Replication-independent change in the frequencies of distinct genome segments of a multipartite virus during its transit within aphid vectors.

Multipartite viruses exhibit a fragmented genome composed of several nucleic acid segments individually packaged in distinct viral particles. The genome of all species of the genus Nanovirus holds eight segments, which accumulate at a very specific and reproducible relative frequency in the host plant tissues. In a given host species, the steady state pattern of the segments' relative frequencies is designated the genome formula and is thought to have an adaptive function through the modulation of gene expression. Nanoviruses are aphid-transmitted circulative non-propagative viruses, meaning that the virus particles are internalized into the midgut cells, transferred to the hemolymph, and then to the saliva, with no replication during this transit. Unexpectedly, a previous study on the faba bean necrotic stunt virus revealed that the genome formula changes after ingestion by aphids. We investigate here the possible mechanism inducing this change by first comparing the relative segment frequencies in different compartments of the aphid. We show that changes occur both in the midgut lumen and in the secreted saliva but not in the gut, salivary gland, or hemolymph. We further establish that the viral particles differentially resist physicochemical variations, in particular pH, ionic strength, and/or type of salt, depending on the encapsidated segment. We thus propose that the replication-independent genome formula changes within aphids are not adaptive, contrary to changes occurring in plants, and most likely reflect a fortuitous differential degradation of virus particles containing distinct segments when passing into extra-cellular media such as gastric fluid or saliva. IMPORTANCE: The genome of multipartite viruses is composed of several segments individually packaged into distinct viral particles. Each segment accumulates at a specific frequency that depends on the host plant species and regulates gene expression. Intriguingly, the relative frequencies of the genome segments also change when the octopartite faba bean necrotic stunt virus (FBNSV) is ingested by aphid vectors, despite the present view that this virus travels through the aphid gut and salivary glands without replicating. By monitoring the genomic composition of FBNSV populations during the transit in aphids, we demonstrate here that the changes take place extracellularly in the gut lumen and in the saliva. We further show that physicochemical factors induce differential degradation of viral particles depending on the encapsidated segment. We propose that the replication-independent changes within the insect vector are not adaptive and result from the differential stability of virus particles containing distinct segments according to environmental parameters.

The genome formula of a multipartite virus is regulated both at the individual segment and the segment group levels.

Differential accumulation of the distinct genome segments is a common feature of viruses with segmented genomes. The reproducible and specific pattern of genome segment accumulation within the host is referred to as the "genome formula". There is speculation and some experimental support for a functional role of the genome formula by modulating gene expression through copy number variations. However, the mechanisms of genome formula regulation have not yet been identified. In this study, we investigated whether the genome formula of the octopartite nanovirus faba bean necrotic stunt virus (FBNSV) is regulated by processes acting at the individual segment vs. viral population levels. We used a leaf infiltration system to show that the two most accumulated genome segments of the FBNSV possess a greater intrinsic accumulation capacity in Vicia faba tissues than the other segments. Nevertheless, processes acting at the individual segment level are insufficient to generate the genome formula, suggesting the involvement of additional mechanisms acting at the supra-segment level. Indeed, the absence of segments with important functions during systemic infection strongly modifies the relative frequency of the others, indicating that the genome formula is a property of the segment group. Together, these results demonstrate that the FBNSV genome formula is shaped by a complex process acting at both the individual segment and the segment group levels.

The nanovirus U2 protein suppresses RNA silencing via three conserved cysteine residues.

Nanoviruses have multipartite, circular, single-stranded DNA genomes and cause huge production losses in legumes and other crops. No viral suppressor of RNA silencing (VSR) has yet been reported from a member of the genus Nanovirus. Here, we demonstrate that the nanovirus U2 protein is a VSR. The U2 protein of milk vetch dwarf virus (MDV) suppressed the silencing of the green fluorescent protein (GFP) gene induced by single-stranded and double-stranded RNA, and the systemic spread of the GFP silencing signal. An electrophoretic mobility shift assay showed that the U2 protein was able to bind double-stranded 21-nucleotide small interfering RNA (siRNA). The cysteine residues at positions 43, 79 and 82 in the MDV U2 protein are critical to its nuclear localization, self-interaction and siRNA-binding ability, and were essential for its VSR activity. In addition, expression of the U2 protein via a potato virus X vector induced more severe necrosis symptoms in Nicotiana benthamiana leaves. The U2 proteins of other nanoviruses also acted as VSRs, and the three conserved cysteine residues were indispensable for their VSR activity.

Insights into the Differential Composition of Stem-Loop Structures of Nanoviruses and Their Impacts.

