Triple Antibody Research Articles

Detection of Citrus psorosis virus in field trees by direct tissue blot immunoassay in comparison with ELISA, symptomatology, biological indexing and cross‐protection tests

Serological detection of Citrus psorosis virus (CPsV) by direct tissue blot immunoassay (DTBIA) and by double (DAS) and triple (TAS) antibody sandwich ELISA, was compared in samples from various citrus varieties growing in the glasshouse and in the field. In young shoots and leaves, CPsV was readily detected by the three procedures, whereas DTBIA detection in old leaves was less consistent. DTBIA detection and ELISA readings in nine different citrus varieties were similar, suggesting that CPsV accumulates to equivalent levels in all of them. In infected field trees from Spain or Italy, CPsV was consistently detected by TAS ELISA, even in samples of old leaves in winter, whereas DTBIA detection in the same trees was reliable only when using young shoots. Detection of CPsV by DTBIA and by DAS and TAS ELISA in previously untested field trees correlated perfectly with psorosis diagnostics based on biological indexing, specifically with the capacity of those sources to cross‐protect against challenge inoculation with psorosis B. Some trees without bark scaling were shown to be psorosis‐infected by biological indexing and to contain CPsV by serological tests; other trees showing psorosis‐like bark or leaf symptoms in the field were shown to be psorosis‐free by biological indexing and also CPsV‐free by serology. This is the first time that the presence of CPsV has been correlated with psorosis infection as diagnosed by biological indexing.

Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies.

Several reports have described the existence of synergy between neutralizing monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 (HIV-1). Synergy between human MAbs b12, 2G12, 2F5, and 4E10 in neutralization of primary isolates is of particular interest. Neutralization synergy of these MAbs, however, has not been studied extensively, and the mechanism of synergy remains unclear. We investigated neutralization synergy among this human antibody set by using the classical approach of titrating antibodies mixed at a fixed ratio as well as by an alternative, variable ratio approach in which the neutralization curve of one MAb is assessed in the presence and absence of a fixed, weakly neutralizing concentration of a second antibody. The advantage of this second approach is that it does not require mathematical analysis to establish synergy. No neutralization enhancement of any of the MAb combinations tested was detected for the T-cell-line-adapted molecular HIV-1 clone HxB2 using both assay formats. Studies of primary isolates (89.6, SF162, and JR-CSF) showed neutralization synergy which was relatively weak, with a maximum of two- to fourfold enhancement between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations. Analysis of b12 and 2G12 binding to oligomeric envelope glycoprotein by using flow cytometry failed to demonstrate cooperativity in binding between these two antibodies. The mechanism by which these antibodies synergize is, therefore, not yet understood. The results lend some support to the notion that an HIV-1 vaccine that elicits moderate neutralizing antibodies to multiple epitopes may be more effective than hereto supposed, although considerable caution in extrapolating to a vaccine situation is required.

Inheritance of resistance to Yam mosaic virus, genus Potyvirus, in white yam (Dioscorea rotundata)

Yam mosaic virus (YMV) causes the most-widespread and economically important viral disease affecting white yam (Dioscorea rotundata) in West Africa. The genetic basis of resistance in white yam to a Nigerian isolate of YMV was investigated in three tetraploid D. rotundata genotypes: TDr 93–1, TDr 93–2 and TDr 89/01444. F1 progeny were produced using TDr 87/00571 and TDr 87/00211 as the susceptible parents. Segregation ratios indicated that a single dominant gene in a simplex condition governs the resistance in TDr 89/01444, while the resistance in TDr 93–2 is associated with the presence of a major recessive gene in duplex configuration. Segregation of progeny of the cross TDr 93–1×TDr 87/00211 fitted a genetic ratio of 2.48:1 resistant:susceptible, which can be expected when two simplex heterozygotes are crossed, indicating the possible modifying effect of the susceptible parent. A triple antibody immunosorbent assay (TAS-ELISA) was used for virus detection in inoculated plants. Slight mosaic symptoms appeared on most resistant individuals, while asymptomatic resistant genotypes with high ELISA (A405) values were observed in all crosses. Such a heterogeneous response suggests the influence of additional modifier genes that segregate in the progeny. The finding that resistance can be inherited as a dominant or recessive character has important implications for YMV resistance breeding.

