The nucleotide sequence of the S RNA of Impatiens necrotic spot virus, a novel tospovirus.

Impatiens necrotic spot virus (INSV) shares a number of properties with tomato spotted wilt virus (TSWV), the type species of the genus tospovirus within the family Bunyaviridae. INSV, however, differs from TSWV in plant host range and serology. In order to define the genomic structure and the taxonomic status of this TSWV-like virus, the nucleotide sequence of its genomic S RNA segment has been determined. The molecular data obtained demonstrate that, like TSWV, INSV has an ambisense S RNA molecule, encoding a non-structural protein in viral sense and the nucleocapsid protein in viral complementary sense. The level of nucleotide sequence homology between their S RNAs, as well as the divergence in amino acid sequence homology of their gene products, confirm previous conclusions from serological studies that INSV and TSWV represent distinct virus species within the newly created genus, tospovirus.     

Serological relationships and purification of bud necrosis virus,a tospovirus occurring in peanut (Arachis hypogaea L.) in India*

A procedure for the purification of a tospovirus which causes bud necrosis disease (BND) of peanut in India is described. The virus contained three polypeptides of 78 kDa, 54 kDa and 31 kDa. In two ELISA procedures the virus failed to react with antisera to tomato spotted wilt virus (TSWV) obtained from different sources and with an antiserum to impatiens necrotic spot virus (INSV). Additionally, in reciprocal tests TSWV and INSV antigens failed to react with antiserum to the virus infecting peanut in India. In electro-blot immunoassay 54 kDa and 31 kDa polypeptides of the virus reacted with the homologous antiserum. None of the heterologous antisera reacted with any of the three viral polypeptides. On the basis of serological differences the virus that causes BND in India is distinct and therefore has been named bud necrosis virus (BNV). This serotype appears to be restricted to Asia.     

Влнянне характера эаселення ноля капустноii совкоii(Barathra brassicae L.) эффективноеть трихограммы (Trichogramma evanescens Westw.) (Lepidoptera,Noctuidae; Hymenoptera,Trichogrammatidae)

TSWV belongs to the genus Tospovirus which was established in the family Bunyaviridae, a family of animal viruses. Besides TSWV, Impatiens necrotic spot virus (INSV) and ground nut bud necrosis virus (GBNV) were established as different Tospovirus species. Tospoviruses have quasispherical particles of 85 nm diametre which are surrounded by a membrane and contain 3 RNA species and 4 structural proteins. In Tospovirus infected plant cells virions were detected in cavaties of the endoplasmatic reticulum and additionally amorphous electron dense material accumulates in infected cells. Defective forms of TSWV lack the ability to form complete virus particles. TSWV is the only plant pathogenic virus that is transmitted by thrips which transmit the virus with different efficiency. The virus has an extensive plant host range of more than 360 different species. The developing symptoms depend on the Tospovirus species, the virulence of the virus strains and the environmental conditions.

Based on the reaction of TSWV isolates with N‐specific polyclonal antisera, 3 serogroups were established. The most frequently used technique for serologically based diagnosis of Tospoviruses is DAS ELISA with N‐specific or preadsorbed antisera against complete virus. For TSWV epidemiology distinct weeds and cultural host plants play an important role for the survival of virus and vector. Breeding for resistance is the most important preventive measure of control.     

Tomato spotted wilt virus (TSWV) in Greece: its incidence following the expansion of Frankliniella occidentalis, and characterisation of isolates collected from various hosts

Leaf samples were collected from plants with tospovirus‐like symptoms from various hosts in different regions of Greece where Thrips tabaci, Frankliniella occidentalis or both vectors occur. The viruses infecting these plants were identified with polyclonal antibodies raised against the N proteins of Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) by ELISA. All samples tested positive for TSWV, but not for INSV. ELISA of thirty three isolates, using monoclonal antibodies against the N protein of TSWV, revealed the existence of five epitopes on the N protein. RT‐PCR tests on a few randomly‐selected isolates, using a pair of universal primers, a pair of primers specific for the L gene and a pair of primers specific for the N gene, as well as sequence analysis of a part of the S gene of one isolate, confirmed the authenticity of the virus isolated as TSWV. Host range studies showed differences in susceptibility, especially among species belonging to the Leguminosae and Cucurbitaceae. The species Beloporone guttata and Coleus sp. are reported for the first time as hosts of the virus, whereas Solanum melongena, Celosia cristata, Dianthus chinensis, Stephanotis floribunda and Catharanthus roseus were identified as new hosts of TSWV in Greece.     