Multipartite viruses package their genomic segments independently and mainly infect plants; few of them target animals. Nanoviridae is a family of multipartite single-stranded DNA (ssDNA) plant viruses that individually encapsidate ssDNAs of ~1 kb and transmit them through aphids without replication in aphid vectors, thereby causing important diseases in host plants, mainly leguminous crops. All of these components constitute an open reading frame to perform a specific role in nanovirus infection. All segments contain conserved inverted repeat sequences, potentially forming a stem-loop structure and a conserved nonanucleotide, TAGTATTAC, within a common region. This study investigated the variations in the stem-loop structure of nanovirus segments and their impact using molecular dynamics (MD) simulations and wet lab approaches. Although the accuracy of MD simulations is limited by force field approximations and simulation time scale, explicit solvent MD simulations were successfully used to analyze the important aspects of the stem-loop structure. This study involves the mutants' design, based on the variations in the stem-loop region and construction of infectious clones, followed by their inoculation and expression analysis, based on nanosecond dynamics of the stem-loop structure. The original stem-loop structures showed more conformational stability than mutant stem-loop structures. The mutant structures were expected to alter the neck region of the stem-loop by adding and switching nucleotides. Changes in conformational stability are suggested expression variations of the stem-loop structures found in host plants with nanovirus infection. However, our results can be a starting point for further structural and functional analysis of nanovirus infection. IMPORTANCE Nanoviruses comprise multiple segments, each with a single open reading frame to perform a specific function and an intergenic region with a conserved stem-loop region. The genome expression of a nanovirus has been an intriguing area but is still poorly understood. We attempted to investigate the variations in the stem-loop structure of nanovirus segments and their impact on viral expression. Our results show that the stem-loop composition is essential in controlling the virus segments' expression level.

Structure-guided mutagenesis of the capsid protein indicates that a nanovirus requires assembled viral particles for systemic infection.

Nanoviruses are plant multipartite viruses with a genome composed of six to eight circular single-stranded DNA segments. The distinct genome segments are encapsidated individually in icosahedral particles that measure ≈18 nm in diameter. Recent studies on the model species Faba bean necrotic stunt virus (FBNSV) revealed that complete sets of genomic segments rarely occur in infected plant cells and that the function encoded by a given viral segment can complement the others across neighbouring cells, presumably by translocation of the gene products through unknown molecular processes. This allows the viral genome to replicate, assemble into viral particles and infect anew, even with the distinct genome segments scattered in different cells. Here, we question the form under which the FBNSV genetic material propagates long distance within the vasculature of host plants and, in particular, whether viral particle assembly is required. Using structure-guided mutagenesis based on a 3.2 Å resolution cryogenic-electron-microscopy reconstruction of the FBNSV particles, we demonstrate that specific site-directed mutations preventing capsid formation systematically suppress FBNSV long-distance movement, and thus systemic infection of host plants, despite positive detection of the mutated coat protein when the corresponding segment is agroinfiltrated into plant leaves. These results strongly suggest that the viral genome does not propagate within the plant vascular system under the form of uncoated DNA molecules or DNA:coat-protein complexes, but rather moves long distance as assembled viral particles.

Nonconcomitant host-to-host transmission of multipartite virus genome segments may lead to complete genome reconstitution.

Because multipartite viruses package their genome segments in different viral particles, they face a potentially huge cost if the entire genomic information, i.e., all genome segments, needs to be present concomitantly for the infection to function. Previous work with the octapartite faba bean necrotic stunt virus (FBNSV; family Nanoviridae, genus Nanovirus) showed that this issue can be resolved at the within-host level through a supracellular functioning; all viral segments do not need to be present within the same host cell but may complement each other through intercellular trafficking of their products (protein or messenger RNA [mRNA]). Here, we report on whether FBNSV can as well decrease the genomic integrity cost during between-host transmission. Using viable infections lacking nonessential virus segments, we show that full-genome infections can be reconstituted and function through separate acquisition and/or inoculation of complementary sets of genome segments in recipient hosts. This separate acquisition/inoculation can occur either through the transmission of different segment sets by different individual aphid vectors or by the sequential acquisition by the same aphid of complementary sets of segments from different hosts. The possibility of a separate between-host transmission of different genome segments thus offers a way to at least partially resolve the genomic maintenance problem faced by multipartite viruses.

Genome-wide analysis of milk vetch dwarf virus and related alphasatellites from garlic and aphids in Shandong Province, China.