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM. About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.

Transmissibility of four tospoviruses by a thelytokous population ofThrips tabaci from Liguria, Northwestern Italy

Studies were carried out on a population ofThrips tabaci Lindeman (Thysanoptera: Thripi-dae) from Liguria to assess its sex-ratio and its ability to transmit four tospoviruses: tomato spotted wilt (TSWV), impatiens necrotic spot, tomato chlorotic spot and groundnut ringspot. The population was composed of females only (therefore thelytokous). The first instar larvae were allowed to acquire the virus for 48 h on infected leaves of datura, basil or pepper, and then reared on cucumber until emergence, which medially occurred 9.5 days after hatching. Transmission capacity was checked using two inoculation access periods (lAPs) of 48 h each on pepper leaf disks.T. tabaci was able to transmit TSWV isolate P105 with an efficiency of 16.7% and 4.4% in the first and second IAP, respectively, and TSWV isolate BR-01 with an efficiency of 2.0%. The onion thrips did not transmit the three other tospoviruses. During the IAPs, almost all adults fed on the leaf disks, producing evident silvery scars. The presence of tospovirus nucleocapsids in thrips was assayed by Triple Antibody Sandwich (TAS) and cocktail ELISA. Not all adults that had transmitted TSWV were positive in the tests, whereas some non-transmitter individuals proved positive. For each of the other tospoviruses, some thrips were positive in at least one test, although none was able to transmit the virus.

First Report of Tobacco streak virus in Strawberry in the Eastern United States.

In a 1998 virus survey (2) conducted on 23 commercial strawberry (Fragaria × ananassa Duchesne) production farms in the state of Maryland, leaf samples from 1,100 randomly sampled plants were sent to the U.S. Department of Agriculture laboratory in Corvallis, OR, for testing by enzyme-linked immunosorbant assays (ELISA). The viruses identified were Strawberry mild yellow edge, Strawberry crinkle, Strawberry veinbanding, Strawberry mottle, and Tomato ringspot viruses, all of which are known in the eastern United States. Tobacco streak virus (TSV) also was identified in 17 of the samples: 12 originated from a 1-year-old planting of 'Sweet Charlie' and 5 from another farm, of which 4 were from a 2-year-old 'Sweet Charlie' planting and 1 was from a 2-year-old 'Delmarvel' planting. Triple antibody sandwich ELISA was used to detect TSV following the procedures described by Finn and Martin (1), except that leaves from test plants were homogenized (1:20, wt/vol, in blocking buffer) and flat bottom microtiter plates (Nunc, Roskilde, Denmark) and goat anti-mouse (polyvalent) alkaline phosphatase conjugate were used in the assays. The absorbance of each well at 405 nm (A405) was read in an ELISA plate reader. Reactions were considered positive if the A405 values were greater than five times the values of healthy samples. The A405 values of healthy samples ranged from 0.0 to 0.04, with values greater than 0.20 considered positive for TSV. An independent determination of TSV was made in plants shipped from Florida to Maryland in 1999. In this instance, leaf samples from 'Sweet Charlie' plants were sent by the Maryland Department of Agriculture to Agdia Inc. (Elkhart, IN), where samples tested positive for TSV.

