Alstroemeria yellow spot virus (AYSV): a new orthotospovirus species within a growing Eurasian clade.

An orthotospovirus distinct from all other orthotospoviruses was isolated from naturally infected alstroemeria plants. Disease symptoms caused by this virus mainly consisted of yellow spots on the leaves based on which the name alstroemeria yellow spot virus (AYSV) was coined. A host range analysis was performed and a polyclonal antiserum was produced against purified AYSV ribonucleoproteins which only reacted with the homologous antigen and not with any other (established or tentative) orthotospovirus from a selection of American and Asian species. Upon thrips transmission assays the virus was successfully transmitted by a population of Thrips tabaci. The entire nucleotide sequence of the M and S RNA segments was elucidated by a conventional cloning and sequencing strategy, and contained 4797 respectively 2734 nucleotides (nt). Simultaneously, a next generation sequencing (NGS) approach (RNAseq) was employed and generated contigs covering the entire viral tripartite RNA genome. In addition to the M and S RNA nucleotide sequences, the L RNA (8865 nt) was obtained. The nucleocapsid (N) gene encoded by the S RNA of this virus consisted of 819 nucleotides with a deduced N protein of 272 amino acids and by comparative sequence alignments to other established orthotospovirus species showed highest homology (69.5% identity) to the N protein of polygonum ringspot virus. The data altogether support the proposal of AYSV as a new orthotospovirus species within a growing clade of orthotospoviruses that seem to share the Middle East basin as a region of origin.

Generic RT-PCR tests for detection and identification of tospoviruses.

A set of tests for generic detection and identification of tospoviruses has been developed. Based on a multiple sequence alignment of the nucleocapsid gene and its 5' upstream untranslated region sequence from 28 different species, primers were designed for RT-PCR detection of tospoviruses from all recognized clades, i.e. the American, Asian and Eurasian clades, and from the small group of distinct and floating species. Pilot experiments on isolates from twenty different species showed that the designed primer sets successfully detected all species by RT-PCR, as confirmed by nucleotide sequence analysis of the amplicons. In a final optimized design, the primers were applied in a setting of five RT-PCR tests. Seven different tospoviruses were successfully identified from diagnostic samples and in addition a non-described tospovirus species from alstroemeria plants. The results demonstrate that the newly developed generic RT-PCR tests provide a relevant tool for broad detection and identification of tospoviruses in plant quarantine and diagnostic laboratories.

The complete nucleotide sequence of chrysanthemum stem necrosis virus.

The complete genome sequence of chrysanthemum stem necrosis virus (CSNV) was determined using Roche 454 next-generation sequencing. CSNV is a tentative member of the genus Tospovirus within the family Bunyaviridae, whose members are arthropod-borne. This is the first report of the entire RNA genome sequence of a CSNV isolate. The large RNA of CSNV is 8955 nucleotides (nt) in size and contains a single open reading frame of 8625 nt in the antisense arrangement, coding for the putative RNA-dependent RNA polymerase (L protein) of 2874 aa with a predicted Mr of 331 kDa. Two untranslated regions of 397 and 33 nt are present at the 5' and 3' termini, respectively. The medium (M) and small (S) RNAs are 4830 and 2947 nt in size, respectively, and show 99 % identity to the corresponding genomic segments of previously partially characterized CSNV genomes. Protein sequences for the precursor of the Gn/Gc proteins, N and NSs, are identical in length in all of the analysed CSNV isolates.

Molecular and biological comparison of two Tomato yellow ring virus (TYRV) isolates: challenging the Tospovirus species concept.

Two strains of Tomato yellow ring virus (TYRV, genus Tospovirus), one from tomato (referred to as TYRV-t) and the other from soybean and potato (denoted TYRV-s), collected from different geographical regions in Iran, were compared. Their genomic S RNA segments differed in size by 55 nucleotides. Comparison of the S RNA intergenic regions revealed the absence of a stretch of 115 nucleotides within the S RNA segment of TYRV-s and, conversely, of 56 nts in that of TYRV-t, apparently a stable genetic difference as it was also found in another isolate of TYRV-s collected from potato. Sequence comparison of the N protein ORFs revealed an identity of 92% between the N proteins of both strains, and the observed strong cross-reaction of TYRV-s in DAS-ELISA with a polyclonal antiserum directed against the TYRV-t N protein confirmed this high identity. Host range analysis revealed several differences, e.g. TYRV-s, but not TYRV-t, being able to systemically infect Nicotiana species, and TYRV-s being localised in tomato. The observed molecular and biological differences of both viruses call into question the currently used criteria for Tospovirus species demarcation.

