COVID-19 among healthcare workers in a specialist infectious diseases setting in Naples, Southern Italy: results of a cross-sectional surveillance study.

A coronavirus disease 2019 (COVID-19) surveillance study was performed in March-April 2020 among asymptomatic healthcare workers (HCWs) at a specialist infectious diseases hospital in Naples, Italy. All HCWs underwent two rounds of molecular and serological testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). One hundred and fifteen HCWs were tested; of these, two cases of infection were identified by reverse transcriptase polymerase chain reaction and two HCWs were SARS-CoV-2 immunoglobulin G seropositive. The overall prevalence of current or probable previous infection was 3.4%. The infection rate among HCWs was reasonably low. Most of the infected HCWs had been asymptomatic for the preceding 30 days, which supports the need for periodic screening of HCWs for COVID-19.

Sequence variation of block III segment identifies three distinct lineages within Eggplant mottled dwarf virus isolates from Italy, Spain and Greece.

Partial polymerase (L) gene sequences of 919 nts, including the conserved segments pre-motif A and motif A of block III, of 20 Eggplant mottled dwarf virus (EMDV) isolates were generated, and trimmed sequences of 889 nts, based on the length of available sequences of other isolates, were used to determine phylogenetic relationships. Phylogenetic reconstructions revealed two divergent lineages, designated as genetic group A (Italian isolates) and group B, with the latter further divided into subgroups BI (Greek isolates) and BII (Spanish isolates). No evidence of recombination signals among sequences was detected, whereas analysis of the nonsynonymous/synonymous ratio indicated strong purifying selection, with codons under negative selection uniformly distributed along the sequences. An RT-PCR-RFLP method able to discriminate EMDV isolates of the two main genetic groups was proposed.

First Report of Cucumber mosaic virus Subgroup IA Isolate Infecting Yucca aloifolia in Italy.

Yucca aloifolia L. (Spanish bayonet), family Asparagaceae, is the type species of the genus Yucca. It is native to Mexico and the West Indies and is appreciated worldwide as an ornamental plant. In 2013, during a survey for viruses in ornamental plants in the Campania region of southern Italy, symptoms consisting of bright chlorotic spots and ring spots 1 to 3 mm in diameter with some necrotic streaks were observed on leaves of two plants of Y. aloifolia growing in a nursery located in the Pignataro Maggiore municipality, Caserta Province. Cucumber mosaic virus (CMV) infection was suspected because the symptoms resembled those caused by CMV in Yucca flaccida (1). A range of herbal plant indicators was inoculated with sap extracts of symptomatic Y. aloifolia plants and developed symptoms indicative of CMV. Furthermore, 30 nm isometric virus particles were observed in the same Y. aloifolia sap extracts by transmission electron microscopy. The identity of the virus was confirmed by positive reaction in ELISA tests with CMV polyclonal antisera (Bioreba) conducted on sap extracts of symptomatic Y. aloifolia plants and systemically infected symptomatic hosts (i.e., Nicotiana tabacum, N. glutinosa, Cucumber sativus cv. Marketer, Solanum lycopersicum cv. San Marzano). The presence of CMV in the two naturally infected Y. aloifolia and other mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with the E.Z.N.A. Plant RNA Kit (Omega Bio-Tek), according to the manufacturer's instructions. RT-PCR was carried out with the ImProm-II Reverse Transcription System first-strand synthesis reaction (Promega) using the primer pair CMV1 and CMV2 (2). These primers amplify part of the CP gene and part of the 3'-noncoding region of CMV RNA3 and were designed to produce amplicons of different sizes to distinguish CMV isolates belonging to subgroups I or II (3). RT-PCR products were obtained from both naturally infected Y. aloifolia and mechanically inoculated plants as well as from PAE1 isolate of CMV (2), used as positive control, but not from healthy plants. Based on the length of the amplicons obtained (487 bp), the CMV isolate from Y. aloifolia (named YAL) belonged to subgroup I (3). The amplified RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen), cloned in the pGEMT vector (Promega), and three independent clones were sequenced at MWG (Ebersberg, Germany). Sequences obtained from the two CMV-infected Y. aloifolia plants were identical. This sequence was deposited at GenBank (Accession No. HG965199). Multiple alignments of the YAL sequence with sequences of other CMV isolates using MEGA5 software revealed highest percentage of identity (98.9%) with the isolates Z (AB369269) and SO (AF103992) from Korea and Japan, respectively. Moreover, the YAL isolate was identified as belonging to subgroup IA, based on the presence of only one HpaII restriction site in the 487-bp sequence, as previously proposed (2). Although CMV seems to not be a major threat currently for the production of Y. aloifolia, because the farming of this plant is performed using vegetative propagation, particular attention should be given to the presence of the virus in donor mother plants in order to avoid the dispersion of infected plants that could serve as sources for aphid transmission to other susceptible plant species. To our knowledge, this is the first report of CMV infection of Y. aloifolia in the world. References: (1) I. Bouwen et al. Neth. J. Plant Pathol. 84:175, 1978. (2) G. Parrella and D. Sorrentino. J. Phytopathol. 157:762, 2009. (3) Z. Singh et al. Plant Dis. 79:713, 1995.