Occurrence of Citrus psorosis virus in Campania, southern Italy

Citrus psorosis virus (CPsV), genus Ophiovirus, is associated with a severe disease of citrus worldwide. Double antibody sandwich (DAS) ELISA using a polyclonal antiserum, and triple antibody sandwich (TAS) ELISAs, employing the IgG monoclonal antibody (mab) 13C5, and the IgM mab 2A3, were used to detect CPsV in orchards of different citrus varieties in Campania, southern Italy. TAS ELISA with 13C5 detected all the infections detected by DAS ELISA. Overall, 14% of trees younger than 15 years were positive, but only 1% of older trees, suggesting that infected propagating material has been increasingly used in recent years, in the absence of certification. Highest infection rates were in younger trees of sweet orange (22.8%) and clementine (18.6%). CPsV could easily be detected at all seasons of the year tested (June–January); these and earlier results indicate that TAS ELISA using 13C5 is a sensitive, broad-spectrum and reliable diagnostic method useful for routine tests and certification programmes. Of 44 field isolates responding strongly to DAS ELISA and 13C5-TAS ELISA, mab 2A3 gave similar results with 29 isolates, but gave low values with the others, thus providing a degree of differentiation among isolates. To confirm that the ELISA tests were indeed detecting CPsV, samples of 42 ELISA-positive plants were analysed by ISEM in a blind test, and in 38 of these, characteristic virus particles were clearly seen. Although CPsV was frequently and consistently detected in the area sampled, bark scaling symptoms were not seen: possible reasons for this are discussed.

Improved detection of citrus psorosis virus using polyclonal and monoclonal antibodies

Citrus psorosis is a serious and widespread disease associated with citrus psorosis virus (CPsV), a novel filamentous negative‐stranded virus in the genus Ophiovirus. Laborious and costly indexing on test plants has been the only routine diagnostic method available, but recently an antiserum usable in double antibody sandwich (DAS) ELISA has been prepared. Here, major improvements to the DAS‐ELISA protocol, a new purification method, and production of two monoclonal antibodies (mabs) to CPsV, an IgG and an IgM are reported. A highly sensitive triple antibody sandwich (TAS) ELISA making use of the mabs is described. In glasshouse citrus the homologous virus was still detectable at a tissue dilution of 1/6250 in DAS and at 1/31250 in TAS‐ELISA. Both the DAS and IgG mab‐TAS formats detected all CPsV isolates so far tested (from Argentina, Italy, Lebanon, Spain and the USA). A few isolates were not detected by the IgM mab.

Biologische Bundesanstalt für Land‐und Forstwirtschaft, Institut für Pflanzenvirologie, Mikrobiologie und biologische Sicherheit, Braunschweig, Germany

Immunoelectron microscopy (IEM) and enzyme‐linked immunosorbent assay (ELISA) studies with polyclonal antibodies (PAbs) indicated that the isolates 1A, Nsaba, and Kpeve of cacao swollen shoot virus (CSSV; genus Badnavirus) are serologically distinct. Out of these isolates, only 1A was reliably detected from infected cacao trees with the homologous PAbs. To improve serological detection of the isolates Nsaba and Kpeve by the application of monoclonal antibodies (MAbs), virion preparations of CSSV‐1A, ‐Nsaba and ‐Kpeve were used to immunize mice. Although immunization with CSSV‐1A yielded a large number of 1A‐specific MAbs, no Nsaba‐ or Kpeve‐specific MAbs were obtained after immunization with these isolates. Instead, the latter exclusively led to the production of MAbs which reacted only with isolate 1A. Epitope characterization of 1A‐specific MAbs using different ELISA procedures, IEM and Western blot experiments showed that three of the 1A‐specific MAbs detected a continuous epitope [sodium dodecyl sulphate (SDS)‐stable] and nine MAbs reacted with conformation‐dependent epitopes (SDS‐labile). Direct adsorption of virus particles to microtitre plates in antigen‐coated plate ELISA affected epitope stability in a similar fashion as SDS treatment of virions. When comparing the sensitivity of CSSV detection by MAbs and PAbs in various ELISA formats, MAbs were slightly more sensitive than PAbs in CSSV detection from plant sap. Using the panel of available 1A‐specific MAbs for triple antibody sandwich‐ELISA analysis of 31 CSSV samples from different cacao growing areas of Ghana revealed the occurrence of four serotypes in the samples containing CSSV‐1A isolates. In addition, the serological reaction patterns of the MAbs correlated well with the geographic origin of the isolates, thus giving further indications about the regional distribution of CSSV 1A‐like isolates in Ghana.