In 2019 and 2020, symptoms of dwarfing, yellowing, and reddening were observed in garlic in open fields in Shandong Province, China. Milk vetch dwarf virus (MDV) was detected in aphids and symptomatic garlic plants using polymerase chain reaction analysis. Furthermore, it was demonstrated using an aphid transmission test that garlic is a natural host of MDV. Rolling-circle amplification was combined with the use of specific primers to amplify the complete genomes of MDV and its related alphasatellites. This is the first report of complete genome sequences of MDV and related alphasatellites from garlic and aphid samples.

ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Rapid and visual detection of milk vetch dwarf virus using recombinase polymerase amplification combined with lateral flow strips.

BACKGROUND: Milk vetch dwarf virus (MDV) is an important ssDNA virus which causes yellowing, stunting and leaf rolling symptoms on legumes. In China, the virus causes great economic losses and has recently been found to infect tobacco. The expansion of its host range and its ability to spread rapidly has given rise to the urgent need for a sensitive, specific and rapid diagnostic assay that can assist in effective disease control. METHODS: Assays based on the polymerase chain reaction combined with lateral flow strip detection (PCR-LFS) and recombinase polymerase amplification combined with LFS (RPA-LFS) were developed targeting the coat protein (CP) gene of MDV. RESULTS: The PCR and RPA assays could detect respectively 103 copies or 101 copies of MDV by agarose gel electrophoresis. The PCR-LFS and RPA-LFS assays developed could both detect as few as 101 copies per reaction at 37 °C. Both methods could detect MDV in crude leaf extracts. CONCLUSIONS: The RPA-LFS assay developed is a rapid, sensitive and specific method for detecting MDV, which is convenient and has great potential for use in the field.

Novel nanovirus and associated alphasatellites identified in milk vetch plants with chlorotic dwarf disease in Iran.

Members of the family Nanoviridae are multi-component single-stranded DNA viruses that infect a variety of plant species. Using a combination of conventional PCR and high throughput sequencing-based approach, we identified a novel nanovirus infecting two symptomatic milk vetch plants (Astragalus myriacanthus Boiss.; family Fabaceae) showing marginal leaf chlorosis, little leaves and dwarfing in Iran. All eight segments (DNA-C, DNA-M, DNA-N, DNA-R, DNA-S, DNA-U1, DNA-U2 and DNAU4) were recovered and Sanger sequenced. The genome of this new nanovirus, hereby referred to as milk vetch chlorotic dwarf virus (MVCDV), shares 62.2-74.7 % nucleotide pairwise identity with the genomes of other nanoviruses. DNA-C, DNA-M, DNA-N, DNA-S components are most closely related to those of black medic leaf roll virus (BMLRV), sharing between 67.8-81.2 % identity. We also identified three nanoalphasatellites (family Alphasatellitidae) associated with the nanovirus which belong to species Faba bean necrotic yellows alphasatellite 1 (genus Subclovsatellite), Faba bean necrotic yellows alphasatellite 2 (genus Fabenesatellite) and Sophora yellow stunt alphasatellite 5 (genus Clostunsatellite). Given the significant diversity of Astragalus spp. in Iran, it is likely that there could be more nanoviruses circulating in these plants and that these may play a role in the spread of these nanovirus to cultivated fabaceous hosts.

Identification of a novel nanovirus in parsley.

Using next-generation sequencing to characterize agents associated with a severe stunting disease of parsley from Germany, we identified a hitherto undescribed virus. We sequenced total RNA and rolling-circle-amplified DNA from diseased plants. The genome sequence of the virus shows that it is a member of the genus Nanovirus, but it lacks DNA-U4. In addition to the seven genomic DNAs of the virus, we identified a second DNA-R and seven distinct alphasatellites associated with the disease. We propose the name "parsley severe stunt associated virus" (PSSaV) for this novel nanovirus.

A multicellular way of life for a multipartite virus.

A founding paradigm in virology is that the spatial unit of the viral replication cycle is an individual cell. Multipartite viruses have a segmented genome where each segment is encapsidated separately. In this situation the viral genome is not recapitulated in a single virus particle but in the viral population. How multipartite viruses manage to efficiently infect individual cells with all segments, thus with the whole genome information, is a long-standing but perhaps deceptive mystery. By localizing and quantifying the genome segments of a nanovirus in host plant tissues we show that they rarely co-occur within individual cells. We further demonstrate that distinct segments accumulate independently in different cells and that the viral system is functional through complementation across cells. Our observation deviates from the classical conceptual framework in virology and opens an alternative possibility (at least for nanoviruses) where the infection can operate at a level above the individual cell level, defining a viral multicellular way of life.

