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The necrotic disorder of freesia (Freesia refracta hyb., Family Iridaceae) was first described in The Netherlands before 1970. In following years, the disorder was widely reported in other European countries and more recently also in the United States and in New Zealand. The presence of the Freesia sneak virus (FreSV, Ophiovirus genus) has been widely associated with the necrotic disease, but some uncertainty remains. Freesia leaves showing necrotic disease were subjected to Ophiovirus purification and the product obtained was analyzed. Several differently shaped virus particles were visualized by TEM and a new virus-like agent, with a ca. 30 kDa coat protein was detected by western blotting. Previously uncharacterized viruses, possibly transmitted by the same vector, might have a role in the disease, at least in mixed infection, and are now thickening the plot about the necrotic disease causal agent.

Tobacco necrosis virus (TNV) was isolated from glasshouse zucchini plants in Liguria, northern Italy, showing yellow spots on young leaves and necrotic symptoms on older leaves, petioles and stems. No other viruses were present in these plants. Experimentally, the virus systemically infected zucchini seedlings grown in naturally infected soil but only local infections were induced when leaves and roots were mechanically inoculated. The virus was identified as serotype D, devoid of satellite particles. This appears to be the first report of TNV in zucchini. Three ELISA procedures for field detection of TNV using polyclonal antibodies for serotype D were compared. Antibodies were conjugated with I) alkaline phosphatase (AP-AB);2) N-Hydroxy-succinimid obiotin (BNHS-Ab); 3) N-biotinyl-6-aminocaproic hydrazide (BACH-Ab). The procedure using BNHS-Ab detected the lowest concentration of the homologous virus, followed by BACH-Ab and AP-Ab. Only with biotinylated antibodies. TNV serotype A was also detected. Zusammenfassung Charakterisierung eines Isolats des Tobacco necrosis virus aus Zucchini Das Tobacco necrosis virus (TNV) wurde in Ligurien (Norditalien) aus Gewachshaus-Zucchinipflanzen isoliert, deren junge Blatter gelbe Flecken aufwiesen und deren altere Blatter, Blattstiele und Stengel Nekrosen zeigten. Die Pflanzen enthielten keine anderen Viren. Zucchinisamlinge, die in naturbch TNV-verseuchtem Boden wuchsen, wurden im Versuch systemisch von dem Virus befallen. Nach mechanischer Inokulation von Blattern und Wurzeln induzierte TNV dagegen nur lokale Infektionen. Das Virus besas keine Satellitenpartikel und wurde als Serotyp D identifiziert. Dies ist scheinbar der erste Bericht von TNV-Befall bei Zucchini. Drei ELISA-Verfahren zum Nachweis von TNV in Feldmaterial, die polyklonale Antikorper fur Serotyp D verwendeten, wurden verglichen. Die Antikorper wurden konjugiert mit: 1) alkalischer Phosphatase (AP-Ab), 2) N-Hydroxy-succinimidobiotin (BNHS-Ab), und 3) N-biotinyl-6-aminocapronhydrazid (BACH-Ab). Bei Verwendung von BNHS-Ab wurde die niedrigste Konzentration des homologen Virus erfast, gefolgt von BACH-Ab und AP-Ab. Nur mit biotinylierten Antikorpem konnte auch der TNV Serotyp A nachgewiesen werden.

Tospovirus serogroups I and III have recently been designated as species, tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV), while the species status of serogroup II isolates remains undefined. Fifteen Tospovirus isolates from ornamental and vegetable crops in Liguria, Italy, were found to belong either to TSWV (seven isolates) or to INSV (eight isolates) on the basis of test-plant reactions, serological techniques using DAS ELISA kits raised against the nucleoproteins of the type members of the two species, and cytopathology. None of them could be assigned to serogroup II using DAS ELISA kits raised against nucleoproteins of this serogroup. Italian isolates representative of the two species reacted in indirect ELISA using a polyclonal antiserum against the entire particle of a TSWV isolate, but with higher intensity for our TSWV isolates than for the INSV isolates. Western blots and dot immunobinding assays confirmed that the nucleoproteins of the two species are unrelated whereas the glycoproteins are related. The cytopathology was similar for two isolates representative of TSWV and INSV, except that the type of filaments encountered was different, and appeared to be characteristic of the species.

