Immunocapture RT-PCR Research Articles

Genome analysis and detection of a Chilean isolate of Grapevine leafroll associated virus-3

The complete genome of the Chilean isolate Cl-766 of Grapevine leafroll-associated virus-3 (GLRaV-3) has been sequenced. This is the first genome sequence obtained from a GLRaV-3 isolate of the Southern hemisphere. The genomic RNA of 17,919 nucleotides contains 13 open reading frames (ORFs) with 5' and 3' untranslated regions (UTR) of 158 and 277 nucleotides, respectively. Comparison with NY1, the only isolate with complete genomic sequence available today, shows 97.6% nucleotide identity between the two isolates. Examination of the genome variability shows that most of the genetic diversity is concentrated in ORF1a. Three additional isolates from different geographic regions of Chile were partially sequenced as well, one which showed sequence divergence with respect to the other local and foreign isolates, indicative of different evolutionary constrains. Immunodetection systems were developed using monoclonal and polyclonal antibodies produced against the recombinant major coat protein of GLRaV-3, providing sensitive and specific detection using a triple antibody sandwich-enzyme linked immunosorbent assay (TAS-ELISA) and an immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) assay.

An Immunocapture RT-PCR Procedure Using Apple stem grooving virus Antibodies Facilitates Analysis of Citrus tatter leaf virus from the Original Meyer Lemon Host.

A magnetic bead-based immunocapture system using polyclonal antiserum against Apple stem grooving virus (ASGV) successfully facilitated polymerase chain reaction (PCR) amplification of sequences from three Citrus tatter leaf virus (CTLV) isolates originally isolated from the citrus host Meyer lemon. Primers designed from a pairwise alignment of genomic sequences of CTLV isolates from lily and from kumquat amplified two nonoverlapping genomic regions of 625 and 1,165 bp (approximately 28% of the CTLV genome) which were cloned and sequenced. Despite being propagated separately in the glasshouse for more than 40 years, the CTLV sequences from separate Meyer lemon sources were identical but had only approximately 80% nucleotide identity with the homologous regions of CTLV genomes of isolates from lily and kumquat. Neighbor-joining phylogenetic analysis indicated the CTLV isolates from Meyer lemon were distinct from but more closely related to CTLV from kumquat than from lily, and these CTLV sequences showed equivalent genetic distances from two ASGV isolates.

Molecular evidence for a new potyvirus species in yam (Dioscorea spp.) on the island of Guadeloupe

Prevalence of yam potyviruses in the French Caribbean islands of Guadeloupe was evaluated in 142 samples collected from yams showing symptoms of mosaic and mottling and belonging to Dioscorea rotundata (52 samples), D. alata (52 samples) and D. trifida (38 samples). Samples were screened by enzyme-linked immunosorbent assay (ELISA) with universal potyvirus monoclonal antibodies (AGDIA) and by immunocapture reverse-transcriptase polymerase chain reaction (IC-RT-PCR) for the specific detection of Yam mosaic virus (YMV) (Bousalem et al., 2000a) and Yam mild mosaic virus (YMMV) (Bousalem et al., 2003). Fifteen percent of the samples were positive in the broad-spectrum ELISA used but negative in both the specific IC-RT-PCR tests used, suggesting one or more other potyviruses might be present in the ELISA-positive samples. The highest proportion was in D. rotundata (15/52; 29%), followed by D. trifida (5/38; 13%), and D. alata (2/52; 4%). In an attempt to characterize those potential yam potyviruses, total RNA was extracted from one D. trifida sample (TGwadE2) and universal potyvirus degenerate primers (Colinet et al., 1994) were used to amplify the core and C-terminal region of the coat protein (CP) and the 3′untranslated region (3′UTR) which are commonly used as markers of genetic relatedness of potyviruses. Sequence information generated (GenBank AY821494) from the cloned fragment was compared with sequences of other potyviruses by a BLAST search and by pairwise sequence comparison carried out on 209 potyviruses sequences aligned by the Clustal algorithm from the MegAlign software of DNASTAR package. The partial amino acid (aa) sequence available for the CP (152 aa) had the highest similarity with Zucchini yellow mosaic virus (66% identity) whereas the 3′UTR (126 nucleotides) had the highest similarity with Potato virus Y (35% identity). Sequence comparisons of the CP (aa) and 3′UTR with YMMV isolates (Bousalem et al., 2003), YMV isolates (Bousalem et al., 2000b) and Japanese yam mosaic virus isolate (JYMV, AB016500) showed the highest similarity at the amino acid level (62·5% identity) with the JYMV CP and highest similarity of the 3′UTR with the YMMV (32% identity). According to molecular taxonomy, TGawdE2 isolate should be regarded as a distinct member of the genus Potyvirus (family Potyviridae) and was tentatively named Dioscorea mosaic virus.

