The Quest to Identify a New Virus Disease of Sunflower from Nebraska.

Between 2010 and 2018, sunflower plants exhibiting virus-like symptoms, including stunting, mottling, and chlorotic ringspots on leaves, were observed from commercial fields and research plots from four sites within three distinct counties of western Nebraska (Box Butte, Kimball, and Scotts Bluff). Near identical symptoms from field samples were reproduced on seedlings mechanically in the greenhouse on multiple occasions, confirming the presence of a sap-transmissible virus from each site. Symptomatic greenhouse-inoculated plants from the 2010 and 2011 Box Butte samples tested negative for sunflower mosaic virus (SuMV), sunflower chlorotic mottle virus (SuCMoV), and all potyviruses in general by ELISA and RT-PCR. Similar viral-like symptoms were later observed on plants in a commercial sunflower field in Kimball County in 2014, and again from volunteers in research plots in Scotts Bluff County in 2018. Samples from both of these years were again successfully reproduced on seedlings in the greenhouse as before following mechanical transmissions. Symptom expression for all years began 12 to 14 days after inoculation as mild yellow spots followed by the formation of chlorotic ringspots from the mottled pattern. The culture from 2014 tested negatively for three groups of nepoviruses via RT-PCR, ruling this group out. However, transmission electron microscopy assays of greenhouse-infected plants from both 2014 and 2018 revealed the presence of distinct, polyhedral virus particles. With the use of high throughput sequencing and RT-PCR, it was confirmed that the infections from both years were caused by a new virus in the tombusvirus genus and was proposed to be called Sunflower ring spot mottle virus (SuRSMV). Although the major objective of this project was to identify the causal agent of the disease, it became evident that the diagnostic journey itself, with all the barriers encountered on the 10-year trek, was actually more important and impactful than identification.

Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana.

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant-microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant-virus interactions in complement to Saccharomyces cerevisiae.

Analysis of a novel RNA virus in a wild northern white-breasted hedgehog (Erinaceus roumanicus).

Tombusviruses are generally considered plant viruses. A novel tombus-/carmotetravirus-like RNA virus was identified in a faecal sample and blood and muscle tissues from a wild northern white-breasted hedgehog (Erinaceus roumanicus). The complete genome of the virus, called H14-hedgehog/2015/HUN (GenBank accession number MN044446), is 4,118 nucleotides in length with a readthrough stop codon of type/group 1 in ORF1 and lacks a poly(A) tract at the 3' end. The predicted ORF1-RT (RdRp) and the capsid proteins had low (31-33%) amino acid sequence identity to unclassified tombus-/noda-like viruses (Hubei tombus-like virus 12 and Beihai noda-like virus 10), respectively, discovered recently in invertebrate animals. An in vivo experimental plant inoculation study showed that an in vitro-transcribed H14-hedgehog/2015/HUN viral RNA did not replicate in Nicotiana benthamiana, Chenopodium quinoa, or Chenopodium murale, the most susceptible hosts for plant-origin tombusviruses.

Identification and characterization of a tombusvirus isolated from Japanese gentian.

The DECS (dsRNA isolation, exhaustive amplification, cloning and sequencing) analysis technique for viral diagnosis detected a tombusvirus in Japanese gentian not displaying severe symptoms. We tentatively named this virus "gentian virus A" (GeVA). GeVA systemically but inefficiently infected Japanese gentian without causing visible symptoms, while it led to severe symptoms in some other plants. The complete genome sequence of GeVA indicated a typical tombusvirus-like structure. Phylogenetic analysis of the deduced amino acid sequences of four tombusvirus-encoded proteins did not reveal other known tombusviruses that were closely-related to GeVA, suggesting that it is a novel tombusvirus.

A simple method to estimate the isoelectric point of modified Tomato bushy stunt virus (TBSV) particles.

We present a simple method to estimate the isoelectric point (pI) of Tomato Bushy Stunt particles. We demonstrate that the combination of agarose gels with different pH buffers can be used to electrophorese the virus particles and their migration patterns can be compared. This method allows us to estimate the pI of the virus particles (wild type, wt, and genetically modified particles) and to monitor the effect of the pI of modified peptide side chains of the viral capsid subunit on the pI of the whole virus particle.

Calcium ions modulate the mechanics of tomato bushy stunt virus.

Viral particles are endowed with physicochemical properties whose modulation confers certain metastability to their structures to fulfill each task of the viral cycle. Here, we investigate the effects of swelling and ion depletion on the mechanical stability of individual tomato bushy stunt virus nanoparticles (TBSV-NPs). Our experiments show that calcium ions modulate the mechanics of the capsid: the sequestration of calcium ions from the intracapsid binding sites reduces rigidity and resilience in ∼24% and 40%, respectively. Interestingly, mechanical deformations performed on native TBSV-NPs induce an analogous result. In addition, TBSV-NPs do not show capsomeric vacancies after surpassing the elastic limit. We hypothesize that even though there are breakages among neighboring capsomers, RNA-capsid protein interaction prevents the release of capsid subunits. This work shows the mechanical role of calcium ions in viral shell stability and identifies TBSV-NPs as malleable platforms based on protein cages for cargo transportation at the nanoscale.

