Cytological and molecular evidence that the whitefly-transmitted Cucumber vein yellowing virus is a tentative member of the family Potyviridae

H. Lecoq1, C. Desbiez1, B. Delécolle1, S. Cohen2 and A. Mansour3

Station de Pathologie Végétale, INRA, Domaine Saint Maurice, BP 94, 84143 Montfavet cedex, France1
Department of Virology, Agricultural Research Organization, The Volcani Center, POB 6, Bet Dagan 50250, Israel2
Horticulture and Plant Protection Department, Faculty of Agriculture, University of Jordan, Amman, Jordan3

Author for correspondence: Hervé Lecoq. Fax +33 4 32 72 28 42. e-mail Herve.Lecoq{at}avignon.inra.fr


   Abstract
Top
Abstract
Main text
References
 
Cucumber vein yellowing virus (CVYV) is widespread in cucurbits in the Middle East. CVYV has filamentous particles and is transmitted by Bemisia tabaci by the semi-persistent mode. It has not yet been assigned to a specific genus or family. Ultramicroscopic observations revealed numerous cylindrical cytoplasmic inclusions in melon and cucumber cells infected by CVYV isolates from Israel and Jordan. Depending on the section orientation, the inclusions appeared as pinwheels or as bundles. In addition, a 1·9 kb DNA fragment was amplified by RT–PCR from CVYV-infected plant extracts using primers designed to detect all potyvirids. Sequence comparisons with the amplified fragment indicated that CVYV is more closely related to Sweet potato mild mottle virus than to any other virus in the family Potyviridae. These results suggest that CVYV can be considered as a tentative new member of the genus Ipomovirus, family Potyviridae.


   Main text
Top
Abstract
Main text
References
 
Cucumber vein yellowing virus (CVYV) causes a severe disease of cucumbers and other cucurbits in the Oriental Mediterranean Basin (Cohen & Nitzany, 1960 ; Harpaz & Cohen, 1965 ; Al-Musa et al., 1985 ; Yilmaz et al., 1989 ). CVYV is reported to have rod-shaped particles, 740–800 nm long with a diameter of 15–18 nm (Sela et al., 1980 ). The virus is readily transmitted mechanically and by the whitefly Bemisia tabaci by the semi-persistent mode (Harpaz & Cohen, 1965 ; Mansour & Al-Musa, 1993 ). Due to the instability of CVYV particles, difficulties have been encountered in obtaining purified virus preparations (Sela et al., 1980 ; Mansour & Hadidi, 1999 ); therefore, very little is known of the biophysical and biochemical properties of CVYV and the virus is still unclassified (Brunt et al., 1996 ; Lecoq et al., 1998 ). However, Sela et al. (1980) have reported that the CVYV coat protein (CP) has a molecular mass of 39 kDa and that the viral nucleic acid is a double-stranded DNA, suggesting that CVYV could be a member of a new virus family.

In order to investigate the taxonomic position of CVYV further, cytopathological, serological and molecular studies were conducted. Cucumber (Cucumis sativus cv. Beit Alfa) and melon (Cucumis melo cv. Védrantais) plants were inoculated mechanically with the CVYV type strain from Israel (CVYV-Isr) or with a CVYV isolate from Jordan (CVYV-Jor) and were kept subsequently in independent screened cages maintained in an insect-proof greenhouse. The two strains induced similar vein-clearing symptoms in cucumber and melon, but CVYV-Jor caused a more severe stunting in cucumber.

For cytological studies, leaf pieces 1 mm across were collected from symptomatic young leaves 3–4 weeks after inoculation and similar samples from healthy plants were used as controls. Samples were fixed with glutaraldehyde, post-fixed with osmium tetroxide and embedded in araldite CY212 (Agar Scientific) (Delécolle, 1978 ). Thin sections were cut with a diamond knife by using an Ultracut E ultramicrotome (Reichert-Jung). Thin sections were stained in 5% uranyl acetate and lead acetate, pH 12, before observations.

Numerous cylindrical cytoplasmic inclusions were observed in CVYV-Isr- and CVYV-Jor-infected melon and cucumber cells. Depending on the section orientation, inclusions appeared as pinwheels (Fig. 1a, c) or as bundles (Fig. 1b, c). Cylindrical inclusions were occasionally observed at the cell periphery, one end being associated with the plasmalemma perpendicular to the cell wall (Fig. 1b). Cylindrical cytoplasmic inclusions are associated specifically with infections by virus species belonging to the family Potyviridae (Shukla et al., 1994 ; Edwardson & Christie, 1996 ). Numerous vesicles were also observed in the cytoplasm, sometimes associated with the cylindrical inclusions (Fig. 1c). Increased vesiculation is also a feature commonly associated with infections by potyvirids (Shukla et al., 1994 ). Only a few scattered virus particles were observed, generally close to the cylindrical inclusions. Neither cylindrical cytoplasmic inclusions nor numerous vesicles were observed in healthy plant samples.