P35 Changes in the system of proteolysis at the growth and metastasis of Lewis lung carcinoma upon development of cisplatin-resistance

The highest estimated prevalence of HCV infection has been reported in Egypt, nearly 12% mostly type 4. Currently, a commercial vaccine to protect this high risk population as well as global HCV infected patients is not available.In the present study, we aim at: (1) examining the viral binding capacities of purified monospecific polyclonal murine antibodies raised against genetically conserved viral protein sequences, i.e. synthetic peptides derived from those sequences located within envelope proteins and (2) assessment of immunogenic properties and safety parameters of those peptides individually and in a vaccine format in mice.Purified IgG Abs from immunized mice were used in immunocapture RT-PCR experiments to test viral neutralization by Abs raised against each of 4 peptides termed p35 (E1), p36 (E2), p37 (E2) and p38 (E2). Swiss mice were immunized with each of the 3 peptides (p35, p37 and p38) which generated neutralizing antibodies in immunocapture experiments. Antibody responses to corresponding peptides were determined using different routes of administration, different adjuvants, different doses and at different time points post-injection. To explore the dose range for future pharmacological studies, three doses namely 50 ng, 10 μg and 50 μg/25 gm mouse body weight were tested for biochemical and histopathological changes in several organs.Murine Abs against p35, p37 and p38 but not p36 showed HCV neutralization in immunocapture experiments. Subcutaneous injection of peptides elicited higher responses than i.m. and i.p. Immunization with Multiple Antigenic Peptide (MAP) form or coupled to Al PO4 elicited the highest Ab responses. Peptide doses of 50 ng/25 gm body weight or less were effective and safe, however dose assessment still requires further study. Histopathological changes were observed in animals that received doses ∼1000 times higher than the potential therapeutic dose.Exploration of humoral immunogenicity, neutralization capacity and safety suggested that the peptides presented herein are candidate vaccine components for further preclinical assessment.

Development of a multiplex immunocapture-RT-PCR for simultaneous detection of BMYV and BChV in plants and single aphids

A multiplex immunocapture-reverse transcription-polymerase chain reaction protocol (mIC-RT-PCR) was successfully developed to improve the detection of Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV) in plants and aphids in single and mixed infections. Viral particles from plant and aphid extracts were enriched by antibody-capture and lysed by heating to release the viral RNA. During the RT-PCR step, 5′ end sequences specific to each virus were amplified and the products analysed by gel electrophoresis; the PCR products corresponding to BMYV and BChV were 440 and 348 bp respectively. The test was evaluated on single aphids carrying BMYV, BChV or both viruses and the results demonstrated that the mIC-RT-PCR is specific and sensitive. Its sensitivity was found to be 10 5 times higher than the TAS-ELISA routinely used for detecting BMYV and BChV and 10 4 times better than RT-PCR when both viruses were present. Eliminating the antibody-capture step to simplify the technique did not affect the sensitivity of the test and a procedure using microtitre plates was developed to allow simultaneous processing of large numbers of samples.

Genetic variability within RNA2 of Grapevine fanleaf virus

The genetic variability of Grapevine fanleaf virus (GFLV) was assessed within RNA2 of nine isolates from Vitis vinifera cv. Volovnik in a vineyard in Slovenia by immunocapture (IC)–reverse transcription (RT)–polymerase chain reaction (PCR)–restriction length fragment polymorphism (RFLP), followed by cloning and sequencing. Four, one, and nine distinct StyI restrictotypes were identified in the 2AHP, 2BMP, and 2CCP genes, respectively, by IC–RT–PCR–RFLP. Each isolate had a specific StyI RFLP profile across the three RNA2-encoded genes. Sequence analysis of cloned RNA2 ORF amplicons obtained by IC–RT–PCR showed mixed infection in four of the nine isolates and a slightly higher nucleotide variability in the 2AHP and 2CCP genes relative to the 2BMP gene. Also, gene 2AHP, unlike genes 2BMP and 2CCP, had a variable size (765–774 nucleotides) and high amino acid diversity (up to 15%). In addition, a recombination event was identified at nucleotide position 220–225 of gene 2AHP in three of the nine isolates. No clear association was apparent between symptomatology and restrictotype composition, phylogenetic clustering, or occurrence of recombination. This study provides new insights into the genetic diversity of GFLV.

