{"id":{"repo_id":"lmu-germany","oai_identifier":"oai:edoc.ub.uni-muenchen.de:1152"},"canonical_url":"https://search.dev.ndltd.org/etd/lmu-germany/oai:edoc.ub.uni-muenchen.de:1152","repository":{"repo_id":"lmu-germany","name":"Ludwig Maxmilians Universität München","base_url":"https://edoc.ub.uni-muenchen.de/cgi/oai2"},"display":{"title":"Klonierung und Mutagenese des Bovinen Herpesvirus Typ 1 als ein infektiöses künstliches bakterielles Chromosom (bacterial artificial chromosome, BAC)","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Trapp, Sascha"],"institution":"Ludwig-Maximilians-Universität","degree_name":null,"degree_level":"thesis.doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2003,"date_issued":"2003-02-07","date_published":"2003-02-07","updated_at":"2026-07-24T02:51:41Z","subjects":[],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://edoc.ub.uni-muenchen.de/1152/","outbound_label":"Repository record","outbound_source":"source_url"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Trapp, Sascha"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:publisher","label":"Institution","values":["Universitätsbibliothek der Ludwig-Maximilians-Universität"]},{"key":"dc:type","label":"Dc Type","values":["doctoralThesis"]},{"key":"thesis:degree_level","label":"Degree Level","values":["thesis.doctoral"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Ludwig-Maximilians-Universität"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.","Das gesamte Genom des BHV-1-Stammes Schönböken wurde erstmals als infektiöses künstliches bakterielles Chromosom (bacterial artificial chromosome, BAC) kloniert. Für die BAC-Konstruktion wurden mini-F-Plasmid-Sequenzen in den offenen Leserahmen (ORF) des Glykoprotein E (gE) inseriert. Durch Transfektion der DNA des resultierenden BAC-Klones pBHV-1DgE in Rindernierenzellen konnte die gE-negative Virusmutante BHV-1DgE rekonstituiert werden. Mit Hilfe der RecE/T-Mutagenese in Escherichia coli wurden auf der Basis des BHV-1-BACs drei mutierte BHV-1-Genome konstruiert, in denen zusätzlich zu gE entweder gG (pBHV-1DgE-gG) oder gM (pBHV-1DgE-gM) oder gK (pBHV-1DgE-gK) deletiert worden war. Nach Transfektion der in Bakterien manipulierten BAC-DNA in eukaryotische Zellen konnten rekombinante Viren mit einer gleichzeitigen Deletion von gE und gG (BHV-1DgE-gG) sowie gE und gM (BHV-1DgE-gM) rekonstituiert werden. Aus der gK-negativen BAC-Mutante pBHV-1DgE-gK konnte jedoch kein Virus generiert werden, was dafür spricht, dass gK bei BHV-1, ebenso wie bei anderen Alphaherpesviren, ein für die Replikation essentielles Genprodukt darstellt. Die Wachstumseigenschaften der übrigen BAC-Mutanten BHV-1DgE, BHV-1DgE-gG und BHV-1DgE-gM wurden in vitro mittels Ein-Schritt-Wachstumskinetik und Plaquegrößenbestimmung genauer analysiert. Die Virustiter der BAC-Mutanten unterschieden sich intra- und extrazellulär kaum voneinander und waren vergleichbar mit Titern des Wildtyps BHV-1 Schönböken. Bei der Bestimmung der Plaquedurchmesser waren BHV-1DgE-spezifische Virus-Plaques um 45% kleiner als die des Wildtyps. Die BAC-Doppelmutanten BHV-1DgE-gG und BHV-1DgE-gM bildeten Plaques, die um 56% (BHV-1DgE-gG) oder 54% (BHV-1DgE-gM) reduziert waren. Diese Beobachtungen bekräftigen einerseits, dass gE, gG und gM die BHV-1-Zell-zu-Zell-Ausbreitung deutlich beeinflussen, andererseits demonstrieren sie, dass eine gleichzeitige Deletion von gE und gG oder gE und gM die Zell-zu-Zell-Ausbreitung nur wenig mehr einschränkt als eine singuläre gE-Deletion. Darüber hinaus konnte durch den Vergleich der Plaquegrößen von BHV-1DgE, BHV-1DgE-gG und BHV-1DgG gezeigt werden, dass gE und gG bei der Zell-zu-Zell-Ausbreitung, anders als bisher vermutet, additiv und nicht synergistisch oder kompensativ agieren. Die hier vorgestellten Ergebnisse illustrieren, dass die Vermehrung und Manipulation eines BHV-1-BACs mit Hilfe des bakteriellen Rekombinationsapparates gegenüber konventionellen Methoden zur Klonierung rekombinanter Viren eine raschere Herstellung und Charakterisierung von BHV-1-Deletionsmutanten ermöglicht. Hieraus folgt, dass mit der neuen Technik BHV-1-Mutanten künftig schneller generiert und auf ihre Eignung als (Marker-)Impfstoff untersucht werden können."]