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Partial sequences of Tomato yellow leaf curl Sardinia virus (TYLCSV) derived from tomato samples collected in Sicily in 1999, 2002 and 2004 indicated the presence of a TYLCSV different from the one previously described as the Sic strain. Here, we report a complete DNA sequence that is classified as belonging to the TYLCSV type strain (Sar strain), confirming the co-existence in Sicily of virus populations of both strains. Moreover, comparisons between this new sequence and those of the two recombinants recently described in Sicily revealed unequivocally (99% identity) that their TYLCSV-derived portion originated from the Sar strain.

Non melanoma skin cancer (NMSC), including basal cell (BCC, 80% of NMSC) and squamous cell carcinoma (SCC, 20%) is the most frequent cancer in Caucasians. Its incidence has dramatically increased in the last years due to sun exposure and immunosuppression: i.e. transplant recipient have a 100-fold and a 10-fold risk increase of developing SCC and BCC, respectively. Sensitive diagnostic techniques have detected a few Human Papillomavirus (HPV) species in lesions of patients affected by these tumours, particularly belonging to the Beta-papillomavirus Genus. This Genus includes viruses with a cutaneous tropism, while high risk HPVs , responsible of almost all cervical cancers in woman, have a mucosal tropism. A few Beta HPVs, such as HPV8 and HPV5, are found associated with skin cancer lesions and are thus considered etiologically related to these pathologies. For preventing mucosal HPV infection, prophylactic vaccines based on the viral L1 major capsid protein, assembled into empty virion like particles are already available. Preventive vaccines targeting non structural viral proteins, such as the oncogenic proteins E6 or E7 are also under study. Instead, for cutaneous HPVs, no vaccine strategies have yet been described. Plants have been proposed as alternative platforms for producing foreign antigens for vaccine production and immunisation purposes. Antigens expressed in plants can be administered in different ways, including direct oral ingestion of plant tissue (edible vaccine). We have recently undertaken a project aimed at obtaining vaccines against cutaneous HPVs, based on the expression in plants of the major L1 capsid and of the E7 protein of HPV 8. To evaluate protein expression in plants tissue, polyclonal antibodies were raised in rabbits against purified GST-L1 and GST-E7 fusion proteins, produced in E. coli. For the expression in plants, 35S-based cassettes for (1) the full length L1 protein, (2) an L1 protein deprived of a putative nuclear localisation signal, (3) the E7 protein and (4) an L1-E7 fusion construct were thus generated and used for both transient and stable expression experiments. For transient expression, infiltration of N. benthamiana leaves with A. tumefaciens carrying the different constructs (agroinfiltration) was carried out. Transgene expression was followed both at the RNA and protein level. Simultaneous infiltration of agrobacteria carrying genes for silencing suppressors, such as p19 from Cymbidium ringspot virus, and/or HC-Pro from Potato virus Y enhanced transgene expression of all constructs. Data concerning the molecular characterisation of N. benthamiana lines transgenic for the L1 and E7 antigens and their derivatives will be presented.

Since autumn 2000 severe and widespread chlorosis, sometimes associated with redness, has been observed in greenhouse tomatoes in different regions of Italy. A total of 104 samples were analysed for Tomato infectious chlorosis virus (TICV), by a one-step RT-PCR procedure. In some areas of central Italy and Sardinia, the symptom was consistently correlated with the presence of TICV. The RT-PCR procedure enabled rapid and reliable detection of TICV from field samples. Sequence analysis of the amplified 501-bp fragment, part of the HSP70 coding region, revealed an identity of 99% with the TICV sequence in the GenBank database. A digoxigenin-labelled DNA probe was also produced and successfully tested in dot blot assays. This is the first report of TICV causing epidemics in Europe.

The sequence of the single-stranded RNA genome of Indian citrus ringspot virus (ICRSV) consists of 7560 nucleotides. It contains six open reading frames (ORFs) which encode putative proteins of 187.3, 25, 12, 6.4, 34 and 23 kDa respectively. ORF1 encodes a polypeptide that contains all the elements of a replicase; ORFs 2, 3 and 4 compose a triple-gene block; ORF5 encodes the capsid protein; the function of ORF6 is unknown. Phylogenetic analysis of the complete genome and each ORF separately, and database searches indicate that ICRSV, though showing some similarities to potexviruses, is significantly different, as in the presence of ORF6, the genome and CP sizes, and particle morphology. These differences favour its inclusion in a new virus genus.

