ABSTRACT
We describe the construction of recombinant Potato virus X (PVX) vectors expressing two different epitopes, ep4 and ep6, from Beet necrotic yellow vein virus (BNYVV). The seven-amino-acid epitopes were expressed as N-terminal coat protein fusions and were displayed on the surface of PVX particles. Particle assembly into full virions was successful even though no wild type coat protein subunits were present, and the epitopes could be detected in crude extracts and purified virus preparations with appropriate antibodies. A construct containing both epitope sequences in tandem was also prepared. The resulting PVX particles could be detected by antibodies against ep4 and ep6, either individually or simultaneously, showing that both epitopes were accessible. In addition mixed infections with PVX vectors containing the individual ep4 and ep6 sequences were carried out. This resulted in the formation of PVX particles displaying ep4 alone, ep6 alone, or both epitopes. These experiments demonstrate for the first time that PVX can be utilized to present multiple epitopes, either tandemly on every coat protein subunit or as heteromultimeric assemblies, both of which could be useful vaccination strategies. The production of epitope-presenting viruses in which every coat protein subunit contains a foreign epitope allows the high-level expression of defined numbers of foreign antigen sites, making such viruses useful standards for immune detection.
Subject(s)
Antigens, Viral/biosynthesis , Genetic Vectors/metabolism , Plant Viruses/immunology , Potexvirus/metabolism , Antigens, Viral/genetics , Capsid Proteins/genetics , Epitopes/biosynthesis , Epitopes/genetics , Genetic Vectors/genetics , Immunohistochemistry , Potexvirus/genetics , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Nicotiana/metabolism , Nicotiana/virologyABSTRACT
The detection of Beet necrotic yellow vein virus (BNYVV) in stored sugar beets by means of monoclonal antibodies or antibody single chain fragments (scFv) often poses problems, because the immunodominant C-terminal epitope of the viral coat protein is readily lost due to proteolysis. Clones which produce scFv specific for protease-stable BNYVV epitopes were selected from two naive phage display libraries. Fusion proteins of the scFv with a human IgG kappa chain (expressed from the newly designed vector pCL) or with alkaline phosphatase,respectively, allow the ELISA detection of BNYVV even in stored sugar beets with a sensitivity which was comparable or often higher than that achieved with polyclonal antibodies.
Subject(s)
Chenopodiaceae/virology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin Fragments/immunology , Plant Viruses/isolation & purification , RNA Viruses/isolation & purification , Antibodies, Monoclonal/immunology , Bacteriophages/genetics , Endopeptidases/metabolism , Epitopes , Humans , Immunoglobulin Fragments/genetics , Molecular Sequence Data , Peptide Library , Plant Diseases/virology , Plant Viruses/immunology , RNA Viruses/immunologyABSTRACT
A tubular virus from onion was found to react with an antiserum to Hypochoeris mosaic virus (HyMV), a putative furovirus. Sequence analysis of its genomic RNAs and further serological tests, however, indicated it to be a tobravirus rather than a furovirus. The reactivity of the HyMV antiserum with several isolates of tobacco rattle virus (TRV) suggests that HyMV itself may be a tobravirus. The deduced amino acid sequences of the putative proteins encoded on RNA 2 of the onion virus isolate (ON) suggest close evolutionary relationships to the TRV isolate TCM from tulip. However, RNA 2 of the ON isolate contains a shorter RNA 1-like sequence on its 3'-end and an additional small ORF upstream of its RNA 1-like part. The sequence of its 315 5'-terminal nucleotides is more similar to that of RNA 2 of the PLB isolate from potato than to that of TCM RNA 2.
Subject(s)
Immune Sera/immunology , Onions/virology , Plant Viruses/genetics , RNA, Viral/chemistry , Amino Acid Sequence , Base Sequence , Microscopy, Electron , Microscopy, Immunoelectron , Molecular Sequence Data , Open Reading Frames , Plant Viruses/immunology , Promoter Regions, Genetic , RNA, Viral/analysisABSTRACT
In dogs with gastric and pancreatic fistulas, we studied the effect of intravenous atropine in doses ranging from 0.9 to 58 nmol X kg-1 X h-1 on the pancreatic secretory response to secretin before and after truncal vagotomy. Truncal vagotomy did not alter the incremental bicarbonate response to secretin. Before and after truncal vagotomy, 7 nmol X kg-1 X h-1 and all higher doses of atropine sulfate significantly decreased the bicarbonate response to low doses (5.2 and 10.3 pmol X kg-1 X h-1) of secretin but had no significant effect on responses to high doses (20.5 and 41 pmol X kg-1 X h-1). The inhibitory potency of the effective doses of atropine did not differ significantly. Secretin did not stimulate pancreatic protein output above basal. Truncal vagotomy reduced protein output basally and during secretin by about 50%. Before and after truncal vagotomy, 7 nmol X kg-1 X h-1 and all higher doses of atropine significantly decreased protein output basally and during secretin. Secretin and truncal vagotomy did not alter basal heart rate. Only the three highest doses (14, 29, and 58 nmol X kg-1 X h-1) of atropine significantly increased heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)
