Klonierung und Mutagenese des Bovinen Herpesvirus Typ 1 als ein infektiöses künstliches bakterielles Chromosom (bacterial artificial chromosome, BAC)

For the first time the complete BHV-1 Schönböken genome was cloned as an infectious bacterial artificial chromosome (BAC), by inserting mini F plasmid sequences into the glycoprotein E (gE) open reading frame (ORF). DNA of the resulting BAC clone pBHV-1DgE was transfected into permissive bovine kidney cells and infectious BHV-1DgE could be recovered. Using RecE/T cloning in Escherichia coli BHV-1 genomes with a deletion of either glycoprotein G (gG) or gM or gK in addition to gE (pBHV-1DgE-gG, pBHV-1DgE-gM and pBHV-1DgE-gK) were generated. The recombinant viruses with a simultaneous deletion of gE and gG (BHV-1DgE-gG) or gE and gM (BHV-1DgE-gM), respectively, were reconstituted after transfection of manipulated BAC DNA into eukaryotic cells. However, no virus could be recovered after transfection of recombinant pBHV-1DgE-gK DNA, suggesting that gK is likewise essential for the replication of BHV-1 as demonstrated for other alphaherpesviruses. Growth properties of the other BAC derived virus mutants BHV-1DgE, BHV-1DgE-gG and BHV-1DgE-gM were analysed in vitro. The mutant viruses exhibited no markedly lowered virus titres compared to wild type strain Schönböken. However, BHV-1DgE specific virus plaques were reduced by 45% compared to BHV-1 Schönböken as assessed by plaque size measurement. Plaques sizes of BHV-1DgE-gG and BHV-1DgE-gM were reduced by 56% (BHV-1DgE-gG) and 54% (BHV-1DgE-gM). The observations made here emphasize the influence of gE, gG and gM on the BHV-1 cell-to-cell spread. Moreover, they clearly demonstrate that viral cell-to-cell spread is slightly more inhibited by a simultaneous deletion of gE and gG or gE and gM than by a single gE deletion. Comparing the plaque sizes of BHV-1DgE, BHV-1DgE-gG and BHV-1DgG it could be shown that gE and gG function independently from each other in cell-to-cell spread, because an additive and no synergistic effect was observed in the gE-gG double deletion mutant. These studies illustrate, that the propagation and manipulation of herpesviruses in bacterial systems provides the advantage of a more rapid and accurate generation and characterisation of BHV-1 deletion mutants over conventional cloning procedures. In conclusion the new technique will allow the fast generation of BHV-1 mutants as well as the investigation for their suitability as future (marker) vaccines.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2003

Author dc:creator

• Trapp, Sascha

Repository record source_url

https://edoc.ub.uni-muenchen.de/1152/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:1152