Beet necrotic yellow vein virus (benyvirus)

<i>Beet necrotic yellow vein virus</i> (<i>benyvirus</i>)

Development of Single-tube Multiplex Immunocapture RT-PCR Assay for Simultaneous Detection of Two Pepper Tobamoviruses

An immunocapture reverse transcription-polymerase chain reaction (IC/RT-PCR) was developed for simultaneous detection of two pepper-infecting RNA viruses, Pepper mud mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV). The assay could be performed in a single tube for simultaneous and sensitive detection of these tobamoviruses. This detection system revealed thousand-fold increase in detection sensitivity compare to ELISA. This method could save time and reagent cost compare to common RT-PCR which needs several reactions and several procedures of viral RNA extractions for the same number of samples.

First Report in Argentina of Plum pox virus Causing Sharka Disease in Prunus.

Sharka disease, caused by Plum pox virus (PPV), is probably the most important disease of stone fruits crops worldwide because of tremendous yield losses from infected trees (1). During November 2004, symptoms resembling sharka disease were observed in a plum and apricot orchard consisting of 5,000 trees in Pocito, San Juan Province, Argentina. Apricot leaves showed chlorotic spots while plum leaves showed chlorotic rings, spots, and irregular edges. Plum fruits were deformed and much smaller than those from symptomless trees. Samples collected from 70 symptomatic trees were tested using double-antibody sandwich enzyme-linked immunosorbent assays with a polyclonal antiserum anti-PPV from BIOREBA (Reinach BL1, Switzerland), and immunosorbent electron microscopy with a polyclonal antiserum from our laboratory made against a recombinant PPV capsid protein (CP). The samples were also tested using double-antibody sandwich indirect enzyme-linked immunosorbent assay using the REAL kit (Durviz, Valencia, Spain) with two different monoclonal antibodies including Mab 5b that recognizes all strains of PPV and Mab 4DG5 that is specific for PPV strain D. Samples were positive with both antibodies in 80% of the cases. Leaf extracts from symptomatic plum samples were also analyzed by immuno-capture reverse-transcription polymerase chain reaction. A 1,209-bp fragment was amplified with specific primers that anneal at the 5' end of the coat protein coding region and the viral 3' end poly A tail. The amplified fragment was cloned and the nucleotide sequence was determined for two of the resulting clones (Gen-Bank Accession Nos. DQ299537 and DQ299538). The sequences were 98% identical with the PPV-strain D from the United States (GenBank Accession No. AF360579) and Germany (GenBank Accession No. X81081). The restriction sites for AluI and RsaI, previously described (2) as typical for the PPV-D strain, were present in the expected positions. To our knowledge, this is the first report of PPV-D in Argentina. Reference: (1) M. Németh. Virus, Mycoplasma, and Rickettsia Disease of Fruit Trees. Martinus Nijhoff Publishers, Dordrecht, the Netherlands, 1986. (2) T. Wetzel et al. J. Virol. Methods 33:355, 1991.

First Report of Pinaceae in Georgia Naturally Infected with Tomato spotted wilt virus