Genetic organisation of Iris yellow spot virus M RNA: indications for functional homology between the G(C) glycoproteins of tospoviruses and animal-infecting bunyaviruses.

The complete nucleotide sequence (4838 nucleotides) of Iris yellow spot virus (IYSV) M RNA indicates, typical for tospoviruses, the presence of two genes in ambisense arrangement. The vRNA ORF codes for the potential cell-to-cell movement (NSm) protein (34.8 kDa) and the vcRNA ORF for the viral glycoprotein (G1/G2) precursor (128.6 kDa). Multiple sequence alignment of the NSm and G1/G2 precursor proteins of IYSV with those of other tospoviruses, showed highest homologies to Peanut bud necrosis virus (PBNV) and Watermelon silver mottle virus (WSMV). The potential cell-to-cell movement protein of tospoviruses is highly conserved (40-70% identity), with the exception of the first 60 N terminal amino acids, a domain that clearly diverged. For the G1 and G2 viral glycoproteins, blast searches revealed a significant homology between the C-terminally located tospoviral G1 (G(C)) protein with the counterpart of the animal-infecting bunyaviruses, suggesting a functional homology for these proteins.

Expression of the movement protein of Tomato spotted wilt virus in its insect vector Frankliniella occidentalis.

Tomato spotted wilt virus (TSWV) is able to infect both its botanical hosts and its insect vector (thrips). In plant tissue the NS(M) protein of TSWV functions as viral movement protein (MP), aggregating into plasmodesma-penetrating tubules to establish cell-to-cell movement. As upon heterologous expression NS(M) was able to form similar tubules on the surface of insect (Spodoptera frugiperda) cells, we have now investigated the expression and cellular manifestation of this protein in infected thrips tissue. It is shown that NS(M), though detectably expressed in both the L2 larval and adult thrips stages, does not aggregate into tubules, indicating that this requirement is associated to its function as MP in plants, and raising the question if NS(M) has a function at all during the insect life cycle of TSWV.

In vivo analysis of the TSWV cap-snatching mechanism: single base complementarity and primer length requirements.

Requirements for capped leader sequences for use during transcription initiation by tomato spotted wilt virus (TSWV) were tested using mutant alfalfa mosaic virus (AMV) RNAs as specific cap donors in transgenic Nicotiana tabacum plants expressing the AMV replicase proteins. Using a series of AMV RNA3 mutants modified in either the 5'-non-translated region or in the subgenomic RNA4 leader, sequence analysis revealed that cleaved leader lengths could vary between 13 and 18 nucleotides. Cleavage occurred preferentially at an A residue, suggesting a requirement for a single base complementarity with the TSWV RNA template, which could be confirmed by analyses of host mRNAs used in vivo as cap donors.

Identification and characterization of a novel tospovirus species using a new RT-PCR approach.

A novel tospovirus serologically distinct from all established tospovirus species was found in Thailand in Physalis minima L. The S RNA of this virus was cloned by a new RT-PCR approach revealing a nucleotide sequence of 3257 nucleotides. The ambisense RNA segment encoded a nonstructural protein (NSs) of 469 amino acids, with a predicted Mr of 53.2 kDa, and a nucleoprotein (N) of 279 amino acids and a Mr of 31.0 kDa, so far the largest N protein known for any tospovirus species. N protein sequence comparisons revealed closet relationship to the species Watermelon bud necrosis virus (58% identity), Watermelon silver mottle virus and Peanut bud necrosis virus (57%) and a distant relationship to Peanut yellow spot virus (23%) and Peanut chlorotic fanspot virus (22%).

Tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells.

The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from the endoplasmic reticulum (ER) to the Golgi complex, where they are retained, a process that could be blocked by tunicamycin. Expression of G2 alone resulted in transport to and retention in the Golgi complex, albeit less efficient, suggesting that G2 contains a Golgi retention signal. G1 alone was retained in the ER, irrespective of whether it contained the precursor's signal sequence or its own N-terminal hydrophobic sequence. Coexpression of G1 and G2 from separate gene constructs resulted in rescue of efficient G1 transport, as the proteins coaccumulated in the Golgi complex, indicating that their interaction is essential for proper targeting to this organelle. The results demonstrate that transport and targeting of the plant TSWV glycoproteins in mammalian BHK cells are strikingly similar to those of animal-infecting bunyavirus glycoproteins in mammalian cells. The observations are likely to reflect the dual tropism of TSWV, which replicates both in its plant host and in its animal (thrips) vector.