First Report of Tomato apical stunt viroid in Tomato in Italy.

During a survey conducted in October 2013 in tomato greenhouses in Diano Marina (Imola Province, northwest Italy), in a single greenhouse, unusual disease symptoms were observed in four out 1,400 (~0.3%) of tomato plants cv. Ingrid, grafted on 'Beaufort' rootstock. Symptoms including shortened apical internodes associated with tiny, deformed, and brittle chlorotic leaves, while ripe fruits appeared reduced in size and pale red. Samples of leaves from the four plants were collected and examined using commercial antisera (Bioreba AG, Reinach, Switzerland) by double antibody sandwich (DAS)-ELISA against Tomato spotted wilt virus, Cucumber mosaic virus, Alfalfa mosaic virus, Tomato/Tobacco mosaic viruses, and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (potygroup test). None of the tested viruses were detected in the four leaf samples. In addition, PCR tests for begomoviruses and phytoplasmas were also negative. In a host range study, the original symptoms, consisting mainly of stunting and chlorosis, were reproduced within ~10 days in tomato seedlings (Momor line), mechanically inoculated at two true leaves stage with sap extract obtained from the four symptomatic tomato plants, whereas no symptoms were observed in Chenopodium amaranticolor, C. quinoa, Nicotiana tabacum (cv. Xanthi nc), N. glutinosa, or Phaseolus vulgaris (cv. Borlotto rosso). Total RNAs extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from symptomatic samples were tested in RT-PCR using pospiviroids generic primers PSTVd-32/33 (1), designed to amplify the whole genome of Potato spindle tuber viroid (PSTVd), Tomato apical stunt viroid (TASVd), and Columnea latent viroid (CLVd), and CEVd-FW/RE primers, designed to amplify the whole genome of Citrus exocortis viroid (CEVd) and TASVd (3). Each of the four samples yielded amplicons of the same size (364 bp) with both primer combinations. The identity of the viroid was then determined by sequencing, on both strands, amplicons obtained from the four symptomatic plants at MWG (Ebersberg, Germany). Sequences obtained were identical, showing the highest nucleotide identity (99.7%) with the TASVd isolate Sj1 (AM777161), identified in Germany on Solanum jasminoides. The sequence was deposited in GenBank (Accession No. HG916812) and the field isolate named To1-IT. Two other cases of pospiviroid infection in tomato in Italy have been reported so far and the viroid species detected were PSTVd (2) and CLVd (4), respectively. To our knowledge, this is the first report of TASVd infecting tomato in Italy. The origin of this infection is still unclear, although based on the biological properties and sequence similarity, the To1-IT isolate probably originated from an ornamental species, most likely S. jasminoides, as recently reported for other tomato TASVd isolates, according to their biological and genetic features (5). References: (1) F. Di Serio. J. Plant Pathol. 89:297, 2007. (2) B. Navarro et al. J. Plant Pathol. 91:723, 2009. (3) N. Önelge. Turk. J. Agric. For. 21:419, 1997. (4) G. Parrella et al. Acta Hortic. 914:149, 2011. (5) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 133:803, 2012.

Dieback and Wilting Caused by Tomato spotted wilt virus in Arctotis × hybrida in Italy.