Immunodiagnostic methods for detection of 5-enolpyruvylshikimate-3-phosphate synthase in Roundup Ready® soybeans

We have developed immunological-based detection methods to support labeling of protein-containing food fractions derived from Roundup Ready® soybeans. Western blotting and enzyme linked immunosorbent assay (ELISA) procedures were developed to measure the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein derived from the Agrobacterium sp. strain CP4 in the major processed fractions derived from Roundup Ready soybean. Expression of the CP4 EPSPS protein confers tolerance to Roundup® herbicide. The western blotting method utilizes a polyclonal goat anti-CP4 EPSPS antibody which specifically binds to CP4 EPSPS followed by detection of bound goat antibody with biotinylated Protein-G. Detection of this complex is accomplished using horseradish-peroxidase (HRP) labeled NeutrAvidin™ and signal development by enhanced chemiluminesence. Data from western blot analyses of these fractions establish that stable epitopes remain after the seed has been subjected to processing conditions typically employed by the food industry, thereby enabling development of an ELISA method. The ELISA for measurement of CP4 EPSPS is a triple antibody sandwich procedure utilizing a monoclonal capture antibody and a polyclonal detection antibody followed by a third biotin labeled monoclonal anti-rabbit antibody. Sandwich formation is detected using HRP labeled NeutrAvidin™ with color development using TMB substrate. In the sandwich ELISA, the immunological activity of CP4 EPSPS was reduced by the extraction method required to solubilize CP4 EPSPS protein from processed fractions. Sensitivity of the CP4 EPSPS ELISA was sufficient to detect CP4 EPSPS protein in processed soybean fractions that contained 2% Roundup Ready soybean mixed with conventional processed soybean fractions, thereby making the ELISA an acceptable method to assess CP4 EPSPS protein in processed soybean fractions. Data on sensitivity, accuracy, precision and specificity, established that the western blot and ELISA methods are appropriate for compliance with the EC Novel Foods Regulation.

Production of Monoclonal Antibodies to Barley Mild Mosaic Virus and Their Use for Strain Differentiation

A panel of five stable hybridoma cell lines secreting mono‐ clonal antibodies (mAbs) were produced using a French mechanically transmitted isolate of barley mild mosaic virus (BaMMV‐MF) as antigen. All mAbs reacted with BaMMV‐MF in two enzyme‐linked immunosorbent assay (ELISA) formats: triple antibody sandwich (TAS)‐ELISA and antigen‐coated plate (ACP)‐ELISA. These mAbs recognized epitopes, present on both degraded virions and intact particles. Four mAbs (5C8, 1D5, 1B12, 1A12) belong to the immunoglobulin (Ig)G class and one mAb (3A9) represents an IgM. The five mAbs were compared in TAS‐ and ACP‐ELISA for reactivity with numerous French isolates. These isolates were detected in TAS‐ and ACP‐ELISA with four mAbs (5C8, 1D5, 1B12, 3A9). In both ELISA systems the mAb 1A12 recognized only an epitope specific for BaMMV‐MF. All mAbs, except 1A12 recognized also the German (BaMMV‐MG), Italian (BaMMV‐I) and Japanese (BaMMV‐Ka1) isolates in both TAS‐ and ACP‐ELISA. The Japanese isolate (BaMMV‐Na1) only reacted with two mAbs (1D5, 5C8) in TAS‐ELISA. Only one mAb (3A9) reacted with BaMMV‐MF, BaMMV‐PF, BaMMV‐I,BaMMV‐MG and BaMMV‐Ka1 in Western blot. These mAbs make it possible to distingish between the three BaMMV serotypes.

