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Viruses can play critical roles in symbioses by initiating horizontal gene transfer, affecting host phenotypes, or expanding their host's ecological niche. However, knowledge of viral diversity and distribution in symbiotic organisms remains elusive. Here we use deep-sequenced metagenomic DNA (PacBio Sequel II; two individuals), paired with a population genomics approach (Pool-seq; 11 populations, 550 individuals) to understand viral distributions in the lichen Umbilicaria phaea. We assess (i) viral diversity in lichen thalli, (ii) putative viral hosts (fungi, algae, bacteria) and (iii) viral distributions along two replicated elevation gradients. We identified five novel viruses, showing 28%-40% amino acid identity to known viruses. They tentatively belong to the families Caulimoviridae, Myoviridae, Podoviridae and Siphoviridae. Our analysis suggests that the Caulimovirus is associated with green algal photobionts (Trebouxia) of the lichen, and the remaining viruses with bacterial hosts. We did not detect viral sequences in the mycobiont. Caulimovirus abundance decreased with increasing elevation, a pattern reflected by a specific algal lineage hosting this virus. Bacteriophages showed population-specific patterns. Our work provides the first comprehensive insights into viruses associated with a lichen holobiont and suggests an interplay of viral hosts and environment in structuring viral distributions.

A putative new virus with sequence similarity to members of the genus Cavemovirus in the family Caulimoviridae was identified in wild chicory (Cichorium intybus) by next-generation sequencing (NGS). The putative new virus was tentatively named “chicory mosaic cavemovirus” (ChiMV), and its genome was determined to be 7,775 nucleotides (nt) long with the typical genome organization of cavemoviruses. ORF1 encodes a putative coat protein/movement polyprotein (1,278 aa), ORF2 encodes a putative replicase (650 aa), and ORF3 encodes a putative transactivator factor (384 aa). The first two putative proteins have 46.2% and 68.7% amino acid sequence identity to the CP/MP protein (YP_004347414) and replicase (YP_004347415), respectively, of sweet potato collusive virus (SPCV). ORF3 encodes a protein with 38.5% amino acid sequence identity to the putative transactivator factor (NP_056849) of cassava vein mosaic virus (CsVMV). The new putative viral genome and those of three cavemoviruses (epiphyllum virus 4 [EpV-4], SPCV, and CsVMV) differ by 24-27% in the nt sequence of the replicase gene, which exceeds the species demarcation cutoff (>20%) for the family.

Sapajus nigritus are non-human primates which are widespread in South America. They are omnivores and live in troops of up to 40 individuals. The oral cavity is one of the main entry routes for microorganisms, including viruses. Our study proposed the identification of viral sequences from oral swabs collected in a group of capuchin monkeys (n = 5) living in a public park in a fragment of Mata Atlantica in South Brazil. Samples were submitted to nucleic acid extraction and enrichment, which was followed by the construction of libraries. After high-throughput sequencing and contig assembly, we used a pipeline to identify 11 viral families, which are Herpesviridae, Parvoviridae, Papillomaviridae, Polyomaviridae, Caulimoviridae, Iridoviridae, Astroviridae, Poxviridae, and Baculoviridae, in addition to two complete viral genomes of Anelloviridae and Genomoviridae. Some of these viruses were closely related to known viruses, while other fragments are more distantly related, with 50% of identity or less to the currently available virus sequences in databases. In addition to host-related viruses, insect and small vertebrate-related viruses were also found, as well as plant-related viruses, bringing insights about their diet. In conclusion, this viral metagenomic analysis reveals, for the first time, the profile of viruses in the oral cavity of wild, free ranging capuchin monkeys.

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.

Cacao swollen shoot virus (CSSV) is causing the major viral disease on cacao (Theobroma cacao) and it is naturally transmitted by mealybugs species. CSSV is restricted to West Africa and is a serious constraint to cacao production, particularly in Ghana. From the high molecular variability of the virus, a complex of 8 viral species has been recognized and the geographical repartition of these species has been done in Côte d'Ivoire, Ghana and Togo. Measures to control the disease are discussed.

Caulimoviridae is a family of non-enveloped reverse-transcribing plant viruses with non-covalently closed circular dsDNA genomes of 7.1–9.8 kbp in the order Ortervirales. They infect a wide range of monocots and dicots. Some viruses cause economically important diseases of tropical and subtropical crops. Transmission occurs through insect vectors (aphids, mealybugs, leafhoppers, lace bugs) and grafting. Activation of infectious endogenous viral elements occurs in Musa balbisiana, Petunia hybrida and Nicotiana edwardsonii. However, most endogenous caulimovirids are not infectious. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caulimoviridae, which is available at ictv.global/report/caulimoviridae.

