Identification of an RNA-silencing suppressor in the genome of Grapevine virus A.

Higher plants use post-transcriptional gene silencing (PTGS), an RNA-degradation system, as a defence mechanism against viral infections. To counteract this, plant viruses encode and express PTGS suppressor proteins. Four of the five proteins encoded by the Grapevine virus A (GVA) genome were screened using a green fluorescent protein (GFP)-based transient expression assay, and the expression product of ORF5 (protein p10) was identified as a suppressor of silencing. ORF5 p10 suppressed local and systemic silencing induced by a transiently expressed single-stranded sense RNA. This protein was active towards both a transgene and exogenous GFP mRNAs. Ectopic expression of GVA-ORF5 by a Potato virus X vector enhanced symptom severity. The findings that p10 markedly reduces the levels of small interfering RNAs (siRNAs) and that the recombinant protein is able to bind single-stranded and double-stranded forms of siRNAs and microRNAs, suggest the existence of a potential mechanism of suppression based on RNA sequestering.

Gastric mucinous adenocarcinoma with cutaneous metastases in a dog: diagnosis by fine-needle aspiration cytology.

A 12-year-old, intact, male mixed-breed dog was presented with anorexia, vomiting and multiple cutaneous nodules on its neck, trunk and hindlimbs. Fine-needle aspiration cytology of the nodules was characterised by a pleomorphic population of cells arranged singly or in small cohesive clusters, embedded in an amorphous mucinous material stained positive by periodic acid-Schiff (PAS). Acinar structures were occasionally found. Cells appeared either small with scant basophilic cytoplasm or large with a histiocytic appearance. Large cells had cytoplasm filled with a PAS-positive granular material. A presumptive diagnosis of cutaneous metastases of a mucinous adenocarcinoma was made. A primary, gastric, signet-ring mucinous adenocarcinoma was confirmed at postmortem examination and by histopathology. To the authors' knowledge, this is the first report of a gastric mucinous adenocarcinoma with cutaneous disseminated metastases in a dog.

Isolation of recombinant antibodies (scFvs) to grapevine virus B.

A panel of 15 recombinant single chain antibodies (scFv) specific to grapevine virus B (GVB) were recovered from a human combinatorial scFv antibody library using the phage display technique against purified virus particles. Two selected scFv-encoding genes were expressed in recombinant Escherichia coli cells as dimeric antibodies. Successful detection of GVB in tissues of herbaceous hosts and grapevine was obtained in a direct binding assay using dimeric scFvs. This reagent was also shown to substitute efficiently for a GVB polyclonal serum in standard DAS-ELISA test used routinely for diagnosis.

Cytological features of canine ovarian tumours: a retrospective study of 19 cases.

The cytological features of 19 histologically confirmed canine ovarian tumours were retrospectively examined. Seven cases were cytologically classified as papillary adenocarcinoma, eight cases as granulosa cell tumours, two cases as mature ovarian teratomas, one case as a dysgerminoma and one case as a mixed granulosa cell tumour/dysgerminoma. On cytology, papillary adenocarcinoma was characterised by a papillary glandular pattern and tight cohesiveness. Granulosa cell tumours showed monolayered clusters of loosely cohesive granulosa cells. Call-Exner-like bodies were found in five of seven cases. Granulosa cells appeared to be heterogeneous and usually contained several intracytoplasmic vacuoles. Teratoma was characterised cytologically by keratin debris (two cases) and a mixture of epithelial cells with sebaceous, basaloid, columnar/palisading or ciliated appearance (one case). The dysgerminoma contained severely atypical round cells admixed with small lymphocytes. The mixed dysgerminoma/granulosa cell tumour had a mixture of germinal and granulosa cells. Cytological diagnosis was in agreement with histopathology in 18 of 19 (94.7 per cent) cases.

Epitope mapping of Grapevine virus A capsid protein.

Previously characterized monoclonal antibodies (MAbs) to Grapevine virus A (GVA) showed a differential reactivity against intact or partially destabilized virus particles [2]. In the present study, this differential reactivity was confirmed and several peptides reacting with a panel of four different antibodies were identified by the PEPSCAN method of epitope mapping. Oligopeptide sequences comprised between coat protein residues 61 (V) and 72 (T) were recognized by all the antibodies tested. One of these peptides (VGPKASK) was also reactive when expressed on recombinant phage particles as a fusion protein with protein pVIII. The specificity of this sequence for antibody binding was also demonstrated by competitive-ELISA using one of the GVA MAbs. The results of this study suggest that GVA particles carry a highly structured epitope centered on a common peptide region of the coat protein sequence.

