Recombinant Semliki Forest virus vector exhibits potential for avian virus vaccine development.

The Semliki Forest virus (SFV) expression system was evaluated as a basis for avian vaccine development. Initial studies indicated that 1-day-old specific pathogen-free (SPF) chicks were susceptible to infection with an infectious strain of SFV, producing SFV-specific antibodies but no clinical disease. One-day-old SPF chicks immunised intramuscularly with recombinant replication-defective SFV (rSFV) particles expressing the Escherichia coli (E. coli) lacZ reporter gene developed high titres of beta-gal- specific antibodies at 4 weeks p.i. after two inoculations. In contrast, significantly lower antibody levels were elicited in chicks immunised with a recombinant SFV-based DNA construct or a conventional CMV promoter-based DNA plasmid. rSFV particles encoding the protective VP2 protein or the VP2/VP4/VP3 polyprotein of infectious bursal disease virus (IBDV) were produced and the expressed antigens were characterised in cell culture. Proteins of the correct size were generated and found to react against a range of IBDV-specific monoclonal antibodies. Immunisation of 1-day-old SPF chicks with rSFV particles encoding the IBDV proteins resulted in specific antibodies being elicited in all birds, neutralising antibodies being induced in some but not all birds.

Semliki Forest virus-based vaccines: persistence, distribution and pathological analysis in two animal systems.

This study has examined the persistence, distribution and pathological changes following intramuscular administration of Semliki Forest virus (SFV) vaccine vectors in mice and chickens. Administration of recombinant SFV RNA particles showed persistence at the injection site of mice up to 7 days, transient detection in secondary lymphoid organs and no dissemination to distal sites. In contrast, administration of a layered SFV DNA/RNA vector and a conventional standard naked DNA vector resulted in long-term persistence at the injection site, plasmid DNA being detected at 8 months post-inoculation in mice. Plasmid DNA was found distributed throughout the body, and tissues distal from the site of injection were positive up to 3 months. A similar pattern was observed in chickens. Mild pathological changes were observed at the injection site only, and plasmid DNA or recombinant RNA was not detected in mouse foetuses. These findings indicate that SFV-based vectors have the potential to be developed as safe vaccines.

Formation of infectious pancreatic necrosis virus-like particles following expression of segment A by recombinant semliki forest virus.

Segment A of the Sp strain (a Norwegian field isolate) of infectious pancreatic necrosis virus (IPNV) was amplified by reverse transcriptase polymerase chain reaction in two stages from RNA isolated from infected cells, and cloned into the Semliki Forest virus (SFV) expression vector pSFV1. Expression and correct processing of IPNV proteins was confirmed by transfection of RNA transcribed from this plasmid into BHK cells. This clone was then used to produce recombinant replication-defective SFV particles (rSFV) expressing the IPNV segment A. Immunofluorescence studies with conformation-dependent monoclonal antibodies to IPNV confirmed that the recombinant proteins produced after infection of the salmonid cell line CHSE-214 with such rSFV retain their antigenicity. Infection of the CHSE cells with the rSFV resulted in the formation of IPNV-like particles, which were similar in size and morphology to IPNV.

Signal recognition particle receptor (SRPR) is downregulated in a rat model of cyclosporin A-induced gingival overgrowth.

Differential display is a powerful technique which can be used to identify those genes whose expression is altered between two or more tissues under investigation. We have applied differential display to a rat model of cyclosporin A-induced gingival overgrowth (CIGO) to identify genes which are differentially expressed as a result of drug treatment. Ten weanling Wistar rats were fed with a pelleted diet containing cyclosporin A (CsA) at 120 mg/kg for 10 d and then 200 mg/kg for a further 30 d prior to culling. Experimental rats were compared with 10 age/sex-matched rats on a control diet. Significant evidence of overgrowth was observed in the interdental papilla between the mandibular first and second molar teeth in the CsA group. Differential display was performed on total cellular RNA extracted from the mandibular buccal gingiva. A cDNA product was isolated which was underexpressed in the overgrowth tissue and demonstrated a 95% sequence homology to the human signal recognition particle receptor (Human Docking Protein). Preliminary studies indicate that this gene is also underexpressed in human CIGO tissue. The method of approach and the potential implications of our findings are discussed.

