Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

ABSTRACT We cloned the sequence encoding murine interleukin-4 (mIL-4), including the secretory signal, into the genome of CVB3/0, an artificially attenuated strain of coxsackievirus B3, at the junction of the capsid protein 1D and the viral protease 2Apro. Two strains of chimeric CVB3 were constructed using, in one case, identical sequences to encode 2Apro cleavage sites (CVB3/0-mIL4/47) on either side of the inserted coding sequence and, in the other case, nonidentical sequences that varied at the nucleotide level without changing the amino acid sequences (CVB3-PL2-mIL4/46). Transfection of HeLa cells yielded progeny viruses that replicated with rates similar to that of the parental CVB3/0 strain, although yields of mIL-4-expressing strains were approximately 10-fold lower than those of the parental virus. Western blot analysis of viral proteins isolated from HeLa cells inoculated with either strain of chimeric virus demonstrated that the chimeric viruses synthesized capsid protein 1D at approximately twofold-higher levels than the parental virus. mIL-4 protein was detected by enzyme-linked immunosorbent assay (ELISA) in HeLa cells inoculated with either strain of chimeric virus. Lysates of HeLa cells inoculated with either chimeric virus induced the proliferation of the mIL-4-requiring murine MC-9 cell line, demonstrating biological activity of the CVB3-expressed mIL-4. Reverse transcription (RT)-PCR analysis of viral RNA derived from sequential passaging of CVB3/0-mIL4/47 in HeLa cells demonstrated deletion of the mIL-4 coding sequence occurring by the fourth passage, while similar analysis of CVB3-PL2-mIL4/46 RNA demonstrated detection of the mIL-4 coding sequence in the virus population through 10 generations in HeLa cells. mIL-4 protein levels determined by ELISA were consistent with the stability and loss data determined by RT-PCR analysis of the passaged viral genomes. Studies of insert stability of CVB3-PL2-mIL4/46 during replication in mice showed the presence of the viral mIL-4 insert in pancreas, heart, and liver at 14 days postinfection. Comparison of the murine antibody responses to CVB3-PL2-mIL4/46 and the parental CVB3/0 strain demonstrated an increased level of CVB3-binding serum immunoglobulin G1 in mice inoculated with CVB3-PL2-mIL4/46.

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First Report of Tomato spotted wilt virus in Blackberry Lily in North America

Plant Disease ◽

10.1094/pdis.2003.87.1.102c ◽

2003 ◽

Vol 87 (1) ◽

pp. 102-102 ◽

Cited By ~ 3

Author(s):

S. Adkins ◽

L. Breman ◽

C. A. Baker ◽

S. Wilson

Keyword(s):

North America ◽

Tomato Spotted Wilt Virus ◽

Amino Acid Sequences ◽

South Florida ◽

N Gene ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Wilt Virus

Blackberry lily (Belamcanda chinensis (L.) DC.) is an herbaceous perennial in the Iridaceae characterized by purple-spotted orange flowers followed by persistent clusters of black fruit. In July 2002, virus-like symptoms including chlorotic ringspots and ring patterns were observed on blackberry lily leaves on 2 of 10 plants in a south Florida ornamental demonstration garden. Inclusion body morphology suggested the presence of a Tospovirus. Tomato spotted wilt virus (TSWV) was specifically identified by serological testing using enzyme-linked immunosorbent assay (Agdia, Elkhart, IN). Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579 base pair region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. Since these 2-year-old plants were grown on-site from seed, they were likely inoculated by thrips from a nearby source. Together with a previous observation of TSWV in north Florida nursery stock (L. Breman, unpublished), this represents, to our knowledge, the first report of TSWV infection of blackberry lily in North America although TSWV was observed in plants of this species in Japan 25 years ago (2). References: (1) S. Adkins, and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) T. Yamamoto and K.-I. Ohata. Bull. Shikoku Agric. Exp. Stn. 30:39, 1977.