Necrotic disorder of freesia is a high impact disease for this attractive ornamental crop and is now widespread in temperate world regions, being reported and studied in Europe, United States, New Zealand and South Korea. The presence of Freesia sneak virus (FreSV, genus Ophiovirus, family Ophioviridae) has been widely associated with the necrotic disease but other viral agents might be involved in producing the heavy necrotic symptomatology of this vegetatively propagated bulbous floral crop, so severely threatened by the disorder. During the 2014 growing season, freesia plants of different cultivars with heavy necrotic symptoms were collected in the Sanremo area, Northern Italy. Symptomatic foliar tissue was used both for mechanical inoculation to the model plant Nicotiana benthamiana and for virus purification using a method applicable to multiple viruses. A total RNA extraction of the sample enriched in viral agents was tested by Sequence-Independent Amplification to get preliminary information about the presence of both known and previously undescribed viruses. In parallel, Next Generation Sequencing (NGS) of total RNA was performed on samples extracted from the inoculated N. benthamiana plants, to more easily identify unknown virus(es), due to the fact that, unlike the freesia genome, the genome of N. benthamiana is available. A total of almost 25 million reads were obtained, then assembled into about 33 thousand contigs. NGS data analysis is currently under elaboration and validation but preliminary results show possible occurrence of previously undescribed DNA and RNA viruses infecting diseased freesia. PCR tests are being implemented to validate detection of such novel virus sequences.

We report a new circular DNA virus identified from a Chinese jujube tree showing mosaic-like symptoms. The genome of this virus is 7194 bp in length and contains five putative open reading frames (ORFs), all on the plus-strand of the genome. The genomic organization, primer binding sites and the sizes of the ORFs were similar to those reported for other badnaviruses (family Caulimoviridae), except for ORF3, which was split into ORF3a and ORF3b with a 70-nt intergenic region. Furthermore, this new virus shares low nucleotide sequence identity (

Endophytic microorganisms are well established for their mutualistic relationship and plant growth promotion through production of different metabolites. Bacteria and fungi are the major group of endophytes which were extensively studied. Virus are badly named for centuries and their symbiotic relationship was vague. Recent development of omics tools especially next generation sequencing has provided a new perspective towards the mutualistic viral relationship. Endogenous virus which has been much studied in animal and are less understood in plants. In this study, we described the endophytic viral population of tea plant root. Viral population (9%) were significantly less while compared to bacterial population (90%). Viral population of tea endophytes were mostly dominated by endogenous pararetroviral sequences (EPRV) derived from Caulimoviridae and Geminiviridae. Subclassification of Caulimoviridae showed the dominance of Badnavirus (42%), Caulimovirus (29%), Soymovirus (3%), Tungrovirus (3%), while Geminviridae was only represented by genus Bagmovirus. Interestingly, the endophytic virome sequence from root also showed the presence of phage virus from order Caudovirales. Identified sequence from Caudovirales were Myoviridae and Siphoviridae. Sequence comparison with viral population of soil and root showed the possibility of horizontal transfer of Caudovirales from soil to root environment. This study will expand the knowledge on endogenous viruses especially for tea plant. This study will also help us to understand the symbiotic integration of viral particle with plant which could be used in broader sense to tackle different agronomic problems.Significance StatementVirus were badly named for centuries and mostly known for their disease-causing abilities. But recent development of omics tools has focused another facet which is symbiotic. This paper discusses about viral community identified from shotgun sequence of tea root samples which are endogenous in origin. Interestingly, we also identified sequences of phage virus from Caudovirales family which possibly have transmitted from soil. Here we also compared the soil virome community with tea virome to establish the hypothesis. This research will broaden the current knowledge on symbiotic relationship of virus and plant.

Members of the Caulimoviridae family are important plant pathogens. These circular double-stranded DNA viruses may integrate into the host genome, although this integration is not required for the viral replication cycle. Here, we describe three complete genomes belonging to a new putative Caulimoviridae genus discovered through air metagenomics., Community of Madrid (Spain), under the AIRBIOTA-CM Program (S2013/MAE-2874).

Many of the recent emerging infectious diseases have occurred due to the transmission of the viruses that have wildlife reservoirs. Arthropods, such as ticks, are known to be important vectors for spreading viruses and other pathogens from wildlife to domestic animals and humans. In the present study, we explored the diversity of viruses in hard ticks (Ixodidae) from select areas of a wildlife-livestock interface ecosystem at Mikumi National Park, Tanzania using a metagenomic approach. cDNA and DNA were amplified with random amplification and Illumina high-throughput sequencing was performed. The high-throughput sequenced data was imported to the CLC genomic workbench and trimmed based on quality (Q = 20) and length (≥ 50). The trimmed reads were assembled and annotated through Blastx using Diamond against the National Center for Biotechnology Information non-redundant database and its viral database. The MEGAN Community was used to analyze and to compare the taxonomy of the viral community. The obtained contigs and singletons were further subjected to alignment and mapping against reference sequences. The viral sequences identified were classified into bacteria, vertebrates, and invertebrates, plants, and protozoans viruses. Sequences related to known viral families; Retroviridae, Flaviviridae, Rhabdoviridae, Chuviridae, Orthomyxoviridae, Phenuiviridae, Totiviridae, Rhabdoviridae, Parvoviridae, Caulimoviridae, Mimiviridae and several Phages were reported. This result indicates that there are many viruses present in the study region, which we are not aware of and do not know the role they have or if they have the potential to spread to other species and cause diseases. Therefore, further studies are required to delineate the viral community present in the region over a large scale.

