Expression of a viral polymerase-bound host factor turns human cell lines permissive to a plant- and insect-infecting virus.

Tospoviruses are the only plant-infecting members of the Bunyaviridae family of ambisense ssRNA viruses. Tomato spotted wilt tospovirus (TSWV), the type-member, also causes mild infection on its main insect vector, Frankliniella occidentalis. Herein, we identified an F. occidentalis putative transcription factor (FoTF) that binds to the TSWV RNA-dependent RNA polymerase and to viral RNA. Using in vitro RNA synthesis assays, we show that addition of purified FoTF improves viral replication, but not transcription. Expression of FoTF deletion mutants, unable to bind the RNA-dependent RNA polymerase or viral RNA, blocks TSWV replication in F. occidentalis cells. Finally, expression of FoTF wild-type turns human cell lines permissive to TSWV replication. These data indicate that FoTF is a host factor required for TSWV replication in vitro and in vivo, provide an experimental system that could be used to compare molecular defense mechanisms in plant, insect, and human cells against the same pathogen (TSWV), and could lead to a better understanding of evolutionary processes of ambisense RNA viruses.

The plant virus Tomato Spotted Wilt Tospovirus activates the immune system of its main insect vector, Frankliniella occidentalis.

Tospoviruses have the ability to infect plants and their insect vectors. Tomato spotted wilt virus (TSWV), the type species in the Tospovirus genus, infects its most important insect vector, Frankliniella occidentalis, the western flower thrips (WFT). However, no detrimental effects on the life cycle or cytopathological changes have been reported in the WFT after TSWV infection, and relatively few viral particles can be observed even several days after infection. We hypothesized that TSWV infection triggers an immune response in the WFT. Using subtractive cDNA libraries to probe WFT DNA macroarrays, we found that the WFT's immune system is activated by TSWV infection. The activated genes included (i) those encoding antimicrobial peptides, such as defensin and cecropin; (ii) genes involved in pathogen recognition, such as those encoding lectins; (iii) those encoding receptors that activate the innate immune response, such as Toll-3; and (iv) those encoding members of signal transduction pathways activated by Toll-like receptors, such as JNK kinase. Transcriptional upregulation of these genes after TSWV infection was confirmed by Northern analysis, and the kinetics of the immune response was measured over time. Several of the detected genes were activated at the same time that viral replication was first detected by reverse transcription-PCR. To our knowledge, this is the first report of the activation of an insect vector immune response by a plant virus. The results may lead to a better understanding of insects' immune responses against viruses and may help in the future development of novel control strategies against plant viruses, as well as human and animal viruses transmitted by insect vectors.

The Identification of the Vector Species of Iris Yellow Spot Tospovirus Occurring on Onion in Brazil.

In Brazil, tospoviruses have been reported in several horticultural and ornamental plants. In the northeast region of Brazil, a tospovirus has emerged as a devastating virus on onion cultures. Based on serology and the sequence of nucleocapsid (N) protein gene, this pathogen was identified as a strain of iris yellow spot tospovirus (IYSV) (1). This virus was first identified on iris and leek in The Netherlands and later on onion in Israel. For an effective integrated management of tospoviruses in Brazil, identification of IYSV vector is essential. Three thrips species, Thrips tabaci, Frankliniella schultzei, and F. occidentalis, that are major vegetable and floral crop pests in the Federal District, Brazil, were tested for their ability to transmit the virus by leaf disk assay (2). All thrips, up to 8 h old, were given an acquisition access period of 48 h at 25°C on IYSV-infected Nicotiana benthamiana plants in Tashiro-cages. Thrips were then reared on uninfected Datura stramonium detached leaves until the adult stage. These adults were transferred individually to microcentrifuge tubes containing an N. benthamiana leaf disk and were incubated for 48 h for virus inoculation. The leaf disks were then incubated 4 more days to allow development of the virus infection, and the presence of virus was evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) with polyclonal antibodies against N protein of IYSV. Adult thrips were also used for direct inoculation to N. benthamiana plants, three thrips per plant. By the leaf disk assay, 45.8% (22 out of 48) of T. tabaci transmitted the virus, but F. schultzei (n = 48) and F. occidentalis (n = 32) did not transmit it. All plants (4 out of 4) directly inoculated by T. tabaci showed symptoms and infection by Dot-ELISA, while no plants inoculated with F. schultzei (n = 5) and F. occidentalis (n = 3) were positive, either by symptom observation or by Dot-ELISA. Only T. tabaci showed potential for a high capacity to transmit the IYSV onion isolate. In the field, considering the host preference of thrips, T. tabaci was considered the most important vector species of IYSV on onion. References: (1) L. Pozzer et al. Plant Dis. (In press.) (2) I. Wijkamp and D. Peters. Phytopathology 83:986, 1993.

First Report of Natural Occurrence of Zucchini Lethal Chlorosis Tospovirus on Cucumber and Chrysanthemum Stem Necrosis Tospovirus on Tomato in Brazil.

During a field survey in 1994, five cucumber (Cucumis sativus) cv. Hokushin plants showing symptom of yellowing, mottling, and vein banding on the leaves were collected from a commercial field of the Federal District. By electron microscopy, quasi-spherical particles with double membrane, typical tospovirus-like particles were found in the infected leaf material. All samples strongly reacted with antibody of zucchini lethal chlorosis tospovirus (ZLCV), but not with antibodies of other to-spoviruses reported in Brazil (1): tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), groundnut ringspot virus (GRSV), chrysanthemum stem necrosis virus (CSNV), or iris yellow spot virusonion isolate (IYSV-BR). The virus was identified as ZLCV, which was first isolated in 1994 from zucchini (Cucurbita pepo) in São Paulo State, Brazil. Tomato (Lycopersicon esculentum) plants showing stem necrosis and necrotic spots and rings on the leaves were collected in Viçosa, Minas Gerais State. By electron microscopy, molecular studies, and enzyme-linked immunosorbent assay with antibodies of the six tospoviruses occurring in Brazil, the virus was identified as CSNV. This virus was first reported in 1995 on a Chrysanthemum sp. in São Paulo State and recently reported in the Netherlands from Dendranthema indicum. This is the first report of the natural occurrence of ZLCV and CSNV on cucumber and tomato, respectively. Reference: (1) A. C. de Ávila et al. 1998. Pages 32-34 in: Int. Symp. on Tospoviruses and Thrips in Floral and Vegetable Crops, 4th.