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Detection of Border Disease Virus in Fetuses, Stillbirths, and Newborn Lambs from Natural and Experimental Infections

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870902100304 ◽

2009 ◽

Vol 21 (3) ◽

pp. 331-337 ◽

Cited By ~ 6

Author(s):

Ana L. García-Pérez ◽

Esmeralda Minguijón ◽

Jesús F. Barandika ◽

Gorka Aduriz ◽

Inés Povedano ◽

...

Keyword(s):

Disease Virus ◽

Experimental Infection ◽

Clinical Symptoms ◽

Pcr Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Border Disease Virus ◽

Rt Pcr ◽

Genotype 4 ◽

Antigen Elisa ◽

Border Disease

The purpose of the present study was to evaluate the use of enzyme-linked immunosorbent assay (ELISA) antigen detection in blood or fetal fluids and reverse transcription polymerase chain reaction (RT-PCR) amplification in tissues for routine laboratory diagnosis of Border disease virus (BDV) infection. Samples from 67 fetuses, 6 stillbirths, and 11 lambs from 25 commercial flocks with suspicion of BDV abortion and 3 fetuses, 7 stillbirths, and 15 lambs obtained from an experimental infection with a local isolate (BDV genotype 4) were investigated. Presence of BDV was detected by RT-PCR in 7.9% of fetuses, 50% of stillbirths, and 50% of lambs from the commercial flocks analyzed, corresponding to 8 of the 25 farms (32%). A similar percentage of the lambs and stillbirths from the experimental infection were positive by RT-PCR of tissue samples (54.5%), and the highest positivity was detected in lymph node, thyroid gland, and kidney. The current study revealed that RT-PCR analysis of stillbirths and lambs with clinical symptoms is more suitable than the analysis of fetuses to confirm the presence of BDV in a flock. Pestiviral antigen was detected by antigen ELISA in a high proportion of fetuses (24/58) and stillbirths (3/4) from commercial flocks, but in lambs, the presence of colostral antibodies masked the detection of the antigen by ELISA. Nevertheless, in lambs from the experimental infection that were not fed colostrum, antigen ELISA was less efficient than RT-PCR in detecting viral presence in stillbirths and lambs. Antigen ELISA is therefore recommended for fetuses with advanced autolysis that can adversely affect RNA integrity.

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Characteristics of a cluster of xylanase genes inFibrobacter succinogenesS85

Canadian Journal of Microbiology ◽

10.1139/w03-024 ◽

2003 ◽

Vol 49 (3) ◽

pp. 171-180 ◽

Cited By ~ 20

Author(s):

Hyun S Jun ◽

Jong K Ha ◽

Laercio M Malburg, Jr. ◽

Ann M Verrinder Gibbins ◽

Cecil W Forsberg

Keyword(s):

Catalytic Domain ◽

Specific Activity ◽

Glycosyl Hydrolase ◽

Amino Acid Sequences ◽

Pcr Analysis ◽

Carbohydrate Binding ◽

Fibrobacter Succinogenes ◽

Rt Pcr ◽

Ph Optimum ◽

Oat Spelt

Xylanase genes xyn10D, xyn10E, and xyn10B, located sequentially on the Fibrobacter succinogenes S85 chromosome, were separately cloned and their properties characterized. Analysis of the sequences documented that xylanases Xyn10D, Xyn10E, and Xyn10B each consist of an N-terminal catalytic domain (glycosyl hydrolase family 10) and a C-terminal carbohydrate-binding module (CBM, family 6) connected by proline-rich linker sequences. The amino acid sequences exhibited similarities of between 53 and 60%. The xyn10D, xyn10E, and truncated xyn10BΔCBM were expressed in Escherichia coli and purified to homogeneity. The purified Xyn10D, Xyn10E, and Xyn10BΔCBM exhibited the same temperature optimum (40°C) and pH optimum (6.5) and the highest specific activity against arabinoxylan, oat spelt xylan, and birchwood xylan, respectively. Xyn10D exhibited an affinity for cellulose and xylan with 47 and 33% binding, respectively, while the truncated Xyn10DΔCBM did not bind to the substrates. The main hydrolysis products of the three xylanases acting on oat spelt xylan and arabinoxylan were xylose and xylobiose. RT-PCR analysis showed that the three genes were co-transcribed as a single transcript. Western immunoblot analysis revealed that the three xylanases were expressed at a very low level by F. succinogenes grown on either glucose or cellulose as the source of carbohydrate.Key words: Fibrobacter succinogenes S85, xylan, xylanase, clustered genes, RT-PCR.