We have characterized the complete genome of a novel circular double-stranded DNA virus, tentatively named Dioscorea nummularia-associated virus (DNUaV), infecting Dioscorea nummularia originating from Samoa. The genome of DNUaV comprised 8139 bp and contained four putative open reading frames (ORFs). ORFs 1 and 2 had no identifiable conserved domains, while ORF 3 had conserved motifs typical of viruses within the family Caulimoviridae including coat protein, movement protein, aspartic protease, reverse transcriptase and ribonuclease H. A transactivator domain, similar to that present in members of several caulimoviridae genera, was also identified in the putative ORF 4. The genome size, organization, and presence of conserved amino acid domains are similar to other viruses in the family Caulimoviridae. However, based on nucleotide sequence similarity and phylogenetic analysis, DNUaV appears to be a distinct novel member of the family and may represent a new genus.

International audience; Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae, and fungi (1, 2). This broad distribution suggests the ancient origin(s) of these viruses, possibly [...]

When human samples are sequenced, many assembled contigs are “unknown”, as conventional alignments find no similarity to known sequences. Hidden Markov models (HMM) exploit the positions of specific nucleotides in protein-encoding codons in various microbes. The algorithm HMMER3 implements HMM using a reference set of sequences encoding viral proteins, “vFam”. We used HMMER3 analysis of “unknown” human sample-derived sequences and identified 510 contigs distantly related to viruses (Anelloviridae (n = 1), Baculoviridae (n = 34), Circoviridae (n = 35), Caulimoviridae (n = 3), Closteroviridae (n = 5), Geminiviridae (n = 21), Herpesviridae (n = 10), Iridoviridae (n = 12), Marseillevirus (n = 26), Mimiviridae (n = 80), Phycodnaviridae (n = 165), Poxviridae (n = 23), Retroviridae (n = 6) and 89 contigs related to described viruses not yet assigned to any taxonomic family). In summary, we find that analysis using the HMMER3 algorithm and the “vFam” database greatly extended the detection of viruses in biospecimens from humans.

Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%–73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant.

Members of the genus Badnavirus have a single non-covalently closed circular double-stranded DNA genome of 7.2-9.2kb. The genome encodes three open reading frames (ORFs) on the positive DNA strand. Canna yellow mottle virus (CaYMV) is a badnavirus that has been described as the etiological cause of yellow mottle disease in canna, although only a 565bp fragment of the genome has been previously reported from cannas. In this report, concentrated virions were recovered from infected canna plants and nucleic acids were extracted. Two full-length sequences represent two badnavirus genomes were recovered and were determined to be 6966bp and 7385bp in length. These DNAs represent a virus strain belonging to Canna yellow mottle virus and a novel species tentatively termed Canna yellow mottle associated virus. Phylogenetic analysis indicates that these two viruses are closely related to sugarcane bacilliform GD virus, pineapple bacilliform comosus virus, banana streak MY virus, and cycad leaf necrosis virus. We also showed naturally grown canna plants to be frequently co-infected by these two badnaviruses along with a potyvirus, Canna yellow streak virus.

International audience; Reverse-transcribing viruses are classified into 5 different families, Retroviridae, Metaviridae, Pseudoviridae, Caulimoviridae, and Hepadnaviridae (1). Retroviruses, hepadnaviruses, and caulimoviruses are full-fledged viruses, whereas metaviruses and pseudoviruses are more often referred to as retrotransposons. Nevertheless [...]

Common bean (Phaseolus vulgaris L.) is the primary source of protein and nutrients in the majority of households in sub-Saharan Africa. However, pests and viral diseases are key drivers in the reduction of bean production. To date, the majority of viruses reported in beans have been RNA viruses. In this study, we carried out a viral metagenomic analysis on virus symptomatic bean plants. Our virus detection pipeline identified three viral fragments of the double-stranded DNA virus Pelargonium vein banding virus (PVBV) (family, Caulimoviridae, genus Badnavirus). This is the first report of the dsDNA virus and specifically PVBV in legumes to our knowledge. In addition two previously reported +ssRNA viruses the bean common mosaic necrosis virus (BCMNVA) (Potyviridae) and aphid lethal paralysis virus (ALPV) (Dicistroviridae) were identified. Bayesian phylogenetic analysis of the Badnavirus (PVBV) using amino acid sequences of the RT/RNA-dependent DNA polymerase region showed the Kenyan sequence (SRF019_MK014483) was closely matched with two Badnavirus viruses: Dracaena mottle virus (DrMV) (YP_610965) and Lucky bamboo bacilliform virus (ABR01170). Phylogenetic analysis of BCMNVA was based on amino acid sequences of the Nib region. The BCMNVA phylogenetic tree resolved two clades identified as clade (I and II). Sequence from this study SRF35_MK014482, clustered within clade I with other Kenyan sequences. Conversely, Bayesian phylogenetic analysis of ALPV was based on nucleotide sequences of the hypothetical protein gene 1 and 2. Three main clades were resolved and identified as clades I–III. The Kenyan sequence from this study (SRF35_MK014481) clustered within clade II, and nested within a sub-clade; comprising of sequences from China and an earlier ALPV sequences from Kenya isolated from maize (MF458892). Our findings support the use of viral metagenomics to reveal the nascent viruses, their viral diversity and evolutionary history of these viruses. The detection of ALPV and PVBV indicate that these viruses have likely been underreported due to the unavailability of diagnostic tools.

Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes.

Mealybugs are well-known sap-sucking insects which transmit plant viruses. They are omnipresent, polyphagous, can cause more damage as pests and are less uncommon as virus vectors. The feeding behavior of these vectors has profound ecological and evolutionary implications for the viruses they transmit, as the acquisition and inoculation of viruses occurs during vector feeding. In most cases, there is an intimate relationship between the virus and its vector, and no transmissions occur without the insects feeding in a specific manner. This feeding behavior often causes considerable economic loss to agriculture through direct damage to crops and via virus transmission. They are considered pests as they feed on the plant juices of economically important crop plants, and also act as vectors for several plant viral diseases. The transmission of the plant virus species belonging to Caulimoviridae and Closteroviridae by different species of mealybugs is furnished in detail in this chapter.

Interactions between microtubules and viruses play important roles in viral infection. The best-characterized examples involve transport of animal viruses by microtubules to the nucleus or other intracellular destinations. In plant viruses, most work to date has focused on interaction between viral movement proteins and the cytoskeleton, which is thought to be involved in viral cell-to-cell spread. We show here, in Cauliflower mosaic virus (CaMV)-infected plant cells, that viral electron-lucent inclusion bodies (ELIBs), whose only known function is vector transmission, require intact microtubules for their efficient formation. The kinetics of the formation of CaMV-related inclusion bodies in transfected protoplasts showed that ELIBs represent newly emerging structures, appearing at late stages of the intracellular viral life cycle. Viral proteins P2 and P3 are first produced in multiple electron-dense inclusion bodies, and are later specifically exported to transiently co-localize with microtubules, before concentrating in a single, massive ELIB in each infected cell. Treatments with cytoskeleton-affecting drugs suggested that P2 and P3 might be actively transported on microtubules, by as yet unknown motors. In addition to providing information on the intracellular life cycle of CaMV, our results show that specific interactions between host cell and virus may be dedicated to a later role in vector transmission. More generally, they indicate a new unexpected function for plant cell microtubules in the virus life cycle, demonstrating that microtubules act not only on immediate intracellular or intra-host phenomena, but also on processes ultimately controlling inter-host transmission.

A distinct caulimovirus, associated with dahlia mosaic, was cloned and sequenced. The caulimovirus, tentatively designated as dahlia common mosaic virus (DCMV), had a double-stranded DNA genome of ca. 8 kb. The genome organization of DCMV was found to be typical of members of the genus Caulimovirus and consisted of six major open reading frames (ORFs), ORFs I-VI, and one minor ORF, ORF VII. Sequence comparisons with the DNA genomes of two known caulimoviruses isolated from dahlia, Dahlia mosaic virus (DMV) and an endogenous caulimovirus, DMV-D10, showed that DCMV is a member of a distinct caulimovirus species, with sequence identities among various ORFs ranging from 25 to 80%.

To test the hypothesis that many viruses remain to be discovered in plants, a procedure was developed to sequence nucleic acids cloned randomly from virus-like particle fractions of plant homogenates. As a test of the efficiency of the procedure we targeted Ambrosia psilostachya, western ragweed, plants growing at the Tallgrass Prairie Preserve of northeastern Oklahoma. Amplifiable nucleic acid was found in the fractions from six of twelve specimens and sequences were characterized from four of them. Evidence was obtained for the presence of viruses belonging to two families (Caulimoviridae, Flexiviridae). Multiple viral species were found in two of the four specimens and their level within the isolated nucleic acid population varied from less than 1-37%. None of the sequences were derived from reported sequences of known viruses. Thus, the analysis of nucleic acid from virus-like particles is a useful tool to expand our knowledge of the universe of viruses to non-cultivated species.

In the plant virus world, there are six genera of plant viruses with dsDNA genomes and six genera with ssDNA (Fauquet et al., 2005). The dsDNA viruses are comprised of 4 genera in the Caulimoviridae, the genus Badnavirus and the genus Tungrovirus. The ssDNA viruses are comprised of four genera in Geminiviridae, and the two genera Nanovirus and Babuvirus in the Nanoviridae. The genera Babuvirus and Badnavirus are not well studied in Asia. However, we recognized the significance of two species, Banana bunchy top virus (BBTV) in the genus Babuvirus and Banana streak virus (BSV) in the genus Badnavirus, during the survey of banana viruses in Asia. Their main characters will be introduced in this mini-review.