When new transgenic plants are obtained following genetic transformation, the putative transformed lines must be checked for the presence of the new genes and, as soon as possible, the number of integrated copies must be estimated. This is normally achieved by Southern analysis, a time-consuming technique which requires several days and several micrograms of plant DNA. In order to overcome these two major constraints and therefore obtain information on the number of integration events in each line early after regeneration, we have tested a novel approach by using a quantitative Real-Time PCR assay. The rationale of the approach consists in the possibility of estimating the transgene copy number of a new plant line by comparison with another line which contains a known number of integrated copies. Two genes must be quantified in parallel experiments, a plant gene and the transgene. This is achieved using PCR reagents (primers and probe) specific for the plant gene, and others specific for the transgene. The plant material used consisted of tomato lines previously transformed with the N gene of Tomato spotted wilt virus: primers and probe specific for a tomato gene (APX), and for the N transgene were designed. As an alternative to the APX-specific reagents, generic plant reagents (t-Leu) were also tested for the quantification of the plant DNA. Preliminary results showed that discrimination between one and two or more integration events is possible, when accurate data are obtained. To improve the accuracy of the measurements a duplex approach, where the two genes are simultaneously quantified in the same reaction tube, is under investigation and data will be presented. The Real-Time quantitative PCR technique requires much less plant DNA than Southern analysis, and therefore it can be performed early after regeneration, when the availability of plant material is limited. Furthermore, it is completed in a few hours. At the present time, however, it cannot be considered as a complete substitute of Southern blots. This new technique can be used for selecting early and rapidly the lines which are probably more interesting than others in the breeding procedures.

Indian citrus ringspot virus (ICRSV) from a Kinnow mandarin orchard at the IARI, New Delhi, India (isolate K1) has flexuous particles 650 nm long, with clearly visible crossbanding. It can be mechanically transmitted to and multiplied in Phaseolus vulgaris cv. Saxa. The genome consists of one ssRNA of about 7.5 kb and the coat protein (CP) is 34 kd in size. The virus has been purified and an antiserum prepared suitable for DAS ELISA. Partial sequencing of the genome, revealing the CP ORF near the 3' end and further sequences upstream, indicates that ICRSV, while morphologically resembling a capillovirus, has no homology with this genus. Absence of serological relationship confirms this. Short amino acid motifs in the CP sequence reveal limited similarities with the genera Potexvirus, Carlavirus, Foveavirus and Allexivirus , but no strong similarity to any one of these, so that the virus does not fall into any known genus. RNA and cDNA probes that detect K1 are vailable, and also primers suitable for RT-PCR. EM decoration tests with an antiserum to K1 revealed the presence, in a Kagzi Kalan lime-lemon hybrid of a virus (KK) with similar morphology but differing consistently from K1 by one serological step. Moreover, in the original Kinnow orchard, we detected a further virus (K2), again with similar morphology, serologically related to K1 but differing by five steps. K2 now in turn appears, in preliminary results, to be a mixture containing yet a further morphologically similar virus, K3. Thus, Indian citrus ringspot disease may be caused by a series of viruses, and while diagnostic methods are now available for K1, these may not immediately solve the problem of field diagnosis.