Full genome sequence analysis and putative host-shifting of Milk vetch dwarf virus infecting tobacco (Nicotiana tabacum) in China.

BACKGROUND: Tobacco production in China has been affected by plant viruses with Milk vetch dwarf virus (MDV) as a recent invader posing serious concern. According to most of the studies, MDV mainly infects hosts from Fabaceae family but in our previous study we reported its infection in tobacco plant (Nicotiana tabacum L.) in Shandong province. FINDINGS: In current study (2016-2017), tobacco plants (Nicotiana tabacum) with severe stunting, yellowing and axillary bunch of new leaves were observed in Zhengning, Gansu province. Isolate GSZN yielded into eight genomic circular single-stranded DNA components while no alphasatellite DNA was obtained. High percent identity of this isolate was recorded in overall nucleotide and amino acid assembly with reported MDV isolates worldwide. Phylogenetic analysis fetched into a separate sub-clade comprising of new isolate along with other tobacco infecting isolates of MDV. While recombination was predicted in DNA-C encoding Clink protein and DNA-U1, which may attribute towards the potential host-shifting phenomenon and ability of this virus to expand its host range. CONCLUSION: To our knowledge this is the first full genome annotation of a Nanovirus, infecting tobacco in natural field conditions, also this is the first extended analysis on host-shifting behavior of MDV.

Subterranean Clover Stunt Virus Revisited: Detection of Two Missing Genome Components.

Subterranean clover stunt virus (SCSV) is a type species of the genus Nanovirus in the family Nanoviridae. It was the first single-stranded DNA plant virus with a multipartite genome, of which genomic DNA sequences had been determined. All nanoviruses have eight genome components except SCSV, for which homologs of two genome components present in all other nanovirus genomes, DNA-U2 and DNA-U4, were lacking. We analysed archived and more recent samples from SCSV-infected legume plants to verify its genome composition and found the missing genome components. These results indicated that SCSV also has eight genome components and is a typical member of the genus Nanovirus.

Rapid detection of milk vetch dwarf virus by loop-mediated isothermal amplification.

Milk vetch dwarf virus (MDV) is a member of the genus Nanovirus, and its genome is composed of multiple circular 1-kb ssDNA components. In this study, we first determined that the diseased tobacco samples obtained in Zhucheng, Shandong Province were naturally infected with MDV using a polymerase chain reaction (PCR) assay. Subsequently, loop-mediated isothermal amplification (LAMP) was developed for the detection of MDV for the first time. The Mg2+ and dNTP concentrations and the reaction temperature and time of the LAMP were optimized to 8 mM, 1.8 mM, 65 °C, and 60 min, respectively. The best ratio of the inner primers (FIP and BIP) to the outer primers (F3 and B3) was 2:1. The LAMP detection limit was 100 times greater than that of PCR. The nucleotide amplification could be clearly observed by adding SYBR Green I. The positive and negative reactions exhibit distinctly different colors in daylight; however, the positive reactions exhibit green fluorescence under a UV lamp. Therefore, the method is stable, sensitive and specific.

Nanovirus DNA-N encodes a protein mandatory for aphid transmission.

Nanoviruses possess a multipartite single-stranded DNA genome and are naturally transmitted to plants by various aphid species in a circulative non-propagative manner. Using the cloned genomic DNAs of faba bean necrotic stunt virus (FBNSV) for reconstituting nanovirus infections we analyzed the necessity of different virus components for infection and transmission by aphids. We found that in the absence of DNA-U1 and DNA-U2 symptom severity decreased, and in the absence of DNA-U1 the transmission efficiency decreased. Most significantly, we demonstrated that the protein encoded by DNA-N (NSP) is mandatory for aphid transmission. Moreover, we showed that the NSP of FBNSV could substitute for that of a distantly related nanovirus, pea necrotic yellow dwarf virus. Altering the FBNSV NSP by adding 13 amino acids to its carboxy-terminus resulted in an infectious but non-transmissible virus. We demonstrate that the NSP acts as a nanovirus transmission factor, the existence of which had been hypothesized earlier.

Alphasatellitidae: a new family with two subfamilies for the classification of geminivirus- and nanovirus-associated alphasatellites.