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Analysis of Amino Acid Sequence Variations and Immunoglobulin E-Binding Epitopes of German Cockroach Tropomyosin

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.5.874-878.2004 ◽

2004 ◽

Vol 11 (5) ◽

pp. 874-878 ◽

Cited By ~ 7

Author(s):

Kyoung Yong Jeong ◽

Jongweon Lee ◽

In-Yong Lee ◽

Han-Il Ree ◽

Chein-Soo Hong ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Single Molecule ◽

Immunoglobulin E ◽

German Cockroach ◽

Amino Acid Sequences ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Ige Binding ◽

Sequence Variations

ABSTRACT The allergenicities of tropomyosins from different organisms have been reported to vary. The cDNA encoding German cockroach tropomyosin (Bla g 7) was isolated, expressed, and characterized previously. In the present study, the amino acid sequence variations in German cockroach tropomyosin were analyzed in order to investigate its influence on allergenicity. We also undertook the identification of immunodominant peptides containing immunoglobulin E (IgE) epitopes which may facilitate the development of diagnostic and immunotherapeutic strategies based on the recombinant proteins. Two-dimensional gel electrophoresis and immunoblot analysis with mouse anti-recombinant German cockroach tropomyosin serum was performed to investigate the isoforms at the protein level. Reverse transcriptase PCR (RT-PCR) was applied to examine the sequence diversity. Eleven different variants of the deduced amino acid sequences were identified by RT-PCR. German cockroach tropomyosin has only minor sequence variations that did not seem to affect its allergenicity significantly. These results support the molecular basis underlying the cross-reactivities of arthropod tropomyosins. Recombinant fragments were also generated by PCR, and IgE-binding epitopes were assessed by enzyme-linked immunosorbent assay. Sera from seven patients revealed heterogeneous IgE-binding responses. This study demonstrates multiple IgE-binding epitope regions in a single molecule, suggesting that full-length tropomyosin should be used for the development of diagnostic and therapeutic reagents.

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The Cryptococcus neoformans GeneDHA1 Encodes an Antigen That Elicits a Delayed-Type Hypersensitivity Reaction in Immune Mice

Infection and Immunity ◽

10.1128/iai.68.11.6196-6201.2000 ◽

2000 ◽

Vol 68 (11) ◽

pp. 6196-6201 ◽

Cited By ~ 26

Author(s):

M. Alejandra Mandel ◽

Greg G. Grace ◽

Kris I. Orsborn ◽

Fredda Schafer ◽

Juneann W. Murphy ◽

...

Keyword(s):

Amino Acid ◽

Cryptococcus Neoformans ◽

Culture Filtrate ◽

Genomic Library ◽

Amino Acid Sequences ◽

Full Length ◽

Delayed Type Hypersensitivity ◽

Pcr Analysis ◽

Rt Pcr ◽

Fruiting Body Formation

ABSTRACT When mice are vaccinated with a culture filtrate fromCryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-lengthDHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5′ rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes(shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded byDHA1. Recombinant DHA1 protein expressed inEscherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.

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Gene expression of Na+/H+ exchanger in zebrafish H+-ATPase-rich cells during acclimation to low-Na+ and acidic environments

AJP Cell Physiology ◽

10.1152/ajpcell.00358.2007 ◽

2007 ◽

Vol 293 (6) ◽

pp. C1814-C1823 ◽

Cited By ~ 115

Author(s):

Jia-Jiun Yan ◽

Ming-Yi Chou ◽

Toyoji Kaneko ◽

Pung-Pung Hwang

Keyword(s):

Amino Acid Sequences ◽

Pcr Analysis ◽

Rt Pcr ◽

Acid Base ◽

Phylogenetic Tree Analysis ◽

Major Player ◽

C Subunit ◽

Proximal Tubular ◽

Acidic Environments

In mammalian nephrons, most of the Na+ and HCO3− is reabsorbed by proximal tubular cells in which the Na+/H+ exchanger 3 (NHE3) is the major player. The roles of NHEs in Na+ uptake/acid-base regulation in freshwater (FW) fish gills are still being debated. In the present study, functional genomic approaches were used to clone and sequence the full-length cDNAs of the nhe family from zebrafish ( Danio rerio). A phylogenetic tree analysis of the deduced amino acid sequences showed that zNHE1–8 are homologous to their mammalian counterparts. By RT-PCR analysis and double/triple in situ hybridization/immunocytochemistry, only zebrafish NHE3b was expressed in zebrafish gills and was colocalized with V-H+-ATPase but not with Na+-K+-ATPase, indicating that H+-ATPase-rich (HR) cells specifically express NHE3b. A subsequent quantitative RT-PCR analysis demonstrated that acclimation to low-Na+ FW caused upregulation and downregulation of the expressions of znhe3b and zatp6v0c (H+-ATPase C-subunit), respectively, in gill HR cells, whereas acclimation to acidic FW showed reversed effects on the expressions of these two genes. In conclusion, both NHE3b and H+-ATPase are probably involved in Na+ uptake/acid-base regulation in zebrafish gills, like mammalian kidneys, but the partitioning of these two transporters may be differentially regulated depending on the environmental situation in which fish are acclimatized.

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Cloning and Characterization of the Zebrafish mll Ortholog.

Blood ◽

10.1182/blood.v110.11.1249.1249 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1249-1249

Author(s):

Blaine W. Robinson ◽

Giuseppe Germano ◽

Yuanquan Song ◽

Rita J. Balice-Gordon ◽

Carolyn A. Felix

Keyword(s):

Amino Acid ◽

Blot Analysis ◽

Temporal Pattern ◽

Southern Blot Analysis ◽

Early Embryogenesis ◽

Amino Acid Sequences ◽

Pcr Analysis ◽

Adult Zebrafish ◽

Wild Type ◽

Rt Pcr

Abstract Introduction: Zebrafish enable studies of early embryogenesis and hematopoiesis like no other animal models. Many zebrafish orthologs of hematopoietic genes have been identified, and zebrafish models of leukemia are emerging but the zebrafish ortholog of MLL, a critical oncogene disrupted by leukemogenic translocations, has not yet been studied. We cloned the complete zmll cDNA and characterized its temporal expression as a framework for further studies of MLL where current knowledge is still incomplete. Methods: Bioinformatic tools were employed to interrogate the existence and relationship of a zmll ortholog and synteny with human MLL. Degenerate RT-PCR was used to determine whether MLL amino acid sequences in domains highly conserved across species could identify the orthologous zebrafish transcript. Cross-species Southern blot analysis was performed to determine if a predicted zmll gene from restriction map simulations of a projected genomic sequence could be detected with a human cDNA probe for the MLL breakpoint cluster region (bcr). The full-length zmll cDNA was obtained using a combination of 5′ RACE PCR, long-range and conventional PCR. The corresponding protein was analyzed in a phylogram tree. The temporal pattern of zmll RNA expression was examined using quantitative (Q) RT-PCR. Results: Bioinformatic analysis using the human MLL protein as the reference sequence identified two putative “similar to MLL proteins” and predicted two proximal transcript sequences on zebrafish chromosome 15. Gene prediction tools suggested a single genomic structure matching both protein sequences. A conserved block of synteny containing several linked genes surrounded the predicted zmll. Furthermore, zmll and human MLL were in the same map order in an uninterrupted segment with the gene for ubiquitination factor E4A. Degenerate RT-PCR analysis of wild-type adult zebrafish RNA based on cross-species amino acid sequences from highly conserved PHD and SET domains amplified the predicted transcript. Cross-species Southern blot analysis with the human probe detected the projected zmll genomic fragments corresponding to the MLL bcr in adult zebrafish genomic DNA. RT-PCR analysis of wild-type adult zebrafish RNA determined that the two predicted “similar to MLL proteins” were from a single gene. The full length 12657 bp, 35-exon zmll cDNA cloned from wild-type 24 hpf zebrafish embryo RNA predicted a 4218 amino acid protein with CXXC, PHD, Bromodomain, FYRN, FYRC, and SET domains and taspase cleavage sites with 45.8% sequence identity and 57.3% similarity to human MLL. Phylogram tree analysis suggested evolutionary divergence of mammals from teleosts but nonetheless conservation of critical functional domains. QRT-PCR demonstrated maternally supplied zmll mRNA during the earliest embryonic developmental timepoints, and expression of zygotic zmll mRNA during embryogenesis and in the zebrafish adult. Conclusions: These results indicate that there is a single zmll gene with highly conserved functional similarity to human MLL. The temporal pattern of expression, including maternal supply of transcript to the embryo, indicates that zmll is important from early embryogenesis through the entire lifespan of the fish. The high evolutionary conservation of critical domains creates the framework to use zebrafish for studying MLL in hematopoiesis and leukemia.