In October 2004, three pine tree seedlings included in an ongoing survey of annual weeds elicited positive reactions for Tomato spotted wilt virus (TSWV [family Bunyaviridae, genus Tospovirus]) using double assay sandwich-enzyme linked immunosorbent assay (DAS-ELISA) (Agdia Inc. Elkhart, IN). All the seedlings appeared healthy with no visible adverse effects from the virus. Over the next 12 months, an additional 1,326 samples of various pine species representing different growth stages were screened for TSWV. Samples were comprised of local populations of Pinus elliottii Engelm., P. taeda L., and P. palustris P. Mill., with the majority (n = 886) of samples being seedlings collected from southern Georgia. Along with the seedlings, needles, stem sections, and roots from saplings, as well as needles from mature trees, were screened for the virus. Of the trees sampled, 5.35% (n = 71) tested positive for TSWV, and of the seedlings 6.77% (n = 60) tested positive. The DAS-ELISA positive threshold was obtained using a figure of three times the average plus two standard deviations of healthy negative pine tissue control absorbance readings at 405 nm. A number of saplings testing positive (n = 6) were marked for further evaluation, and the needles from these saplings consistently screened positive for TSWV in subsequent testing. Furthermore, several samples were processed in modified burlese funnels to detect the possible presence of thrips. No thrips were ever identified in any of the burlese funnel collections. Different tissue types (needles, roots, stem sections, and reproductive organs) were screened, but the virus was only detected in needles. This suggests that local infections are only at feeding sites of viruliferous thrips. The known thrips vectors for TSWV are not considered to be pine feeders, and there is no indication that pine trees are a reproductive reservoir for any local thrips species. However, pine-feeding thrips may also feed on known weed hosts, thus pines could be a perennial reservoir. Mechanical inoculations from surface-sterilized infected pine needles onto known TSWV indicator plants (Nicotiana glutinosa L., N. benthamiana, and Emilia sonchifolia L. (DC)) were inconsistent. Successful transmission occurred 24% of the time. To further verify serological data, total RNA extracts of pine sap were purified and subjected to immunocapture-reverse transcriptase-polymerase chain reaction (IC-RT-PCR) using primers specific to the nucleocapsid gene of TSWV (1). IC-RT-PCR was used due to the inability to obtain useful total RNA from the pine tissues. This may be due to a secondary metabolite interfering with the total RNA extraction protocol. The IC-RT-PCR products were analyzed with electrophoresis using 0.01% ethidium bromide stain in a 0.8% agarose gel. Amplicons produced at the expected size (bp = 774) were considered positive for TSWV. Several were sequenced and were consistent with known, local TSWV isolates. There is no indication that TSWV is detrimental to pine trees, but considering the widespread distribution of the genus Pinus and the potential of serving as a reservoir of TSWV, it may play a role in the overall epidemiology of TSWV in southern Georgia. Reference: (1) R. K. Jain et al. Plant Dis. 82:900, 1998.

Cloning and sequence analysis of coat protein gene for characterization of sugarcane mosaic virus isolated from sugarcane and maize in Thailand

Three isolates of sugarcane mosaic virus (SCMV) from sugarcane and maize in Thailand, were selected and used as viral sources for coat protein (CP) genes cloning by immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR). The CP gene sequences and their deduced amino acids were determined. The sequences of three cloned CP genes, UT6TH-sgc and UD7TH-sgc from sugarcane, and SBC2TH-mz from maize were compared with other previously reported sequences of SCMV-CP gene and their phylogeny were studied. The analysis revealed that Thai SCMV CP gene contained 942 nucleotides encoded for coat protein MW of 33.65 kDa. The nucleotide sequence identity among cloned CPs from three isolates were 98-99% to each other. The N-terminus of the Thai SCMV CP contained a distinctive sequence, especially at nucleotide positions 28-46 of the N-terminal variable 70 amino acid residues which discriminated Thai SCMV from all previously reported SCMV isolates. Thai SCMV were placed in a separate branch of SCMV-MDB cluster which is closely related to most of SCMV isolates from maize, and distinct from the other cluster containing sugarcane-SCMV strains.

Utility of recombinant integrin αvβ6 as a capture reagent in immunoassays for the diagnosis of foot-and-mouth disease

Recombinant integrin αvβ6 was evaluated as a capture ligand in a sandwich ELISA for the detection and serotyping of foot-and-mouth disease (FMD) virus. Our routinely applied method employs seven serotype-specific rabbit polyclonal antibodies as capture ligands and seven serotype-specific guinea pig polyclonal antibodies as detecting reagents. The recombinant integrin bound FMD virus of all seven serotypes but not that of another vesicular disease, swine vesicular disease (SVD). Considerable heterotypic cross-reactions were evident when using the integrin capture ligand in combination with guinea pig detecting antibodies but totally type-specific reactions resulted when serotype-specific monoclonal antibodies (mabs) were used instead of the guinea pig reagents. The specificity of reaction of the integrin capture/mab detector combination was superior to that of our routinely employed rabbit/guinea pig ELISA and offers an improvement for test interpretation. As a universal trapping reagent for all FMD virus serotypes the αvβ6 recombinant protein also has the potential for application in other test procedures for viral identification (e.g. pen-side chromatographic strip-tests, biosensors, immunocapture RT-PCR, antigenic characterization procedures and monoclonal antibody profiling of emerging field virus strains) and in antibody detection assays employed for the diagnosis of FMD.