A virus was isolated in Italy from Tamus communis in which it caused symptoms of red mosaic and leaf distortion. The virus, for which the name tamus red mosaic virus (TRMV) is suggested, was differentiated from other potexviruses or possible potexviruses on the basis of host range, particle properties and serology. In crude preparations, TRMV had elongated particles of modal length 550–560 nm and diameter of 13 nm. TRMV was easily purified from Chenopodium quinoa and had a buoyant density of 1·34 g/cm3 in CsCl and 1·27 g/cm-3 in Cs2SO4. An antiserum to TRMV was obtained with a titre of 1/1024 in slide precipitin tests. TRMV was related to, but distinct from, tamus latent virus, a potexvirus isolated from Tamus communis in the UK, and more distantly related to potato virus X, cactus virus X and commelina virus X. Polyacrylamide gel electrophoresis (PAGE) indicated that the virus particles contained a single protein species of estimated molecular weight 25·2 kDa. Enzymatic digestions, agarose gel electrophoresis and infectivity of the isolated nucleic acid indicated that the virus has a single major molecule of single-stranded positive-sense RNA of estimated molecular weight 2120 kDa (ca. 6·4 kb).

An undescribed virus, here named ranunculus white mottle virus, was isolated in Italy from cultivated ranunculus showing mottle and distortion of leaves. The virus was mechanically transmissible to several herbaceous hosts. In negative stain, the particles appeared as circularised supercoiled threads 3 nm in diameter of different contour lengths; in some conditions the circles collapsed to form linear pseudobranched structures 9 nm in diameter. Immunolabeling of thin sections showed that viral antigen was widely distributed in the cytoplasm of parenchyma cells. The virus was not serologically related to the morphologically similar tenuiviruses, citrus psorosis-ringspot virus and tulip mild mottle mosaic virus. A major 43 kDa protein was present in purified preparations and in infected plant tissue, as also was a minor 28 kDa protein, serologically related to the major one. Nucleic acids extracted from purified particles consisted of at least three RNAs, of approximately 7.5, 1.8 and 1.5 kb, which appeared partly in single- and partly in double-stranded form. Purified preparations, but not viral RNAs, when mechanically inoculated, were infectious. Host range, tissue tropism, particle morphology and coat protein size place the virus closest to citrus psorosis-ringspot and tulip mild mottle mosaic viruses. These three viruses in turn show similarities with the Tenuiviruses and Bunyaviridae.

Broad bean wilt virus (BBWV), first isolated from broad bean (Vicia faba) in Australia (Stubbs, 1947), is the type member of the genus Fabavirus. Fabaviruses are aphid-transmitted viruses infecting a wide range of plants worldwide. Cooper (1994) noted that this genus includes a number of poorly characterized isolates with different common names and from many different plant species. We shall try to clarify this rather confused situation. BBWV isolates were divided by Uyemoto and Provvidenti (1974) into two serotypes (I and II). The type strain belongs to serotype I. Nasturtium ringspot virus (NRSV) (Smith, 1950), petunia ringspot virus (PeRSV) (Rubio-Huertos, 1959), parsley virus 3 (PV3) (Frowd and Tomlinson, 1970, 1972), and P.O. pea streak virus (Kim and Hagedorn, 1959) are isolates or strains closely related to BBWV serotype I. BBWV serotype II was first detected in Plantago lanceolata in the United States (Uyemoto and Provvidenti, 1974), and later found in several other plant species. A possible third serotype was reported from Australia (Shukla and Gough, 1983), and an isolate from Cynara scolymus was reported to differ from both recognized serotypes: Migliori (1993) named it artichoke French latent virus (AFLV). Lamium mild mosaic virus (LMMV) (Lovisolo, 1957) was isolated in the United Kingdom from Lamium album and a few other species of Labiatae (Lovisolo, 1957; Lisa et al., 1982), but has not been reported since. It is distantly serologically related to BBWV, but distinct from it, and is regarded as another fabavirus.

Nicotiana benthamiana was transformed with the nucleoprotein (N) gene of an Italian isolate of tomato spotted wilt virus (TSWV). Forty-five T1 primary transformant lines were analyzed for the expression of N protein and for resistance to TSWV and three other tospoviruses: impatiens necrotic spot virus (INSV), groundnut bud necrosis virus (GBNV), and groundnut ringspot virus (GRSV). Thirteen of these lines were further characterized. Resistance to all TSWV isolates tested was found in two lines. The expression of the transgene (N mRNA) was lower in these resistant lines than in any of the susceptible lines, and the transgene N protein was either absent or present below detectable levels. These lines were susceptible to the other tospoviruses tested, but they developed symptoms milder than controls when inoculated with GRSV. Some of the lines producing high levels of N protein showed delays (of 2-3 weeks) in symptom expression with at least one of the TSWV isolates tested and symptom delay or attenuation with INSV or GRSV (or both). From our results it appears that high expression of TSWV N protein retards, in some cases, disease development by TSWV and INSV. In contrast, the lack of detectable expression of the transgenic N protein, accompanied by limited production of N transcripts, conferred TSWV-specific resistance.

