Study on the mechanism of Bioelectric Recognition Assay: evidence for immobilized cell membrane interactions with viral fragments.

The Bioelectric Recognition Assay (BERA) is a whole-cell based biosensing system that detects the electric response of cultured cells, suspended in a gel matrix, to various ligands, which bind to the cell and/or affect its physiology. Previous studies have demonstrated the potential application of this method for rapid, inexpensive detection of viruses in a crude sample. However, the understanding, so far, of the fundamental processes that take place during cell-virus interactions within the probe has been rather limited. In the present study, we combined electrophysiological and fluorescence microscopical assays, so that we can prove that animal and plant cells immobilized in BERA sensors respond to different viruses primarily by changing their membrane potential. The response of immobilized cells against different viruses did not depend on the virus ability to penetrate the cell, but was modified after binding each virus to a virus-specific antibody or removal of its coat protein after treatment with a protease. Consequently, we were able to assay the presence of a virus in its complete form or fragments thereof. Combination of immunological recognition with the electrophysiological response of immobilized cells allows for a considerable increase of the specificity of the BERA biosensory assay. In addition, rather than simply detect the presence of a protein or genomic sequence, the method can help gain information on the bioactivity of a virus.

First Report of Citrus tristeza virus in Greece.

Large-scale surveys of Citrus spp. for the presence of Citrus tristeza virus (CTV) by the Ministry of Agriculture in Greece began in 1995. Over 26,000 trees have been tested by enzyme-linked immunosorbent assay and immunoprinting (2). In summer 2000, the first CTV-infected sweet orange cv. Lane Late tree grafted on CTV-tolerant Carrizo citrange was found in Argolis County, Peloponnese. This tree belonged to a batch of CAC propagation material (20 trees) illegally introduced from Spain in 1994, which was subsequently traced and found to be infected (45%). A follow-up search of trees grafted with the above material was undertaken in the two concerned regions (Argolis and Chania-Crete), and more than 3,500 trees have been removed. Extensive surveys continue to identify and destroy new infections. Few cases (15 of 16,800) of natural transmission to cultivars other than cv. Lane Late have been found. All of these have been close to the initially infected trees in the Argolis area. Surveys in spring 2001 were extended to certified propagation material of Clemenpons mandarin on Carrizo citrange imported from Spain, and 7 of 1,038 plants were infected (0.64%). The virus was successfully graft-transmitted to sweet orange cv. Madame Vinous and sweet lime seedlings, where it was identified by immunoprinting and reverse transcription-polymerase chain reaction (1). Mild vein clearing symptoms appeared on both indicators. Vein clearing on sweet lime was also accompanied by leaf cupping. To our knowledge, this is the first report of CTV in Greece. References: (1) A. Sambade et al. J. Virol. Methods 87:25, 2000. (2) C. Vela et al. J. Gen. Virol. 67:91, 1986.

The application of the bioelectric recognition assay for the detection of human and plant viruses: definition of operational parameters.

The bioelectric recognition assay (BERA) is a novel biosensory method based on a unique combination of a group of cells, their immobilization in a matrix that preserves their physiological functions and the expression of the cell interaction with viruses as a change in electrical properties. A BERA sensor consists of an electroconductive, tube-like probe containing components of immobilized cells in a gel matrix. Cells are selected to specifically interact with the virus under detection. In this way, when a positive sample is added to the probe, a characteristic, 'signature-like' change in electrical potential occurs upon contact between the virus and the gel matrix. In the present study, we demonstrate that BERA can be used for the detection of viruses in humans (hepatitis C virus) and plants (tobacco and cucumber viruses) in a remarkably specific, rapid (1-2 min), reproducible and cost-efficient fashion. The sensitivity of the virus detection with BERA (0.1 ng) is equal or even better than with advanced immunological, cytological and molecular techniques, such as the reverse transcription polymerase chain reaction. Moreover, a good storability of the sensors can be achieved without affecting their performance. The potential use of portable BERA biosensors in medicine, for mass screening purposes, as well as for the detection of biological warfare agents without prior knowledge of a specific receptor-molecule interaction is discussed.