First Report of Citrus tristeza virus in the State Union of Serbia and Montenegro.

Citrus production in the State Union of Serbia and Montenegro has a strategic importance to the agricultural sector. Approximately 400,000 trees are now grown in the major citrus producing region, which is the Montenegrin Coastal Region. Satsuma mandarins and lemons grafted on Poncirus trifoliata are the most cultivated varieties. In December 2003, eight samples taken from the coastal region close to the towns of Bar and Ulcinj were analyzed using enzyme-linked immunosorbent assay (ELISA) with SP7 antibodies produced at Universidade do Algarve, Portugal (3). Further analysis was done using immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) targeting the entire coat protein (CP) gene (forward primer CTV1: 5'- ATGGACGACGAAACAAAGAA-3' and reverse primer CTV10: 5'-ATCAACGTGTGTTGAATTTCC-3'). Using both techniques, seven of eight samples analyzed were found to be infected by Citrus tristeza virus (CTV), including samples from five trees that exhibited chlorosis, gummosis, and fruit deformation, and two trees that were symptomless. When analyzed using agarose gel electrophoresis, PCR products from the positive samples consisted of a single amplicon of the expected size for the CP gene compared with a positive control. The PCR products of two samples were TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells and the CP inserts were analyzed using single-strand conformation polymorphism (SSCP) and DNA sequencing. In both cases, the SSCP analysis of several clones showed a variety of different patterns, suggesting the occurrence of infections with a mixture of genomic variants. Sequence analysis of different variants showed a CP gene with 669 nucleotides having greater than 90% nucleotide identity to most CTV CP gene sequences available in GenBank. A genomic variant (GenBank Accession No. AY764154) was closely related (98.5% nucleotide identity) to the T30 mild strain from Florida (GenBank Accession No. AF260651). However, other sequences obtained showed only 93% nucleotide identity with this variant and were closely related to other CP gene sequences obtained from Croatian isolates. A previous report (1) refers to the existence of CTV-infected Satsuma plants illegally introduced in Italy from the former Yugoslavia. The presence of CTV in the former Yugoslavia was later confirmed (2) but in a region that became part of the Croatian Republic. To our knowledge, this is the first report of CTV in the State Union of Serbia and Montenegro, although a relationship with Croatian isolates cannot be excluded. Although a very small number of samples were analyzed in this study, CTV appears to be very common in the Satsuma orchards. This could be due to the traditional use of the trifoliate rootstock that prevents the appearance of tristeza decline, thus enabling the unnoticed propagation of infected material. Because the kind of symptoms observed in five trees are not typical of CTV and two infected trees were symptomless, the virus is probably not responsible for the symptoms observed in the field.

Occurrence of Stem-Pitting Strains of Citrus tristeza virus in Croatia.

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30'N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field-infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kaštela, Split, Metković (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33°C. Eight months later, the bark of terminal twigs was peeled off, and the wood was examined for the occurrence of pits. Typical tristeza stem-pitting (SP) was observed in four isolates originating from cvs. Fukumoto navel, Washington navel, and Ichimaru Satsuma and C. wilsonii. The bark from the infected sources was analyzed using immunocapture reverse transcription-polymerase chain reaction (RT-PCR) with primers CTV1 and CTV10 (1), targeting the whole coat protein (CP) gene. The PCR products of the expected size (669 nucleotides) were obtained and TA cloned (pGEM-T Easy Vector; Promega, Madison, WI) in E. coli cells. Thirty-two clones harboring the CTV CP gene were sequenced. Two of the SP isolates contained four genomic variants that differed an average of 2.0% from the severe SwO SP strains SY568 and Nuaga (4) from California and Japan, respectively. The other two SP isolates contained four variants that differed as little as 1.6% from the severe SwO SP from India, CTV-Puna, and CTV-Bangalore (2). The net average distance between these two clusters of sequences is 5.2%. One sequence from each of the four SP isolates was deposited in GenBank (Accession Nos. AY791841 to AY791844). These findings were confirmed by direct observation of SP symptoms in a Satsuma orchard in the Neretva Valley during the spring of 2004. No other conspicuous symptoms that could be attributed to CTV were observed in the field. Most Satsumas were introduced to the Neretva Region from Japan between 1964 and 1984. Together with the fact that the related Nuaga strain was also isolated from Satsumas in Japan (4), our results suggest that SwO SP strains were introduced into Croatia at the same time and have been spreading for several decades. It has been generally believed that this kind of CTV strains either do not exist in the Mediterranean basin or, when found (e.g., Spain), are immediately eradicated. The findings reported here suggest that the epidemiological scenario for the Mediterranean Basin requires revision.

Use of crude sap for one-step RT-PCR-based assays of Bean yellow mosaic virus and the utility of this protocol for various plant?virus combinations

One-step reverse transcription-polymerase chain reaction (one-step RT-PCR) was successfully adapted to detect Bean yellow mosaic virus in the crude sap of infected dwarf gentian plants (Gentiana scabra). The virus was detected directly from crude sap at dilutions up to 106-fold (W/V), which is 100 times more sensitive than indirect enzyme-linked immunosorbent assay or bioassay after mechanical inoculation, and equally as sensitive as immunocapture RT-PCR. This one-step RT-PCR method using crude sap was also useful for detecting 16 virus species from ten genera with isometric, filamentous, and rod-shaped particles from 32 plant species in 15 families.

Discrimination between tobamoviruses and their pathotypes for L-gene-mediated resistance in green pepper (Capsicum annuum L.) by reverse transcription-polymerase chain reaction

A new method to discriminate between tobamoviruses and their pathotypes that infect green pepper (Capsicum annuum L.) was developed using reverse transcription-polymerase chain reaction (RT-PCR). The P0 pathotype (Tobacco mosaic virus, Tomato mosaic virus, and Tobacco mild green mosaic virus) and the P1 pathotype (Paprika mild mottle virus) were distinguished by RT-PCR using primers specific to each pathotype. However, the P1,2 and P1,2,3 pathotypes of Pepper mild mottle virus (PMMoV) could not be distinguished from each other using this procedure. The P1,2 and P1,2,3 pathotypes were differentiated by RT-nested PCR, in which a DNA fragment was first produced by RT-PCR using primers containing conserved sequences of PMMoV. The product was then used as the DNA template in a second PCR using primers specific to each pathotype. An immunocapture (IC) RT-PCR method was developed based on results from this study, which facilitated detection of tobamoviruses in pepper plants, seeds, and field soils and allowed the identification of their pathotypes.

Comparison of TAS-ELISA, dot and tissue blot, ISEM and immunocapture RT-PCR assays for the detection of Yam mosaic virus in yam tissues

The performance of five techniques for detecting Yam mosaic virus (YMV), genus Potyvirus, in Dioscorea rotundata was evaluated. Triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA), dot (DBI) and direct tissue blot immunoassays (DTBI), immunosorbent electron microscopy (ISEM) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) detected YMV in stems, leaf petioles, leaves and tubers. Younger leaves were more suitable for the detection of YMV using TAS-ELISA and DBI. DBI was as sensitive as TAS-ELISA when the detecting antibody was YMV-M24. The IC-RT-PCR technique was five times more sensitive than the purely protein-based serological detection methods. TAS-ELISA, DTBI and symptomatology were more suitable in detecting YMV in yam tubers than IC-RT-PCR and ISEM because of their sensitivity and reliability. The DTBI assays were very useful for the detection of YMV in yam tubers and tuber sprouts.

Detection and subgrouping of Cucumber mosaic virus isolates by TAS-ELISA and immunocapture RT-PCR

Eight mouse hybridoma cell lines secreting monoclonal antibodies (MAbs) against Cucumber mosaic virus (CMV) were produced. Analysis of the specificities of the MAbs against CMV isolates by triple antibody sandwich (TAS)-ELISA demonstrated that four MAbs were specific for subgroup I (S-I) isolates and two for subgroup II (S-II) isolates, whereas another two MAbs could detect both S-I and S-II isolates. TAS-ELISA and immunocapture RT-PCR (IC-RT-PCR) methods were then established for reliable and efficient detection and subgrouping of CMV isolates using the produced MAbs. When 197 field samples collected from six provinces in China were tested by TAS-ELISA, 130 samples were found to be infected by CMV. Among them, 121 samples were infected by S-I isolates (93.1%) and another nine samples by S-II isolates (6.9%). In IC-RT-PCR using the MAbs and specific primers in the region of the coat protein ( CP) gene, samples shown to contain S-I isolates by TAS-ELISA gave one specific band about 500 nucleotides in length, whereas samples containing S-II isolates produced a single band with the length of approximately 600 nucleotides. The validity and reliability of the results of TAS-ELISA and IC-RT-PCR was confirmed by sequencing and phylogenetic analysis of nearly full-length CP genes of the isolates.

DETECTION AND MOLECULAR CHARACTERIZATION OF GRAPEVINE FANLEAF VIRUS AND GRAPEVINE LEAFROLL-ASSOCIATED VIRUS 3 IN JORDAN

Grapevine fanleaf virus (GFLV) and Grapevine leafroll-associated virus 3 (GLRaV-3) have been detected by Double-Antibody Sandwich (DAS)-ELISA in six locations where grapevines are commonly grown in Jordan. Using pairs of specific primers, fragments of the RNA-dependent RNA polymerase (RdRp) gene of GLRaV-3 and the coat protein (CP) gene of GFLV were amplified from symptomatic grapevine tissues by Immunocapture- Reverse Transcriptase-Polymerase Chain Reaction (IC-RT-PCR). After cloning and sequencing, sequences of the amplified fragments were deposited in Genbank. Alignment analysis revealed that the amplified CP gene fragment of the Jordanian isolate of GFLV (GFLV-JOR) shared sequence identity between 82 and 88% with 11 GFLV isolates from different parts of the world. High sequence identity (95-99%) was observed between the amplified fragment of RdRp gene of GLRaV-3 from Jordan (GLRaV-3-JOR) and isolates from Brazil, USA, Czech Republic and China.

First Report of Vidalia Onion (Allium cepa) Naturally Infected with Tomato spotted wilt virus and Iris yellow spot virus (Family Bunyaviridae, Genus Tospovirus) in Georgia.

Vidalia onion is an important crop in Georgia's agriculture with worldwide recognition as a specialty vegetable. Vidalia onions are shortday, Granex-type sweet onions grown within a specific area of southeastern Georgia. Tomato spotted wilt virus (TSWV) has been endemic to Georgia crops for the past decade, but has gone undetected in Vidalia onions. Tobacco thrips (Frankliniella fusca) and Western flower thrips (Frankliniella occidentalis) are the primary vectors for TSWV in this region, and a number of plant species serve as reproductive reservoirs for the vector or virus. Iris yellow spot virus (IYSV), an emerging tospovirus that is potentially a devastating pathogen of onion, has been reported in many locations in the western United States (2,4). Thrips tabaci is the known vector for IYSV, but it is unknown if noncrop plants play a role in its epidemiology in Georgia. During October 2003, a small (n = 12) sampling of onions with chlorosis and dieback of unknown etiology from the Vidalia region was screened for a variety of viruses, and TSWV and IYSV infections were serologically detected. Since that time, leaf and bulb tissues from 4,424 onion samples were screened for TSWV and IYSV using double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) with commercial kits (Agdia Inc., Elkhart, IN). Samples were collected from 53 locations in the Vidalia region during the growing season between November 2003 and March 2004. Plants exhibiting stress, such as tip dieback, necrotic lesions, chlorosis or environmental damage were selected. Of these, 306 were positive for TSWV and 396 were positive for IYSV using positive threshold absorbance of three times the average plus two standard deviations of healthy negative onion controls. Positive serological findings of the onion tissues were verified by immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) for TSWV (3) and RT-PCR for IYSV (1). In both instances, a region of the viral nucleocapsid (N) gene was amplified. The PCR products were analyzed with gel electrophoresis with an ethidium bromide stain in 0.8% agarose. Eighty-six percent (n = 263) of the TSWV ELISA-positive samples exhibited the expected 774-bp product and 55 percent (n = 217) of the IYSV ELISA-positive samples exhibited the expected 962-bp product. The reduced success of the IYSV verification could be attributed to the age and deteriorated condition of the samples at the time of amplification. Thrips tabaci were obtained from onion seedbeds and cull piles within the early sampling (n = 84) and screened for TSWV by the use of an indirect-ELISA to the nonstructural (NSs) protein of TSWV. Of the thrips sampled, 25 were positive in ELISA. While the incidence of IYSV and TSWV in the Vidalia onion crop has been documented, more research is needed to illuminate their potential danger to Vidalia onions.