Studies were made to identify sources of resistance to yam viruses in Dioscorea alata. Forty genotypes of D. alata were evaluated in both the field and in the screenhouse for reactions to the yam viruses: Yam mosaic virus (YMV), genus Potyvirus; Dioscorea alata virus (DAV), genus Potyvirus; Cucumber mosaic virus (CMV), genus Cucumovirus; and Dioscorea alata bacilliform virus (DaBV), genus Badnavirus. The D. alata genotypes were planted in the field and subsequently scored for virus symptom severity. All the genotypes were also planted in an insect-proofed screenhouse, and challenged mechanically and by vectors for susceptibility to each of the viruses. Analysis of variance ( [smallcapital anova]) of the symptom severity scores showed that the genotypes responded differently (P < 0.01) to virus disease in the field. Field evaluation also showed that TDa 291 (a landrace genotype from Puerto Rico), TDa 87/01091, TDa 96-4, TDa 95-163 and TDa 289 from Nigeria, and TDa 95-25 (a landrace genotype from Ghana), had a low virus disease symptom rating. Overall screening results showed that two D. alata genotypes (TDa 289 and TDa 291) are good sources of resistance to YMV, DAV and CMV, and that they are tolerant to DaBV.

Abstract An appreciation of the risks caused by emergent plant viruses is critical in tropical areas that rely heavily on agriculture for subsistence and rural livelihood. Molecular ecology, within 10 years, has unraveled the factors responsible for the emergence of several of the economically most important tropical plant viruses: Rice yellow mottle virus (RYMV), Cassava mosaic geminiviruses (CMGs), Maize streak virus (MSV), and Banana streak virus (BSV). A large range of mechanisms—most unsuspected until recently—were involved: recombination and synergism between virus species, new vector biotypes, genome integration of the virus, host adaptation, and long-distance dispersal. A complex chain of molecular and ecological events resulted in novel virus-vector-plant-environment interactions that led to virus emergence. It invariably involved a major agricultural change: crop introduction, cultural intensification, germplasm movement, and new genotypes. A current challenge is now to complement the analysis of the causes by an assessment of the risks of emergence. Recent attempts to assess the risks of emergence of virulent virus strains are described.

The delivery of the double-stranded DNA viral genome into the nucleus is a critical step for the type member of Caulimoviridae, cauliflower mosaic virus (CaMV). The nucleocapsid (NC) of CaMV is directly involved in this process. A nuclear localization signal located at the N-terminus of the NC was shown to be exposed at the surface of the virion. This nuclear localization signal appears to be important to direct the virus to the nuclear pore complex. The nuclear targeting of the NC needs to be tightly regulated because the process of virus assembly, which also involves the viral NC, occurs in the cytosol. It is now accepted that the N- and C-terminal extensions of the viral NC precursor are efficient regulatory sequences that determine the localization of the viral NC in infected leaves. Proteolytic maturation and phosphorylation of the N- and C-terminal extensions are also important in the regulation of this process. Despite these recent discoveries, the transport of CaMV toward and into the nucleus during early events in the infection cycle remains unclear. In this review, we summarize recent advances that explain the mechanisms of targeting of the CaMV genome to the nucleus and extract from other related animal and plant viruses mechanisms that could hint at the possible strategies used by CaMV to enter the nucleus.

▪ Abstract Sequences of various DNA plant viruses have been found integrated into the host genome. There are two forms of integrant, those that can form episomal viral infections and those that cannot. Integrants of three pararetroviruses, Banana streak virus (BSV), Tobacco vein clearing virus (TVCV), and Petunia vein clearing virus (PVCV), can generate episomal infections in certain hybrid plant hosts in response to stress. In the case of BSV and TVCV, one of the parents contains the integrant but is has not been seen to be activated in that parent; the other parent does not contain the integrant. The number of integrant loci is low for BSV and PVCV and high in TVCV. The structure of the integrants is complex, and it is thought that episomal virus is released by recombination and/or reverse transcription. Geminiviral and pararetroviral sequences are found in plant genomes although not so far associated with a virus disease. It appears that integration of viral sequences is widespread in the plant kingdom and has been occurring for a long period of time.

Plant viruses are most frequently transmitted from one host plant to another by vectors. In noncirculative vector transmission, the virus does not process through a cycle within the vector body. Instead, upon acquisition by the vector, viruses are retained in the mouth parts or the anterior gut; from there, they will be subsequently released in a new host plant. Two molecular strategies have been described for the virus—vector interaction. In the capsid strategy, the virus coat interacts directly with binding sites in the vector mouth parts, whereas an additional nonstructural protein, designated helper component (HC), is required in the helper strategy. The HC and virus particles can be acquired sequentially, and this property introduces the possibility that an HC acquired first by the vector assists the transmission of virus particles located in the same cell, in other cells, or even in other host plants probed by the vector. Such a phenomenon is here called HC-transcomplementation. Surprisingly, the existing definition of HC does not explicitly include the concept of HC-transcomplementation, and it is generally omitted in the literature in any consideration of the virus biology other than the molecular interaction with the vector. Here we propose an extended definition of HC and emphasize the concept of HC-transcomplementation that distinguishes the helper strategy from any other type of vector transmission and may have consequences at the level of the virus population genetics and evolution.