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Isolation and analysis of sox9 gene derived from yellow catfish Pelteobagrus fulvidraco

Chinese Journal of Agricultural Biotechnology ◽

10.1079/cjb200693 ◽

2006 ◽

Vol 3 (2) ◽

pp. 109-114 ◽

Cited By ~ 2

Author(s):

Yu Ju-Hua ◽

Li Jian-Lin ◽

Cao Li-Ping ◽

Wu Ting-Ting ◽

Yang Hong

Keyword(s):

Sex Differentiation ◽

Amino Acid Sequences ◽

Ovary Development ◽

Pcr Analysis ◽

Pelteobagrus Fulvidraco ◽

Rt Pcr ◽

Yellow Catfish ◽

Chain Reaction ◽

Traditional Taxonomy ◽

Polymerase Chain

AbstractSox9 is one of the important genes related to sex differentiation and determination. Two partial cDNAs encoding sox9 were derived from brain, testis and ovary of yellow catfish Pelteobagrus fulvidraco using reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis revealed that the ovarian sox9a2 was different from sox9a1 derived from brain and testis. The identity rate of cDNA was 77%, that of amino acids was 84%, and the different nucleotides were distributed in different sites all over the cDNA. This finding confirmed that there were transcripts from two genes. Based on the homology of the amino-acid sequences for sox9 of other animals, the similarity for sox9a1 from yellow catfish and sox9 of other animals averaged 86%. The similarity for sox9a2 reached a mean of 83%. The phylogenetic analysis revealed yellow catfish sox9a1 and sox9a2 were clustered into the fish branch. And yellow catfish sox9a2 was different from the other fish sox9. There was no significant branch including all sox9 derived from testis and brain or including all ovarian sox9. The genetic distance was the same with the results of the traditional taxonomy. RT-PCR analysis indicated that sox9a1 was expressed in male and female yellow catfish forebrain as well as in gonads, whereas sox9a2 was only expressed in ovaries. The function of sox9a2 in female sex formation and ovary development needs to be studied in depth.

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First Report on the Natural Occurrence of Beet chlorosis virus in Poland

Plant Disease ◽

10.1094/pdis-91-3-0326c ◽

2007 ◽

Vol 91 (3) ◽

pp. 326-326 ◽

Cited By ~ 2

Author(s):

A. Kozlowska-Makulska ◽

M. S. Szyndel ◽

J. Syller ◽

S. Bouzoubaa ◽

M. Beuve ◽

...

Keyword(s):