Application of Phage Display in Selecting Tomato spotted wilt virus-Specific Single-Chain Antibodies (scFvs) for Sensitive Diagnosis in ELISA.

ABSTRACT A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis.

Infection of barley protoplasts with rice hoja blanca tenuivirus. Brief report.

A barley protoplast system has been established that supports replication of Rice hoja blanca tenuivirus (RHBV). Following polyethylene glycol-mediated RHBV inoculation of barley protoplasts, newly synthesized viral RNAs and proteins could be detected. Time course analyses revealed de novo synthesis of genome length viral RNA4, as well as subgenomic-sized RNA4 molecules of both polarities. Two proteins, N and NS4, encoded by viral complementary RNA3 and viral RNA4 respectively, were detected by Western immunoblot analysis. The barley protoplast system thus constitutes a promising tool for in vivo studies of the sequential steps involved in the multiplication cycle of RHBV.

Alfalfa mosaic virus RNAs serve as cap donors for tomato spotted wilt virus transcription during coinfection of Nicotiana benthamiana.

Tomato spotted wilt virus (TSWV) was shown to use alfalfa mosaic virus (AMV) RNAs as cap donors in vivo during a mixed infection in Nicotiana benthamiana. By use of nested reverse transcription-PCR, TSWV N and NSs mRNAs provided with capped leader sequences derived from all four AMV RNAs could be cloned and sequenced. The sequence specificity of the putative TSWV endonuclease involved is discussed.

Tomato spotted wilt virus particle morphogenesis in plant cells.

A model for the maturation of tomato spotted wilt virus (TSWV) particles is proposed, mainly based on results with a protoplast infection system, in which the chronology of different maturation events could be determined. By using specific monoclonal and polyclonal antisera in immunofluorescence and electron microscopy, the site of TSWV particle morphogenesis was determined to be the Golgi system. The viral glycoproteins G1 and G2 accumulate in the Golgi prior to a process of wrapping, by which the viral nucleocapsids obtain a double membrane. In a later stage of the maturation, these doubly enveloped particles fuse to each other and to the endoplasmic reticulum to form singly enveloped particles clustered in membranes. Similarities and differences between the maturation of animal-infecting (bunya)viruses and plant-infecting tospoviruses are discussed.

Increase of tospoviral diversity in Brazil with the identification of two new tospovirus species, one from chrysanthemum and one from zucchini.

ABSTRACT During a survey conducted in several different regions of Brazil, two unique tospoviruses were isolated and characterized, one from chrysanthemum and the other from zucchini. The chrysanthemum virus displayed a broad host range, whereas the virus from zucchini was restricted mainly to the family Cucurbitaceae. Double-antibody sandwich-enzyme-linked immunosorbent assay and western immunoblot analyses demonstrated that both viruses were serologically distinct from all reported tospovirus species including the recently proposed peanut yellow spot virus and iris yellow spot virus (IYSV) species. The nucleotide sequences of the nucleocapsid (N) genes of both viruses contain 780 nucleotides encoding for deduced proteins of 260 amino acids. The N proteins of these two viruses displayed amino acid sequence similarities with the previously described tospovirus species ranging from 20 to 75%, but they were more closely related to each other (80%). Based on the biological and molecular features, these viruses are proposed as two new tospovirus species, designated chrysanthemum stem necrosis virus (CSNV) and zucchini lethal chlorosis virus (ZLCV). With the identification of CSNV and ZLCV, in addition to tomato spotted wilt virus, groundnut ring spot virus, tomato chlorotic spot virus, and IYSV, Brazil harbors the broadest spectrum of tospovirus species reported.

Characterization of a Tospovirus Isolate of Iris Yellow Spot Virus Associated with a Disease in Onion Fields in Brazil.