In winter 2012, some potted plants of African daisy (Arctotis × hybrida L., family Asteraceae) cv. Hannah, propagated by rooted stem cuttings and cultivated for commercial purposes in a greenhouse located at Albenga (Liguria region, Italy), were noticed for a rapid dieback, generalized reddening, following by an irreversible wilting. Around 130 plants on a total of 3,000 cultivated plants showed symptoms (4 to 5%). One gram of fresh leaves, each collected from three different symptomatic plants, was ground in 4 ml of cold (∼5°C) sodium phosphate 0.03 M buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of carborundum (600 mesh). The inoculum was rubbed on healthy indicator herbaceous plants and inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24/18°C day/night. Healthy and buffer inoculated plants were also included in the test and used as negative control in the subsequent serological and molecular analysis. Sap-inoculated plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in Chenopodium amaranticolor and C. quinoa, yellowing and stunting following by systemic necrosis and death of the plants in tomato (Solanum lycopersicum cv. San Marzano), necrotic local lesions following by systemic necrotic patterns and leaf deformation in tobacco (Nicotiana tabacum cv. Xanthi nc.) and N. glutinosa, necrotic local lesions in petunia (Petunia × hybrida cv. Pink Beauty). No symptoms were recorded on buffer inoculated plants. Leaf samples from both symptomatic hosts and the three original symptomatic African daisy plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus (CMV) and tospoviruses (Tospovirus broad-spectrum, Serogroups I, II, and III, Bioreba AG, Switzerland). Positive reaction was obtained with Tospo-groups antibodies, but not with the CMV ones. Total RNA was extracted from infected leaves of African daisy with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to reverse transcription (RT)-PCR by using the tospovirus universal primers BR60/BR65 that amplify part of the nucleocapsid protein gene (1). Target amplicons of 454 bp were produced for all samples tested. The PCR products were cloned and sequenced on both strands (one clone per amplicon cloned). The resulting sequences were 100% identical, so a single sequence was deposited in GenBank (HF913777). The sequence showed highest homology (99%) with the Tomato spotted wilt virus (TSWV) tomato isolate NJ-JN from South Korea (HM581936). The identity of the virus infecting African daisy was further confirmed by sequencing amplicons obtained by RT-PCR using primers partially covering the movement protein gene of TSWV (2). The sequence obtained (HF913776) showed the highest homology (99%) with three TSWV isolates: a tomato isolate from Spain (AY744493), a pepper isolate from South Korea (AB663306), and again the tomato NJ-JN isolate from South Korea (HM581936). To our knowledge, this is the first natural report of TSWV infecting African daisy plants. Moreover, since this ornamental is often cultivated with other flowering plants, it can act as reservoir for the virus that can infect other ornamentals and crops, considering that TSWV has a very broad host range (3). This result also represents the first finding of TSWV in the genus Arctotis, family Asteraceae, the greater botanical family of TSWV hosts (3). References: (1) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (2) M. M. Finetti et al. J. Plant Pathol. 84:145, 2002. (3) G. Parrella et al. J. Plant Pathol. 85:227. 2003.

Araujia sericifera New Host of Alfalfa mosaic virus in Italy.

Araujia sericifera Brot. (Fam. Apocynaceae) is an evergreen climbing plant native of South America, originally introduced in Europe as an ornamental. In spring 2012, virus-like symptoms including bright yellow mosaic of calico-type and leaf distortion were observed in three A. sericifera plants growing in an abandoned field located in Pomigliano d'Arco (Campania region, Italy). Leaves from the three plants were collected and examined using commercial antisera (Bioreba AG, Reinach, Switzerland) by double antibody sandwich (DAS)-ELISA against Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (Potygroup test). Only AMV was detected serologically in the three A. sericifera samples. The virus was mechanically transmitted from the ELISA-positive samples to four plants each of Chenopodium quinoa, C. amaranticolor, tobacco (Nicotiana tabacum cv. Xanthi nc), cowpea (Vigna unguiculata, cv. Black eyes), basil (Ocimum basilicum, cv. Gigante), and tomato (Solanum lycopersicum cv. San Marzano), using chilled 0.03 M sodium phosphate buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). Inoculated plants were kept in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, plants showed the following symptoms after 1 to 3 weeks, consistent with symptoms caused by AMV (1): chlorotic local lesions following by mosaic in C. quinoa and C. amaranticolor, reddish local lesions following by mosaic in cowpea, necrotic local lesions followed by systemic necrosis in tomato, bright yellow mosaic (calico type) in basil, and mosaic and strong deformation of the apical leaves in tobacco. The presence of AMV in ELISA-positive A. sericifera and host plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primers for the coat protein gene (CP) previously used for the molecular characterization of AMV isolates (2). An Italian isolate of AMV from Lavandula stoechas (GenBank Accession No. FN667967) and RNA extracted from a healthy A. sericifera plant were used as positive and negative controls, respectively. An amplicon of the correct predicted size (∼750 bp) was obtained from each of the infected plants assayed, and that derived from A. sericifera isolate Ars2 was purified (QIAqick PCR Purification Kit, Qiagen), cloned in pGEMT easy vector (Promega, Fitchburg, WI) and sequenced (HF570950). Sequence analysis of the CP gene, conducted with MEGA5 software, revealed the highest nucleotide identity of 98% (99% amino acid identity) with the AMV isolate Tef-1 (FR854391), an isolate belonging to subgroup I (3). To our knowledge, this is the first report of AMV infecting A. sericifera in Italy. Since A. sericifera is considered an invasive plant, in continuous expansion to new areas in Italy and in other European countries, particular attention should be paid to the possibility that this species may play a role in the epidemiology of aphid-transmitted viruses such as AMV and CMV, representing a threat to susceptible crops growing nearby. References: (1) G. Marchoux et al. Page 163 in: Virus des Solanacées. Quae éditions, Versailles, 2008. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000. (3) G. Parrella et al. Plant Dis. 96:249, 2012.

First Report of China Rose (Hibiscus rosa-sinensis) as a Host of Alfalfa mosaic virus in Spain.