A functional capsid protein (CP) is essential for host plant infection and insect transmission in monopartite geminiviruses. We studied two defective genomic DNAs of tomato yellow leaf curl virus (TYLCV), Sic and SicRcv. Sie, cloned from a field-infected tomato, was not infectious, whereas SicRcv, which spontaneously originated from Sie, was infectious but not whitefly transmissible. A single amino acid change in the CP was found to be responsible for restoring infectivity. When the amino acid sequences of the CPs of Sic and SicRcv were compared with that of a closely related wild-type virus (TYLCV-Sar), differences were found in the following positions: 129 (P in Sic and SicRcv, Q in Sar), 134 (Q in Sic and Sar, H in SicRcv) and 152 (E in Sic and SicRcv, D in Sar). We constructed TYLCV-Sar variants containing the eight possible amino acid combinations in those three positions and tested them for infectivity and transmissibility. QQD, QQE, QHD, and QHE had a wild-type phenotype, whereas PHD and PHE were infectious but nontransmissible. PQD and PQE mutants were not infectious; however, they replicated and accumulated CP, but not virions, in Nicotiana benthamiana leaf discs. The Q129P replacement is a nonconservative change, which may drastically alter the secondary structure of the CP and affect its ability to form the capsid. The additional Q134H change, however, appeared to compensate for the structural modification. Sequence comparisons among whitefly-transmitted geminiviruses in terms of the CP region studied showed that combinations other than QQD are present in several cases, but never with a P129.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) which infects species in the families Cucurbitaceae and Solanaceae (Padidam et al., 1995; Mizutani et al., 2011). Begomoviruses are transmitted by the whitefly Bemisia tabaci in a persistent manner (Rosen et al., 2015). In October 2015, severe symptoms not previously reported by growers in the horticultural area of the Province of Trapani (Sicily, Italy) were observed on zucchini squash (Cucurbita pepo) in open fields. The symptoms included yellow mosaic, severe leaf curling, swelling of veins of young leaves, shortening of internodes, roughness of the skin of fruit and reduced fruit size; the symptoms were reminiscent of those caused by begomoviruses. Total DNA was extracted from young leaves of 22 plants by phenol/chloroform extraction and ethanol precipitation. PCR was performed with the A1F/A1R primer pair (Mizutani et al., 2011) for the DNA-A component and the pair described by Ruiz et al. (2015) for the DNA-B component to amplify a ~1200-bp fragment of DNA-A and a ~890 bp fragment of DNA-B, respectively. All 10 samples were positive by PCR with both primer pairs. No amplification products were obtained using primers specific for the monopartite begomoviruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus (Davino et al., 2008). DAS-ELISA analysis for Cucumber mosaic virus, Papaya ring spot virus and Zucchini yellow mosaic virus (Loewe Phytodiagnostica, Germany) yielded negative results. The amplification products of DNA-A and DNA-B components from three plants were purified using the UltraClean® PCR Clean-Up kit (Mo-Bio, USA) and the nucleotide sequences were determined in both orientations using an ABI 3130XL Genetic Analyzer (Life Technologies, USA). The sequences obtained from the three samples showed 99% identity. A Basic Local Alignment Search Tool (BLAST) analysis showed an identity >99% with sequences of DNA-A and DNA-B of Tomato leaf curl New Delhi virus (ToLCNDV) isolates recently reported in Spain on zucchini (GenBank Accession Nos. KF749224 and KF749227) and tomato (KM977733 and KM977734). The DNA-A and DNA-B sequences of one sample were deposited in GenBank (KU145141 and KU145142). Sicily is an important region for horticulture in the Mediterranean basin and Europe in general. With the possible opening of a free trade area in countries bordering the Mediterranean basin, Sicily may become a transit centre for flow of plant materials, potentially carrying new pathogens, between Europe and North Africa. To our knowledge, this is the first report of ToLCNDV in Italy, as well as the first identification of a bipartite begomovirus in Italian crops.

Partial sequences of Tomato yellow leaf curl Sardinia virus (TYLCSV) derived from tomato samples collected in Sicily in 1999, 2002 and 2004 indicated the presence of a TYLCSV different from the one previously described as the Sic strain. Here, we report a complete DNA sequence that is classified as belonging to the TYLCSV type strain (Sar strain), confirming the co-existence in Sicily of virus populations of both strains. Moreover, comparisons between this new sequence and those of the two recombinants recently described in Sicily revealed unequivocally (99% identity) that their TYLCSV-derived portion originated from the Sar strain. © 2010 Springer-Verlag.

