[Show abstract][Hide abstract] ABSTRACT: A virus with filamentous particles ca. 700 nm long, denoted Fig latent virus 1 (FLV-1) is widespread in Apulian (southern Italy) fig orchards, in trees showing or not mosaic symptoms and in symptomless seedlings. The virus was transmitted by sap inoculation to a very restricted range of herbaceous hosts without inducing apparent symptoms and
was transmitted through fig seeds to a very high percentage (80 to 100 %). It was successfully purified from root tissues of infected figs. A virus-specific antiserum raised in rabbits, proved useful for its detection in fig leaf dips by immunosorbent electron microscopy (ISEM), Western Blot, dot immuno-binding (DIBA), ELISA. The viral genome
structure resembles that of members of the genus Trichovirus in the family Flexiviridae.
[Show abstract][Hide abstract] ABSTRACT: Several dsRNA bands (approx. 0.6-7 kbp in size) were recovered from tissues of mosaic-diseased fig seedlings which contained the enveloped round structures known as double membrane bodies (DMBs). blast analysis of a 4353 and a 1120 nt sequence from the two largest RNA segments showed homology with the polymerase and the putative glycoprotein precursor genes of negative-sense single-stranded RNA viruses of the family Bunyaviridae. Negative- and positive-sense riboprobes designed from both RNA segments hybridized to two bands of approximately 7 and 2.3 kbp in Northern blots of dsRNAs. Thus, these segments were identified as putative RNA-1 and RNA-2 of a novel virus for which the name fig mosaic virus (FMV) is proposed. Identity levels of predicted amino acids of the protein encoded by FMV RNA-1 with those of species of the family Bunyaviridae and European mountain ash ringspot-associated virus (EMERaV) were 28 and 54 %, respectively. RNA-2 showed 38 % identity at the amino acid level only with EMARaV. RNA-1 segment contained five conserved motifs (A-E) and an endonucleolytic centre of comparable genes of L RNA of bunyaviruses and EMARaV RNA-1. In a phylogenetic tree constructed with RdRp sequences, EMARaV grouped with FMV in a clade distinct from those of all bunyavirus genera. The consistent association of DMBs with mosaic symptoms and the results of molecular investigations strongly indicate that DMBs are particles of FMV, the aetiological agent of fig mosaic disease.
Journal of General Virology 04/2009; 90(Pt 5):1281-8. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The transmission through seeds of Fig latent virus 1 (FLV-1), a putative member of the genus Trichovirus, was investigated. Batches of seedlings of different age (1-yearold and 4-weeks-old) and geographical origin (Italy, Greece and Turkey) were analyzed by immunosorbent electron microscopy (ISEM) using a virus-specific antiserum.
[Show abstract][Hide abstract] ABSTRACT: Though fig mosaic disease has been known for decades but the causal agent has been elusive. Here we present data on the incidence of at least four new viruses isolated from fig trees exhibiting mosaic symptoms.
21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops; 01/2009
[Show abstract][Hide abstract] ABSTRACT: A virus with filamentous particles ca. 700 nm long,
denoted Fig latent virus 1 (FLV-1) is widespread in
Apulian (southern Italy) fig orchards, in trees showing
or not mosaic symptoms and in symptomless seedlings.
This virus was transmitted by sap inoculation to a very
restricted range of herbaceous hosts without inducing
apparent symptoms. It was successfully purified from
root tissues of infected figs. A virus-specific antiserum
raised in rabbits, proved useful for its detection in fig
leaf dips by immunosorbent electron microscopy. The
cytology of infected cells was little affected. Bundles of
filamentous particles were observed in the cytoplasma
of parenchyma cells of infected fig trees and seedlings.
The viral genome is a single-stranded positive-sense
RNA with an estimated size of ca. 8,000 nt, 6,620 of
which have been sequenced, starting from the
polyadenylated 3’ terminus. Genomic RNA consists of
four open reading frames encoding, in the 5’→3’ direction,
the replication-associated proteins (ORF 1), a 43
kDa putative movement protein (ORF 2), the 46 kDa
coat protein (ORF 3), and a 12 kDa protein with nucleic
acid binding properties. The viral genome structure
and organization resembles that of members of the
genus Trichovirus, family Flexiviridae and, indeed, FLV-
1 clusters with trichoviruses in phylogenetic trees constructed
with coat protein sequences. However, a distinct
difference with all members of the genus rests with
the size of the coat protein subunits (46 versus 22-27
kDa) and the presence of ORF 4, which is present only
in three tentative species of this genus.