China rose (Hibiscus rosa-sinensis L.) is an ornamental plant grown throughout the tropics and subtropics. In June 2011, a China rose plant (sample CV-1) showing bright yellow "aucuba"-type mosaic, mainly at the center of the leaves, was found in a public garden in Caleta de Vélez (Málaga Province, southern Spain). Electron microscope examination of negatively stained preparations from the symptomatic plant revealed the presence of semispherical to bacilliform virus-like particles of 30 to 56 × 16 nm. Sap extracts also reacted positively in double-antibody sandwich (DAS)-ELISA to antiserum against Alfalfa mosaic virus (AMV) (Bioreba AG, Reinach, Switzerland). RNA of this sample was extracted with the RNeasy Kit (Qiagen, Valencia, CA) and tested by reverse-transcription (RT)-PCR with AMV specific primers (2), using AMV and GoTaq Master Mixes (Promega, Madison, WI) for cDNA synthesis and amplification, respectively. After cloning and sequencing, the ~750-bp DNA fragment was confirmed as the coat protein (CP) gene of AMV (GenBank Accession No. HE591387) with the highest nucleotide identity of 96% to AMV isolates belonging to subgroup IIA (e.g., GenBank Accession No. AJ130707). Sap from affected leaves of sample CV-1 was mechanically inoculated onto herbaceous indicator plants (Chenopodium amaranticolor, C. quinoa, and Ocimum basilicum). Both Chenopodium species developed chlorotic local lesions followed by mosaic within 3 days after inoculation, and O. basilicum showed bright yellow mosaic of calico type 2 weeks postinoculation. These symptoms are consistent with those reported for AMV in these hosts (1). Virus infection in the inoculated plants was confirmed by DAS-ELISA and RT-PCR. To gain insight on the prevalence and genetic variability of AMV in China rose, a survey was carried out in nearby locations in the provinces of Málaga (14 samples from Torre del Mar and 5 samples from Rincón de la Victoria) and Granada (12 samples from La Herradura). Leaf samples were analyzed by tissue blot hybridization with an AMV-specific digoxigenin-labeled RNA probe obtained from the RNA 1 of the Spanish isolate Tec1 (3), and only two samples from Torre del Mar tested positive. One of these samples (TM-2) was used to amplify by RT-PCR the AMV CP gene that was cloned and sequenced (GenBank Accession No. HE591386). The highest nucleotide identity of the TM-2 CP gene (98%) was with the subgroup IIB Spanish isolate Tec1, whereas identity with the CV-1 isolate was 95%. Nevertheless, phylogenetic analysis (neighbor-joining method) showed that both CV-1 and TM-2 isolates belong to the recently proposed AMV subgroup IIB (3), which includes the Tec1 isolate and two other isolates from ornamental plants, Phlox paniculata from the United States (GenBank Accession No. DQ124429) and Viburnum lucidum from Spain (GenBank Accession No. EF427449). These results show that AMV subgroup IIB is emerging as a complex cluster of virus isolates that currently are reported to infect only ornamentals. To our knowledge, this is the first report of AMV naturally occurring in China rose. References: (1) G. Marchoux et al. Page 163 in: Virus des Solanacées. Quae éditions, Versailles, 2008. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000. (3) G. Parrella et al. Arch. Virol. 156:1049, 2011.

First Record of Alfalfa mosaic virus in Teucrium fruticans in Italy.

