Volume 1 Number 2 2007

CONTENTS AND ABSTRACTS

Gabriella Kazinczi, József Horváth, András Takács (Hungary) Tospoviruses on Ornamentals (pp 142-162)

Full Text [PDF]

Invited Review: The ornamental industry has experienced tremendous growth in the past 50 years and has developed into one of the major economic branches of modern agriculture. Tospoviruses are one of the major pathogens of ornamentals all over the world, reducing their aesthetic value and profitability. The rapid emergence and marked geographic spread of tospoviruses is due to the enhancement of the international trade of propagative materials and their final products. The distribution of tospoviruses was generally preceeded by a rapid expansion of their efficient new vector, Frankliniella occidentalis. In this review, we give a short account of the history and distribution of tospoviruses, and describe the host range, genetic and transmission studies, isolation and detection methods. Finally conventional and transgenic means for their control are described.

Shun-Fang Cheng, Jen-Wen Lin, I-Hsuan Chen, Yau-Heiu Hsu, Ching-Hsiu Tsai (Taiwan) The Identification of cis- and trans-Acting Elements in the Infection Cycle of Bamboo mosaic virus (pp 163-169)

Full Text [PDF]

Invited Mini-Review: Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus with flexuous rod morphology which belongs to the potexvirus group. To study the infection cycle of BaMV at the molecular level, a full-length infectious cDNA clone was constructed for in vivo protoplast and plant transfection assays. For in vitro transcription assays, RdRp replicase complex extracted from infected plants was used. From the results derived from these in vivo and in vitro analyses, we identified that a cloverleaf-like structure in the 3¢ UTR of the BaMV genome is involved not only in viral RNA accumulation in cells but also in viral systemic movement. A major stem-loop contains two cis-acting elements, one is a potexviral conserved hexamer motif which is important for the accumulation of viral products in protoplasts and the other is the polyadenylation motif which is involved in minus-strand RNA synthesis and in regulating the length of the poly(A) tail. To identify the trans-acting factors required for efficient accumulation of viral products in plants two techniques were employed. The first one is conventional chromatography to isolate the proteins that bind to the RNA genome. The second is cDNA-amplified fragment length polymorphism (cDNA-AFLP) to identify the host gene expression profiles that show up- or down-regulated patterns corresponding to BaMV infection. We found that chloroplast phosphoglycerate kinase (PGK), a gluconeogenic enzyme binds to the poly(A) of BaMV RNA and using virus induced gene silencing, a powerful tool for functional analysis, we found that chloroplast PGK is required for efficient accumulation of BaMV in plants. Using the combination of cDNA-AFLP and VIGS, we will identify novel genes that regulate the accumulation of viral products in plants.

Maria do Rosário Félix, Joana M.S. Cardoso, Solange Oliveira, Maria Ivone E. Clara (Portugal) Biological and Molecular Characterization of Olive latent virus 1 (pp 170-177)

Full Text [PDF]

Invited Mini-Review: Olive latent virus 1 (OLV-1) belongs to the Necrovirus genus, Tombusviridae family and is pathogenic to olive, citrus and tulip plants. It is easily mechanically transmissible to indicator plants causing necrotic lesions and can be transmitted through the soil into the plant roots in the absence of biological vectors. Infected cells contain virus aggregates, inclusions made up of excess of viral coded peptides and extensive vesiculation in the cytoplasm. The virions are isometric with ca. 30 nm, possess a monopartite single-stranded positive-sense RNA genome sized 3700 nt with 5 open reading frames (ORFs) and small inter cistronic regions. ORF 1 encodes a polypeptide with a molecular weight of 23 kDa and the read through of its amber stop codon results in ORF 1 RT that encodes the virus RNA dependent RNA polymerase with 82 kDa. ORF2 and ORF3 encode two small peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in the virus cell-to-cell movement. ORF 4 is located in the 3′-terminal and encodes a protein with 30 kDa identified as the viral coat protein. The complete genomic sequences of two well characterized OLV-1 isolates (obtained from citrus and olive) are similar, revealing an overall nucleotide sequence identity of 95%. The electrophoretic profile of the dsRNAs recovered from infected tissues exhibits three major species with ca. 3.7, 1.5, and 1.3 kbp. Application of molecular techniques based on PCR and on dot blot hybridization has been successfully used for routine diagnosis of OLV-1 infections.

Paula F. Tennant (Jamaica), Gustavo A. Fermin (Venezuela), Marcia E. Roye (Jamaica) Viruses Infecting Papaya (Carica papaya L.): Etiology, Pathogenesis, and Molecular Biology (pp 178-188)

Full Text [PDF]

Invited Review: During the last few decades, over 20 plant virus species capable of infecting papaya (Carica papaya L.) have emerged in tropical and subtropical regions. The extent and severity of disease symptoms vary widely from minor or unapparent to reduced tree vigor, yield, and impaired fruit quality. Existing data on molecular characteristics of viruses infecting papaya also vary; while phenotypic data are available for the majority, the genotype of many have not been characterized and partial or complete nucleotide sequences have not been determined. As a result, some virus species are classified in recognized taxa while others are tentatively assigned to genera or have not been sufficiently distinguished from viruses in recognized genera so as to form a new genus. This paper presents an overview of the virus species capable of infecting papaya, diseases they elicit, genetic structure and diversity, and factors contributing to their emergence where molecular data are available.

