. benthamiana and the natural hosts tomato and pepper developed severe symptoms upon agroinfiltration with pToMMV, which had a strong infectivity. Electron micrographs indicated that a large number of rigid rod-shaped ToMMV virions were observed from the agroinfiltrated N. benthamiana leaves. Finally, our results also confirmed that tomato plants inoculated with pToMMV led to a high infection rate of 100% in 4–5 weeks post-infiltration (wpi), while pepper plants inoculated with pToMMV led to an infection rate of 40–47% in 4–5 wpi. This is the first report of the development of a full-length infectious cDNA clone of ToMMV. We believe that this infectious clone will enable further studies of ToMMV genes function, pathogenicity and virus–host interaction.

Species of Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named “ambiviruses” with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.

In this work, we determined the diversity and dynamics of the gut virome of infants during the first year of life. Fecal samples were collected monthly, from birth to one year of age, from three healthy children living in a semi-rural village in Mexico. Most of the viral reads were classified into six families of bacteriophages including five dsDNA virus families of the order Caudovirales, with Siphoviridae and Podoviridae being the most abundant. Eukaryotic viruses were detected as early as two weeks after birth and remained present all along the first year of life. Thirty-four different eukaryotic virus families were found, where eight of these families accounted for 98% of all eukaryotic viral reads: Anelloviridae, Astroviridae, Caliciviridae, Genomoviridae, Parvoviridae, Picornaviridae, Reoviridae and the plant-infecting viruses of the Virgaviridae family. Some viruses in these families are known human pathogens, and it is surprising that they were found during the first year of life in infants without gastrointestinal symptoms. The eukaryotic virus species richness found in this work was higher than that observed in previous studies; on average between 7 and 24 virus species were identified per sample. The richness and abundance of the eukaryotic virome significantly increased during the second semester of life, probably because of an increased environmental exposure of infants with age. Our findings suggest an early and permanent contact of infants with a diverse array of bacteriophages and eukaryotic viruses, whose composition changes over time. The bacteriophages and eukaryotic viruses found in these children could represent a metastable virome, whose potential influence on the development of the infant’s immune system or on the health of the infants later in life, remains to be investigated.

The genera Blunervirus, Cilevirus and Higrevirus are classified in the recently created family Kitaviridae. Kitaviruses possess positive-sense RNA segmented genomes: bi-, tri-, or quadripartite for cile-, higre- and blunerviruses, respectively. Based on phylogenetic reconstructions using different protein sequences, kitaviruses likely emerged from recombination and/or reassortment events of two important plant virus families: Virgaviridae and Bromoviridae. Furthermore, a small predicted structural protein, present in all kitaviruses and some unclassified invertebrate viruses, may suggest arthropod-infecting viruses as evolutionary ancestors of kitaviruses. Despite the genomic differences, kitaviruses share important epidemiological aspects such as symptomatology, lack of systemic movement and mite-mediated transmission.

Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus, family Alphaflexiviridae), potyvirus (Potyvirus, family Potyviridae), and tobamovirus (Tobamovirus, family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies.