Two tomato geminivirus species co-exist in protected crops in Sicily, Tomato yellow leaf curl Sardinia virus (TYLCSV, found in 1989) and Tomato yellow leaf curl virus (TYLCV, found in 2002), and mixed infections have been detected. In a field survey conducted in 2004, the viral intergenic region (IR) was amplified from infected plants, and molecules apparently hybrid between the two species were found, but only in plants where one or both parental species were also present. Two of these hybrids, named 2/2 and 2/5, were isolated and infectious clones were obtained. They were both readily whitefly-transmitted to tomato plants; clone 2/5 produced symptoms typical of TYLCSV and TYLCV, while clone 2/2 produced more severe symptoms, with leaves showing downward curling and rugosity. Sequence analysis showed that both 2/2 and 2/5 are newly generated hybrids, with two recombination sites each. One site, common to both hybrids, is in the stem-loop of the IR. The other is close to the 3'-end of the CP ORF in 2/5 and within the Rep ORF in 2/2. Thus, the 2/2 hybrid virus has a hybrid Rep protein, with the 202 amino-terminal aa from TYLCV and the remaining 155 aa from TYLCSV. Replication assays in leaf disc indicated a lower replicative capacity with respect to parental viruses, a fact that might help to explain why plants infected only by a recombinant have not been found so far.

Tomato yellow leaf curl disease (TYLCD) is well known in Mediterranean countries, where it has been causing severe losses in tomato crops for decades. Until recently, two viruses (with several isolates) in the genus Begomovirus, family Geminiviridae, have been associated with the epidemics: Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV). However, recombinants between these, such as Tomato yellow leaf curl Malaga virus (TYLCMalV), are spreading, and new methods for detecting all viruses present in the region are needed. By considering all DNA sequences available of viruses causing TYLCD in the Mediterranean basin, a PCR/RFLP protocol was developed that amplifies the intergenic region in a multiplex reaction, followed by digestion with AclI (=Psp1406I) restriction enzyme. This procedure generates an easily recognizable pattern on gels, with DNA fragments of specific size for each virus species and each recombinant: 800 bp for TYLCSV, 410 bp for TYLCV, 570 bp for TYLCMalV and the other detected recombinants, 640 bp for hypothetical recombinants of different type. This new method gives, with a single reaction, an overview of the species present in the sample and will be useful for screening the causal agents of TYLCD, as well as in breeding programs for resistance.

Pepino mosaic virus (PepMV), a member of the genus Potexvirus , was first described in 1980 on pepino ( Solanum muricatum ) and was later isolated from different wild species of the genus Solanum (formerly Lycopersicon ) (Soler et al ., 2002). PepMV has been reported in several European countries and in North America, causing disease in tomato. In July 2008, plants of basil ( Ocimum basilicum ) showing interveinal chlorosis on young leaves were observed in three greenhouses in Sicily, in the area where tomato plants were found to be infected by PepMV in 2005 (Davino et al ., 2006). The disease was transmitted mechanically to Nicotiana benthamiana , producing chlorotic spots and leaf deformation. Electron microscopy of negativelystained preparations from basil and N. benthamiana leaves, both with symptoms, revealed the presence of flexuous, filamentous virus particles, typical of potexviruses. Leaves from 100 plants with or without symptoms were analysed by double-antibody sandwich (DAS) ELISA using a polyclonal antibody to PepMV (Loewe Biochemica). The presence of the symptoms was correlated with positive ELISA results. Total RNA was extracted from the same samples using an RNeasy Plant Mini Kit (Qiagen) and analysed by RT-PCR using the PepMV-specific primers PepMV-TGB-F and PepMVUTR-R (Mumford & Metcalfe, 2001). The 840 bp RT-PCR product was

The geminivirus Tomato yellow leaf curl virus (TYLCV) was reported for the first time in Italy in 2002. We have followed its spread in Sicily, where Tomato yellow leaf curl Sardinia virus (TYLCSV), another tomato-infecting geminivirus, is endemic and has been causing severe crop losses since 1989. The presence of the two viruses was monitored in the main tomato growing area, the Ragusa province, analyzing samples with yellow leaf curling symptoms. At first (spring-summer 2002) both viruses were always found in mixed infections, but in 2003 and 2004 18-35% of plants were found infected by TYLCV alone and 8-28% by TYLCSV alone, with 41-69% carrying both viruses. TYLCV has spread quickly in the area, demonstrating, as in other parts of the world, its high virulence and invasiveness; however it has not, so far, completely displaced TYLCSV. An infectious clone of TYLCV from Sicily (TYLCV-IT) was sequenced. The nucleotide sequence was 97% identical to other TYLCV strains of the 'severe' type, found in many countries worldwide.