Family Caulimoviridae comprises seven genera of plant viruses whose members replicate by reverse transcription and whose virions contain double-stranded deoxyribonucleic acid. In a recent survey of the international scientific community, Cauliflower mosaic virus (CaMV), the type species of the family, was ranked sixth in the world in a list of the most scientifically or economically important plant viruses. This notoriety is due purely to the major conceptual advances that have been made in plant virology using CaMV as a model pathogen. However, several relatively lesser known viruses in the family, such as Rice tungro bacilliform virus and Cacao swollen shoot virus, are very serious constraints to crop production in tropical regions of the world. In this article, information on the taxonomy, replication cycle, vector transmission, epidemiology and disease management of this important group of plant viruses is summarised. Key Concepts: The Caulimoviridae is the only family of plant viruses with a double-stranded deoxyribonucleic acid (dsDNA) genome, and in common with all viral retroelements, incorporates a reverse transcription step in the replication cycle. Cauliflower mosaic virus is the type species of the family, and has been a very important model plant virus for elucidating fundamental aspects of virus replication, cell-to-cell movement and aphid transmission. Members of the Caulimoviridae are most prominent in tropical regions, where they cause serious diseases such as rice tungro, cacao swollen shoot and banana streak disease. The most important component of a control programme for these viruses is to reduced inoculum levels by using clean planting material, avoiding overlapping crops, and removing diseased plants and alternative hosts of the virus. In some host species, infection can arise as a consequence of activation of viral DNA that is integrated in the nuclear genome of the plant. Keywords: plant viruses; retroelement; pararetrovirus; reverse transcription; cauliflower mosaic virus; rice tungro; cacao swollen shoot; tropical crop

International audience; Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.

Rice tungro bacilliform virus (RTBV) is a reverse-transcribing DNA virus which, in association with an RNA virus, Rice tungro spherical virus (RTSV), is responsible for rice tungro disease (22), the most important viral disease of rice in South and Southeast Asia. In rice tungro, RTBV induces most of the symptoms (yellowing and reddening of the leaves, stunting of rice plants) and RTSV is mainly involved in the transmission of both viruses via the green leafhopper Nephotettix virescens (5). RTBV is the type and only known member of the “RTBV-like viruses” genus, which has been classified in the Caulimoviridae family comprising caulimoviruses, badnaviruses, and two other genera (29, 31). The plant viruses which belong to this family have many features in common with retroviruses and are also often referred to, together with the human and animal hepadnaviruses, as pararetroviruses (23, 40, 42). The bacilliform RTBV particles are elongated icosahedrons with a diameter of 30 nm and a length of approximately 130 nm, which varies with the virus isolate (22). The RTBV genome is a circular double-stranded DNA molecule of about 8 kbp, containing two site-specific discontinuities resulting from the replication process by reverse transcription and four large open reading frames (ORFs) (Fig. ​(Fig.1A)1A) (1, 17, 39). The corresponding proteins, P1, P2, P3, and P4, are synthesized by specialized translation mechanisms (10–12) from a pregenomic RNA which is used as the template for viral replication and also serves as a polycistronic mRNA (22). FIG. 1 Schematic representations of the RTBV genome and P3 polyprotein. (A) Genome organization. Viral DNA is represented by a thin double line with the sites of the two discontinuities (Δ1 and Δ2) indicated. The thick arrows outside the DNA ... The roles of P1 (24 kDa) and P4 (46 kDa) are still unknown. P3 is a large polyprotein of 196 kDa (Fig. ​(Fig.1B).1B). Sequence comparisons with retroviral and other pararetroviral proteins suggest that P3 contains domains corresponding to the movement protein (MP), coat protein (CP), aspartic protease (PR), reverse transcriptase (RT), and RNase H (RH), ordered from the N terminus to the C terminus (17, 26, 39, 45). The viral protease is at least partly responsible for the processing of P3. The cleavage sites at the N- and C-terminal extremities of the RT-RH domain have been characterized. It has been demonstrated that the PR-RT-RH polyprotein can be processed to yield two proteins of 55 and 62 kDa (p55 and p62) when expressed in insect cells from the 3′ part of gene III (27). First reports indicated that RTBV particles contain two major CP species of 33 and 37 kDa (p33 and p37) (39). The N terminus of p33 was determined to be at amino acid 502. Considering its size and the position of its N-terminal residue within P3, p33 should contain, in its C-terminal region, the basic domain and the Cys-His motif which are conserved in plant pararetrovirus CPs. This motif is the equivalent of the zinc finger motif of retroviral Gag proteins and consequently is thought to be involved in specific RNA binding during packaging of the pregenomic RNA into virions (40). Recently, Marmey et al. (30) showed that RTBV virions contain only a single coat protein species of 37 kDa, with the second peptide (of 34 kDa) most probably being a degradation product of the 37-kDa protein generated during virus purification. Amino acids 477 and 791 of P3 were deduced, from mass spectral analysis, to correspond to the N- and C-terminal residues, respectively, of the 37-kDa coat protein (p37). ORF II encodes a 12-kDa protein (P2) for which no definite function has been assigned. P2 of RTBV and of the badnavirus commelina yellow mottle virus (CoYMV) were shown to be associated with purified virions (3, 22; A. Druka and R. Hull, personal communication). P2 of RTBV and of the badnavirus cacao swollen shoot virus (CSSV) were also described as sequence-nonspecific nucleic acid binding proteins (24, 25). The C termini of RTBV and CSSV P2, which possess basic, hydrophobic, and proline residues, support the nucleic acid binding activity. Such residues are also present at the C termini of caulimovirus gene III products and of bacterial histone-like proteins (34). Moreover, the C-terminal extremity of cauliflower mosaic virus (CaMV) P3 possesses a nonspecific nucleic acid binding activity (33, 34), suggesting a common role for this protein and the P2 of RTBV or badnaviruses in their respective life cycles. To investigate the role of RTBV P2, we searched for possible interactions between this protein and other RTBV proteins. P2 was shown to interact with the CP domain of P3 both in the yeast two-hybrid system and in vitro. We have characterized this interaction and identified peptide motifs involved in the binding on both proteins. To evaluate the importance of this interaction in the context of viral infection, we introduced point mutations within gene II of the RTBV genome and investigated the infectivity of these mutants by agroinoculation of rice plants. Our results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3.

