title: Generation of an infectious clone of duck enteritis virus (DEV) and of a vectored DEV expressing hemagglutinin of H5N1 avian influenza virus.
abstract: We report on the generation of an infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus (DEV) and a vectored DEV vaccine expressing hemagglutinin (H5) of high pathogenicity H5N1 avian influenza virus (AIV). For generation of the DEV BAC, we inserted mini-F vector sequences by homologous recombination in lieu of the UL44 (gC) gene of DEV isolate 2085. DNA of the resulting in recombinant virus v2085-GFPΔgC was electroporated into Escherichia coli and a full-length DEV BAC clone (p2085) was recovered. Transfection of p2085 into chicken embryo cells resulted in DEV-specific plaques exhibiting green autofluorescence. A gC-negative mutant, v2085ΔgC, was generated by deleting mini-F vector sequences by using Cre-Lox recombination, and a revertant virus v2085ΔgC-R was constructed by co-transfection of p2085 with UL44 sequences. Finally, AIV H5 was inserted into p2085, and high-level H5 expression of the v2085_H5 virus was detected by indirect immunofluorescence and western blotting. Plaque area measurements showed that v2085ΔgC plaques were significantly increased (12%) over those of parental 2085 virus or the v2085ΔgC-R revertant virus (ANOVA, P<0.05), while plaque areas of the H5- or GFP-expressing DEV mutants were significantly smaller. There was no significant difference between DEV with respect to virus titers determined after trypsinization titration of infected cells, while virus titers of infected-cell supernatants revealed significant reductions in case of the gC-negative viruses of more than 700-fold when compared to parental 2085 or v2085ΔgC-R. Cell-associated virus titers of gC-negative DEV also showed significant reduction of 50-500-fold (ANOVA, P<0.05). We conclude that (i) absence of DEV gC results in increased plaque sizes in vitro, (ii) gC plays a role in DEV egress, and (iii) generation of an infectious DEV clone allows rapid generation of vectored vaccines.
authors.affiliation: Institut für Virologie, Freie Universität Berlin, Philippstr. 13, Haus 18, 10115 Berlin, Germany.
authors.forename: Jichun
authors.initials: J
authors.lastname: Wang
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Osterrieder
mesh_terms: D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics* / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000276:immunology*; D007252:Influenza Vaccines / Q000235:genetics* / Q000276:immunology; D025922:Mardivirus / Q000235:genetics* / Q000472:pathogenicity*; D011995:Recombination, Genetic; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology; D019562:Viral Load; D010948:Viral Plaque Assay; D014774:Virulence
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D014614:Vaccines, Synthetic