Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library.

Immunomodulation by the ectopic expression of intracellular antibodies ('intrabodies') has a great potential for interfering with physiological or pathological functions in vivo in a highly specific manner. One of the major obstacles of this technology is the inability of most antibodies to properly fold and function in the reducing environment of the cytoplasm, which prevents the formation of essential disulfide bonds. We wished to assess the intracellular performance of antibodies derived from a semi-synthetic single-chain variable fragment (scFv) phage display library ('F8 library') built on a thermodynamically stable single-framework scaffold. To this purpose, we chose to modulate the infection of a pandemic plant pathogen, the cucumber mosaic virus (CMV). After in vitro 'biopanning' on immobilized virions, two scFvs were biochemically characterized, showing high affinity toward the antigen. They were transiently expressed at high yields as soluble molecules in the cytoplasm of Nicotiana benthamiana plants. Subsequently, they were expressed in the cytoplasm of transgenic tomato plants. Challenge with high viral dose showed that both scFvs were able to elicit a phenotypic effect and led to the identification of a transgenic line fully resistant to infection. In these plants, the scFv binds the virus in the inoculated leaves preventing viral long distance movement. This work represents the first demonstration that the 'F8 library' can be directly screened in vitro to rapidly isolate antigen-specific scFvs that act as effective intrabodies in vivo. These antibodies represent powerful tools to interfere with several intracellular targets, modulating pathogen infectivity and/or cellular metabolism.

Detection of Tomato spotted wilt virus in its vector Frankliniella occidentalis by reverse transcription-polymerase chain reaction.

A method for rapid and reliable detection of Tomato spotted wilt virus (TSWV) (Tospovirus, Bunyaviridae) in its vector Frankliniella occidentalis (Thysanoptera Thripidae) would be a useful tool for studying the epidemiology of this virus. A RT-PCR method developed for this purpose is reported. The method was tested on thrips involved in laboratory transmission trials and on thrips collected in the field, whose capability to transmit TSWV was checked previously by leaf disk assays. The RT-PCR results were consistent with the results obtained by the leaf disk assays. Among thrips involved in laboratory experiments, 97% of the adults that transmitted TSWV were positive by RT-PCR; as did some non-transmitter adults reacted, whereas among field-collected thrips only the individuals able to transmit were positive by RT-PCR. In addition, healthy thrips were allowed to feed as adults on virus-infected leaves for 48 h, and then examined by RT-PCR immediately or after starving or feeding on virus-free plants for various times, to determine if virus ingested (but not transmissible) was also detectable. The virus was detectable immediately after the feed or within 12 and 24 h for individuals starved or fed on virus-free plants, respectively, but not after those periods. Thus, the method could detect rapidly and reliably the virus in vectors from the field, providing 24 h of starving to avoid positive RT-PCR results from thrips simply carrying the virus.

Transmission by Olpidium brassicae of Mirafiori lettuce virus and Lettuce big-vein virus, and Their Roles in Lettuce Big-Vein Etiology.

ABSTRACT Big-vein disease occurs on lettuce worldwide in temperate conditions; the causal agent has been presumed to be Lettuce big-vein virus (LBVV), genus Varicosavirus, vectored by the soilborne fungus Olpidium brassicae. Recently, the role of LBVV in the etiology of big-vein disease has been questioned because a second soilborne virus, Mirafiori lettuce virus (MiLV), genus Ophiovirus, has been found frequently in big-vein-affected lettuce. LBVV and MiLV, detectable and distinguishable by enzyme-linked immunosorbent assay using specific antisera, were tested for their ability to be transmitted from lettuce to lettuce by mechanical inoculation of sap extracts, or by zoospores of O. brassicae, and to cause big-vein disease. Both viruses were mechanically transmissible from lettuce to herbaceous hosts and to lettuce, but very erratically. LBVV was transmitted by O. brassicae but lettuce infected with only this virus never showed symptoms. MiLV was transmitted in the same manner, and lettuce infected with this virus alone consistently developed big-vein symptoms regardless of the presence or absence of LBVV. With repeated mechanical transmission, isolates of both viruses appeared to lose the ability to be vectored, and MiLV appeared to lose the ability to cause big-vein symptoms. The recovery of MiLV (Mendocino isolate, from Cali-fornia) from stored O. brassicae resting spores puts the earliest directly demonstrable existence of MiLV at 1990.

Field Isolates of Tomato spotted wilt virus Overcoming Resistance in Pepper and Their Spread to Other Hosts in Italy.

Isolates of Tomato spotted wilt virus (TSWV) severely and systemically infecting commercial pepper cultivars with resistance introgressed from Capsicum chinense PI152225 were found in Albenga (northwestern Italy) in July 2000. Experimentally, these resistance-breaking (RB) isolates overcame the resistance in C. chinense PI152225, but they produced infection in other hosts similarly to non-RB isolates from the same area. The RB isolates were indistinguishable from TSWV by serology and electron microscopy, and they were efficiently transmitted by Frankliniella occidentalis. Such isolates were recovered on the same farm in tomato, pepper, and artichoke 2 and 12 months later, suggesting natural spread from the resistant plants and survival. The RB isolates survived in experimental mixed infections with a non-RB isolate in susceptible pepper and C. chinense, but cross-protection in pepper acted against them. Commercial TSWV-resistant pepper but not resistant tomato cultivars from different companies were susceptible to these RB isolates after mechanical inoculation. Similar isolates were not detected among TSWV samples collected from 1993 to 2000 in the area. The management of TSWV and thrips using resistant pepper cultivars is discussed.