JOURNAL OF PLANT PATHOLOGY 01/2009; 91(3):555-564. · 0.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is a major pathogen of grapevine. A previously described single-chain fragment variable (scFv) antibody (scFvLR3), directed against the coat protein (CP) of GLRaV-3, was expressed in Escherichia coli and used to develop a diagnostic ELISA kit. The antibody was fused to the light chain constant domain of human immunoglobulin to create the bivalent reagent C(L)-LR3, which was purified from the periplasmic fraction, giving a yield of ~5 mg/l. The sensitivity of the reagent against recombinant GLRaV-3 CP was 0.1 ng. The sensitivity, specificity and durability of the reagent was similar to a commercial kit. The C(L)-LR3 showed a weak cross-reaction in immune electron microscopy assays to GLRaV-1 and -7, but not with the phylogenetically more distant GLRaV-2. A fully recombinant kit was developed with the inclusion of a recombinant GLRaV-3 CP expressed in bacteria, thus avoiding problems associated with virus propagation and purification. This system represents a rapid, simple, sensitive and standardized diagnostic protocol for GLRaV-3 detection.
Archives of Virology 01/2009; 154(1):19-26. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Grapevine phloem-limited isometric virus (GPLIV) is the name proposed for a non mechanically-transmissible virus found in Italian and Tunisian grapevines. In density gradient centrifugation purified virus preparations sedimented as two components: T, made up of empty protein shells, and B, composed of intact nucleoprotein particles. B particles had a buoyant density of 1.45 g/cm3 at equilibrium in CsCl and contained 35% RNA consisting of a single molecule with an apparent size of 7.4 kb. The coat protein consisted of a single species with a mol.wt of 28,000 daltons. Purified virus preparations did not infect herbaceous hosts by manual inoculation. A specific antiserum with a titre of 1: 64 raised in rabbits, was used for identification of, GPLIV in field-grown Tunisian grapevines and in leafroll-affected Italian vines before and after heat treatment. Although heat treatment eliminated the virus from the majority of the plants, leafroll symptoms persisted in several GPLIV-free vines, indicating that there is no clear-cut relationship between GPLIV and this disease.ZusammenfassungEinige Eigenschaften eJnes im Phloem begrenzten, nicht mechanisch iibertragbaren WeinstockvirusDer Name grapevine phloem-limited isometric virus [GPLIV) wird für ein nicht mechanisch übertragbares, in Italien und Tunesien vorkommendes Virus vorgeschlagen, Nach Dichtegradiemenzentnfugation konnten zwei Komponenten aus gereinigten Virusprparaten festgestellt werden: T, die aus leeren Proteinhüllen bestand, und B, die ganze Nucieoproteinpartikein enthielt. Die B-Komponenten hatten eine schwimmende Dichte von 1,45 g/cm3 im Gleichgewicht mit CsCl und emhielten 35 % RNS, die aus eincm einzelnen Molekül mit einer scheitibaren Größe von 7,4 kb bestand. Das Hüllprotein setzte sich aus einer Art mit einem Mokkulargewicht von 28 000 Dalton zusammen. Gereinigte Virusprparaie waren nicht in der Lage, Wirtspflanzen nach einer kiinsthchen Inokulation zu lnfizieren. Ein aus Kaninchen gewonnenes, spezifisches Antiserum, mit einem Titer von 1: 64, wurde bei der Feststellung vom CPLIV in Weinstöcken aus Feldern in Tunesien und in Reben in Italien. die geroiite Blatter aufwiesen, vor und nach einer Hitzebehandlung angewandt. Obwohl Nach einer Hitzebehandlung das Virus in fast alien Pflanzen nicht mehr nachzuweisen war, wurden die gerollten Blttersymptome bei einigen GPLIV-freien Reben nicht aufgehoben. Dies deutet darauf hin, daß es keinen eindeutigen Zusammenhang zwischen dem GPLIV und dieser Krankheit gibt.