Lily mottle virus (LMoV), genus Potyvirus, an important virus of lily that also causes flower-breaking in tulip (1), is considered to have a natural host range restricted to the family Liliaceae. In 1996, escarole (Cichorium endivia L. var. latifolium LAM) plants growing in fields close to Torino, Italy, and showing mosaic and necrotic spots on outer leaves were infected by a potyvirus related to LMoV. The virus was identified by immunoelectron microscopy (IEM) done on experimentally infected Nicotiana benthamiana and Chenopodium quinoa. The virus isolated from escarole (LMoV-E) had an experimental host range similar to that of lily isolates of LMoV, although species within the Liliaceae were not tested. LMoV-E systemically infected all nine escarole cultivars and six of seven endive cultivars (C. endivia L. var. crispum LAM) but did not infect any of six lettuce and two chicory cultivars (C. intybus L. var. foliosum HEGI). Symptoms ranged from mild to severe mosaic and were generally more severe on escarole than on endive. Some of the same escarole, endive, and lettuce cultivars were inoculated with a typical LMoV isolate from lily (Le97/49, from A. F. L. M. Derks, the Netherlands), which induced mild systemic infections in only one escarole and one endive cultivar. Both cultivars were also susceptible to LMoV-E. LMoV-E was purified from N. benthamiana, and an antiserum was prepared. IEM decoration titer experiments were done with LMoV-E and four other LMoV isolates from Liliaceae and their homologous antisera. Heterologous titers ranging from identity to serological differentiation index values of 2 to 4 were obtained, confirming the identity of the escarole isolate as a LMoV strain and indicating considerable serological variability among LMoV isolates. In a field survey of endive and escarole crops in 1998, in the area where LMoV-E was first identified, the virus was again detected by enzyme-linked immunosorbent assay in 4 of 80 escarole plants tested. LMoV-E appears to be a LMoV strain particularly adapted to escarole. To our knowledge, this is the first report of LMoV identified in a naturally infected host outside monocotyledonous plants. Reference: (1) E. L. Dekker et al. J. Gen. Virol. 74:881, 1993.

SUMMARY A virus associated with a mosaic disease in melons in the Ourmia district in northern Iran was mechanically transmissible to a wide range of test plants but was not transmitted using aphids, the whitefly Trialeurodes vaporariorum or the mite Tetranychus urticae. Its rather stable particles somewhat resembled those of both geminiviruses and alfalfa mosaic virus; they were 18·5 nm in mean width, parallel-sided, and of several discrete lengths, 30 nm and 37 nm being the shortest and commonest lengths. Both ends of all particles were sharply triangular in profile. The particles contained linear single-stranded RNA of three sizes, estimated as 0·91,0·35 and 0·32, all × 106 molecular weight, and two coat proteins of 26·3 and 23·3 × 103 estimated molecular weight. The virus, named Ourmia melon virus, appears to be the first described representative of a new virus group.

White clover cryptic viruses (WCCV) 1, 2, and 3 were purified from apparently healthy plants with yields of about 200 μg of virus/kg of tissue. After isopycnic centrifugation in CsCl, WCCV 1 and 2 formed homogeneous bands at densities of 1.392 and 1.375 g/ml, respectively, whereas WCCV 3 was not recovered. Particles of WCCV 1 and 3 were 34 nm in diameter, without obvious morphological subunits, whereas WCCV 2 particles were 38 nm in diameter, with prominent morphological subunits. Antisera with titers up to 1:2048 were obtained against WCCV 1 and 2. Each of the three viruses contained two segments of linear dsRNA ( M r from 1.03 to 1.49 X 10 −6 . Virus-like particles, probably those of WCCV 1 and 2 were detected in low numbers in the cytoplasm of about 1/10 of the parenchyma cells of carrier plants examined by thin sectioning. The particles of WCCV 1 and 2 were not transmitted by grafting or mechanical inoculation, but were seed transmitted. Evidence is given that WCCV 1 and 2 multiply in their carrier plants, and are not viruses of associated parasitic or commensal organisms.