Induction of S phase and apoptosis by the human papillomavirus type 16 E7 protein are separable events in immortalized rodent fibroblasts.

The HPV16 E7 oncoprotein neutralizes several cell cycle checkpoints, favouring the entry of quiescent cells into S phase. This activity is mediated in part by association of E7 with the pocket proteins and consequent activation of E2F transcription factors. In addition, HPV16 E7 protein is able to promote apoptosis. In this study we demonstrate that the ability to induce apoptosis is a common property of E7s belonging to both benign and malignant HPV types. The E7-induced apoptosis is mediated by inactivation of pRb, whilst neutralization of the other two pRB-related proteins, p107 and 130, is not sufficient to trigger apoptosis. Moreover, we show that certain point mutations in the conserved region 1 (CR1) of HPV16 E7 abolish the induction of apoptosis without altering the ability to stimulate S phase. Thus, these two E7-mediated cellular events, apoptosis and S phase entry, can be separated in immortalized rodent fibroblasts. Our findings demonstrate that the E7-mediated pRb destabilization is not required for its ability to drive quiescent cells into S phase and to induce apoptosis. Finally, expression of E7 proteins in NIH3T3, which lack a functional p19ARF, does not lead to p53 accumulation, indicating that the E7 impacts upon additional cellular pathways to promote apoptosis.

Infectious cDNA clones of two grapevine viruses.

Full-length cDNA copies of the genomes of Grapevine virus A (GVA) and Grapevine virus B (GVB) under the control of bacteriophage T7 promoter have been synthesized, which were refractory to cloning in Escherichia coli. However, both transcribed cDNAs were infectious when mechanically inoculated to Nicotiana plants. A full-length cDNA copy of GVB was engineered in pCass2, a plasmid containing a partially duplicated copy of the Ca35S promoter, but was rather unstable in Escherichia coli. No infection of Nicotiana plants was obtained following mechanical inoculation but detached Nicotiana leaves, inoculated by particle bombardment, supported the multiplication of a GVB isolate seemingly identical to the wild-type used for cloning. Nicotiana seedling inoculated with sap expressed from these leaves became infected showing typical GVB symptoms. Transient transcription of Ca35S driven cDNA clones was also detected by RT-PCR in leaves of the grapevine hybrid LN33 following inoculation by particle bombardment. The availability of infectious cDNA clones of GVA and GVB constitutes a tool for the study of genome expression and pathogenesis, and for the ultimate establishment of the aetiological role of these viruses.

The nucleotide sequence of RNA1 and RNA2 of olive latent virus 2 and its relationships in the family Bromoviridae.

The complete nucleotide sequence of RNA1 and RNA2 of olive latent virus 2 (OLV-2), a virus with quasi-spherical to bacilliform particles and a non-polyadenylated tripartite ssRNA genome, was determined. RNA1 consists of 3126 nucleotides and contains a single open reading frame (ORF) coding for a polypeptide with a molecular mass of 102689 Da (p1a). RNA2 is also a monocistronic molecule, 2734 nt in length, coding for a polypeptide with a molecular mass of 90631 Da (p2a). The translation products of RNA1 and RNA2 possess the motifs proper to helicase, methyltransferase (RNA1) and RNA polymerase (RNA2), suggesting that both are involved in the replication of the viral RNA. The similarities found between OLV-2 and members of the Bromoviridae in some properties and in the sequences of all genomic products (including p1a and p2a) are strongly indicative that it belongs in this family. OLV-2, however, did not show a direct relationship with any of the current genera in the family. Rather, it revealed homologies in diverging directions with one or other of the Bromoviridae genus, thus qualifying as the possible representative of a new taxon in this family.

Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease.

The gram negative, microaerophilic bacterium Helicobacter pylori colonizes the human gastric mucosa and establishes a chronic infection that is tightly associated with atrophic gastritis, peptic ulcer, and gastric carcinoma. Cloning of the H. pylori cytotoxin gene shows that the protein is synthesized as a 140-kD precursor that is processed to a 94-kD fully active toxin. Oral administration to mice of the purified 94-kD protein caused ulceration and gastric lesions that bear some similarities to the pathology observed in humans. The cloning of the cytotoxin gene and the development of a mouse model of human gastric disease will provide the basis for the understanding of H. pylori pathogenesis and the development of therapeutics and vaccines.