[Show abstract][Hide abstract] ABSTRACT: Water samples were collected from some rivers of Southern Italy whose water is normally used for irrigation and checked for the presence of plant viruses. The assayes were carried out by centrifuging at 5000x g one liter of water, resuspending the sediments in phosphate buffer and testing their infectivities on Chenopodium quinoa Willd. It was possible to isolate tobacco mosaic virus, cucumber mosaic virus and two more viruses not identified yet.
[Show abstract][Hide abstract] ABSTRACT: Mosaic disease (FMD) is a cosmopolitan disorder of fig (Ficus carica),
characterized by various patterns of discolouration (mosaic, chlorotic
mottling and blotching, vein banding, ringspots, line patterns) and
malformation (twisting, puckering, rosetting) of the leaves. Infected plants
have reduced vigour and may bear small mottled fruits. FMD has a viral aetiology but its causal agents are still under scrutiny.
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Different patterns of chlorotic to yellowish mottling and deformation were observed in leaves of field-grown fig trees in Mexico and South Africa. Potted rooted cut- tings from both sources grown under glasshouse condi- tions displayed chlorotic blotching, vein clearing and banding of the leaves. Electron microscope observations of thin-sectioned tissues from symptomatic leaves showed that parenchyma cells of both Mexican and South African samples contained the double-membrane bodies (DMBs) typically associated with fig mosaic disease (FMD). Also, accumulations of filamentous semi-rigid and of very flexuous virus-like particles were observed in parenchyma or phloem cells, respectively. RT-PCR assays of total nucleic acids extracted from leaf tissues of both sources using primers designed to amplify the HSP70 gene of the putative closteroviruses Fig leaf mottle-associ- ated virus 1 (FLMaV-1) and Fig leaf mottle-associated virus 2 (FLMaV-2) gave a positive response only for FLMaV-1. DMBs have been observed previously in the symptomatic Mexican fig source. However, our results are the first records of FMD and associated DMBs in South Africa and of both FLMaV-1 and an unidentified filamentous semi-rigid virus in Mexico and South Africa.
[Show abstract][Hide abstract] ABSTRACT: Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was observed in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata.
Archives of Virology 08/2005; 150(7):1369-81. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RACE-PCR using a denatured dsRNA extract from
an olive plant infected by Olive leaf yellowing-associated
virus (OLYaV), extended a previously sequenced
large fragment of the viral genome by 854 nt downstream
in the heat shock protein 90 (HSP90). This sequence
showed from 45 to 53% identity at the nucleotide
level with that of putative HSP90s of different
members of the family Closteroviridae. In phylogenetic
trees constructed with published HSP90 and RdRp sequences,
OLYaV was well separated from other viruses,
as was Little cherry virus 1 (LChV-1), another unassigned
species in the family Closteroviridae. A polyclonal
antiserum was raised to a recombinant protein encoded
by the HSP70 gene in fusion with sequence encoding
the maltose binding protein. In Western blots of extracts
from infected olive tissues, IgGs purified from
this antiserum reacted with a polypeptide with a Mr of
ca. 65 kDa, which was identified as the putative viral
HSP70. Closterovirus-like flexuous particles with distinct
cross banding and a maximum length of ca. 2000
nm were seen in extracts from infected roots and leaves
of rooted olive cuttings. The cytopathology of infected
leaves was similar to that induced by other members of
the family Closteroviridae. However, the new information
is still insufficient for the ultimate classification of
JOURNAL OF PLANT PATHOLOGY 01/2005; 87(3):223-228. · 0.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Different Prunus species from Southern Italy with stem pitting symptoms were assayed by RT-PCR for the presence of Plum bark necrosis stem pitting-associated virus (PBNSPaV). DNA of the expected size was am- plified from cortical scrapings of almond, peach, plum, apricot, and sweet cherry. The test was repeated monthly on various tissues throughout a year. Different protocols for total nucleic acid extraction and RT-PCR amplification were compared. Virus particles of an Ital- ian apricot isolate (APR-SP1) were purified and used to produce antiserum that reacted in IEM and ELISA with particles of an American PBNSPaV isolate. How- ever, the antiserum did not detect PBNSPaV in crude plant extracts.