View larger version (193K):
[in this window]
[in a new window]
 
Fig. 1. Cylindrical cytoplasmic inclusions typical of cucumber plants infected by CVYV-Isr (a, b) or CVYV-Jor (c). Depending on the section orientation, inclusions appear as pinwheels (a, c) or bundles (b, c) and are sometimes disposed perpendicular to the cell wall (cw). Note the numerous vesicles in the cytoplasm or associated with the inclusions. Bars, 0·5 µm.

 
The cytological observations outlined above suggest that a virus belonging to the family Potyviridae was present in tissue infected by CVYV-Isr and CVYV-Jor. To rule out the possibility that an aphid-borne potyvirus was contaminating the CVYV cultures, three series of experiments were conducted. CVYV-infected cucumber and melon plant extracts were tested in double-antibody sandwich (DAS)-ELISA with antisera raised against the five most common potyviruses encountered in cucurbits in the Mediterranean region (Lecoq et al., 1998 ): Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV), Moroccan watermelon mosaic virus (MWMV), Zucchini yellow fleck virus (ZYFV) and Zucchini yellow mosaic virus (ZYMV). No positive reaction was observed with CVYV-infected plant extracts in three replicate tests (data not shown). In addition, CVYV-infected plant extracts did not react in antigen-trapped indirect ELISA with monoclonal antibody PTY 1, which detects most aphid-transmitted potyviruses but which does not detect potyvirids transmitted by other vectors with the exception of Ryegrass mosaic virus (RGMV) (Jordan, 1992 ; Richter et al., 1995 ) (data not shown). Finally, attempts to transmit CVYV-Isr and CVYV-Jor by the aphid Myzus persicae by the non-persistent mode were unsuccessful (data not shown).

Further experiments were conducted with the ‘potyvirid’ primers designed by Gibbs & Mackenzie (1997) , which are able to amplify genome fragments of aphid-, mite-, fungus- and whitefly-transmitted potyvirids. Total RNA was extracted from 50 mg infected, symptomatic young cucumber or melon leaves with TRI-Reagent (Molecular Research Center), resuspended in 20 µl DEPC-treated H2O and heated for 5 min at 65 °C prior to reverse transcription. Reverse transcription and PCR were performed following a procedure derived from Gibbs & Mackenzie (1997) . cDNA was synthesized from 15% of the total RNA in a 20 µl reaction mixture using the Promega cDNA synthesis protocol and 1 µM of a primer derived from ‘potyvirid’ primer 1 of Gibbs & Mackenzie (1997) , Poty(dT)3' (5' CACGGATCCTTTTTTTTTTTTTTTTTV 3'). DNA encoding the 3' part of the viral genome was amplified in 25 µl of the PCR mixture described by Gibbs & Mackenzie (1997) with 2 µl of the cDNA and Taq polymerase instead of Tth polymerase. Primers Poty(dT)3' and Poty5' (5' CCACGGATCCGGBAAYAAYAGYGGDCARCC 3') were used for amplification. PCR cycles were performed as described by Gibbs & Mackenzie (1997) in a PEC2400 thermocycler (Perkin Elmer). A 1·9 kb fragment was amplified from CVYV-Isr- and CVYV-Jor-infected cucumber or melon extracts but not from extracts of healthy cucumber or melon plants.

The 1·9 kb fragment amplified from CVYV-Isr-infected plant tissue was ligated into a linearized and T-tailed pBlueScript plasmid (Stratagene). Plasmid DNA was purified from Escherichia coli DH10B and sent for double-strand sequencing to Genome Express (Grenoble, France). At least two sequence readings were obtained for each nucleotide. The deduced amino acid sequence of CYVY-Isr was compared to sequences in the GenBank database by using the FASTA procedure of Pearson & Lipman (1988) . Sequence alignments and distance matrices were obtained with CLUSTAL W (Thompson et al., 1994 ). A PAM Dayhoff matrix was used to calculate distance trees with the NEIGHBOR algorithm of the PHYLIP package (Felsenstein, 1989 ). Graphics were generated for the unrooted distance tree with the TreeView application (Page, 1996 ).

The deduced amino acid sequence of CVYV was aligned with sequences from other potyvirids belonging to the whitefly-transmitted genus Ipomovirus (Sweet potato mild mottle virus, SPMMV), the aphid-transmitted genera Potyvirus [Potato virus Y (PVY) and ZYMV] and Macluravirus (Maclura mosaic virus, MacMV), the fungus-transmitted genus Bymovirus (Barley yellow mosaic virus, BaYMV) and the mite-transmitted genera Rymovirus (Agropyron mosaic virus, AgMV) and Tritimovirus (Wheat streak mosaic virus, WSMV). An unrooted tree was obtained using the amino acid sequences of the core of the CP of these potyvirids and those of Barley mild mosaic virus (BaMMV), Brome streak mosaic virus (BSMV), Narcissus latent virus (NLV), PRSV, RGMV, Sweet potato feathery mottle virus (SPFMV), Turnip mosaic virus (TuMV) and WMV.