A new application of monolithic supports: the separation of viruses from one another.

The emergence of next-generation "deep" sequencing has enabled the study of virus populations with much higher resolutions. This new tool increases the possibility of observing mixed infections caused by combinations of plant viruses, which are likely to occur more frequently than previously thought. The biological impact of co-infecting viruses on their host has yet to be determined and fully understood, and the first step towards reaching this goal is the separation and purification of individual species. Ion-exchange monolith chromatography has been used successfully for the purification and concentration of different viruses, and number of them have been separated from plant homogenate or bacterial and eukaryotic lysate. Thus, the question remained as to whether different virus species present in a single sample could be separated. In this study, anion-exchange chromatography using monolithic supports was optimized for fast and efficient partial purification of three model plant viruses: Turnip yellow mosaic virus, Tomato bushy stunt virus, and Tobacco mosaic virus. The virus species, as well as two virus strains, were separated from each other in a single chromatographic experiment from an artificially mixed sample. Based on A260/280 ratios, we were able to attribute specific peaks to a certain viral morphology/structure (icosahedral or rod-shaped). This first separation of individual viruses from an artificially prepared laboratory mixture should encourage new applications of monolithic chromatographic supports in the separation of plant, bacterial, or animal viruses from all kinds of mixed samples.

Complete genome sequence and biological characterization of Moroccan pepper virus (MPV) and reclassification of Lettuce necrotic stunt virus as MPV.

Moroccan pepper virus (MPV) and Lettuce necrotic stunt virus (LNSV) have been steadily increasing in prevalence in central Asia and western North America, respectively, over the past decade. Recent sequence analysis of LNSV demonstrated a close relationship between the coat proteins of LNSV and MPV. To determine the full extent of the relationship between LNSV and MPV, the genomes of three MPV isolates were sequenced and compared with that of LNSV. Sequence analysis demonstrated that genomic nucleotide sequences as well as virus-encoded proteins of the three MPV isolates and LNSV shared 97% or greater identity. A full-length clone of a California LNSV isolate was developed and virus derived from infectious transcripts was used to evaluate host plant reactions under controlled conditions. Symptoms of LNSV matched those described previously for MPV on most of a select series of host plants, although some differences were observed. Collectively, these molecular and biological results demonstrate that LNSV should be classified as MPV within the family Tombusviridae, genus Tombusvirus, and confirm the presence of MPV in North America.

Genetic analysis and mapping of resistance to lettuce dieback: a soilborne disease caused by tombusviruses.

A diverse collection of modern, heirloom and specialty cultivars, plant introduction (PI) accessions, and breeding lines of lettuce were screened for susceptibility to lettuce dieback, which is a disease caused by soilborne viruses of the family Tombusviridae. Susceptibility was evaluated by visual symptom assessment in fields that had been previously shown to be infested with Lettuce necrotic stunt virus. Of the 241 genotypes tested in multiple field experiments, 76 remained symptom-free in infested fields and were therefore classified as resistant to dieback. Overall, resistant genotypes were as prevalent among modern cultivars as in heirloom cultivars or primitive germplasm. Within modern germplasm, however, all crisphead (iceberg) cultivars were resistant, while all romaine cultivars were susceptible. Using enzyme-linked immunosorbent assay, tombusviruses were detected in leaves of some plants of resistant genotypes that were grown in infested fields, suggesting that symptom-free plants are not immune to viral infection. The inheritance of resistance was studied for 'Salinas', a modern iceberg cultivar, and PI 491224, the progenitor of recently released romaine germplasm with resistance to lettuce dieback. Resistance was conferred by a dominant allele at a single locus in both genotypes. The tombusvirus resistance locus from 'Salinas', Tvr1, was mapped in an intraspecific Lactuca sativa population to a location that corresponds to linkage group 2 on the consensus map of Lactuca. The largest cluster of resistance genes in lettuce, the Dm1/Dm3 cluster, is found on this linkage group; however, the precise position of Tvr1 relative to this cluster has not yet been determined. To our knowledge, Tvr1 is the first tombusvirus resistance gene identified for any plant host.

Evaluation of various species demarcation criteria in attempts to classify ten new tombusvirus isolates.