Macrophomina phaseolina is an important necrotrophic phytopathogenic fungus and cause extensive damage in many oilseed crops. Twelve M.phaseolina isolates with diverse biological phenotypes were selected for a high-throughput sequencing-based metatranscriptomic and bioinformatics analysis to identify viruses infecting M.phaseolina. The analysis identified 40 partial or nearly complete viral genome segments, 31 of which were novel viruses. Among these viral sequences, 43% of the viral genomes were double-stranded RNA (dsRNA), 47% were positive single-stranded RNA (ssRNA+), and the remaining 10% were negative sense-stranded RNA (ssRNA−). The 40 viruses showed affinity to 13 distinct viral lineages, including Bunyavirales (four viruses), Totiviridae (three viruses), Chrysoviridae (five viruses), Partitiviridae (four viruses), Hypoviridae (one virus), Endornaviridae (two viruses), Tombusviridae (three viruses), Narnaviridae (one virus), Potyviridae (one virus), Bromoviridae (one virus), Virgaviridae (six viruses), ‘Fusagraviridae’ (five viruses), and Ourmiavirus (four viruses). Two viruses are closely related to two families, Potyviridae and Bromoviridae, which previously contained no mycovirus species. Moreover, nine novel viruses associated with M.phaseolina were identified in the family Totiviridae, Endornaviridae, and Partitiviridae. Coinfection with multiple viruses is prevalent in M.phaseolina, with each isolate harboring different numbers of viruses, ranging from three to eighteen. Furthermore, the effects of the viruses on the fungal host were analyzed according to the biological characteristics of each isolate. The results suggested that M.phaseolina hypovirus 2, M.phaseolina fusagravirus virus 1-5 (MpFV1-5), M.phaseolina endornavirus 1-2 (MpEV1-2), M.phaseolina ourmia-like virus 1-3 (MpOLV1-3), M.phaseolina mitovirus 4 (MpMV4), and M.phaseolina mycobunyavirus 1-4 (MpMBV1-4) were only detected in hypovirulent isolates. Those viruses associated with hypovirulence might be used as biological control agents as an environmentally friendly alternative to chemical fungicides. These findings considerably expand our understanding of mycoviruses in M.phaseolina and unvailed the presence of a huge difference among viruses in isolates from different hosts in distant geographical regions. Together, the present study provides new knowledge about viral evolution and fungus-virus coevolution.

Background Physostegia chlorotic mottle virus (PhCMoV; genus: Alphanucleorhabdovirus, family: Rhabdoviridae) and tomato brown rugose fruit virus (ToBRFV; genus: Tobamovirus, family: Virgaviridae) are newly emerging plant viruses that have a dramatic effect on tomato production. Among various known virus-control strategies, RNAi-mediated defence has shown the potential to protect plants against various pathogens including viral infections. Micro(mi)RNAs play a major role in RNAi-mediated defence. Methods Using in silico analyses, we investigated the possibility of tomato-encoded miRNAs (TomiRNA) to target PhCMoV and ToBRFV genomes using five different algorithms, i.e., miRanda, RNAhybrid, RNA22, Tapirhybrid and psRNATarget. Results The results revealed that 14 loci on PhCMoV and 10 loci on ToBRFV can be targeted by the TomiRNAs based on the prediction of at least three algorithms. Interestingly, one TomiRNA, miR6026, can target open reading frames from both viruses, i.e., the phosphoprotein encoding gene of PhCMoV, and the two replicase components of ToBRFV. There are currently no commercially available PhCMoV- or ToBRFV-resistant tomato varieties, therefore the predicted data provide useful information for the development of PhCMoV- and ToBFRV-resistant tomato plants.

Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae within which few RNA viruses have been documented. By combining total RNA sequencing with sequence and structural-based homology detection, we identified 18 novel RNA viruses in cultured samples from two major groups of microbial algae: the chlorophytes and the chlorarachniophytes. Most of the RNA viruses identified in the green algae class Ulvophyceae were related to the Tombusviridae and Amalgaviridae viral families commonly associated with land plants. This suggests that the evolutionary history of these viruses extends to divergence events between algae and land plants. Seven Ostreobium sp-associated viruses exhibited sequence similarity to the mitoviruses most commonly found in fungi, compatible with horizontal virus transfer between algae and fungi. We also document, for the first time, RNA viruses associated with chlorarachniophytes, including the first negative-sense (bunya-like) RNA virus in microalgae, as well as a distant homolog of the plant virus Virgaviridae, potentially signifying viral inheritance from the secondary chloroplast endosymbiosis that marked the origin of the chlorarachniophytes. More broadly, these data suggest that the scarcity of RNA viruses in algae results from limited investigation rather than their absence.

