Richard Kormelink

Multiplication of tomato spotted wilt virus in its insect vector, Frankliniella occidentalis.

The accumulation of two proteins, the nucleocapsid (N) protein and a non-structural (NSs) protein both encoded by the S RNA of tomato spotted wilt virus (TSWV), was followed in larvae during development and in adults of Frankliniella occidentalis after ingesting the virus for short periods on infected plants. The amounts of both proteins increased, as shown by ELISA and Western blot analysis, within 2 days above the levels ingested, indicating multiplication of TSWV in these insects. Accumulation of these proteins and of virus particles was further confirmed by in situ immunolabelling of the salivary glands and other tissues of adult thrips. The accumulation of large amounts of N and NSs protein, the occurrence of several vesicles with virus particles in the salivary glands and the massive numbers of virus particles in the salivary gland ducts demonstrate that the salivary glands are a major site of TSWV replication. The occurrence of virus particles in the salivary vesicles is indicative of the involvement of the Golgi apparatus in the maturation of the virus particles and its transport to the salivary ducts.

The nucleotide sequence of the M RNA segment of tomato spotted wilt virus, a bunyavirus with two ambisense RNA segments

The complete sequence of the tomato spotted wilt virus (TSWV) M RNA segment has been determined. The RNA is 4821 nucleotides long and has an ambisense coding strategy similar to that of the S RNA segment. The M RNA segment contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted size of 33.6K, and one in the viral complementary sense which encodes the precursor to the G1 and G2 glycoproteins, with a predicted size of 127.4K. Both ORFs are expressed via the synthesis of subgenomic mRNAs that possibly terminate at a stable hairpin structure, located in the intergenic region. The precursor for the glycoproteins contains a sequence motif (RGD) which is characteristic of cellular attachment domains. Significant sequence homology was found between the G1 glycoproteins of members of the genus Bunyavirus and a corresponding region in the glycoprotein precursor of TSWV, indicating a close evolutionary relationship between these viruses. With the elucidation of the M RNA sequence, the complete nucleotide sequence of TSWV has been determined. TSWV represents the first member of the Bunyaviridae shown to contain two ambisense RNA segments.

Classification of tospoviruses based on phylogeny of nucleoprotein gene sequences

The nucleotide sequences of the nucleoprotein (N) genes of seven tospovirus isolates representing three serogroups were determined and used to establish phylogenetic parameters to delineate species within the Tospovirus genus of the Bunyaviridae. A high sequence divergence (55.9% identity at the nucleotide level) was observed between isolates of serogroup I (tomato spotted wilt virus) and isolates of serogroup III (Impatiens necrotic spot virus). The serogroup II isolates take an intermediate position. Their N genes have 75% identity with those of serogroup I isolates and 57% with those of serogroup III isolates. Whereas the isolates within serogroups I or III have almost identical sequences, the two isolates BR-03 and SA-05 of serogroup II diverged significantly from each other (82.1% sequence identity). The results obtained support the conclusion that, in addition to the species TSWV and INSV, the serogroup II isolates BR-03 and SA-05 have to be considered as distinct species within the genus Tospovirus for which the names tomato chlorotic spot virus and groundnut ringspot virus, respectively, are proposed.

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.

The nonstructural protein (NSs) encoded by the ambisense S RNA segment of tomato spotted wilt virus is associated with fibrous structures in infected plant cells

The open reading frame located in the viral strand of the ambisense S RNA of tomato spotted wilt virus (TSWV), was cloned into transfer vector pAc33DZ1 and inserted downstream of the polyhedrin promoter in the Autographa californica nuclear polyhedrosis virus genome. Recombinant baculoviruses were obtained that showed a high-level expression of a 52.4-kDa protein corresponding to the inserted TSWV gene. The viral protein thus produced was purified and injected into rabbits to raise antibodies. Western immunoblot analyses of extracts from TSWV-infected plants demonstrated that the 52.4-kDa TSWV-specific polypeptide represents a nonstructural protein (denoted NSs), being absent in purified virus particles. Immunogold labeling of tissue sections of TSWV-infected Nicotiana rustica plants showed that this protein was, depending on the virus isolate, either found dispersed throughout the cytoplasm or associated with fibers which appeared as elongated flexible filaments or paracrystalline rods.