Bioelectric recognition assay (BERA).

A novel biosensory method has been developed for the determination of various chemical and biological molecules by assessing their electrophysiological interactions with a group of cells and cell components immobilized in a gel matrix that preserves their 'physiological' functions. The method was applied for the detection of: (i) hepatitis C virus in human blood samples; (ii) plant viruses; and (iii) a herbicide (glyphosate) in aqueous solutions. It was able to rapidly (assay time 3-5 min) and specifically detect the molecules in question at a concentration lower than 100 pg/ml, among other compounds f similar structure. The potential use of BERA biosensors for a rapid and cost-efficient molecule determination without prior knowledge of a specific receptor-molecule interaction is discussed.

First Report of Tomato yellow leaf curl virus on Tomato Crops in Greece.

In late summer 2000, tomato (Lycopersicon esculentum Mill.) grown in greenhouses in Ierapetra, Tympaki, and Chania (Crete) showed leaf curling, reduced leaf size, yellowing, shortened internodes, and a bushy appearance. More than 30 ha of tomato greenhouses were affected and the disease incidence ranged from 15 to 60% with estimated crop losses of over $500,000. Similar symptoms were observed in tomato samples from Marathon (Attiki) and Southern Peloponnese. All greenhouses with infected plants were infested with high populations of Bemisia tabaci (Gennadius), which were also observed outside the greenhouses on several weeds. Tomato symptoms were similar to those caused by Tomato yellow leaf curl virus (TYLCV). The assumed virus could not be transmitted mechanically but successful transmission was obtained by grafting onto healthy tomato plants. Over 100 samples of symptomatic tomato plants collected from Crete and southern Peloponnese gave positive reactions when tested by ELISA using monoclonal antibodies to TYLCV-European (Adgen Ltd). The serological results were confirmed by PCR using two pairs of primers, universal degenerate (1) and MA 13 and MA 17 (2), amplifying different parts of the virus genome. The restriction fragment length polymorphism (RFLP) analysis (AluI, HaeIII, and TaqI) of the 541 bp amplicon obtained with the degenerate primers showed patterns similar to TYLCV-Is (Israeli species). The second pair of primers gave the expected 348 bp product, which was sequenced. Sequence comparisons revealed 99% identity with TYLCV-Is (EMBL no. X15656, X76319). The resulting sequence was at least 97.7% identical to sequences of TYLCV isolates from the Dominician Republic (EMBL no. AF024715), Cuba (EMBL no. AJ223505), Portugal (EMBL no. AF105975), Iran (EMBL no. AJ13271), and Spain (EMBL no. AF071228). The disease appeared for the first time in 1992 in Tymbaki, but was limited to very few plants in one glasshouse. However, the cause was not determined. To our knowledge, this is the first report of TYLCV of the Begomovirus genus in Greece. References: (1) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (2) J. Navas-Castillo et al. J. Virol. Methods 75:195, 1998.

A New Ilarvirus Isolated from Grapevine in Greece.