Improved Diagnostic Techniques for Tomato Yellow Leaf Curl Virus in Tomato Breeding Programs

Breeding for tomatoes resistant to tomato yellow leaf curl geminivirus (TYLCV) is difficult, mainly due to the lack of an accurate system for selecting resistant plants. The variable nature of the genotypes used (wild or wild-derived genotypes and advanced breeding lines) makes the development of a reproducible selection procedure necessary during the breeding program. Serological and nucleic acid-based diagnostic techniques for detecting TYLCV were evaluated for their sensitivity, reliability, and possibility of quantification in order to select the combination of techniques that provides the most rapid and accurate characterization of the resistance level of each item. Squash blot followed by the more sensitive polymerase chain reaction (PCR) detection of inconclusive samples is recommended for the screening of new resistant sources among asymptomatic Lycopersicon spp., as they accumulate extremely low viral concentrations that cannot be detected by serological methods. A triple antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA), modified to reduce the background, can be used in large-scale field screening of advanced breeding lines if combined with the more sensitive squash blot, which is less affected by the age and state of the tissue. Hybridization methods were also more appropriate to assess viral distribution throughout the plant than TAS-ELISA or even PCR, which fails to reliably detect TYLCV in tissues like roots.

First Report of East African Cassava Mosaic Begomovirus in Ghana.

Virus species causing cassava mosaic disease have been categorized into three classes based on their reaction with monoclonal antibodies (MAbs) and their distribution (2). These viruses have different, scarcely overlapping distribution: African cassava mosaic begomovirus (ACMV) occurs in Africa west of the Rift Valley and in South Africa; East African cassava mosaic (EACMV) occurs in Africa east of the Rift Valley and in Madagascar; and Indian cassava mosaic virus (ICMV) occurs in India and Sri Lanka (2). During 1998, surveys were conducted in farmers' fields in Ghana to assess the incidence and reaction of local cassava cultivars to cassava mosaic disease. Leaf samples from symptomatic plants were indexed by triple antibody sandwich-enzyme-linked immunosorbent assay with crude extracts and monoclonal antibodies obtained from the International Institute of Tropical Agriculture (IITA). Each sample was assayed with monoclonal antibody SCR 23, which detects ACMV and EACMV, SCR 33, which detects ACMV, and SCR 58, which detects ICMV. None of the samples reacted with SCR 58. Two of the samples collected from the western region of Ghana produced strong reactions with MAb SCR23 but did not react with ACMV-specific MAb SCR 33. This result was consistent in three separate experiments conducted on the samples, confirming that the virus was EACMV and not ACMV. The results extend the work by Ogbe et al. (1) and provide further evidence of the occurrence of EACMV in west Africa.

First Report of Soilborne Wheat Mosaic Virus in the United Kingdom.

In April 1999, severe soilborne wheat mosaic virus (SBWMV) symptoms were observed in five fields of winter wheat (Triticum aestivum, cvs. Consort, Equinox, and Savannah) on one farm in Wiltshire, UK. Affected plants were markedly stunted and had a pale mosaic on their leaf sheaths that developed into bright yellow, parallel streaks on the leaves as they unfolded. Symptomatic plants were found in discrete, elliptical patches ranging in size from a few square meters to nearly a hectare. During May and June, symptoms became less marked as temperatures increased and were restricted to lower leaves. SBWMV was positively identified in all five fields (60 to 170 plants per field) by double (W. Huth, BBA-Braunschweig, Germany; Sanofi Phyto-Diagnostics, Paris) and triple (T. Wilson, SCRI, Dundee, UK) antibody sandwich enzyme-linked immunosorbent assay and by reversetranscription polymerase chain reaction (2). Identification was confirmed by immunoelectron microscopy, including protein-A gold labeling, which revealed bipartite, rod-shaped particles typical of SBWMV. Neither wheat spindle streak mosaic virus nor barley yellow dwarf virus was detected in the field samples, nor was SBWMV detected in any other field subsequently sampled, despite a survey of the surrounding area. Wheat is the most important economic crop in the United Kingdom (≈1.9 million hectares are grown annually, yielding ≈16 million tonnes), but its position is threatened by the economic impact of SBWMV, which has decreased yields by up to 50% in the United States (1).

Characteristics and Distribution of Potato Latent Carlavirus (Red LaSoda Virus) in North America.