The amplified fragment contained the 3' part of the CVYV genome, including the C-terminal part of the polymerase (NIb) coding region, the CP coding region and the 3' non-coding extremity. Sequence comparison with virus databases showed that CVYV-Isr exhibited 43% amino acid sequence identity to the Ipomovirus SPMMV, while the identity to other potyvirids did not exceed 32% for tritimoviruses and 27% for potyviruses. No significant sequence identity was observed outside the family Potyviridae. Fig. 2 presents the CLUSTAL W alignment of the C-terminal 559 amino acids of CVYV-Isr with the corresponding parts of seven potyvirids representing the six genera included in this family. The NIb–CP cleavage site and the limit of the variable N-terminal part of CP were determined approximately by sequence comparisons and are underlined in Fig. 2. The DAG triplet conserved in the N-terminal part of the CP of most aphid-transmitted potyviruses was not present in the sequence of the putative CVYV CP. However, two amino acids that are highly conserved among potyvirids in the core of the CP and involved in virion assembly (Jacquet et al., 1998 ), R449 and D494, were present in the sequence of CVYV-Isr.



View larger version (134K):
[in this window]
[in a new window]
 
Fig. 2. Multiple alignment of the C-terminal 559 amino acids of CVYV-Isr polyprotein with the corresponding protein sequences of seven potyvirids. Sequences were aligned by using CLUSTAL W. Residues identical to the CVYV-Isr sequence are shaded. The double-underlined region locates the putative NIb–CP cleavage sites, while the single-underlined region locates the limit of the variable N-terminal parts of the CP of these potyvirids. Conserved amino acids involved in virion assembly are indicated by asterisks.

 
An unrooted tree obtained by the neighbour-joining method with the amino acid sequences of the core CP of 16 potyvirids is shown in Fig. 3. Six sequence clusters were obtained, concurrent with the classification of the Potyviridae. CVYV-Isr appeared to belong to the same cluster as SPMMV; this group is more closely related to Tritimovirus than to Potyvirus, Rymovirus or Bymovirus.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3. Phylogenetic analysis of the core of the CP of CVYV-Isr and those of 15 species belonging to the Potyviridae. The phylogenetic neighbour-joining tree was produced by using CLUSTAL W and the NEIGHBOR procedure of the PHYLIP package. Ellipses have been added to highlight the different genera. Bar, calculated genetic distance of 10.

 
Our attempts to obtain preparations of CVYV-Isr or CVYV-Jor that were sufficiently pure to allow an unequivocal determination of the composition of the viral nucleic acid failed. This is probably due to the very labile nature of the virions (Sela et al., 1980 ; Mansour & Hadidi, 1999 ). However, the obtention of a 1·9 kb DNA fragment by using the ‘potyvirid’ primers (Gibbs & Mackenzie, 1997 ) through RT–PCR but not through direct PCR with total nucleic acid extracts from infected plants (data not shown) strongly suggests that the CVYV nucleic acid is RNA, rather than double-stranded DNA as determined by Sela et al. (1980) . The numerous cylindrical cytoplasmic inclusions observed in CVYV-infected plants clearly associate this virus with the family Potyviridae. Indeed, these inclusions have been accepted for over 25 years as an intrinsic and characteristic feature of the former group Potyvirus, which is now the family Potyviridae (Shukla et al., 1994 ). Sequence data further confirmed that CVYV is more closely related to the family Potyviridae than to any other known virus family. The molecular grouping with SPMMV revealed by the unrooted distance tree (Fig. 3) and similarities in their biological properties (transmission by the whitefly Bemisia tabaci) strongly support the suggestion that CVYV should be considered as a tentative member of the genus Ipomovirus (Pringle, 1999 ). However, in DAS-ELISA, CVYV-Isr- and CVYV-Jor-infected plant extracts did not react with an antiserum against SPMMV kindly provided by J. Vetten (BBA, Braunschweig, Germany). The absence of serological relationships and the low percentage of amino acid identity (46%) in the core part of the CP between CVYV and SPMMV suggest that these two viruses should be considered as distinct species (Shukla et al., 1994 ). Recently, a whitefly-transmitted potyvirid that infected squash was described in the Sultanate of Oman (Zouba et al., 1998 ). Whether this virus is distinct from or a strain of CVYV is not known. However, this finding indicates that tentative ipomoviruses along with other whitefly-borne viruses (criniviruses and geminiviruses) constitute a major component of the complex pathosystem affecting cucurbit crops in the Mediterranean and subtropical regions (Lecoq et al., 1998 ).