The Teucrium genus (Lamiaceae family) contains ~300 species of evergreen and deciduous shrubs with some species widely used as ornamental plants in rock gardens. During the springs of 2010 and 2011, some plants of Teucrium fruticans L., also known as "tree germander", growing singly in pots in a Ligurian nursery (Savona Province, northern Italy), were noted for a bright yellow calico mosaic on the leaves (~1% of ~2,000 plants inspected exhibited symptoms). Preliminary electron microscope observations of leaf-dips showed semispherical to bacilliform particles, consistent with Alfamovirus and Oleavirus, in preparations obtained only from leaves of symptomatic plants. Three symptomatic and two asymptomatic plants were checked for Cucumber mosaic virus or Alfalfa mosaic virus (AMV) in protein A sandwich (PAS)-ELISA with commercial kits (Bioreba, Reinach, Switzerland) and Olive latent virus 2 (OLV2) by immunodecoration of virus particles with an OLV2 antiserum produced against an Italian OLV2 isolate. Symptomatic plants were positive only to AMV and all asymptomatic plants were negative to all viruses checked. The virus was successfully transmitted mechanically to Chenopodium amaranticolor and Ocimum basilicum that reacted, as expected for infections caused by AMV (1), with a chlorotic local lesion followed by mosaic and bright yellow mosaic, respectively. The disease was transmitted also by grafting an infected scion on healthy T. fruticans. Symptoms appeared after ~3 weeks in one plant of six grafted. AMV infection in a symptomatic grafted plant was verified by PAS-ELISA, confirming that bright yellow mosaic symptoms observed in T. fruticans were induced by an isolate of AMV. Immunocapture reverse transcription (IC-RT)-PCR assay, following the protocol described by Wetzel et al. (4), was performed on leaf extracts from one symptomatic plant using a polyclonal serum raised against a French isolate of AMV, provided by H. Lot (INRA, Station de Pathologie Végétale, Avignon, France). Specific AMV primer pair was used in the RT-PCR reactions (2). A DNA fragment of ~750 bp, covering the entire coat protein gene (CP), was obtained after IC-RT-PCR. The amplicon was gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI), cloned into pGEMT-easy vector (Promega) and two independent clones sequenced on both strands at MWG Biotech (Ebersberg, Germany). The consensus sequence was submitted to EMBL (No. FR854391). Pairwise comparison of the AMV-T. fruticans isolate CP sequence (named Tef-1) with those of AMV reference isolates revealed the maximum (98.0 to 97.3%) nucleotide identities with isolates belonging to subgroup I, 95.5 to 94.0% identities with subgroup IIA isolates, and 95.6% identity with the subgroup IIB isolate Tec-1 (3). Among subgroup I isolates, Tef-1 had the maximum CP nucleotide identity with the CP gene belonging to an AMV isolate identified in 2010 in Lavandula stoechas in the same geographic area, suggesting a common origin for these two viral isolates. Overall results clearly indicate that an AMV isolate was the causal agent of the calico-type mosaic observed in T. fruticans. To our knowledge, this is the first report of T. fruticans as a natural host of AMV. References: (1) G. Marchoux et al. Page 163 in: Virus des Solanacées. Quae éditions, Versailles, 2008. (2) G. +Parrella et al. Arch. Virol. 145:2659, 2000. (3) G. Parrella et al. Arch. Virol. 156:1049, 2011. (4) T. Wetzel et al. J. Virol. Methods 39:27, 1992.

First Record and Complete Nucleotide Sequence of Alfalfa mosaic virus from Lavandula stoechas in Italy.

During spring 2009, lavender plants (Lavandula stoechas L.) showing a bright yellow mosaic of calico type and light stunting were observed in a commercial nursery in Liguria Province in northern Italy. Of 300 plants inspected, ~2% were symptomatic. Preliminary observations of leaf sap with the transmission electron microscope revealed bacilliform virus-like particles in three symptomatic plants, whereas no virus-like particles were observed in asymptomatic plants. The same symptomatic plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus, and Alfalfa mosaic virus (AMV). All three plants reacted positively against AMV antibodies, but not the other antibodies. A crude sap extract obtained from a single symptomatic plant, hereafter referred to as the Lst isolate, was prepared by macerating 1 g of fresh leaves in 4 ml of sodium phosphate 0.03 M, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). Sap extract was mechanically inoculated onto a set of herbaceous hosts. Inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in bean (Phaseolus vulgaris L., cv. Borlotto) and cowpea (Vigna unguiculata L., cv. Black eye); necrotic local lesions followed by systemic necrosis in broad bean (Vicia faba L., cv. Super Simonia) and tomato (Solanum lycopersicum L., cv. San Marzano); and bright yellow mosaic (calico type) in basil (Ocimum basilicum L., cv. Gigante). To sequence the entire genome of the Lst isolate, total RNA was extracted from infected samples with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to AMV-specific reverse transcription-PCR by using four primer pairs for each genomic RNA of overlapping oligonucleotides according to the complete genome sequence of AMV 425L isolate (GenBank No. L00163 for RNA1, X01572 for RNA2, and K03542 for RNA3). The 5'- and 3'-terminals regions of each RNA were amplified with the strategy described by Lozano et al. (1) and specific AMV oligonucleotides designed for the corresponding viral RNA. The complete genome of the AMV-Lst isolate comprised 3,643 nucleotides for RNA1 (No. FN667965), 2,593 nucleotides for RNA2 (No. FN667966), and 2,038 nucleotides for RNA3 (No. FN667967). Comparative sequence analyses revealed that the AMV-Lst isolate from Italy shared overall nucleotide sequence identities with the AMV isolate 425L of 97.1, 95.5, and 94.7% for RNA1, 2, and 3, respectively. P1 and P2 replicase genes and the movement protein and coat protein (CP) genes of AMV-Lst isolate showed, respectively, 97.2, 95.1, 96.2, and 97.8% identity with those from the 425L isolate. The AMV-Lst CP gene was shorter by nine nucleotides compared with the CP gene of 425L. A phylogenetic tree, obtained with neighbor-joining and maximum likelihood methods, showed that the Lst isolate grouped within subgroup I of AMV isolates confirmed that the differences between subgroups I and II correlate mainly with the geographic origin of isolates (2). Lst represents the first Italian isolate of AMV completely sequenced, and to our knowledge, this is the first report of this virus in L. stoechas. References: (1) G. Lozano et al. Arch. Virol. 151:581, 2006. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000.