A carlavirus (code name RLSV and subsequently named potato latent carlavirus, PotLV) with serological and biological characteristics different from those of potato M carlavirus (PVM) and potato S carlavirus (PVS) was detected in the potato (Solanum tuberosum) cultivar Red LaSoda by the Scottish Agricultural Science Agency in 1992. During a routine electron microscope testing of accessions in the Vancouver Collection of Virus-Free Potatoes growing in the California winter test in 1993, a filamentous rod-shaped virus similar to PVS and PVM was found in a Red LaSoda clone from Nebraska. The virus was isolated and purified. The monoclonal antibody, MAb 4E12, which is highly specific to the PotLV virus, was developed. From 1994 to 1998, the accessions in the Vancouver Collection of Virus-Free Potatoes were assayed by triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) using the 4E12 MAb. Seven accessions tested from 1994 to 1996 were infected with PotLV. None of the 270 and 267 accessions in this collection tested positive for this virus in 1997 and 1998, respectively. In 1997 and 1998, the 137 accessions in the U.S. National Varietal Collection maintained at Presque Isle, Maine, were also assayed using the 4E12 MAb. The cultivars High Plains, Platte, and Red LaSoda were the only accessions that tested positive for PotLV. Nicotiana benthamiana, N. megalosiphon, and N. occidentalis are new systemic hosts for PotLV. TAS-ELISA with the 4E12 MAb is now part of the standardized test for PotLV in Canada.

First Report of East African Cassava Mosaic Begomovirus in Nigeria.

Cassava (Manihot esculenta Crantz) is an important food crop in sub-Saharan Africa. One of the major production constraints is cassava mosaic disease caused by African cassava mosaic (ACMV) and East African cassava mosaic (EACMV) begomoviruses. ACMV is widespread in its distribution, occurring throughout West and Central Africa and in some eastern and southern African countries. In contrast, EACMV has been reported to occur mainly in more easterly areas, particularly in coastal Kenya and Tanzania, Malawi, and Madagascar. In 1997, a survey was conducted in Nigeria to determine the distribution of ACMV and its strains. Samples from 225 cassava plants showing mosaic symptoms were tested with ACMV monoclonal antibodies (MAbs) in triple antibody sandwich enzyme-linked immunosorbent assay (1). Three samples reacted strongly with MAbs that could detect both ACMV and EACMV. One of them did not react with ACMV-specific MAbs while the other two reacted weakly with such MAbs. With polymerase chain reaction (2), the presence of EACMV and a mixture of EACMV and ACMV in the respective samples was confirmed. These samples were collected from two villages: Ogbena in Kwara State and Akamkpa in Cross River State. Co-infection of some cassava varieties with ACMV and EACMV leads to severe symptoms. More importantly, a strain of mosaic geminivirus known as Uganda variant arose from recombination between the two viruses (2). This report provides evidence for the presence of EACMV in West Africa.

First Report of Tomato Yellow Leaf Curl Virus in Réunion Island.