Nanoviruses and geminiviruses are circular, single stranded DNA viruses that infect many plant species around the world. Nanoviruses and certain geminiviruses that belong to the Begomovirus and Mastrevirus genera are associated with additional circular, single stranded DNA molecules (~ 1-1.4 kb) that encode a replication-associated protein (Rep). These Rep-encoding satellite molecules are commonly referred to as alphasatellites and here we communicate the establishment of the family Alphasatellitidae to which these have been assigned. Within the Alphasatellitidae family two subfamilies, Geminialphasatellitinae and Nanoalphasatellitinae, have been established to respectively accommodate the geminivirus- and nanovirus-associated alphasatellites. Whereas the pairwise nucleotide sequence identity distribution of all the known geminialphasatellites (n = 628) displayed a troughs at ~ 70% and 88% pairwise identity, that of the known nanoalphasatellites (n = 54) had a troughs at ~ 67% and ~ 80% pairwise identity. We use these pairwise identity values as thresholds together with phylogenetic analyses to establish four genera and 43 species of geminialphasatellites and seven genera and 19 species of nanoalphasatellites. Furthermore, a divergent alphasatellite associated with coconut foliar decay disease is assigned to a species but not a subfamily as it likely represents a new alphasatellite subfamily that could be established once other closely related molecules are discovered.

Small Bottleneck Size in a Highly Multipartite Virus during a Complete Infection Cycle.

Multipartite viruses package their genomic segments independently and thus incur the risk of being unable to transmit their entire genome during host-to-host transmission if they undergo severe bottlenecks. In this paper, we estimated the bottleneck size during one infection cycle of Faba bean necrotic stunt virus (FBNSV), an octopartite nanovirus whose segments have been previously shown to converge to particular and unequal relative frequencies within host plants and aphid vectors. Two methods were used to derive this estimate, one based on the probability of transmission of the virus and the other based on the temporal evolution of the relative frequency of markers for two genomic segments, one frequent and one rare (segment N and S, respectively), both in plants and vectors. Our results show that FBNSV undergoes severe bottlenecks during aphid transmission. Further, even though the bottlenecks are always narrow under our experimental conditions, they slightly widen with the number of transmitting aphids. In particular, when several aphids are used for transmission, the bottleneck size of the segments is also affected by within-plant processes and, importantly, significantly differs across segments. These results indicate that genetic drift not only must be an important process affecting the evolution of these viruses but also that these effects vary across genomic segments and, thus, across viral genes, a rather unique and intriguing situation. We further discuss the potential consequences of our findings for the transmission of multipartite viruses.IMPORTANCE Multipartite viruses package their genomic segments in independent capsids. The most obvious cost of such genomic structure is the risk of losing at least one segment during host-to-host transmission. A theoretical study has shown that for nanoviruses, composed of 6 to 8 segments, hundreds of copies of each segment need to be transmitted to ensure that at least one copy of each segment was present in the host. These estimations seem to be very high compared to the size of the bottlenecks measured with other viruses. Here, we estimated the bottleneck size during one infection cycle of FBNSV, an octopartite nanovirus. We show that these bottlenecks are always narrow (few viral particles) and slightly widen with the number of transmitting aphids. These results contrast with theoretical predictions and illustrate the fact that a new conceptual framework is probably needed to understand the transmission of highly multipartite viruses.

Nanovirus-alphasatellite complex identified in Vicia cracca in the Rhône delta region of France.

Nanoviruses are multi-component plant-infecting single-stranded DNA viruses. Using a viral metagenomics-informed approach, a new nanovirus and two associated alphasatellite molecules have been identified in an uncultivated asymptomatic Vicia cracca plant in the Rhône region of France. This novel nanovirus genome includes eight genomic components (named DNA-R, DNA-S, DNA-M, DNA-C, DNA-N, DNA-U1, DNA-U2 and DNA-U4) and, across all components, shares < 66% pairwise sequence identity with other nanovirus genomes. The two associated alphasatellites share 62% identity with each other and < 81% identity will all other nanovirus-associated alphasatellites.

Molecular characterization of faba bean necrotic yellows viruses in Tunisia.

Faba bean necrotic yellows virus (FBNYV) (genus Nanovirus; family Nanoviridae) has a genome comprising eight individually encapsidated circular single-stranded DNA components. It has frequently been found infecting faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) in association with satellite molecules (alphasatellites). Genome sequences of FBNYV from Azerbaijan, Egypt, Iran, Morocco, Spain and Syria have been determined previously and we now report the first five genome sequences of FBNYV and associated alphasatellites from faba bean sampled in Tunisia. In addition, we have determined the genome sequences of two additional FBNYV isolates from chickpea plants sampled in Syria and Iran. All individual FBNYV genome component sequences that were determined here share > 84% nucleotide sequence identity with FBNYV sequences available in public databases, with the DNA-M component displaying the highest degree of diversity. As with other studied nanoviruses, recombination and genome component reassortment occurs frequently both between FBNYV genomes and between genomes of nanoviruses belonging to other species.