Insects are the host and vector of diverse viruses including those that infect vertebrates, plants, and fungi. Recent wide-scale transcriptomic analyses have uncovered the existence of a number of novel insect viruses belonging to an alphavirus-like superfamily (virgavirus/negevirus-related lineage). In this study, through an in silico search using publicly available insect transcriptomic data, we found numerous virus-like sequences related to insect virga/nege-like viruses. Phylogenetic analysis showed that these novel viruses and related virus-like sequences fill the major phylogenetic gaps between insect and plant virga/negevirus lineages. Interestingly, one of the phylogenetic clades represents a unique insect-infecting virus group. Its members encode putative coat proteins which contained a conserved domain similar to that usually found in the coat protein of plant viruses in the family Virgaviridae. Furthermore, we discovered endogenous viral elements (EVEs) related to virga/nege-like viruses in the insect genomes, which enhances our understanding on their evolution. Database searches using the sequence of one member from this group revealed the presence of EVEs in a wide range of insect species, suggesting that there has been prevalent infection by this virus group since ancient times. Besides, we present detailed EVE integration profiles of this virus group in some species of the Bombus genus of bee families. A large variation in EVE patterns among Bombus species suggested that while some integration events occurred after the species divergence, others occurred before it. Our analyses support the view that insect and plant virga/nege-related viruses might share common virus origin(s).

Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae in few RNA viruses have been documented to date. By combining meta-transcriptomic approaches with sequence and structural-based homology detection, followed by PCR confirmation, we identified 18 novel RNA viruses in two major groups of microbial algae – the chlorophytes and the chlorarachniophytes. Most of the RNA viruses identified in the green algae class Ulvophyceae were related to those from the families Tombusviridae and Amalgaviridae that have previously been associated with plants, suggesting that these viruses have an evolutionary history that extends to when their host groups shared a common ancestor. In contrast, seven ulvophyte associated viruses exhibited clear similarity with the mitoviruses that are most commonly found in fungi. This is compatible with horizontal virus transfer between algae and fungi, although mitoviruses have recently been documented in plants. We also document, for the first time, RNA viruses in the chlorarachniophytes, including the first observation of a negative-sense (bunya-like) RNA virus in microalgae. The other virus-like sequence detected in chlorarachniophytes is distantly related to those from the plant virus family Virgaviridae, suggesting that they may have been inherited from the secondary chloroplast endosymbiosis event that marked the origin of the chlorarachniophytes. More broadly, this work suggests that the scarcity of RNA viruses in algae most likely results from limited investigation rather than their absence. Greater effort is needed to characterize the RNA viromes of unicellular eukaryotes, including through structure-based methods that are able to detect distant homologies, and with the inclusion of a wider range of eukaryotic microorganisms.Author summaryRNA viruses are expected to infect all living organisms on Earth. Despite recent developments in and the deployment of large-scale sequencing technologies, our understanding of the RNA virosphere remains anthropocentric and largely restricted to human, livestock, cultivated plants and vectors for viral disease. However, a broader investigation of the diversity of RNA viruses, especially in protists, is expected to answer fundamental questions about their origin and long-term evolution. This study first investigates the RNA virus diversity in unicellular algae taxa from the phylogenetically distinct ulvophytes and chlorarachniophytes taxa. Despite very high levels of sequence divergence, we were able to identify 18 new RNA viruses, largely related to plant and fungi viruses, and likely illustrating a past history of horizontal transfer events that have occurred during RNA virus evolution. We also hypothesise that the sequence similarity between a chlorarachniophyte-associated virga-like virus and members of Virgaviridae associated with plants may represent inheritance from a secondary endosymbiosis event. A promising approach to detect the signals of distant virus homologies through the analysis of protein structures was also utilised, enabling us to identify potential highly divergent algal RNA viruses.

Xrn1 is a major 5ʹ-3ʹ exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3ʹ untranslated region of several beny- and cucumovirus RNAs harbors a so-called ‘coremin’ motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence to deduce putative structural features responsible for Xrn1 stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5ʹ proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3ʹ proximal hairpin loop sequence is variable. The sequence of the 10-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae, Potyviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn1-stalling motifs are more widespread among RNA viruses than previously realized.

Fungal viruses (mycoviruses) are frequently found in several plant pathogenic fungi. This study was aimed at examining mycoviral sequences in the fungus Podosphaera prunicola, the causal agent of powdery mildew disease in sweet cherry. Double-stranded RNA (dsRNA) preparations of fungal conidia and mycelium scraped from infected cherry leaves were subjected to next-generation sequencing (NGS). The quality filtered NGS reads were assembled de novo into contigs and subjected to BLASTn and BLASTx analyses against sequences available in public databases. The results showed the presence of mycovirus-related RNA sequences. Four of these sequences, ranging in size between 1915 and 2334 nucleotides (nt), contained a single open reading frame (ORF) encoding a protein with similarity to the RNA-dependent RNA polymerase of viruses in the family Partitiviridae. Three additional sequences, ranging in size between 1736 and 2104 nt, contained a single ORF encoding a putative protein similar to the capsid protein of viruses in the family Partitiviridae. Another sequence of 11,449 nt containing four putative ORFs showed similarity to the genome of Macrophomina phaseolina tobamo-like virus, an unclassified mycovirus in the family Virgaviridae. These analyses lead to the conclusion that the eight sequences represent genome segments of five novel mycoviruses.

Ever since their discovery just about 56 years ago in the cultivated mushroom Agaricus bisporus, many more viruses infecting fungi have been identified in a wide range of fungal taxa. With mostly being asymptomatic, especially the ones that are detrimental to their phytopathogenic hosts are intensively studied due to their considerable importance in developing novel plant protection measures. Contrary to the rapid accumulation of notable data on viruses of plant pathogenic microfungi, much less information have hitherto been obtained in regards to the viruses whose hosts are macrofungi. According to the current literature, only more than 80 distinct viruses bearing either linear dsRNA or linear positive sense ssRNA genome and infecting a total number of 34 macrofungal species represented with four Ascomycota and 30 Basidiomycota have been identified so far. Among these 34 macrofungal species, 14 are cultivated edible and wild edible mushroom species. According to the 10th ICTV (International Committee on Taxonomy of Viruses) Report, macrofungal viruses with linear dsRNA genome are classified into five families (Partitiviridae, Totiviridae, Chrysoviridae, Endornaviridae and Hypoviridae) and macrofungal viruses with linear positive sense ssRNA genome are classified into seven families (Betaflexiviridae, Gammaflexiviridae, Barnaviridae, Narnaviridae, Virgaviridae, Benyviridae and Tymoviridae). In this review, following a brief overview of some general characteristics of fungal viruses, an up to date knowledge on viruses infecting macrofungal hosts were presented by summarizing the previous, recent and prospective studies of the field.

It has been believed for a long time that the transfer and fixation of genetic material from RNA viruses to eukaryote genomes is very unlikely. However, during the last decade, there have been several cases in which “virus-to-host” gene transfer from various viral families into various eukaryotic phyla have been described. These transfers have been identified by sequence similarity, which may disappear very quickly, especially in the case of RNA viruses. However, compared to sequences, protein structure is known to be more conserved. Applying protein structure-guided protein domain-specific Hidden Markov Models, we detected homologues of the Virgaviridae capsid protein in Schizophora flies. Further data analysis supported “virus-to-host” transfer into Schizophora ancestors as a single transfer event. This transfer was not identifiable by BLAST or by other methods we applied. Our data show that structure-guided Hidden Markov Models should be used to detect ancestral virus-to-host transfers.

The use of metagenomics for virome characterization and its implementation for wastewater analyses, including wastewater-based epidemiology, has increased in the last years. However, the lack of standardized methods can led to highly different results. The aim of this work was to analyze virome profiles in upstream and downstream wastewater samples collected from four wastewater treatment plants (WWTPs) using two different library preparation kits. Viral particles were enriched from wastewater concentrates using a filtration and nuclease digestion procedure prior to total nucleic acid (NA) extraction. Sequencing was performed using the ScriptSeq v2 RNA-Seq (LS) and the NEBNext Ultra II RNA (NB) library preparation kits. Cleaned reads and contigs were annotated using a curated in-house database composed by reads assigned to viruses at NCBI. Significant differences in viral families and in the ratio of detection were shown between the two library kits used. The use of LS library showed Virgaviridae, Microviridae and Siphoviridae as the most abundant families; while Ackermannviridae and Helleviridae were highly represented within the NB library. Additionally, the two sequencing libraries produced outcomes that differed in the detection of viral indicators. These results highlighted the importance of library selection for studying viruses in untreated and treated wastewater. Our results underline the need for further studies to elucidate the influence of sequencing procedures in virome profiles in wastewater matrices in order to improve the knowledge of the virome in the water environment., This study was supported by the “VIRIDIANA” project AGL2017-82909 (AEI/FEDER, UE) funded by Spanish Ministry of Science, Innovation and Universities, CPS project 2018CPS10 from the Center for Produce Safety Grant Agreement, and the APOTI grant (APOTIP/2018/007) from the Generalitat Valenciana. WR was supported by a postdoctoral fellowship from Generalitat Valenciana (APOSTD/2018/150). EC-F is recipient of a predoctoral contract from the MICINN, Call 2018.

Capsid proteins (CPs) of (+)RNA-containing plant viruses are multifunctional proteins involved in many stages of viral infection cycle, in addition to their main function of virus capsid formation. For example, the tobamoviral CP ensures virus systemic transport in plants and defines the virus-host interactions, thereby influencing the virus host range, virus infectivity, pathogenicity, and manifestation of infection symptoms. Hordeiviruses and tobamoviruses belong to the Virgaviridae family and have rod-shaped virions with a helical symmetry; their CPs are similar in structure. However, no non-structural functions of hordeiviral CPs have been described so far. In this study, we assayed possible non-structural functions of CP from the barley stripe mosaic virus (BSMV) (hordeivirus). To do this, the genome of turnip vein clearing virus (TVCV) (tobamovirus) was modified by substituting the TVCV CP gene with the BSMV CP gene or its mutants. We found that BSMV CP efficiently replaced TVCV CP at all stages of viral infection. In particular, BSMV CP performed the role of tobamoviral CP in the long-distance transport of the chimeric virus, acted as a hypersensitive response elicitor, and served as a pathogenicity determinant that influenced the symptoms of the viral infection. The chimeric tobamovirus coding for the C-terminally truncated BSMV CP displayed an increased infectivity and was transported in plants in a form of atypical virions (ribonucleoprotein complexes).

Viruses represent one of the most important threats to agriculture. Several viral families include highly stable pathogens, which remain infective and can be transported long distances in water. The diversity of plant viruses in wastewater remains understudied; however, their potential impact is increasing with the increased irrigation usage of reclaimed wastewater. To determine the abundance, diversity and biological relevance of plant viruses in wastewater influents and effluents we applied an optimized virus concentration method followed by high-throughput sequencing and infectivity assays. We detected representatives of 47 plant virus species, including emerging crop threats. We also demonstrated infectivity for pathogenic and economically relevant plant viruses from the genus Tobamovirus (family Virgaviridae), which remain infective even after conventional wastewater treatment. These results demonstrate the potential of metagenomics to capture the diversity of plant viruses circulating in the environment and expose the potential risk of the uncontrolled use of reclaimed water for irrigation.

Xrn1 is a major 5′-3′ exoribonuclease involved in the RNA metabolism of many eukaryotic species. RNA viruses have evolved ways to thwart Xrn1 in order to produce subgenomic non-coding RNA that affects the hosts RNA metabolism. The 3’ untranslated region of several beny-and cucumovirus RNAs harbors a so-called ‘coremin’ motif that is required for Xrn1 stalling. The structural features of this motif have not been studied in detail yet. Here, by using in vitro Xrn1 degradation assays, we tested over 50 different RNA constructs based on the Beet necrotic yellow vein virus sequence, to deduce putative structural features responsible for Xrn1-stalling. We demonstrated that the minimal benyvirus stalling site consists of two hairpins of 3 and 4 base pairs respectively. The 5’ proximal hairpin requires a YGAD (Y = U/C, D = G/A/U) consensus loop sequence, whereas the 3′ proximal hairpin loop sequence is variable. The sequence of the 9-nucleotide spacer that separates the hairpins is highly conserved and potentially involved in tertiary interactions. Similar coremin motifs were identified in plant virus isolates from other families including Betaflexiviridae, Virgaviridae and Secoviridae (order of the Picornavirales). We conclude that Xrn-stalling motifs are more widespread among RNA viruses than previously realized.

Plant pathogenic single strand positive-sense RNA viruses with the tripartite genome are classified into two families: Bromoviridae and Virgaviridae. Family Bromoviridae contains four genera Bromo, Cucumo, Alfamo, and Ilarviruses characterized by icosahedral particles. By contrast family Virgaviridae contains only one genus, Hordeivirus, with tripartite genome and characterized by helical particles. Unlike in monopartite plant viruses, packaging in tripartite RNA viruses requires a well-orchestrated process to ensure that viral progeny is selectively encapsidated and distributed optimally into three or four different viral capsids. Among the tripartite RNA viruses mentioned above, brome mosaic virus (BMV), the type member of the genus bromovirus, has been extensively used as a model system to unravel the mechanism of genome packaging. Using the available research data on BMV, this review is focused in updating the readers on how various macromolecular interactions (e.g. packaging signals) and biological factors (i.e. type of host plant) modulate genome packaging. The review also offers new directions of research to further our knowledge on the genome packaging in tripartite viruses.

Isolates of an Australian indigenous virus, Yellow tailflower mild mottle virus (YTMMV-Kalbarri), and an exotic virus,Pelargonium zonate spot virus (PZSV-SW13), are described from Anthocercis ilicifolia subsp. ilicifolia (red-striped tail-flower, family Solanaceae), a species endemic to Western Australia. This is the first report of either virus fromthis plant species. The complete genome sequences of YTMMV-Kalbarri and of PZSV-SW13 were obtained. YTMMV-Kalbarri shared 97% nucleotide pairwise identity with the sequence of the type isolate YTMMV-Cervantes. Thesequence PZSV-SW13 shared greatest sequence identity with the partial sequence of an Australian isolate of PZSV, alsofrom a wild plant, and with a sunflower-derived isolate of PZSV from Argentina. An experimental host range studywas done of YTMMV-Kalbarri using cultivated and wild solanaceous and non-solanaceous plants. Most solanaceousplants became systemically infected, with symptoms of systemic infection ranging from symptomless to whole plantnecrosis. Based on these studies, it is suggested that YTMMV has the potential to become a pathogen of commercialspecies of Solanaceae. This study provides further evidence that PZSV is present in wild plants in Australia, in this casean indigenous host species, and possible routes by which it invaded Australia are discussed.Keywords: anulavirus, indigenous plant virus, plant virus ecology, tobamovirus, virus invasion, virus emergenceAnthocercis (family Solanaceae, subfamily Nicotianoi-deae) is a genus of 15 plant species endemic to southernAustralia (Haegi, 1986). Previously, a tobamovirus(genus Tobamovirus, family Virgaviridae) was isolatedfrom A. littoria (yellow tailflower), a spindly shrub, 3 min height, that grows along the coastline in calcareoussand, limestone ridges and sand dunes of southwesternAustralia, but the virus was not detected from A. viscosa(sticky tailflower) plants growing in Albany, 400 km tothe south. The new tobamovirus was named Yellow tail-flower mild mottle virus (YTMMV; Wylie et al., 2014).Pelargonium zonate spot virus (PZSV; genus Anulavi-rus, family Bromoviridae) was first isolated from Pelar-gonium zonale (Geraniaceae) in Italy (Quacquarelli G Luis-Arteaga & Cambra,2000; Gebre-Selassie et al., 2002; Finetti-Sialer & Galli-telli, 2003; Liu & Sears, 2007; Escriu et al., 2009; Gulat-i-Sakhuja et al., 2009; Lapidot et al., 2010; Blouin et al.,2013; Giolitti et al., 2014). Its geographical rangeincludes much of Europe and the Americas. In Australia,PZSV was recently described for the first time in Cakilemaritima (Brassicaceae), a self-introduced exotic weedspecies (Luo et al., 2010).This study describes the complete genome sequences ofnew isolates of YTMMV and PZSV that co-infected a wildplant of Anthocercis ilicifolia subsp. ilicifolia (red-stripedtailflower), a new host species for both viruses. Althoughdetails of the host range of PZSV have been published, thepotential host range of YTMMV is not known. The natu-ral hosts of YTMMV live at the interface between wildand managed systems, and consequently the opportunityexists for the virus to expand its host range into cultivatedplant species. Thus, an experimental host range study wasundertaken of the new YTMMV isolate and the type YT-MMV isolate, and the potential of YTMMV to emerge asa pathogen of commercial importance is discussed. Fur-ther, it is speculated as to how PZSV may have enteredAustralia, given that it has not yet been detected there incommercial plantings.

The Plasmodiophorida (Phytomyxea, Rhizaria) are a group of protists that infect plants. Of this group, Spongospora subterranea causes major problems for the potato industry by causing powdery scab and root galling of potatoes and as vector for the Potato mop-top virus (PMTV) (genus Pomovirus, family Virgaviridae). A single tuber isolate (SSUBK13) of this uncultivable protist was used to generate DNA for Illumina sequencing. The data were assembled to a draft genome of 28.08 Mb consisting of 2,340 contigs and an L50 of 280. A total of 10,778 genes were predicted and 93% of the BUSCO genes were detected. The presented genome assembly is only the second genome of a plasmodiophorid. The data will accelerate functional genomics to study poorly understood interaction of plasmodiophorids and their hosts.

An unusual novel plant virus provisionally named goji berry chlorosis virus (GBCV) was isolated from goji berry plants (Lycium chinense Miller) showing chlorosis symptoms and its complete genome sequence was determined. The viral genome consists of a positive-sense single-stranded RNA of 10,100 ribonucleotides and contains six open reading frames (ORFs). Electron microscopy showed that the viral genome is packaged as a filamentous particle with an average length of approximately 850 nm. Phylogenetic analysis and amino acid similarity analysis of the encoded ORFs revealed that this new virus could be classified in an intermediate position between the families Benyviridae and Virgaviridae. The GBCV 200-kDa replicase (ORF1) is more similar to benyvirus replicases than to virgavirus replicases, while its 17-kDa coat protein (CP, ORF2) is more closely related with virgavirus CPs than benyvirus CPs. ORF3 was predicted to produce a C-terminally extended protein from ORF2 via frameshifting. While ORF4 (45-kDa), ORF5 (44-kDa), and ORF6 (16-kDa) have no apparent sequence homology with other known viruses, ORF5 is predicted to encode a movement protein (MP) that is phylogenetically related to the furovirus MP and ORF6 was experimentally proven to encode a viral suppressor of RNA silencing. These unusual characteristics suggest that GBCV may represent an evolutionary link between the families Benyviridae and Virgaviridae and indicate the existence of a novel, unidentified virus group.

International audience; In the global context of arboviral emergence, deep sequencing unlocks the discovery of new mosquito-borne viruses. Mosquitoes of the species Culex pipiens, C. torrentium, and C. hortensis were sampled from 22 locations worldwide for transcriptomic analyses. A virus discovery pipeline was used to analyze the dataset of 0.7 billion reads comprising 22 individual transcriptomes. Two closely related 6.8 kb viral genomes were identified in C. pipiens and named as Culex pipiens associated tunisia virus (CpATV) strains Ayed and Jedaida. The CpATV genome contained four ORFs. ORF1 possessed helicase and RNA-dependent RNA polymerase (RdRp) domains related to new viral sequences recently found mainly in dipterans. ORF2 and 4 contained a capsid protein domain showing strong homology with Virgaviridae plant viruses. ORF3 displayed similarities with eukaryotic Rhoptry domain and a merozoite surface protein (MSP7) domain only found in mosquito-transmitted Plasmodium, suggesting possible interactions between CpATV and vertebrate cells. Estimation of a strong purifying selection exerted on each ORFs and the presence of a polymorphism maintained in the coding region of ORF3 suggested that both CpATV sequences are genuine functional viruses. CpATV is part of an entirely new and highly diversified group of viruses recently found in insects, and that bears the genomic hallmarks of a new viral family.