In September 1997, stunting, reduced leaf size, leaf curling, and yellow margins were observed on tomato plants on a farm on the south coast of Réunion, a French island belonging to the Mascarenes archipelago. To our knowledge, these symptoms appeared to be characteristic of a tomato yellow leaf curl virus (TYLCV) infection. Diseased plants gave positive reactions with a triple antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA), using ADGEN antibodies specific for begomoviruses (1). The serological results were confirmed by polymerase chain reaction (PCR) with a pair of degenerate primers-MP16, 5'-CCTCTAGATAATATTAC(C/T)(G/T)(G/A)(A/T)(T/G)G(G/A)CC-3' and MP82, 5'-CGGAATTC(T/C)TGNAC(C/T)TT(G/A)CANGGNCC(T/C)T C(G/A)CA-3'-designed by Malla Padidam (ILTAB, San Diego, CA) to amplify a region of the A component of begomoviruses, between the intergenic conserved nonanucleotide sequence (TAATATTAC) and the first 5' quarter of the capsid protein gene. A 500-bp PCR product was obtained from a symptomatic plant but not from a healthy looking one. After cloning the PCR product in a pGEM-T Easy vector (Promega, Madison, WI) and sequencing it with plasmid-specific primers (SP6, T7), the sequence was compared with the sequences of the NCBI data base, with the use of BLAST. Nineteen sequences among those producing the highest scoring segment pairs were compared with each other and with the 500-bp PCR product from Réunion by the Clustal method of MegAlign (DNASTAR, London). The Réunion sequence (AJ010790) was at least 94% similar to sequences of TYLCV isolates from the Dominican Republic (AF024715), Cuba (AJ223505), and Israel (X15656, X76319 for the mild clone). Based on these results, it appeared that the analyzed tomato plant was infected by a geminivirus isolate belonging to the Israeli species of TYLCV. A preliminary survey was carried out from December 1997 to April 1998 in both outdoor and protected tomato crops. Infected plants were detected by TAS-ELISA in 52 of the 123 locations visited. Severe economic losses were observed: 14 locations with 60 to 100% yield reduction and 11 locations with 40 to 60% yield reduction. All the infected samples were collected in the leeward coast, which is the driest region of the island. Although Bemisia tabaci (Gennadius) has been recorded since 1938 in Réunion (2), it has been observed on tomato crops only since 1997 and population levels were low compared with those of Trialeurodes vaporariorum Westwood. During the first six months of 1998, B. tabaci was found on Euphorbia heterophylla L., Lantana camara L., Solanum melongena L., S. nigrum L., and Phaseolus vulgaris L. These host plants often occur near infected tomato crops.

Studies on a Nigerian isolate of banana streak badnavirus: II. Effect of intraplant variation on virus accumulation and reliability of diagnosis by ELISA

SummaryMonitoring of banana streak badnavirus (BSV) antigens and symptoms in naturally BSV‐infected plantain and banana (Musa spp.) plants showed a great variation in symptom expression, distribution and relative concentration of BSV between and within plants. Expression and distribution of symptoms was erratic within individual leaves as well as between different leaves of the same plant. The concentration of BSV antigens detected by triple antibody sandwich enzyme linked immunosorbent assay (TAS‐ELISA) varied in different plant parts including leaf lamina, midrib and pseudostem, roots and young ‘cigar’ leaf. The concentration of BSV antigens was high in symptomatic tissues but was low or below the limits of detection in most asymptomatic tissues. During ‘hot dry’ seasons when symptoms were not fully expressed, the concentration of BSV antigens in leaf tissues declined drastically, often below the detection limit of TAS‐ELISA. These results suggested that for more reliable detection of BSV antigens by TAS‐ELISA, it is advisable to index plants using composite tissue samples comprising as many leaves as possible for each plant and collected during cool and/or rainy seasons when symptom expression is generally severe.

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

SummaryA Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)‐ELISA, triple antibody sandwich (TAS)‐ELISA, antigen coated plate (ACP)‐ELISA, and protein‐A coated antibody sandwich (PAS)‐ELISA. TAS‐ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP‐ELISA and PAS‐ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV‐infected plants from asymptomatic plant samples than did TAS‐ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS‐ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV‐indexing of field samples, tissue sampling should be done during the rainy season when most BSV‐infected plants express severe symptoms.

Sexual differences in transmission and accumulation of tomato spotted wilt virus in its insect vector Frankliniella occidentalis (Thysanoptera : Thripidae)

Sexual differences in the transmission and accumulation of tomato spotted wilt virus (TSWV) were investigated in adults of the western flower thrips, Frankliniella occidentalis. Both TSWV transmission rates to petunia leaf disks and the triple antibody sandwich enzyme-1inked immunosorbent assay (TAS-ELISA)-positive rates showed no significant difference between the sexes. In ELISA-positive thrips, however, males transmitted TSWV to leaf disks with a significantly higher efficiency than females : 81% in males and 47% in females. Apparently, TSWV-transmitters had higher ELISA values than non-transmitters, and the average ELISA value of female transmitters was approximately 2 times that of male transmitters. These results suggest that TSWV may have a higher propagation rate in females than in males, whereas the efficiency of the virus transmission by males may be greater than that by females if they become viruliferous adults. Potential causes for these differences are discussed.