The nucleic acid of carnation cryptic virus has been identified as double-stranded RNA on the basis of (i) susceptibility to RNase in 0.1 × SSC (SSC = 0.15 M NaCl-0.015 M sodium citrate buffer, pH 7.0) and resistance to RNase in 2 × SSC and to DNase, (ii) reaction with an antiserum containing antibodies to dsRNA, and (iii) electron microscopic appearance in nondenaturing media similar to that of the dsRNA of maize rough dwarf virus. Upon electrophoresis in 5% polyacrylamide gels carnation cryptic virus RNA separated into three major segments plus a fourth minor component, and had a total molecular weight of about 4 × 106.

SUMMARY Small isometric virus-like particles were found in low concentration in apparently healthy carnations of the Mediterranean, miniature and Chinese type but not in eleven Sim cultivars tested. Most carnations containing these particles were from Italy but some were from France and the USA. The particles were not transmitted by grafting or by mechanical inoculation but were seed-transmitted to a large proportion of seedlings. Antisera to partially purified particles were obtained. The particles did not react with antisera to twenty-eight isometric plant viruses or virus-like particles but were serologically related to similar particles found in carnations in England, Holland and Israel. When negatively stained, the particles were isometric with a diameter of about 29 nm and a rounded rather than angular profile, but without clear substructure; some particles were penetrated by the stain. The particles remained intact in neutral sodium phosphotungstate. After isopycnic centrifugation in CsCl solution, preparations of particles formed a main band of mean density 1.377 g/ml and other fainter bands that varied in intensity and position in different preparations. In thin sections of carnations, no virus-like particles or cytological abnormalities were observed.

La microscopie électronique de particules purifiées d'un virus trouvé sur D. sanguinalis au Vanuatu a permis de déterminer son appartenance au groupe des geminivirus. Les préparations de particules virales contiennent une protéine de poids moléculaire égal à 27500 et des ADN monocaténaires circulaire et linéaire de 2350 nucléotides. Des particules géminées ont été trouvées dans les coupes fines de cellules infectées à l'intérieur de structures cristallines à la fois dans le noyau et dans le cytoplasme. Ce virus est sérologiquement apparenté au virus de la striure du maïs

Publisher Summary It is interesting that at this relatively late stage in virus research, new kinds of viruses continue to come to light. Cryptic viruses successfully evaded detection until recently because they induce no or, perhaps in some cases, very slight disease symptoms, are not transmissible in the ordinary way, and have particles present in such low concentrations as to escape casual discovery. The fact that cryptic viruses appear to cause no economically important disease has tended to give them low research priority. Nevertheless they are of interest as they contain double-stranded (ds) RNA, present only in another very different group of plant viruses, and appear unable to pass from carrier to non-carrier cells across a graft union. The latter is a decidedly non-virus like property. They are also of practical importance as they may be responsible for misleading results with methods for the detection of single-stranded RNA viruses based on the presence in plants of the double-stranded replicative forms.

Maize rough dwarf virus, a reolike virus, was purified from roots of maize by clarification with the fluorocarbon 1,1,2-trifluoro-1,2,2-trichloroethane (Freon 113) or with carbon tetrachloride followed by sucrose density gradient centrifugation. The product consisted mostly of complete particles, but some subviral particles were present. The virus was spherical, varying from 63 to 70 nm in diameter under different staining conditions; it had a double capsid and typical reovirus structure with, probably, 92 morphological units in the outer capsid. In addition, each particle possessed 12 projecting spikes (A spikes) each some 11 nm long, one at each 5-fold symmetry axis. Various physical or chemical treatments stripped off the A spikes and part of the outer capsid to give a spherical 50–57 nm subviral particle possessing 12 previously hidden spikes (B spikes) each about 8 nm long, coaxial with the A spikes. Each B spike was implanted on a differentiated part of the inner capsid here called a baseplate. Treatment of whole virus or subviral particles with aqueous neutral potassium phosphotungstate produced smooth subviral particles without B spikes, and longer treatment with phosphotungstate or brief treatment with n-butanol also removed the nucleic acid to give ghosts exhibiting internal structures. Detached B spikes were composed of 5 morphological subunits surrounding a central hole. Infectivity of preparations was tested by injecting them into adult females of the planthopper vector Laodelphax striatellus, which were later screened for production duction of virus symptoms on maize and barley. Crude and purified preparations containing the large virus particles were infectious; Freon-treated preparations possessed the highest infectivity. Subviral preparations made by chloroform treatment were not or very slightly infectious and smooth subviral particles made by treatment with phosphotungstate were not infectious.