Devapriya Das (India), Luke Simon (UK/India), K.S. Shankarappa, P. Narayanaswamy, C.N. Lakshminarayana Reddy, K.T. Rangaswamy (India) Cloning, Sequencing and Identification of a Banana Bunchy Top Virus Isolate from Bangalore (pp 189-192)

Full Text [PDF]

Original Research Paper: Banana bunchy top disease is one of the most devastating viral diseases of bananas (Musa spp.) in Asia. It is wide-spread in all banana-producing areas and causes serious problems in most countries. Banana bunchy top viruses (BBTV) are isomeric particles with a single coat protein and contain six single-stranded, circular DNA components. PCR amplification was performed on isolates from Bangalore with primers specific to coat protein, which amplified a DNA fragment of 1058 bp. The PCR amplified products of coat protein gene of BBTV were cloned into ptz57R/T vector and sequenced. Sequence analysis resulted in a sequence of 1058 nucleotides which correspond to the DNA II component of the BBTV genome. The sequence comparison resulted in a maximum similarity (97%) with the isolates obtained from India and a minimum similarity (68.1%) with the isolates obtained form Taiwan. Based on the sequence analysis it can be inferred that the isolate from Bangalore was characterized under ‘S’ type (Severe strain) of BBTV that causes distinct bunched atrophy of leaves and dwarfness.

Haggag Salah Zein, Mona Hashem Hussein (Egypt), Kazutaka Miyatake (Japan) Characterization of Cucumber mosaic virus Monoclonal Antibodies (mAbs) Specificity, Neutralization of Infectivity and Gene Sequence (pp 193-200)

Full Text [PDF]

Original Research Paper: Monoclonal antibodies (mAbs) were produced against the coat protein of subgroups I and II of two Japanese strains of Cucumber mosaic virus, namely pepo- and m2-CMV. We used direct ELISA to determine the apparent dissociation constants (Kd), which were 0.041-27.9 nM. The mAbs were screened for their ability to neutralize virus infectivity in vitro. Infectivity was almost completely inhibited when virus preparations were mixed in vitro with the homologous mAb prior to inoculation. Aggregation of CMV virions following incubation with mAbs was demonstrated using an immunobinding assay. The long-term goal of expressing these antibody genes is to make the recipient biovital and to exhibit virus resistance. V gene sequences of the mAbs specific subgroup I, V_L expressed from a germline family V_κ2, gene bd2, while the V_H genes were derived from germline family V_HJ558, namely gene V_HJ558.45. On the other hand, the V gene sequence of subgroup II-specific mAbs, V_L, expressed from a different germline family V_κ1A, gene bb1.1, while V_H genes derived from same germline family V_HJ558 resulted in a different gene, V130.3. The deduced amino acid sequences, i.e. V_H and V_L chains, established from our mAbs-specific subgroups I and II of Japanese strains shared a total nucleotide identity with previously reported antibodies specific to CMV. We concluded that high-affinity CMV-binding antibodies could arise without extensive somatic hypermutation in the variable-region genes because of the expression of appropriate HCDR3s. The present information permits us to analyze the relationship between antibody H and L chain genes and the antigenic domains on antigen.

Haggag Salah Zein (Egypt), Kazutaka Miyatake (Japan) Cloning, Sequencing and Expression of Immunoglobulin Variable Regions of Murine Monoclonal Antibodies Specific to Cucumber mosaic virus coat protein (pp 201-207)

Full Text [PDF]

Original Research Paper: A mouse monoclonal antibody (mAb) prepared by hybridoma technology could recognize the Cucumber mosaic virus (CMV) coat protein. The F_ab fragments (antigen-binding site, one complete light chain, and part of one heavy chain) genes encoding the light chain and the Fd (a monovalent antigen-binding fragment consisting of a complete light chain paired with the variable region and the first constant domain of the heavy chain) region of the heavy chain of the mAb were cloned and expressed in Escherichia coli. The F_ab fragment was produced by subjecting the heavy and light chain antibody genes of the pepo-4 cell line to reverse transcription-polymerase chain reaction, then subcloning the products in the pGEM-T easy vector. Sequence analyses of the F_ab fragment revealed that the light and heavy chains belong to the Vк2 and V_H1/V_HJ558 germline gene family with GenBank accession numbers EF672211 and EF672197, respectively. The pARA7 expression vector was designed for the expression of F_ab in the periplasmic space. Recombinant F_ab fragments were purified and analyzed by indirect ELISA. These results suggest that the recombinant F_ab-4 antibody produced by E. coli acts as a source for the generation of F_ab with very stable and specific expression.

Anis Ben Amar (Germany/Tunisia), Alexandra Bassler (Germany), Abdelwahed Ghorbel, Ahmed Mliki (Tunisia), Gabriele Krczal, Thierry Wetzel (Germany) Determination of the Length of the Poly(A) Tails of the Arabis mosaic nepovirus Genomic RNAs (pp 208-210)

Full Text [PDF]

Short Communication: The genomic RNAs 1 and 2 of the NW isolate of the Arabis mosaic nepovirus were polyguanylated and amplified by RT-PCR, to determine the lengths of their poly(A) tails. Primers specific of ArMV-NW RNAs 1 or 2 were used in combination with a primer designed to hybridize at the junction poly(A)-poly(G) introduced at the end of the poly(A) tail during the polyguanylation procedure of the viral RNAs. The RT/PCR products were cloned and sequenced. The poly(A) tails in the different clones ranged from 10 to 81 adenosine residues for RNA 1, and from 10 to 120 adenosine residues for RNA 2, revealing an unexpected variability in the length of the ArMV genomic RNAs poly(A) tails.