Expression of the apoptosis-suppressing gene BCL-2 in pheochromocytoma is associated with the expression of C-MYC.

It has become increasingly clear that deregulation of programmed cell death is a critical component in multistep tumorigenesis. Previous studies have demonstrated a high frequency of Bcl-2 expression in tumors arising from cells derived from the neural crest and in tumor cell lines of neural origin. The present investigation was undertaken to determine whether similar molecular events occur in human pheochromocytoma. With the aim of determining the potential role of apoptosis in the pathogenesis of this tumor, we assessed proto-oncogene Bcl-2 and c-myc protein products as well as Bcl-2 messenger RNA levels in a collection of such tumors. Western blot analysis revealed that such tumors expressed the 26 kDa Bcl-2 (5 of 8 cases) and the 64 kDa c-Myc (7 of 8 cases) proteins. Northern blot analysis detected the Bcl-2 transcripts in 6 of 8 tumors. Immunoperoxidase staining, using a monoclonal anti-Bcl-2 antibody, was positive in 18 (82%), including 5 malignant tumors, of the 22 specimens examined. This Bcl-2 immunoreactivity was seen in 14 of 18 (78%) sporadic tumors, including 2 that were extra-adrenal, and all familial tumors. Of the 22 tumor samples examined for c-Myc protein, 20 (91%) tumors were positive. Our results suggest that deregulation of programmed cell death may be a critical component in the multistep tumorigenesis of human pheochromocytoma. The genetic complementation of simultaneously deregulated Bcl-2 and c-myc may be implicated in this process.

Paraffin-embedded tissue as a source of RNA for gene expression analysis in oral malignancy.

OBJECTIVE: To assess the feasibility of using archival oral mucosal tissue to examine gene expression at the ribonucleic acid (RNA) level. MATERIALS AND METHODS: We describe the isolation of RNA from 8 microns sections of formalin-fixed paraffin-embedded oral mucosal tissue. RNA was reverse transcribed and three candidate genes amplified by polymerase chain reaction (PCR). The ribosomal protein S14 gene is a housekeeping gene which has been used as an internal standard in several quantitative PCR protocols. The thymidine kinase (TK) gene is expressed at low levels in most tissues and, with a well-documented genomic organisation, is a useful tool for discrimination between genomic DNA and cDNA. The RI alpha gene is reported to be overexpressed in many cancer cell lines, in malignant tissue and in vitro transformed cells. RESULTS: The S14 gene, the TK gene and the RI alpha gene of the cAMP-dependent protein kinase (PKA) were amplified successfully from formalin-fixed paraffin-embedded tissue sections. The TK primer pair is a useful additional tool in the unambiguous identification of RNA-derived species. CONCLUSION: RNA suitable for reverse transcribed (RT)-PCR was extracted from archival oral mucosal tissue. This should permit rapid sequence analysis of transcribed tumor suppressor genes and oncogenes in this material. Furthermore, the RT-PCR approach described may allow quantification of gene expression in oral mucosal archival material processed in a standard fashion.

Transcriptional analysis and genome expression of chicken anaemia virus.

Strand-specific riboprobes representative of either strand of the chicken anaemia virus (CAV) replicative form (RF) DNA indicated that only one strand of the RF was transcribed to produce a major 2.0 kb transcript and that the encapsidated DNA strand was of negative sense. Primer extension analysis located a single transcriptional start site at nucleotide position 360 of the CAV sequence. Amplification, cloning and sequencing of the 3' end of the major transcript revealed the polyadenylation site at nucleotide position 21. Northern blot analysis using a series of genomic probes indicated that the 2.0 kb transcript was devoid of splicing and identified a non-transcribed region of the genome. This non-transcribed region was shown to possess promoter activity, enhancing the expression of the human growth hormone reporter gene in a transient gene expression assay. These observations suggest a simple strategy of genome expression involving a functional polycistronic message.

Some biological and physico-chemical properties of porcine circovirus.

Some important biological and physico-chemical characteristics of porcine circovirus are reported. These include a study on the host distribution in nature, levels of colostrum-derived antibodies in piglets from sero-positive sows and the susceptibility of a range of cell cultures to infection with this virus. The results of haemagglutination studies, resistance to pH 3, chloroform and heat are also reported as are comparative buoyant densities and sedimentation coefficients of porcine circovirus and chicken anaemia virus.