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Klonierung und Mutagenese des Bovinen Herpesvirus Typ 1 als ein infektiöses künstliches bakterielles Chromosom (bacterial artificial chromosome, BAC)"]}]}],"canonical_facts":{"dc:creator":["Trapp, Sascha"],"dc:description.abstract":["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.","Das gesamte Genom des BHV-1-Stammes Schönböken wurde erstmals als infektiöses künstliches bakterielles Chromosom (bacterial artificial chromosome, BAC) kloniert. Für die BAC-Konstruktion wurden mini-F-Plasmid-Sequenzen in den offenen Leserahmen (ORF) des Glykoprotein E (gE) inseriert. Durch Transfektion der DNA des resultierenden BAC-Klones pBHV-1DgE in Rindernierenzellen konnte die gE-negative Virusmutante BHV-1DgE rekonstituiert werden. Mit Hilfe der RecE/T-Mutagenese in Escherichia coli wurden auf der Basis des BHV-1-BACs drei mutierte BHV-1-Genome konstruiert, in denen zusätzlich zu gE entweder gG (pBHV-1DgE-gG) oder gM (pBHV-1DgE-gM) oder gK (pBHV-1DgE-gK) deletiert worden war. Nach Transfektion der in Bakterien manipulierten BAC-DNA in eukaryotische Zellen konnten rekombinante Viren mit einer gleichzeitigen Deletion von gE und gG (BHV-1DgE-gG) sowie gE und gM (BHV-1DgE-gM) rekonstituiert werden. Aus der gK-negativen BAC-Mutante pBHV-1DgE-gK konnte jedoch kein Virus generiert werden, was dafür spricht, dass gK bei BHV-1, ebenso wie bei anderen Alphaherpesviren, ein für die Replikation essentielles Genprodukt darstellt. Die Wachstumseigenschaften der übrigen BAC-Mutanten BHV-1DgE, BHV-1DgE-gG und BHV-1DgE-gM wurden in vitro mittels Ein-Schritt-Wachstumskinetik und Plaquegrößenbestimmung genauer analysiert. Die Virustiter der BAC-Mutanten unterschieden sich intra- und extrazellulär kaum voneinander und waren vergleichbar mit Titern des Wildtyps BHV-1 Schönböken. Bei der Bestimmung der Plaquedurchmesser waren BHV-1DgE-spezifische Virus-Plaques um 45% kleiner als die des Wildtyps. Die BAC-Doppelmutanten BHV-1DgE-gG und BHV-1DgE-gM bildeten Plaques, die um 56% (BHV-1DgE-gG) oder 54% (BHV-1DgE-gM) reduziert waren. Diese Beobachtungen bekräftigen einerseits, dass gE, gG und gM die BHV-1-Zell-zu-Zell-Ausbreitung deutlich beeinflussen, andererseits demonstrieren sie, dass eine gleichzeitige Deletion von gE und gG oder gE und gM die Zell-zu-Zell-Ausbreitung nur wenig mehr einschränkt als eine singuläre gE-Deletion. Darüber hinaus konnte durch den Vergleich der Plaquegrößen von BHV-1DgE, BHV-1DgE-gG und BHV-1DgG gezeigt werden, dass gE und gG bei der Zell-zu-Zell-Ausbreitung, anders als bisher vermutet, additiv und nicht synergistisch oder kompensativ agieren. Die hier vorgestellten Ergebnisse illustrieren, dass die Vermehrung und Manipulation eines BHV-1-BACs mit Hilfe des bakteriellen Rekombinationsapparates gegenüber konventionellen Methoden zur Klonierung rekombinanter Viren eine raschere Herstellung und Charakterisierung von BHV-1-Deletionsmutanten ermöglicht. Hieraus folgt, dass mit der neuen Technik BHV-1-Mutanten künftig schneller generiert und auf ihre Eignung als (Marker-)Impfstoff untersucht werden können."],"dc:format.medium":["application/pdf"],"dc:publisher":["Universitätsbibliothek der Ludwig-Maximilians-Universität"],"dc:title":["Klonierung und Mutagenese des Bovinen Herpesvirus Typ 1 als ein infektiöses künstliches bakterielles Chromosom (bacterial artificial chromosome, BAC)"],"dc:type":["doctoralThesis"],"thesis:degree_level":["thesis.doctoral"],"thesis:institution_name":["Ludwig-Maximilians-Universität"]},"updated_at":"2026-07-24T02:51:41Z"}