Sugar Beet ◽

Beta Vulgaris ◽

Amino Acid Sequences ◽

Enzyme Linked Immunosorbent Assay ◽

Natural Occurrence ◽

Rt Pcr ◽

First Report ◽

Beet Western Yellows Virus ◽

Access Period ◽

Commercial Sugar

Yellowing symptoms on sugar beet (Beta vulgaris L.) are caused by several viruses, especially those belonging to the genus Polerovirus of the family Luteoviridae, including Beet mild yellowing virus (BMYV) and Beet western yellows virus (BWYV), and recently, a new species, Beet chlorosis virus (BChV), was reported (2). To identify Polerovirus species occurring in beet crops in Poland and determine their molecular variability, field surveys were performed in the summer and autumn of 2005. Leaves from symptomatic beet plants were collected at 26 localities in the main commercial sugar-beet-growing areas in Poland that included the Bydgoszcz, Kutno, Lublin, Poznań, Olsztyn, and Warszawa regions. Enzyme-linked immunosorbent assay (ELISA) tests (Loewe Biochemica GmbH, Sauerlach, Germany) detected poleroviruses in 23 of 160 samples (approximately 20 samples from each field). Multiplex reverse-transcription polymerase chain reaction (RT-PCR) (1) (GE Healthcare S.A.-Amersham Velizy, France) confirmed the presence of poleroviruses in 13 of 23 samples. Nine of twenty sugar beet plants gave positive reactions with BChV-specific primers and three with primers specific to the BMYV P0 protein. Two isolates reacted only with primer sets CP+/CP, sequences that are highly conserved for all beet poleroviruses. Leaf samples collected from three plants infected with BChV were used as inoculum sources for Myzus persicae in transmission tests to suitable indicator plants including sugar beet, red beet (Beta vulgaris L. var. conditiva Alef.), and Chenopodium capitatum. All C. capitatum and beet plants were successfully infected with BChV after a 48-h acquisition access period and an inoculation access period of 3 days. Transmission was confirmed by the presence of characteristic symptoms and by ELISA. Amino acid sequences obtained from each of four purified (QIAquick PCR Purification kit, Qiagen S.A., Courtaboeuf, France) RT-PCR products (550 and 750 bp for CP and P0, respectively) were 100% identical with the CP region (GenBank Accession No. AAF89621) and 98% identical with the P0 region (GenBank Accession No. NP114360) of the French isolate of BChV. To our knowledge, this is the first report of BChV in Poland. References: (1) S. Hauser et al. J. Virol. Methods 89:11, 2000. (2) M. Stevens et al. Mol. Plant Pathol. 6:1, 2005.

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Natural Infection of Canavalia ensiformis with Tobacco mosaic virus in Venezuela

Plant Disease ◽

10.1094/pdis.2004.88.6.681c ◽

2004 ◽

Vol 88 (6) ◽

pp. 681-681 ◽

Cited By ~ 1

Author(s):

E. Marys ◽

E. Ortega ◽

O. Carballo ◽

C. Ramis

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Natural Infection ◽

Amino Acid Sequences ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Electron Microscopic ◽

Crop Species ◽

Jack Bean ◽

Canavalia Ensiformis

Jack bean (Canavalia ensiformis) is a valuable green manure and cover-crop species. In late summer of 2002, jack bean plants showing severe stunting, leaf mosaic, mottling, distortion, and general yellowing were observed in fields located in Maracay, Aragua State, Venezuela. Sap from symptomatic leaves was used to mechanically inoculate healthy jack bean, and field symptoms were successfully reproduced. Similar inoculations on Nicotiana tabacum var. Sansum resulted in mosaic symptoms and leaf distortion. Electron microscopic examination of leafdip preparations showed filamentous rods resembling those of a tobamovirus. Tobacco mosaic virus (TMV) was specifically identified with TMV-specific polyclonal antibody (PVAS-958, ATTC) in enzyme-linked immunosorbent assay. Sequence analysis of a coat protein gene (CP) fragment amplified using reverse transcription-polymerase chain reaction (RT-PCR) with primers TMV-CP-F and TMV-CP-R (1) from total RNA confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 450-bp region of the RT-PCR product were 96 to 99% and 98 to 100% identical, respectively, to the TMV CP gene in GenBank Accession Nos. J02415 and X68110. On the basis of foliar symptoms, incidence of TMV in jack bean was more than 50% in this experimental field. The source of infection is not known. Because TMV is reported to be seedborne in many other plant species, testing jack bean seed stocks for TMV infection could have important implications on the future control of the virus. To our knowledge, this is the first report of natural infection of jack bean by a tobamovirus. Reference: (1) N. J. Spence et al. Eur. J. Plant Pathol. 107:633, 2001.

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