   Acknowledgments
 
We thank Dr J. Vetten for his gift of SPMMV antigens and antibodies.


   Footnotes
 
The GenBank accession number of the sequence reported in this paper is AF233429.


   References
Top
Abstract
Main text
References
 
Al-Musa, A. M., Qusus, S. J. & Mansour, A. N. (1985). Cucumber vein yellowing virus on cucumber in Jordan.Plant Disease 69, 361.

Brunt, A., Crabtree, K., Dallwitz, M., Gibbs, A. & Watson, L. (1996). Viruses of Plants. Description and Lists from the VIDE Database. Wallingford, UK: CAB International.

Cohen, S. & Nitzany, F. E. (1960). A whitefly transmitted virus of cucurbits in Israel.Phytopathologia Mediterranea 1, 44-46.

Delécolle, B. (1978). Essais de rationalisation des méthodes de préparation d’échantillons végétaux pour la microscopie électronique: problème des précipités parasites.Cellular and Molecular Biology 23, 431-436.[Medline]

Edwardson, J. R. & Christie, R. G. (1996). Cylindrical Inclusions. Bulletin 894. Gainesville, FL: University of Florida Agricultural Experiment Station.

Felsenstein, J. (1989). PHYLIP – Phylogeny Inference Package (version 3.2). Cladistics 5, 164-166.

Gibbs, A. & Mackenzie, A. (1997). A primer pair for amplifying part of the genome of all potyvirids by RT–PCR.Journal of Virological Methods 63, 9-16.[Medline]

Harpaz, I. & Cohen, S. (1965). Semipersistent relationship between cucumber vein yellowing virus (CVYV) and its vector, the tobacco whitefly (Bemisia tabaci Gennadius).Phytopathologische Zeitschrift 54, 240-248.

Jacquet, C., Delecolle, B., Raccah, B., Lecoq, H., Dunez, J. & Ravelonandro, M. (1998). Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.Journal of General Virology 79, 1509-1517.[Abstract]

Jordan, R. (1992). Potyviruses, monoclonal antibodies, and antigenic sites.Archives of Virology Supplementum 5, 81-95.[Medline]

Lecoq, H., Wisler, G. & Pitrat, M. (1998). Cucurbit viruses: the classics and the emerging. In Cucurbitaceae ’98. Evaluation and Enhancement of Cucurbit Germplasm, pp. 126-142. Edited by J. D. McCreight. Alexandria, VA: ASHS Press.

Mansour, A. & Al-Musa, A. (1993). Cucumber vein yellowing virus; host range and virus vector relationships.Journal of Phytopathology 137, 73-78.

Mansour, A. N. & Hadidi, N. (1999). Cucumber vein yellowing virus: purification and serological studies.Dirasat Agricultural Sciences 26, 8-14.

Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on personal computers.Computer Applications in the Biosciences 12, 357-358.[Medline]

Pearson, W. R. & Lipman, D. J. (1988). Improved tools for biological sequence comparison.Proceedings of the National Academy of Sciences, USA 85, 2444-2448.[Abstract]

Pringle, C. R. (1999). Virus Taxonomy – 1999. The universal system of virus taxonomy, updated to include the new proposals ratified by the International Committee on Taxonomy of Viruses during 1998. Archives of Virology 144, 421-429.[Medline]

Richter, J., Rabenstein, F., Proll, E. & Vetten, H. J. (1995). Use of cross-reactive antibodies to detect members of the Potyviridae. Journal of Phytopathology 143, 459-464.

Sela, I., Assouline, I., Tanne, E., Cohen, S. & Marco, S. (1980). Isolation and characterization of a rod-shaped, whitefly-transmissible, DNA- containing plant virus.Phytopathology 70, 226-228.

Shukla, D. D., Ward, C. W. & Brunt, A. A. (1994). The Potyviridae. Wallingford, UK: CAB International.

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Research 22, 4673-4680.[Abstract]

Yilmaz, M. A., Ozaslan, M. & Ozaslan, D. (1989). Cucumber vein yellowing virus in Cucurbitaceae in Turkey.Plant Disease 73, 610.

Zouba, A. A., Lopez, M. V. & Anger, H. (1998). Squash yellow leaf curl virus: a new whitefly-transmitted poty-like virus.Plant Disease 82, 475-478.

Received 24 February 2000; accepted 6 June 2000.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Lecoq, H.
Articles by Mansour, A.
Articles citing this Article
PubMed
PubMed Citation
Articles by Lecoq, H.
Articles by Mansour, A.
Agricola
Articles by Lecoq, H.
Articles by Mansour, A.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS