Enhancement of resistance to potato leafroll virus multiplication in potato by combining the effects of host genes and transgenes.

Four potato clones with host gene-mediated resistance to potato leafroll virus (PLRV) multiplication were transformed with the PLRV coat protein (CP) gene. Plants of lines expressing high levels of transcript were highly resistant to PLRV multiplication; virus concentration was only 20-40 ng/g of leaf, which is approximately 1% of the concentration reached in susceptible cultivars. The effects of the transgenic and host-derived resistance genes appear to be additive.

Developments in the understanding of the particle structure of tobraviruses.

Particles of tobraviruses resemble those of tobacco mosaic tobramovirus (TMV) in having helical symmetry and in being rod-shaped. However, isolated tobravirus coat protein and TMV coat protein respond to changes in the ionic strength and pH of the solute in contrasting ways. The types of aggregate formed in solutions of coat protein also differ which may be related to differences in the apparent mechanism of reconstitution of virus particles from isolated protein and RNA. The amino acid sequences of tobravirus and tobramovirus coat proteins have been shown to be similar in some regions known to be important for the structure of TMV particles. These alignments also show that tobravirus proteins are larger than tobramoviral proteins in part because of extra residues at the C-terminus. Tobravirus particles give a signal in proton NMR spectroscopy but TMV particles do not. The signal is caused by segmental mobility of the C-terminal peptide. This difference between TMV and tobraviruses may be related to a property not shared by tobraviruses and TMV and it is therefore speculated that the mobile C-terminal peptide of tobravirus coat proteins may be important in the transmission of tobravirus particles by nematode vectors.

How important is genome division as a taxonomic criterion in plant virus classification?

The number of nucleic acid components that constitute a virus genome has been used as an important discriminatory character in defining groups of plant viruses. However, with some virus groups, in particular potyviruses, recent results of nucleotide sequencing have reinforced previously deduced tentative relationships among viruses with different numbers of genome parts. A convenient solution is to classify these different types into groups or genera within a family (e.g. Potyvirus and Bymovirus in the family Potyviridae).

Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries.

Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC(L) for expression as fusion proteins with human kappa constant region (scFv-C(L)). Expression of these constructs in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c-myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.

A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA.

A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.

Synthetic antigen from a Peptide library can be an effective positive control in immunoassays for the detection and identification of two geminiviruses.

ABSTRACT Phage-displayed peptides were selected from the Cys 1 random phage display peptide library that bound strongly to the monoclonal antibody (MAb) SCR 20. The binding peptides were fused to the N-terminus of the phage protein pVIII. Preparations of the phage were shown to be effective as controls for the functionality of the SCR 20 MAb in both enzyme-linked immunosorbent assays and dot blot immunoassays. UV irradiation that eliminated phage infectivity did not greatly alter the antigenicity. Peptides displayed on phage are quick and cheap to prepare, and preparations can be standardized to ensure comparability among different assays. The peptide library approach can be readily extended for use with other MAbs to obtain inexpensive and safe standardized positive control reagents for use in immunoassays to diagnose plant disease.

Fusion proteins of single-chain variable fragments derived from phage display libraries are effective reagents for routine diagnosis of potato leafroll virus infection in potato.

ABSTRACT A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to potato leafroll virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (C(L)), an amphipathic helix (Zip), a combination of C(L) and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvC(L) fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting antibodies in TAS-ELISA. The limit of detection of purified PLRV for the different scFvC(L) fusion proteins ranged from 250 to 5 ng/ml; that for SCR3 is 5 ng/ml. Of the 11 scFv, 4 cross-reacted with some other luteoviruses. Several scFvC(L) and scFvC(L)Zip fusion proteins trapped PLRV from extracts of infected potato leaves as effectively as the polyclonal antibody preparation. Four scFv fusion proteins were used in a stem print assay to detect PLRV, and the results were similar to those obtained in tests using SCR3. The scFvC(L) fusion proteins retained activity for at least 6 months at 4 degrees C, and all scFv fusion proteins were fully active on reconstitution after lyophilization. A fully recombinant ELISA was devised that detected PLRV in extracts of infected potato, with results comparable to those obtained using the standard TAS-ELISA. The advantages of using scFv fusion proteins for the routine detection of plant viruses include the ability to produce large quantities of reagents cheaply in bacterial fermenters and to incorporate them into standardized tests.

Aphid Acquisition and Cellular Transport of Potato leafroll virus-like Particles Lacking P5 Readthrough Protein.

ABSTRACT Lepidopteran cells (Spodoptera frugiperda) produced isometric virus-like particles (VLP) when infected with a recombinant baculovirus Ac61 that contained the Potato leafroll virus (PLRV) coat protein gene modified with an N-terminal histidine tag (P3-6H). Cells infected with AcFL, a recombinant baculovirus that expressed cDNA copies of the PLRV genome RNA, did not produce virus-like particles (VLP). In cell lines doubly infected with Ac61 and AcFL, VLP were formed that contained PLRV-RNA packaged in P3-6H coat protein (FL). Both the P3-6H and the FL particles were morphologically indistinguishable from particles of PLRV despite the fact that they lacked the P5 readthrough protein present in wild-type PLRV. When aphids (Myzus persicae) were fed on, or injected with, purified PLRV, or VLP of either type (FL or P3-6H) and examined by electron microscopy, no differences were observed among treatments for particle endocytosis, transcellular transport, or exocytosis at the aphid midgut or accessory salivary glands. Particles were observed in the salivary canals and in the salivary duct leading out of the aphid. These results suggest that P5 readthrough protein of PLRV may not be essential for cellular transport of virus through aphid vectors.

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA.

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.

Development and application of an indirect competitive enzyme-linked immunoassay for aflatoxin m(1) in milk and milk-based confectionery.

High-titer rabbit polyclonal antibodies to aflatoxin M(1) (AFM1) were produced by utilizing AFM1-bovine serum albumin (BSA) conjugate as an immunogen. An indirect competitive enzyme-linked immunosorbent assay was standardized for estimating AFM1 in milk and milk products. To avoid the influence of interfering substances present in the milk samples, it was necessary to prepare AFM1 standards in methanol extracts of certified reference material (CRM) not containing detectable AFM1 (< 0.05 ng/g). The reliability of the procedure was assessed by using CRM with AFM1 concentrations of < 0.5 and 0.76 ng/g. Also, assays of milk samples mixed with AFM1 ranging in concentration between 0.5 and 50 ng/L gave recoveries of > 93%. The relative cross-reactivity with aflatoxins (AF) and ochratoxin A, assessed as the amount of AFM1 necessary to cause 50% inhibition of binding, was 5% for AFB1 and much less for AFB2, AFG1, and AFG2; there was no reaction with ochratoxin A. AFM1 contamination was measured in retail milk and milk products collected from rural and periurban areas in Andhra Pradesh, India. Of 280 milk samples tested, 146 were found to contain < 0.5 ng/mL of AFM1; in 80 samples it varied from 0.6 to 15 ng/mL, in 42 samples from 16 to 30 ng/mL, and in 12 samples from 31 to 48 ng/mL. Most of the milk samples that contained high AFM1 concentrations were obtained from periurban locations. The results revealed a significant exposure of humans to AFM1 levels in India and thus highlight the need for awareness of risk among milk producers and consumers.

Occurrence of aflatoxins and ochratoxin A in Indian poultry feeds.

From 1998 to 2001, 216 ingredients intended for incorporation into chicken feed, which included groundnut cake, maize, millets, rice bran, sorghum, soybean, sunflower, and mixed feeds, were assayed for aflatoxins and ochratoxin A contamination using an indirect competitive enzyme-linked immunosorbent assay. Thirty-eight percent of the samples were contaminated with aflatoxins and 6% with ochratoxin A. The incidence scores of aflatoxin contamination in excess of 10 microg/kg were 41 of 95 for maize, 18 of 30 for mixed feeds, 10 of 37 for groundnut, 6 of 29 for sorghum, 5 of 10 for sunflower, 3 of 14 for rice bran, and 1 of 8 for millet. Ochratoxin A contamination, in excess of 10 microg/kg, was found in 9 of 29 sorghum samples, 1 of 27 groundnut samples, 1 of 14 rice bran samples, 1 of 10 sunflower samples, and 2 of 8 millet samples. Ochratoxin A was not found in maize and mixed feeds. None of the three soybean samples contained ochratoxin A. This is the first report, to our knowledge, of co-occurrence of aflatoxins and ochratoxin A in Indian poultry feeds. The results confirm the importance of analysis of ingredients before incorporating them into mixed feeds.

Occurrence of Tobacco streak virus on Peanut (Arachis hypogaea) in India.

A virus disease of peanut (groundnut, Arachis hypogaea L.), characterized by necrosis of the stem and terminal leaflets followed by death, caused severe crop losses in Andhra Pradesh, India during the rainy season of the year 2000. The disease was referred to as peanut stem necrosis disease (PSND). Cowpea (Vigna unguiculata, cv. C-152) and Phaseolus vulgaris (cv. Topcrop) were found to be suitable for propagating the virus. In laboratory inoculation tests, the virus was found to infect a large number of plants. In laboratory tests, the virus was transmitted by the thrips Frankliniella schultzei. Virus particles were purified by differential centrifugation and sucrose density gradient centrifugation from infected cowpea plants and were used to elicit the production of a rabbit polyclonal antiserum with high titer. Extracts of infected plants reacted with antiserum to Tobacco streak virus (TSV). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from purified virus particles showed them to contain a major protein of 28 kDa and a minor, though prominent, protein of 57 kDa. Gel electrophoresis of RNA extracted from virus particles resolved it into four species with estimated sizes of 3.7, 3.1, 2.2, and 0.9 kb. Complementary DNA (cDNA) was made using as template a sample of the 2.2-kb RNA 3 and as primer an oligonucleotide complementary to sequence in RNA 3 of TSV. Following second strand synthesis, the cDNA was cloned in pBluescript and the nucleotide sequence was obtained for 868 nt of the cDNA. The sequence was 88.4% identical to the sequence in RNA 3 of TSV (strain WC). The results indicate that the causal agent of PSND is TSV. The same virus also was found to cause sunflower necrosis, an economically important disease in India. Studies on the epidemiology of PSND and the identification of virus-resistant peanut genotypes have been initiated to devise strategies to control PSND.

[Hemodynamic modifications in splenic circulation studied by echo-Doppler during pregnancy]. / Modificaciones hemodinámicas de la circulación esplácnica estudiada por eco-Doppler durante la gestación.

INTRODUCTION: Pregnancy produces multiple changes in the mother's body, most of which have been studied. However, changes in hepatosplenic circulation are not well-known. The routine use of ultrasonography and of echo-Doppler has created new possibilities for the knowledge of splenic circulation during pregnancy. MATERIAL AND METHOD: We studied 30 healthy pregnant women who had given their informed consent. To evaluate the morphological and hemodynamic changes that might occur in the splenic vessels during pregnancy and immediate puerperium, laboratory investigations, obstetric and hepatic ultrasonography, and hepatosplenic echo-Doppler were performed between weeks 8-12, 20-24, and 32-36, as well as in the immediate puerperium. RESULTS: The caliber of the vena porta and its tributaries, as well as that of its intrahepatic branches, increased while the caliber of the suprahepatic vessels slightly decreased during pregnancy. The hemodynamic changes detected by Doppler ultrasonography were: progressive flattening of the pulsed Doppler trace of the suprahepatic vessels during the course of pregnancy; a progressive reduction in mean portal velocity, which was more marked in the third trimester, and a decrease in the markers of resistance in the hepatic artery and superior mesenteric artery. CONCLUSIONS: Hemodynamic changes in hepatosplenic circulation are produced during pregnancy that can be safely and effectively evaluated in real time by ultrasonography and echo-Doppler. Knowledge of these changes is required to evaluate these vessels in pathological conditions.

The effect of protoplast concentration during inoculation on the infection of tobacco protoplasts.

When tobacco protoplasts were inoculated with tobacco rattle virus, the proportion infected was inversely related to the protoplast concentration between 0.5 X 10(5) and 4 X 10(5) per milliliter when the inocula contained phosphate or Tris-HCL buffers, but was independent of protoplast concentration when using citrate buffer. The decrease in infection obtained at high protoplast concentrations in phosphate was diminished by increasing the poly-L-ornithine (PLO) concentration. At usual PLO concentrations, inocula in phosphate produced more infection that those in citrate, but at some suboptimal PLO concentrations the position was reversed. These effects may result from differences in the reaction between PLO and the different buffer anions.