[Show abstract][Hide abstract] ABSTRACT: Vein necrosis (VN), a virus-like disease latent in all European grapevine cultivars and in most American root-stock species and hybrids, induces necrosis of the veinlets of its specific indicator Vitis rupestris x Vitis berlandieri 110 R at the abaxial side of the leaf blade. VN is very com-mon in southern Italy, e.g. 109 out of 218 of the putative grapevine clones selected during sanitary improvement programmes in the last few years indexed positive in 110 R. As assessed by ELISA, the same vines had a very low rate of infection (< 4 %) by major detrimental viruses (GFLV, GVA, GVB, GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-7, GFkV) commonly looked for during selection. When the VN-posi-tive 110 R indicators were checked by PCR and Western blot for the presence of Grapevine rupestris stem pitting-associated virus (GRSPaV) a strikingly high association (98 %) was observed between this virus and VN symptoms. Likewise, all 72 mother plants of Vitis rupestris used as indicators in indexing trials and recently discovered to be infected by GRSPaV, induced VN reactions after grafting onto 110 R. By contrast, no VN reactions developed in 110 R top-grafted on a single GRSPaV-free V. rupestris. Moreo-ver, GRSPaV was consistently detected in the symptomatic lower leaves of the shoots of infected 110 R vines, but not in the symptomless upper leaves of the same shoots. These findings strongly suggest that GRSPaV is involved in the aetiology of VN. K e y w o r d s: grapevine, vein necrosis, GRSPaV, V. rupestris, PCR, Western blot, Foveavirus.
[Show abstract][Hide abstract] ABSTRACT: SUMMARY A filamentous virus was isolated by mechanical sap transmission from GF 305 seedlings showing asteroid ringspots to Nicotiana occidentalis. The GF 305 seedlings were graft-inoculated with buds from an apricot tree cv Mistikawi, from Palestine. The electrophoretic dsRNA pattern from symptomatic GF 305 and N. occidentalis showed a major band of about 9.5 kbp. Bundles of elon- gated virus-like particles were present in the cytoplasm of infected N. occidentalis. Dissociated coat protein from partially purified virus preparations consisted of a single subunit with an estimated Mr of ca. 50 kDa. RT-PCR us- ing primers specific for Apricot latent virus (ApLV) RNA amplified a DNA product of about 200 bp from infected GF 305 and N. occidentalis plants. Sequence analysis of this fragment showed 90-93% identity in the encoded amino acid sequence with corresponding sequences from different ApLV isolates. This is the first record of ApLV from the southern Mediterranean. An ApLV-spe- cific digoxygenin-labelled riboprobe was produced and used for molecular hybridisation tests. A survey of apri- cot orchards in Southern Italy did not show the presence of ApLV.
[Show abstract][Hide abstract] ABSTRACT: SUMMARY In a recent survey of the sanitary status of stone fruits in Palestine, a virus (isolate PL27) was transmitted to Nicotiana occidentalis by mechanical inoculation from Japanese plum. Purified virus consisted of quasi-spheri- cal particles 26-35 nm in diameter which sedimented as 3-4 components in density gradient centrifugation. Virus coat protein had an estimated molecular mass of ca. 25 kDa, while four encapsidated RNA bands were visible in electrophoregrams of purified nucleic acid. The nucleotide sequence of a 385 bp PCR-generated amplicon from RNA-3 was determined showing 99% identity at the nucleic acid level and 98.9% similarity at the aminoacid sequence level with a previously charac- terized RNA-3 region of American plum line pattern virus (APLPV). A digoxigenin-labelled probe was syn- thesized which specifically recognized virus isolates in extracts from herbaceous and woody samples but did not cross-hybridize with Prunus necrotic ringspot virus (PNRSV), Apple mosaic virus (ApMV), and Prune dwarf virus (PDV). An antiserum to PL27 was raised, which recognized homologous antigens but not PDV and PNRSV. An ELISA kit prepared with this antiserum was successfully used for the detection of PL27 in in- fected Prunus species. Based on particle morphology, biological, serological, and molecular properties, PL27 was identified as an isolate of APLPV.