Petunia vein-clearing virus (PVCV) is a plant pararetrovirus that has some features of retrotransposons. It encapsidates dsDNA and has isometric particles and inclusion bodies similar to those of caulimoviruses. The PVCV genome of 7205 bp has two large ORFs in the transcribed strand and a methionine tRNA primer-binding site in its 663-bp intergenic region. The N-terminal position of the large protein (126 kDa) encoded by ORF I has similarity to the movement protein of caulimoviruses. Toward the C-terminus of this same polyprotein are the two distinctive sequence elements [HHCC and DD(35)E] of the integrase function of retroviruses and retrotransposons. ORF II of PVCV encodes a protein of 125 kDa with domains for an RNA-binding element, common to thegaggene of retroelements, followed by consensus sequences for an acid protease, reverse transcriptase, and ribonuclease H. Hence, thegagequivalent (capsid protein) andpolgene of PVCV are part of the same polyprotein. Phylogenetic comparison of the reverse transcriptase of PVCV with that of various other retroelements grouped PVCV between caulimoviruses and the Ty3/gypsyretrotransposons, suggesting that PVCV is a divergent member of the caulimoviruses.

Virus diseases are serious constraints to the productivity and profitability of a wide range of tropical crops. Identification of the causal viruses and understanding their epidemiology is the key to estimating the incidence and economic impact of the diseases they cause, and to devising virus management strategies. Epidemics result from interactions between virus, host plant, vector and environmental factors, and every epidemic can be considered to be a unique pathosystem in which each of the components contributes to the epidemic, and in which none are limiting. Pathogen diagnosis is the key to managing diseases, and we list 18 crops, their main virus/viroid diseases and pathosystem descriptors for each. Six types of pathosystem are described on the basis of the mode of spread, and the different management strategies applicable to each are discussed. The Ninth Report of the International Committee on Taxonomy of Viruses (2012) is an essential reference for virologists working with tropical crops. Key Concepts: Identification of viruses or viroids is the key to managing the diseases they cause. Koch's rules are applied to establish causal associations between viruses/viroids and disease. Viruses/viroids are identified by their biological, morphological and genetic properties using classical and molecular methods. The universal database for viruses/viroids published by the International Committee on Taxonomy of Viruses is the resource for classification. Rapid diagnostic methods rely on nucleotide sequence comparisons with publicly available data bases. Epidemics are driven by virus, vector, host, environment, time and human activity. Vector–virus interaction is the main determinant of the rate, range, timing and pattern of disease spread. Viruses/viroids of tropical plants have unique pathosystems. A full description of a pathosystem is required to sustainably manage virus epidemics. Management of epidemics can be achieved by destabilising or down-regulating one or more components of the relevant pathosystem. Keywords: Geminiviridae; Potyviridae; Tospovirus; Caulimoviridae; Nanoviridae; Pospiviroidae; Sobemovirus; taxonomy; pathosystem; management

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.

Endogenous members of the family Caulimoviridae have now been found in the genomes of many plant species. Although these sequences are usually fragmented and rearranged and show varying degrees of decay, the genomes of the ancestral viruses can often be reassembled in silico, allowing classification within the existing viral taxonomic framework. In this paper, we describe analyses of endogenous members of the family Caulimoviridae in the genomes of Oryza sativa, Nicotiana tabacum and Solanum spp. and on the basis of phylogeny, genome organization and genetic distance within the pol gene, propose two new virus genera called Orendovirus and Solendovirus. A system of nomenclature for endogenous virus sequences in plants is also proposed.