Sequence Diversity of the Nucleoprotein Gene of Peanut bud necrosis virus Isolates from South India

Peanut bud necrosis virus (PBNV), genus Tospovirus (family Bunyaviridae), is an important virus infecting peanut and other crops in South India. PBNV isolates naturally infecting groundnut, brinjal, tomato, black gram, field bean, cowpea, cotton, jute, taro and Calotropis plants were collected from different regions of South India and characterized. Infection was confirmed by direct antigen‐coating enzyme‐linked immunosorbent assay (DAC‐ELISA) using PBNV‐specific antiserum. The coat protein gene was further amplified using PBNV coat protein‐specific primers. The amplicon (830 bp) was cloned and sequenced; sequence analysis revealed that the N gene shared 93–100% and 95–100% sequence identity with PBNV at the nucleotide and amino acid levels, respectively.     

Occurrence of Different Tospoviruses in Vegetable Crops in Argentina

Significant damage on vegetable crops by tospoviruses had occurred sporadically in Argentina in the past but since 1994, severe outbreaks have been recorded every year. The crops that have been most affected, tomato, lettuce, and pepper, were surveyed in the provinces of Mendoza and Buenos Aires in 1994–95 and 1995–96. A few weeds and miscellaneous crops were also collected. A total of 543 samples showing symptoms typical for tospoviruses were analysed by double-antibody sandwich-enzyme-linked immunosorbent assay with polyclonal antibodies to groundnut ringspot tospovirus (GRSV), impatiens necrotic spot tospovirus (INSV) and tomato spotted wilt tospovirus (TSWV). The 339 samples collected in 1995–96 were also assayed for tomato chlorotic spot tospovirus (TCSV). In addition, the incidence of tospoviruses in tomato crops was assessed in 41 farms representing 310 ha and 10 cultivars. GRSV was identified in 222 samples (40.8%), TSWV in 194 samples (32.7%), TCSV in 50 samples (14.7%), INSV was not detected and 77 samples did not react with the antisera used. TSWV was found to prevail in Buenos Aires and GRSV in Mendoza. Mixed infections were not found in this survey. In tomato crops the mean incidence of tospoviruses was 33%. These results show that the disease formerly assigned to TSWV, is caused by at least three tospoviruses.     

The appearance of new polyadenylated nuclear RNA sequences during rat-liver regeneration

Polyadenylated RNA populations from normal and 16-hour regenerating rat-liver nuclei were compared by heterologous hybridisation reactions with cDNA and unique DNA probes. Whereas unique DNA hybridisations did not show differences between the RNA populations, comparisons by cDNA hybridisation showed that about 10--15% by weight of polyadenylated sequences present in the nuclei of 16-hour regenerating rat livers were not found in the polyadenylated nuclear RNA of normal rat livers. These regenerating-specific nuclear cDNA sequences were isolated and characterised; the experiments showed that the complexity of the new sequences was 1-2 x 10(7) nucleotides (equivalent to 5,000--10,000 RNA sequences of 2,000 nucleotides in length) and that they were probably not potential messenger RNA sequences.     

Production and use of cDNA clones from arabis mosaic virus

Arabis mosaic virus (AMV) genomic RNAs were converted to dsDNA and cloned into bacterial plasmids. Insert sizes of cDNA clones ranged from 0·2 to 3·2 kbp. Restriction enzyme mapping identified clones representing at least 90% of the RNA-2 genome. A 0·9 kbp clone specific to RNA-1 was also identified. Northern blot hybridisations of AMV RNAs with clones from either RNA-1 or RNA-2 showed no cross reactions. The sensitivity of virus detection in dot hybridisation was 15 pg of purified genomic RNA and 40 pg of purified virus particles. The possibility of using cDNA clones for the detection of AMV in strawberry sap was demonstrated. Two AMV dsRNAs corresponding to genomic RNAs in size were isolated from infected cucumber plants and reacted in hybridisation experiments.     