Isolated laryngeal leishmaniasis in an immunocompetent patient: a case report.

Visceral leishmaniasis, a protozoan disease caused by Leishmania infantum, is endemic in the Mediterranean basin, especially southern and Tyrrhenian Italy. Its aetiological agent can also sporadically cause isolated laryngeal localization in at-risk patients (i.e., heavy smokers, immunocompromised patients). This rare localization is often pauci-symptomatic and thus can easily escape diagnosis. A case of isolated leishmaniasis limited to the left vocal cord in an immunocompetent Italian male without significant risk factors, randomly discovered upon histological examination, is described herein. We inquire how many patients affected by non-specific symptoms such as dysphonia and live in countries where Leishmania infantum infection is reported, could be truly affected by Leishmania spp infection.

Development of improved molecular methods for the detection of deformed wing virus (DWV) in honeybees (Apis mellifera L.) and mites ( Varroa destructor Oud.).

A simple and rapid method for the extraction of total nucleic acid from honeybee and mite, useful either as template for RT-PCR or in nucleic acids hybridization, was developed. Sensitivity of the methods were evaluated up to 10(9) and 10(6) dilution of TNAs extracted from a single honeybee, for reverse transcriptase polymerase chain reaction and molecular hybridization respectively. The two diagnostic methods developed could be useful for the study of the molecular biology and the pathology of DWV. For practical applications dot-blot hybridization could be used in order to study the incidence of DWV in honeybees populations. The method is enough sensitive, rapid and less affected by contamination problems compared to RT-PCR and thus it could be applied to the sanitary certification of honeybees and their products.

Typing of tomato yellow leaf curl viruses and their vector in Italy.

A molecular survey of TYLCV/TYLCSV and their associated vector Bemisia tabaci, was performed during 2004-2005 in five regions of Southern Italy: i.e. Sardinia (one locations), Sicily (one location), Calabria (three locations), Campania (two locations) and Basilicata (one location). A total of 71 tomato samples were checked for virus infection and for the presence of the vector. Degenerate primers allowing the amplification of the coat protein gene of both TYLCSV and TYLCV isolates were designed. PCR fragments were then digested with restriction endonuclease Ava II, which was expected to cut TYLCSV differently from TYLCV. Results clearly suggested that in all the inspected Italian regions the two viruses are widespread and present in single plant both alone and in mixed infections. The identity of the two viruses was confirmed by total or partial sequencing of field isolates. Concerning the populations of the B. tabaci associated with TYLCD epidemics, the molecular characterization of COI gene (citocrome oxidase I) indicated that Q biotype was the most prevalent biotype. This fact might be the result of the large use of some insecticides against which Q biotype populations easily develop resistances, as already confirmed in some countries of Mediterranean basin.

Detection and characterization of tobacco mild green mosaic virus (TMGMV) large type isolate from trailing petunia in France.

During 2003 and 2004, unusual viral symptoms were observed on Surfinia trailing petunias in protected cultivations of Southern France. Symptoms consisted in yellow mosaic and distortion of the leaves accompanied by vein necrosis in some samples. The flowers were deformed and showed light colour break of the petals. Electron microscope observation of negatively stained leaf-dip from symptomatic leaves showed straight rod-shaped virus particles of about 300 nm in length. Sap extracts reacted in double-immunodiffusion tests by forming weak precipitin bands with antisera against Tomato mosaic virus (ToMV) and Tobacco mild green mosaic virus (TMGMV). However, symptoms developed on host range after mechanical inoculation suggested that ToMV was not involved in the disease. By using specific primer pairs designed to amplify the coat protein (CP) genes of ToMV and TMGMV in reverse transcriptase-polymerase chain reaction (RT-PCR), expected amplicon was obtained only with TMGMV primer pair. The identity of the virus was also confirmed by using a specific TMGMV riboprobe in dot-blot hybridization assays of symptomatic leaf extracts. The nucleotide sequence of TMGMV CP of the isolate from trailing petunia, named TMGMV-Pt, was determined and compared with those available from EMBL. The percentage of nucleotide identity was 97-98% compared with those of other isolates. Further molecular and biological characterization revealed that TMGMV-Pt belonging to the large type group of TMGMV isolates. In fact, the 3' UTR region of TMGMV-Pt consisted of 360 nucleotides, comprising of a 147 base repeat, as reported only for TMGMV large type isolates. Moreover, symptoms development observed on a differentially host range, used to distinguish between large type and small type isolates, confirmed that TMGMV-Pt belonging to the large type group of isolates. Only one commercial variety of trailing petunia out of 12 tested remained symptomless after mechanical inoculation with TMGMV-Pt. This highlights the potential risk that TMGMV could represent to petunia cultivations. To our knowledge this is the first report of a natural infection by TMGMV in trailing petunia.