The usefulness of various suggested species demarcation criteria was compared in attempts to determine the taxonomic status of ten new tombusvirus isolates. Five of them (Lim 1, 2, 3, 5 and 6) were obtained from different sources of commercially grown statice (Limonium sinuatum), two (Gyp 1 and 2) from different sources of commercially grown Gypsophila paniculata and three from water samples, i.e. from a small river (Schunter) in Northern Germany, from a brook (near Dossenheim) in Southern Germany and from the groundwater in a Limonium production glasshouse in the Netherlands (Lim 4). The immunoelectron microscopical decoration test allowed a quick preliminary assignment of various isolates to several known tombusviruses. A more precise analysis of the relationships was achieved by comparing the deduced amino acid sequences of the coat proteins. Sequence as well as serological data suggested that eight of the isolates should be classified as strains or variants of either Carnation Italian ringspot virus, Grapevine Algerian latent virus, Petunia asteroid mosaic virus or Sikte waterborne virus, respectively, whereas the 9th isolate (Lim 2) appears to represent a distinct new tombusvirus species. The case of the 10th isolate (Lim 5) illustrates the classification problems experienced when the properties of a virus place it close to the more or less arbitrary man-made borderline between virus species and virus strains. The coat protein gene sequences were also determined for some viruses for which these data had not yet been available, i.e. Neckar river virus, Sikte waterborne virus and Eggplant mottled crinkle virus. The sequences of the coat protein gene and also of ORF 1 of the latter virus proved to be almost identical to the corresponding genome regions of the recently described Pear latent virus, which for priority reasons should be renamed. Criteria which have been suggested in addition to serology and sequence comparisons for tombusvirus species demarcation, i.e. differences in natural and in experimental host ranges, in cytopathological features and in coat protein size, appear to be of little value for the classification of new tombusviruses.

The complete nucleotide sequence and synthesis of infectious RNA of genomic and defective interfering RNAs of TBSV-P.

The complete nucleotide sequences of the genome of the pepper isolate of tomato bushy stunt Tombusvirus (TBSV-P), and its defective interfering (DI) RNAs were determined. The genome of TBSV-P is a linear single-stranded monopartite RNA molecule of positive polarity, 4776 nucleotides long and has an organisation identical to that reported for other tombusviruses. In vitro transcripts of the genome were highly infectious, and it could support replication of the DI RNAs associated with the wild type virus. Two DI RNAs were found in the infected leaves of Nicotiana clevelandii, whose sequences were completely derived from the genomic RNA. The longest DI RNA (DI-5) has 550 nucleotides (nt), while the shorter DI RNA (DI-4) composed of 463 nt, both of them were formed by essentially the same genomic sequence blocks. Since host specificity of TBSV-P and other tombusviruses with available infectious cDNA clones is different, it is feasible to carry out gene exchange studies to determine viral host specificity factors for tombusviruses.

New satellite RNAs, but no DI RNAs, are found in natural populations of tomato bushy stunt tombusvirus.

A collection of 57 field isolates of the tombusvirus tomato bushy stunt virus was obtained from eggplant and tomato during 1994-1997 and was examined for the presence of defective interfering (DI) RNA species by Northern blot hybridization and RT-PCR. No DI RNA species were detected associated with any of the field TBSV isolates. However, serial passaging of two field isolates in Nicotiana clevelandii at high multiplicity of infection resulted in the rapid generation of DI-like RNA species, indicating that the absence of DI RNAs in natural populations of the virus was not due to the inability of the TBSV field isolates to generate them in a suitable host. The results indicate that DI RNAs may not play a role in modulating natural TBSV infections in the hosts examined. In 4 of 57 isolates analyzed we have detected less than full-length RNAs and we show here that they are true satellite RNAs. Two different satellite RNA species were detected, named TBSV sat RNAs B1 (822 nt) and B10 (612 nt). TBSV sat RNAs lack significant open reading frames and do not present sequence homology except in a central box that is also conserved in TBSV-Ch genomic RNA and in all the DI RNAs derived from it. TBSV sat RNA B10 attenuated the symptoms induced by the helper virus in N. clevelandii while sat RNA B1 did not modify the symptoms. This is the first report of sat RNAs associated with TBSV and the first time that sat RNAs are associated with natural tombusvirus infections.

Immunodetection, expression strategy and complementation of turnip crinkle virus p28 and p88 replication components.

The plus-sense RNA genome of turnip crinkle virus (TCV) encodes at its 5' end a 28-kDa protein of unspecified function. Readthrough suppression of the p28 stop codon allows for the production of an 88-kDa product which is required for genome replication. Immunological analysis of the expression of p28 and p88 demonstrated that: (i) the genome directs the synthesis of polypeptides of approximately 28 and 88 kDa, (ii) the 88-kDa protein is immunologically related to p28, consistent with p88 being a readthrough product, and (iii) p28, but not p88, is detectable in vivo. An in vivo assay, in which readthrough is linked to the expression of a beta-glucuronidase reporter gene, showed that readthrough of the p28 amber stop codon occurs with an efficiency of approximately 1%. A similar efficiency of readthrough was observed when an altered context from the nonviable TCV mutant, mA2, containing a disrupted secondary structure (FfFa) spanning the p28 termination codon, was tested. This result suggests that the defective phenotype of mA2 is likely not linked to an alteration in readthrough efficiency. Additional studies demonstrated that complementation occurs in coinoculations with two nonviable TCV mutants, RT and APA, which are unable to express either p28 or p88, respectively. This result verifies that p28 is essential for TCV genome replication and provides the first definitive evidence for the role of a 5'-proximal open reading frame for any member of the family Tombusviridae.