Background The giant panda (Ailuropoda melanoleuca) is a vulnerable mammal herbivore living wild in central China. Viral infections have become a potential threat to the health of these endangered animals, but limited information related to these infections is available. Methods Using a viral metagenomic approach, we surveyed viruses in the feces, nasopharyngeal secretions, blood, and different tissues from a wild giant panda that died from an unknown disease, a healthy wild giant panda, and 46 healthy captive animals. Results The previously uncharacterized complete or near complete genomes of four viruses from three genera in Papillomaviridae family, six viruses in a proposed new Picornaviridae genus (Aimelvirus), two unclassified viruses related to posaviruses in Picornavirales order, 19 anelloviruses in four different clades of Anelloviridae family, four putative circoviruses, and 15 viruses belonging to the recently described Genomoviridae family were sequenced. Reflecting the diet of giant pandas, numerous insect virus sequences related to the families Iflaviridae, Dicistroviridae, Iridoviridae, Baculoviridae, Polydnaviridae, and subfamily Densovirinae and plant viruses sequences related to the families Tombusviridae, Partitiviridae, Secoviridae, Geminiviridae, Luteoviridae, Virgaviridae, and Rhabdoviridae; genus Umbravirus, Alphaflexiviridae, and Phycodnaviridae were also detected in fecal samples. A small number of insect virus sequences were also detected in the nasopharyngeal secretions of healthy giant pandas and lung tissues from the dead wild giant panda. Although the viral families present in the sick giant panda were also detected in the healthy ones, a higher proportion of papillomaviruses, picornaviruses, and anelloviruses reads were detected in the diseased panda. Conclusion This viral survey increases our understanding of eukaryotic viruses in giant pandas and provides a baseline for comparison to viruses detected in future infectious disease outbreaks. The similar viral families detected in sick and healthy giant pandas indicate that these viruses result in commensal infections in most immuno-competent animals. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0308-0) contains supplementary material, which is available to authorized users.

Recent studies have declared that members of the ssDNA virus family Microviridae play an important role in multiple environments, as they have been found taking a dominant position in the human gut. The aim of this study was to analyze the overall composition of the gut virome in coronary heart disease (CHD) patients, and try to discover the potential link between the human gut virome and CHD. Viral metagenomics methods were performed to detect the viral sequences in fecal samples collected from CHD inpatients and healthy persons as controls. We present the analysis of the virome composition in these CHD patients and controls. Our data shows that the virome composition may be linked to daily living habits and the medical therapy of CHD. Virgaviridae and Microviridae were the two dominant types of viruses found in the enteric virome of CHD patients. Fourteen divergent viruses belonging to the family Microviridae were found, twelve of which were grouped into the subfamily Gokushovirinae, while the remaining two strains might represent two new subfamilies within Microviridae, according to the phylogenetic analysis. In addition, the genomic organization of these viruses has been characterized.

Cucumber green mottle mosaic virus (CGMMV) is seed borne virus belonging to the genus Tobamovirus into family Virgaviridae, which transmit mechanically during agricultural practice, seed and through water. In this study, based on virus description cucurbits fields were observed to detect CGMMV. Therefore, biological purification was performed to recognise the type of symptoms incited by each isolates. Accordingly, local lesion host plants were inoculated with virus isolates. Following, the samples having CGMMV symptoms are selected to RNA extraction and molecular tests. Furthermore, to detect different strains of CGMMV, a pair of specific primers corresponding to full coat protein and a part of movement protein gene was applied to amplify a 523 bp fragment in reverse transcription polymerase chain reaction (RT-PCR). Eventually, an expected band with 523 bp length was revealed in nine isolates by RT-PCR. It is a first report of CGMMV from north-west of Iran by molecular methods.

Tobamovirus is a group of viruses that have become serious pathogens of crop plants. As part of a study informing risk of wild plant virus spill over to crops, we investigated the capacity of a solanaceous-infecting tobamovirus from an isolated indigenous flora to adapt to new exotic hosts. Yellow tailflower mild mottle virus (YTMMV) (genus Tobamovirus, family Virgaviridae) was isolated from a wild plant of yellow tailflower (Anthocercis littoria, family Solanaceae) and initially passaged through a plant of Nicotiana benthamiana, then one of Nicotiana glutinosa where a single local lesion was used to inoculate a N. benthamiana plant. Sap from this plant was used as starting material for nine serial passages through three plant species. The virus titre was recorded periodically, and 85% of the virus genome was sequenced at each passage for each host. Six polymorphic sites were found in the YTMMV genome across all hosts and passages. At five of these, the alternate alleles became fixed in the viral genome until the end of the experiment. Of these five alleles, one was a non-synonymous mutation (U1499C) that occurred only when the virus replicated in tomato. The mutant isolate harbouring U1499C, designated YTMMV-δ, increased its titre over passages in tomato and outcompeted the wild-type isolate when both were co-inoculated to tomato. That YTMMV-δ had greater reproductive fitness in an exotic host than did the wild type isolate suggests YTMMV evolution is influenced by host changes.