Schoelz, J. E., Wiggins, B. E., Wintermantel, W. M., and Ross, K. 2006. Introgression of a tombusvirus resistance locus from Nicotiana edwardsonii var. Columbia to N. clevelandii. Phytopathology 96:453-459. A new variety of Nicotiana, N. edwardsonii var. Columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. Columbia was developed from a hybridization between N. glutinosa and N. clevelandii, the same parents used for the formation of the original N. edwardsonii. However, in contrast to the original N. edwardsonii, crosses between Columbia and either of its parents are fertile. Thus, the inheritance of virus resistance genes present in N. glutinosa could be characterized by using Columbia as a bridge plant in crosses with the susceptible parent, N. clevelandii. To determine how virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic virus (TMV). Our genetic evidence indicated that the entire chromosome containing the N gene was introgressed into N. clevelandii to create an addition line, designated N. clevelandii line 19. Although line 19 was homozygous for resistance to TMV, it remained susceptible to Tomato bushy stunt virus (TBSV) and Cauliflower mosaic virus (CaMV) strain W260, indicating that resistance to these viruses must reside on other N. glutinosa chromosomes. We also developed a second addition line, N. clevelandii line 36, which was homozygous for resistance to TBSV. Line 36 was susceptible to TMV and CaMV strain W260, but was resistant to other tombusviruses, including Cucumber necrosis virus, Cymbidium ringspot virus, Lettuce necrotic stunt virus, and Carnation Italian ringspot virus.

The non-enveloped bacilliform viruses are the second group of plant viruses known to possess a genome consisting of circular double-stranded DNA. We have characterized the viral transcript and determined the complete sequence of the genome of Commelina mellow mottle virus (CoYMV), a member of this group. Analysis of the viral transcript indicates that the virus encodes a single terminally-redundant genome-length plus 120 nucleotide transcript. A fraction of the transcripts is polyadenylated, although the majority of the transcript is not polyadenylated. Analysis of the genome sequence indicates that the genome is 7489 bp in size and that the transcribed strand contains three open reading frames capable of encoding proteins of 23, 15 and 216 kd. The function of the 25 and 15 kd proteins is unknown. Similarities between the 216 kd polypeptide and the cauliflower mosaic virus coat protein and protease/reverse transcriptase polyprotein suggest that the 216 kd polypeptide is a polyprotein that is proteolytically processed to yield the virion coat protein, a protease, and replicase (reverse transcriptase and ribonuclease H). Each strand of the CoYMV genome is interrupted by site-specific discontinuities. The locations of the 5'-ends of these discontinuities, and the presence and location of a region on the CoYMV transcript capable of annealing with the 3'-end of cytosolic initiator methionine tRNA are consistent with replication by reverse transcription. We have demonstrated that a construct containing 1.3 CoYMV genomes is infective when introduced into Commelina diffusa, the host for CoYMV, using Agrobacterium-mediated infection.

An isolate of banana streak virus (BSV) that does not also occur as an integrant in the Musa balbisiana genome was sought in order to investigate the biological role of BSV in the evolution of either the Musa genome or of the virus itself. We isolated BSV virions from a Musa acuminata siamea accession from Vietnam and sequenced the entire viral genome. The molecular organization is similar to that described for other BSV but slightly larger (7801 bp vs. 1611-7568 bp), and ORF I has a non-conventional start codon. This genome was sufficiently different to propose it as a member of a distinct species named Banana streak virus strain acuminata Vietnam (BSAcVNV).

Background Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV)-like sequences (ERTBVs) have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response. Results We have collected ERTBVs from the rice genomes. They contain rearranged structures and no intact ORFs. The identified ERTBV segments were shown to be phylogenetically divided into three clusters. For each phylogenetic cluster, we were able to make a consensus alignment for a circular virus-like structure carrying two complete ORFs. Comparisons of DNA and amino acid sequences suggested the closely relationship between ERTBV and RTBV. The Oryza AA-genome species vary in the ERTBV copy number. The species carrying low-copy-number of ERTBV segments have been reported to be extremely susceptible to RTBV. The DNA methylation state of the ERTBV sequences was correlated with their copy number in the genome. Conclusions These ERTBV segments are unlikely to have functional potential as a virus. However, these sequences facilitate to establish putative virus that provided information underlying virus integration and evolutionary relationship with existing virus. Comparison of ERTBV among the Oryza AA-genome species allowed us to speculate a possible role of endogenous virus segments against its related disease.

Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is a multifunctional protein essential for basic replication of CaMV. It also plays a role in viral pathogenesis in crucifer and solanaceous host plants. Deletion mutagenesis revealed that N- and C-terminal parts of Tav are not essential for CaMV replication in transfected protoplasts. Two deletion mutants having only minimal defects in basic replication were infectious in turnips but only with highly attenuated virulence. This was shown to be due to delayed virus spread within the inoculated leaves and to the upper leaves. Unlike the wild-type virus, the mutant viruses successfully spread locally without inducing a host defense response in inoculated Datura stramonium leaves, but did not spread systemically. These results provide the first evidence that a Tav domain required for avirulence function in solanaceous plants is not essential for CaMV infectivity but has a role in viral virulence in susceptible hosts.