Potato virus Y CFH, a putative recombinant isolate from Capsicum chinense cv. Habanero.

Ornamental plants of Chili pepper, Capsicum chinense cv. Habanero, with symptoms of leaf mosaic, necrotic rings on fruits and necrotic stems were observed in June 2003 in a private garden in the province of Naples (Italy). Preliminary serological characterisation allowed the association of these symptoms with infections by Potato virus Y (PVY). The virus was isolated on Nicotiana tabacum cv. Xanthi and characterised by mechanical inoculation on herbaceous hosts and molecular characterisation of the P1 and the coat protein (CP) genes. Symptoms produced on indicator plants were generally consistent with those described for PVY. The identity of PVY was further confirmed by reaction with PVYN, PVYC and PVYO specific monoclonal antibodies: the isolate reacted only with the PVYC specific Mab. Immuno capture reverse transcription polymerase chain reaction (IC-RT-PCR) was performed on extracts of PVY-CFH infected N. tabacum cv. Xanthi plants, using two couples of primers specifically designed out of the P1 and the CP coding regions of the so far fully sequenced PVY isolates. PCR products were then cloned into pCRII-TOPO vector using TOPO-TA cloning kit (Invitrogen) and sequenced. Sequence analysis suggests that PVY-CFH originated from a recombination event involving a virus of the PVYO type and another parental virus, maybe resembling the PVYNP isolates, given the reasonably high similarity shared by PVY-CFH and, respectively, non potato PVY isolates in the CP coding region, PVYO isolates in the P1 coding region. Evidence for the existence of such a recombination comes, apart from similarity analysis, by the different locations of CFH within phylogenetic trees constructed from P1 and CP genomic regions.

Recessive resistance genes against potyviruses are localized in colinear genomic regions of the tomato ( Lycopersicon spp.) and pepper ( Capsicum spp.) genomes.

Resistance against both Potato virus Y (PVY) and Tobacco etch virus (TEV) was identified in the wild tomato relative Lycopersicon hirsutum PI247087. Analysis of the segregation ratio in F(2)/F(3) and BC(1) interspecific progenies indicated that a single recessive gene, or two very tightly linked recessive loci, are involved in resistance to both potyviruses. This locus was named pot-1. Using amplified fragment length polymorphism markers and a set of L. hirsutum introgression lines, pot-1 was mapped to the short arm of tomato chromosome 3, in the vicinity of the recessive py-1 locus for resistance to corky root rot. Because of the occurrence of phenotypically similar genes in pepper ( Capsicum spp.), the comparative genetics of resistance to potyviruses between tomato and pepper was investigated. Unlike most of the comparative genetic studies on resistance genes, pot-1 was tightly flanked by the same restriction fragment length polymorphism (RFLP) markers than the pvr2/pvr5 locus for resistance to PVY and TEV from pepper. These results may indicate that recessive resistance genes against potyviruses evolve less rapidly than the majority of the dominant genes cloned so far, and consequently may belong to a different family of resistance genes.

First Report of an Isolate of Pelargonium zonate spot virus in Commercial Glasshouse Tomato Crops in Southeastern France.

In summer 2000, symptoms similar to Pelargonium zonate spot virus (PZSV) were observed for the first time on tomato plants in southeastern France. The plants were from commercial glasshouse fresh-market crops. Symptoms observed were chlorotic mottling with bright yellow distinct rings on leaves and curved line patterns on stems. Fruit symptoms included chlorotic and necrotic spotting, marked concentric ring patterns, and distortions. Diagnosis was made from symptomatic leaves and fruits by mechanical inoculation on a set of host plants. Local chlorotic and necrotic lesions were observed on Chenopodium amaranticolor, C. quinoa, Cucumis sativus cv. Marketer, Cucumis melo cv. Vedrantais, Phaseolus vulgaris cv. Pinto, Vicia faba cv. D'Aguadulce, Vigna unguiculata cv. Black Eye, and systemic symptoms were observed on Capsicum annuum cvs. Yolo Wonder, Yolo Y, Florida VR2, and Criollo de Morelos 334, Datura stramonium, Lycopersicon esculentum cvs. Momor and Stevens, L. hirsutum (PI 134417 and PI 247087), Nicotiana benthamiana, N. clevelandii, N. tabacum cv. Xanthi nc, Ocimum basilicum cv. Latino, Petunia hybrida cv. Rose du ciel, and Physalis floridana. No reaction was observed on Pisum sativum cv. Douce Provence, Salvia splendens cv. Etna, or Zinnia elegans cv. Liliput. Symptoms on tomato of PZSV, Parietaria mottle virus (PMoV), and Tomato spotted wilt virus (TSWV) are similar, particularly those elicited in fruits. Therefore, the field samples were checked using double-antibody sandwich enzyme-linked immunosorbent assay against antisera of the type-strain of PZSV and tomato strain of PMoV and their homologous antigenes, which were supplied by D. Gallitelli and P. Roggero respectively, and our antiserum of TSWV. Electron microscopy of negatively stained preparations from leaves of tomato and D. stramonium showed that the sap contained very few paraspheric shaped particles, 26 to 29 nm in diameter. Three isolates collected from two different regions (Vaucluse and Bouches du Rhône) showed a very close serological relationship with the Italian type-strain of PZSV and tested negative against antisera of PMoV and TSWV. The French isolates were biologically different from the type-strain, but were similar to the Spanish strain of PZSV because they infected D. stramonium, N. benthamiana, O. basilicum, and V. unguiculata (2). Moreover, in transverse tissue sections, virions were not observed in the nucleus and tubular structures, unlike the Italian isolates, (1) but were present in the cytoplasm and particularly in the mesophyll cells. There are only a few records of the occurrence and distribution of PZSV in Mediterranean countries. References: (1) M. A Castellano and G. P Martelli. Phytopathol. Mediterr. 20:64, 1981. (2) M. Luis-Arteaga. Plant Dis. 84:807, 2000.