Functional Entry of Baculovirus into Insect and Mammalian Cells Is Dependent on Clathrin-Mediated Endocytosis

ABSTRACT Entry of the budded virus form of baculoviruses into insect and mammalian cells is generally thought to occur through a low-pH-dependent endocytosis pathway, possibly through clathrin-coated pits. This insight is primarily based on (immuno)electron microscopy studies but requires biochemical support to exclude the use of other pathways. Here, we demonstrate using various inhibitors that functional entry of baculoviruses into insect and mammalian cells is primarily dependent on clathrin-mediated endocytosis. Our results further suggest that caveolae are somehow involved in baculovirus entry in mammalian cells. A caveolar endocytosis inhibitor, genistein, enhances baculovirus transduction in these cells considerably.

The Tomato Yellow Leaf Curl Virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases.

Tomato Yellow Leaf Curl Virus Disease incited by Tomato yellow leaf curl virus (TYLCV) causes huge losses in tomato production worldwide and is caused by different related begomovirus species. Breeding for TYLCV resistance has been based on the introgression of multiple resistance genes originating from several wild tomato species. In this study we have fine-mapped the widely used Solanum chilense–derived Ty-1 and Ty-3 genes by screening nearly 12,000 plants for recombination events and generating recombinant inbred lines. Multiple molecular markers were developed and used in combination with disease tests to fine-map the genes to a small genomic region (approximately 70 kb). Using a Tobacco Rattle Virus–Virus Induced Gene Silencing approach, the resistance gene was identified. It is shown that Ty-1 and Ty-3 are allelic and that they code for a RNA–dependent RNA polymerase (RDR) belonging to the RDRγ type, which has an atypical DFDGD motif in the catalytic domain. In contrast to the RDRα type, characterized by a catalytic DLDGD motif, no clear function has yet been described for the RDRγ type, and thus the Ty-1/Ty-3 gene unveils a completely new class of resistance gene. Although speculative, the resistance mechanism of Ty-1/Ty-3 and its specificity towards TYLCV are discussed in light of the function of the related RDRα class in the amplification of the RNAi response in plants and transcriptional silencing of geminiviruses in plants.

Generation of envelope and defective interfering RNA mutants of tomato spotted wilt virus by mechanical passage.

During a series of mechanical transfers of tomato spotted wilt virus, two distinct types of mutants were generated. Firstly, a morphologically defective isolate was obtained which had lost the ability to produce the membrane glycoproteins and, as a consequence, was not able to form enveloped particles. Analysis of the genomic RNAs of this isolate suggested that this defect was caused by either point mutations or very small deletions in the medium genomic RNA segment. Secondly, isolates were obtained which had accumulated truncated forms of the large (L) RNA segment. These shortened L RNA molecules most likely represented defective interfering RNAs, since they replicated more rapidly than full-length L RNA and their appearance was often associated with symptom attenuation. Defective L RNAs of different sizes were generated after repeated transfers, and hybridization analysis using L RNA-specific cDNA probes showed that the internal regions deleted varied in length. The presence of defective L RNAs in nucleocapsid fractions as well as in enveloped virus particles indicates that all defective molecules retained the sequences required for replication, encapsidation by nucleocapsid proteins and packaging of the nucleocapsid into virus particles.

Distinct levels of relationships between tospovirus isolates.

SummaryThe taxonomic relations of a number of tospovirus isolates, collected in different geographical areas and from different host plants, were studied. To delineate these isolates, properties such as susceptibility of a limited range of host plants, symptomatology, cytopathology, nucleocapsid composition, serology of their nucleocapsid proteins, and nucleotide sequence homology were compared. The results show that isolates which have previously been discriminated as members of three different serogroups, should in fact be regarded as representatives of at least three distinct virus species in the tospovirus genus.

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.