Members of the ribosomal protein S6 (RPS6) family act as pro-viral factor for tomato spotted wilt orthotospovirus infectivity in Nicotiana benthamiana

To identify host factors for tomato spotted wilt orthotospovirus (TSWV), a virus-induced gene silencing (VIGS) screen using tobacco rattle virus (TRV) was performed on Nicotiana benthamiana for TSWV susceptibility. To rule out any negative effect on the plants’ performance due to a double viral infection, the method was optimized to allow screening of hundreds of clones in a standardized fashion. To normalize the results obtained in and between experiments, a set of controls was developed to evaluate in a consist manner both VIGS efficacy and the level of TSWV resistance. Using this method, 4532 random clones of an N. benthamiana cDNA library were tested, resulting in five TRV clones that provided nearly complete resistance against TSWV. Here we report on one of these clones, of which the insert targets a small gene family coding for the ribosomal protein S6 (RPS6) that is part of the 40S ribosomal subunit. This RPS6 family is represented by three gene clades in the genome of Solanaceae family members, which were jointly important for TSWV susceptibility. Interestingly, RPS6 is a known host factor implicated in the replication of different plant RNA viruses, including the negative-stranded TSWV and the positive-stranded potato virus X.     

Impatiens necrotic spot virus infection and feeding behavior of nematode-parasitized western flower thrips

The effects of parasitism by the nematode Thripinema nicklewoodi Siddiqi (Tylenchida: Allantonematidae) on tospovirus infection and feeding behavior of Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) were studied in the laboratory. In an initial experiment, nematode parasitism reduced the numbers of adult thrips that were positive to Impatiens necrotic spot virus (INSV) by enzyme-linked immunosorbent assay (ELISA) compared to controls. Three hypotheses on possible mechanisms causing this reduction were tested. H₁: nematodes have a tendency to penetrate healthy thrips rather than INSV-infected thrips; H₂: parasitized first instar thrips are less able to acquire virus during feeding; or H₃: INSV replication is suppressed in parasitized thrips. H₁ and H₂ were proven false as we found no difference in nematode attack rate between healthy thrips and thrips that have taken up INSV and no difference in virus uptake or feeding activity between parasitized and non-parasitized larval thrips. H₃ was not supported by data from our tests (P=0.07) but remains the explanation most worthy of future investigation. Interestingly, INSV transmission was not affected by nematode parasitism even though it reduced feeding activity of adult female thrips by 81% on leaves, 38% on pollen, and 22% on honey. However, despite lowered total feeding, probing by parasitized thrips (in honey) was not reduced, and this may explain why lowered feeding did not result in lowered virus transmission.     

A single nucleotide change within a plant virus satellite RNA alters the host specificity of disease induction

Some RNA plant viruses contain satellite RNAs which are dependent upon their associated virus for replication and encapsidation. Some cucumber mosaic virus satellite RNAs induce chlorosis on any of several host plants, including either tobacco or tomato. The exchange of sequence domains between cDNA clones of chlorosis-inducing and non-pathogenic satellite RNAs delimited the chlorosis domain for both tobacco and tomato plants to the same region. Site-directed mutagenesis of one nucleotide (149) within this domain changed the host plant specificity for a chlorotic response to satellite RNA infection from tomato to tobacco. Within the chlorosis domain, three conserved nucleotides are either deleted or altered in all satellite RNAs that do not induce chlorosis. Deletion of one of these nucleotides (153) did not affect satellite RNA replication but rendered it non-pathogenic. Thus, two single nucleotides have been identified which play central roles in those interactions between the virus, its satellite RNA and the host plant, and together result in a specific disease state.     

Characterisation of bacterial clones containing DNA sequences derived from Xenopus laevis vitellogenin mRNA.

A 1700 nucleotide DNA sequence derived from Xenopus vitellogenin mRNA has been cloned in the bacterial plasmid pBR322. The identity of the cloned sequence was verified in two ways. Firstly, the plasmid DNA was shown to hybridise to an RNA of the correct size (6,700 nucleotides). This was shown by in situ hybridisation to electrophoretically separated RNA and also by the formation of "R-loops" with purified vitellogenin mRNA. Then, using a novel procedure in which plasmid DNA covalently bound to diazotised paper is used to select complementary mRNA sequences, the cloned sequence was shown to hybridise to an mRNA which directed the synthesis of vitellogenin when translated in a reticulocyte lysate cell-free system.