Functional expression of the gene cu, encoding the phytotoxic hydrophobin cerato-ulmin, enables Ophiostoma quercus, a nonpathogen on elm, to cause symptoms of Dutch elm disease.

We studied the involvement of the phytotoxic hydrophobin cerato-ulmin (CU) in pathogenesis and virulence of Dutch elm disease (DED) by expressing its encoding gene (cu) in Ophiostoma quercus, a nonpathogenic species on elm closely related to the DED pathogens O. ulmi and O. novo-ulmi. The production of the toxin was quantitatively determined in culture filtrates and in mycelial extracts of the transformants. Production of CU in vitro was associated with the ability to cause typical DED symptoms, consisting of foliar yellow and wilting and vascular tissue discoloration on a moderately resistant elm genotype. The presence of CU was monitored by enzyme-linked immunosorbent assay in symptomatic leaves of plants inoculated with O. quercus transformants expressing CU and found to be associated with wilt symptoms. In general, the virulence of the cu-expressing transformants, as measured in terms of vascular discoloration and percentage of defoliation, was lower than that of the mildly pathogenic isolate E2 of O. ulmi. However, one transformant (C39) displayed a virulence level intermediate between that of E2 and 182, a highly virulent isolate of O. novo-ulmi. Our results indicate that CU production influences virulence in nonaggressive strains of Ophiostoma fungi.

Evidence for two distinct subgroups of alfalfa mosaic virus (AMV) from france and italy and their relationships with other AMV strains Brief report.

The nucleotide sequence of the putative coat protein open reading frame of seven previously uncharacterized AMV strains from Italy and France was determined and aligned with comparable sequences of other AMV strains (425 L, 425 M, YSMV, S, VRU, 15/64 and Da). The data set of AMV sequences was used to determine phylogenetic relationships by both a stochastic (stationary Markov model) and a deterministic method (maximum-parsimony) of analysis. The topology of the trees obtained with the two methods was essentially the same showing that all AMV strains clustered in two monophyletic groups. Close clustering of Italian strains in subgroup I and of French strains in subgroup II seems to suggests the effect of geographic distinctiveness of evolutionary dynamics of these AMV strains. This separation did not correlate with differences in host range or symptoms (necrotic or non necrotic) induced in tomato but rather it reflected variations in the amino acid sequence of their CP, which might be related to structural properties of virus particles. A simple and rapid procedure based on the reverse transcriptase-polymerase chain reaction (RT-PCR) followed by ezymatic digestion (RFLP) was developed to identify and classify AMV isolates into the two subgroups. The method applied to a number of other AMV isolates from Italy and France supported their division in two distinct subgroups. This RT-PCR RFLP method may be useful way to investigate the dynamics of AMV populations in nature.

[Thrombocytopenia with bilateral absence of the radius (TAR syndrome). Reports of 2 clinical cases]. / Trombocitopenia con assenza bilaterale del radio (sindrome TAR). Osservazione di due casi clinici.

Thrombocytopenia with bilateral absent radii (TAR syndrome) is a hereditary condition with a recessive autosomic character. It is characterised by a series of hematological, skeletal and cardiac alterations, and also by various clinical manifestations. The frequency of this syndrome is very low. During the first months of life, prognosis is related to the severity of hemorrhage, but it improves after two years. Diagnosis is at times complex owing to the similarity between this and other syndromes. Therapy, especially during the first year of life, is based on blood transfusion. The paper describes clinical findings in two subjects admitted to the Istituto di Clinica Pediatrica. The two cases presented diverse degrees of hematological and skeletal involvement and followed different clinical evolutions.