In 1994, characteristic viruslike symptoms on grapevine were reported in the collection of the Grapevine Institute in Athens, Greece, on the hybrid Baresana × Baresana. The symptoms were sharp angular mosaic, leaf crinkle, and little leaf. The affected vines showed gradual decline and severe stunting or death. Such vines produced abortive flowers or very few berries with smaller, wrinkled, and nongerminating seeds. Serological testing, by enzyme-linked immunosorbent assay (ELISA), of the affected vines against the most common grapevine viruses Alfalfa mosaic, Arabis mosaic, Grapevine fanleaf, Grapevine fleck, Grapevine A, Rasberry ringspot, and grapevine leafroll-associated viruses gave negative results. A virus was isolated from affected grapevine young leaves by mechanical inoculation of Gomphrena globosa and single lesioned. The virus host range included G. globosa (local and systemic dark red or necrotic lesions), Chenopodium quinoa (necrotic local lesions and systemic mottle), and three tobacco cultivars (sharp necrotic local lesions, 1 to 3 mm in diameter). Pollination of C. quinoa with pollen from infected plant gave about 30% infected seedlings. The virus was purified from C. quinoa by differential centrifugation using 0.02 M phosphate buffer pH 8.0, containing 0.01 M DIECA and 0.01 M sodium thioglycolate as extraction buffers. In a purified preparation, quasisphaerical virus particles of about 29 nm were observed. Electrophoretic mobility of the viral coat protein showed a molecular weight of 30 kDa. Using purified preparations, an antiserum was obtained with a titer of 1:1024 in microprecipitin test and an optimum IgG dilution in ELISA of 1:10,000 for maximum absorption at OD405 nm Using degenerate primers designed from homologous regions in RNA-2 corresponding to a fragment of the polymerase gene of Ilarviruses, the expected 381-bp polymerase chain reaction product was obtained. This product was cloned and sequenced. Comparisons with sequence data from the homologous regions of RNA-2 of other known Ilarviruses, showed that the sequence of the above 381-bp amplicon shared 72% sequence similarity with Tobacco streak virus, 67% of Citrus variegation virus and Spinach latent virus, 66% of Asparagus virus 2 and Elm mottle virus, and 65% of Citrus leaf rugose virus. Based on the above data, it is concluded that the isolated virus is an Ilarvirus with closest similarity to Tobacco streak virus. From the relative bibliography (1-3) it appears that the virus reported here is different from Grapevine line pattern virus, a possible Ilarvirus, previously reported from Hungary. References: (1) J. Lehoczky et al. Kertgazdasag 19:61, 1987. (2) J. Lehoczky et al. Phytoparasitica 17:59, 1989. (3) J. Lehoczky et al. Phytopathol. Medit. 31:115, 1992.

First Report of Occurrence of Potato Tuber Necrotic Ringspot Disease in Greece.

Potato tuber necrotic ringspot disease (PTNRD), caused by potato Y potyvirus isolate NTN (PVYNTN) of the N virus strain group, was first described in Hungary in 1980. It has spread throughout Europe, with the most recent reports from Portugal and Italy in 1997 to 1998 (1). Superficial necrotic ringspot areas on tubers, typical of PTNRD, were first observed in commercial potato fields of the Nevrokopi region in northern Greece in 1994. Affected cultivars included Timate and, to a lesser extent Spunta, the original seed of which came from the Netherlands. PVY was identified from all tubers tested by indexing on Nicotiana tabacum and subsequent testing of the plants by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA). The potato seed lots were rejected by the local authorities. PTNRD reappeared in the same area in 1998 in a more aggressive manner. Cultivar Hermes, imported from Scotland, was most affected, with very severe symptoms in 80% of the tubers. Symptoms appeared in early September, 40 days after defoliation of the plants. Other cultivars were affected at lower rates; cv. Spunta showed typical symptoms and cvs. Fabola, Santana, and Irvila exhibited atypical cracks and blisters. In all cases, PVY was isolated in N. tabacum and its presence confirmed by DAS-ELISA. Immunocapture-reverse transcription-polymerase chain reaction with specific PVYNTN primers (2) detected the characteristic 835-bp product in all cultivars tested. The incidence of PTNRD seems to be expanding in northern Greece, where it has become a threat to potato production. PTNRD symptoms were also observed in southern Greece (Ahaia) in experimental "crossing" fields of seed stocks. In this case, the disease seems to have spread especially in cv. Marfona. References: (1) L. Tomassoli et al. Plant Dis. 82:350, 1998. (2) H. L. Wiedemann and E. Maiss. Z. Pflanzenkrankh. Pflanzenschutz 103:337, 1996.

The ammonium sulfate activation of phosphorylase b.

The ammonium sulfate activation of phosphorylase b has been studied. Ammonium sulfate, when present in high concentrations, induces properties of phosphorylase a in phosphorylase b, such as an enhanced affinity for AMP, a reversal of the glucose-6-P inhibition and enzyme tetramerization. The data are consistent with the interpretation that sulfates bind to the Ser-14 site and the sulfate-protein interactions at this site are responsible for activation of phosphorylase b.