A tospovirus from onion causing a disease known as "sapeca" by growers in Brazil was characterized. Symptoms on onion consisted of numerous eyelike spots on the leaves and flower stalks resulting in flower abortion. Nicotiana benthamiana and N. rustica were the only systemic hosts experimentally found. Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) experiments demonstrated that this virus was serologically related to iris yellow spot virus (IYSV), a tospovirus recently described in the Netherlands. This virus, from onion, based on an amino acid sequence identity of 90.5% for the N gene protein, is regarded as a strain of IYSV and is designated IYSVBR This 10% divergence in the nucleocapsid protein may represent an adaptation of the virus to distinct ecological niches.

Molecular characterization of tomato spotted Wilt virus defective interfering RNAs and detection of truncated L proteins.

Junction sites of 25 different defective interfering (DI) RNAs of tomato spotted wilt virus (TSWV) were characterized. The DI RNAs varied in size from 2.0 to 5.2 kilobases (kb) and contained a single internal deletion. The absence of DI RNAs smaller than 2 kb suggested a size constraint for the survival of TSWV DI RNAs. This hypothesis was reinforced by the finding of a dimeric DI RNA formed by two 1.6-long monomers linked head to tail. Three types of junction sites were found, one type originating from a simple deletion; the second type contained a few extra nucleotides of unknown origin; and the third type contained a stretch of three to five nucleotides, originally occurring at both sides of the deletion and of which one was deleted. In 19 of the 25 DI RNAs studied, the original reading frame was maintained, suggesting a selective preference of DI RNAs with translational potency. Truncated proteins encoded by these DI RNAs were indeed detected in the nucleocapsid preparations. Folding studies of the complete L RNA revealed that the calculated minimal energy of folding was at 16 degreesC lower than at 23 degrees, indicating a higher stability of this molecule at low temperatures. The results suggest an involvement of locally folded secondary structures in the process of deletion, rather than the requirement of certain sequences around the deletion point. The DI RNA generation in TSWV is essentially, as discussed, similar to the process of RNA recombination described in many viruses.

Molecular and serological characterization of iris yellow spot virus, a new and distinct tospovirus species.

ABSTRACT A new tospovirus was identified in iris cultivations in the Netherlands. Both serological comparisons and sequence determination of the S RNA demonstrate that this virus represents a new and distinct species, belonging to a separate serogroup, and for which the name iris yellow spot virus (IYSV) is proposed. The disease symptoms on iris are characterized by yellow spots on the leaves. Its experimental host range is very narrow and, in addition to iris, only includes Nicotiana benthamiana and Datura stramonium. The nucleoprotein of IYSV shows only 30 to 44% sequence identity with those of other tospoviruses identified so far; the highest homology being found with the tospovirus species of serogroup IV.

Binding of Tomato Spotted Wilt Virus to a 94-kDa Thrips Protein.

ABSTRACT Using protein blot assays, a 94-kDa thrips protein was identified that exhibited specific binding to tomato spotted wilt virus (TSWV) particles. Renaturation of the 94-kDa protein, which is conserved among the two major vector species of TSWV, Frankliniella occidentalis and Thrips tabaci, was crucial for its virus-binding properties, whereas under the same conditions no specific binding was observed with aphid (Myzus persicae) proteins. The 94-kDa protein species was present in all developmental stages of both vectoring thrips, whereas it was present mainly in the adult stage of a nonvectoring thrips species, Parthenothrips dracenae. Using antibodies against the different TSWV structural proteins, the G2 envelope glycoprotein was identified as the viral determinant involved. Because the virus-binding protein is present throughout the thrips body, but not in the gut, it may represent a receptor protein involved during circulation of the virus through its vector but probably not during viral uptake in the midgut.

A protoplast system for studying tomato spotted wilt virus infection.

A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.

Serological comparison of tospoviruses with polyclonal antibodies produced against the main structural proteins of tomato spotted wilt virus.

A new purification procedure for the tospoviruses of serogroups II and III was developed. SDS-polyacrylamide gel electrophoresis of purified tomato spotted wilt virus (TSWV; serogroup I), groundnut ringspot virus (GRSV: serogroup II), tomato chlorotic spot virus (TCSV; serogroup II) and impatiens necrotic spot virus (INSV; serogroup III) showed that the glycoprotein G2 of serogroup II members differs significantly in size from that of the serogroup I virus. Western immunoblot analysis using polyclonal antisera produced against purified glycoproteins TSWV-G1 and G2 as well as peptides of hydrophilic sequences of TSWV-G1 and G2 expressed in Escherichia coli demonstrated a higher homology amongst G1 of different serogroups than for G2. These results are supported by comparing the glycoprotein gene sequences of different serogroups.