{"id":{"repo_id":"fu-berlin","oai_identifier":"oai:refubium.fu-berlin.de:fub188/52394"},"canonical_url":"https://search.dev.ndltd.org/etd/fu-berlin/oai:refubium.fu-berlin.de:fub188/52394","repository":{"repo_id":"fu-berlin","name":"Freie Universität Berlin","base_url":"https://refubium.fu-berlin.de/oai/request"},"display":{"title":"Systematic Enhancement of Human Herpesvirus 6A Reconstitution: Bridging Technical Refinement and Host Determinants of Virus Replication","abstract":"Human herpesvirus 6A (HHV-6A) is a betaherpesvirus that presents substantial challenges for research due to its strong propensity for latency and slow replication. This thesis addresses the critical bottleneck of efficient recombinant HHV-6A reconstitution from bacterial artificial chromosome (BAC) constructs, a process historically hampered by low transfection efficiencies and prolonged propagation duration. We demonstrated that pretreatment with dimethyl sulfoxide (DMSO) and exonuclease V (ExoV), coupled with refined post-nucleofection cell recovery, increased transfection efficiencies up to 30%. We identified coordinated stimulation strategies that markedly enhanced virus replication. Notably, combining the hypoxia-mimetic IOX2 and hydrocortisone (HC) drastically reduced reconstitution time to two weeks, providing the first evidence that the hypoxic response plays a role in HHV-6A replication. Furthermore, we elucidated the suppressive role of interferon (IFN) signaling in virus reconstitution, highlighting the potential of JAK1/JAK2 inhibition via ruxolitinib (RUX) to improve viral yields. By systematically optimizing nucleofection and stimulation conditions, we developed a robust and reproducible methodology that significantly enhances virus reconstitution. Our refined method, validated across multiple experimental conditions, facilitates the generation of recombinant HHV-6A mutants, including a novel reporter virus for detailed studies of virus replication kinetics and gene functions. Collectively, these advances provide a powerful platform for mechanistic studies of HHV-6A biology and lay the groundwork for future investigations into herpesvirus latency, reactivation and antiviral strategies","abstract_html":"Human herpesvirus 6A (HHV-6A) is a betaherpesvirus that presents substantial challenges for research due to its strong propensity for latency and slow replication. This thesis addresses the critical bottleneck of efficient recombinant HHV-6A reconstitution from bacterial artificial chromosome (BAC) constructs, a process historically hampered by low transfection efficiencies and prolonged propagation duration. We demonstrated that pretreatment with dimethyl sulfoxide (DMSO) and exonuclease V (ExoV), coupled with refined post-nucleofection cell recovery, increased transfection efficiencies up to 30%. We identified coordinated stimulation strategies that markedly enhanced virus replication. Notably, combining the hypoxia-mimetic IOX2 and hydrocortisone (HC) drastically reduced reconstitution time to two weeks, providing the first evidence that the hypoxic response plays a role in HHV-6A replication. Furthermore, we elucidated the suppressive role of interferon (IFN) signaling in virus reconstitution, highlighting the potential of JAK1/JAK2 inhibition via ruxolitinib (RUX) to improve viral yields. By systematically optimizing nucleofection and stimulation conditions, we developed a robust and reproducible methodology that significantly enhances virus reconstitution. Our refined method, validated across multiple experimental conditions, facilitates the generation of recombinant HHV-6A mutants, including a novel reporter virus for detailed studies of virus replication kinetics and gene functions. Collectively, these advances provide a powerful platform for mechanistic studies of HHV-6A biology and lay the groundwork for future investigations into herpesvirus latency, reactivation and antiviral strategies","abstract_has_math":false,"creators":["Reich, Jana"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2026,"date_issued":"2026","date_published":"2026","updated_at":"2026-08-21T16:44:52Z","subjects":["human herpesvirus 6","viral replication","reconstitution","stimuli","enhancers","laboratory methods"],"languages":["eng"],"rights":[],"rights_urls":["https://www.fu-berlin.de/sites/ub/publizieren/refubium/rechtliches/index.html"],"identifier_entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://dx.doi.org/10.17169/refubium-52124"],"render_values":[{"text":"http://dx.doi.org/10.17169/refubium-52124","href":"http://dx.doi.org/10.17169/refubium-52124","code":true}]}]},"links":{"outbound_url":"https://refubium.fu-berlin.de/handle/fub188/52394","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"source_record":{"url":"https://refubium.fu-berlin.de/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arefubium.fu-berlin.de%3Afub188%2F52394","prefix":"dim"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Reich, Jana"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2026"]},{"key":"dc:type","label":"Dc Type","values":["doc-type:doctoralThesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["human herpesvirus 6","viral replication","reconstitution","stimuli","enhancers","laboratory methods"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights.uri","label":"Rights URI","values":["https://www.fu-berlin.de/sites/ub/publizieren/refubium/rechtliches/index.html"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://refubium.fu-berlin.de/handle/fub188/52394","http://dx.doi.org/10.17169/refubium-52124"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Human herpesvirus 6A (HHV-6A) is a betaherpesvirus that presents substantial challenges for research due to its strong propensity for latency and slow replication. This thesis addresses the critical bottleneck of efficient recombinant HHV-6A reconstitution from bacterial artificial chromosome (BAC) constructs, a process historically hampered by low transfection efficiencies and prolonged propagation duration. We demonstrated that pretreatment with dimethyl sulfoxide (DMSO) and exonuclease V (ExoV), coupled with refined post-nucleofection cell recovery, increased transfection efficiencies up to 30%. We identified coordinated stimulation strategies that markedly enhanced virus replication. Notably, combining the hypoxia-mimetic IOX2 and hydrocortisone (HC) drastically reduced reconstitution time to two weeks, providing the first evidence that the hypoxic response plays a role in HHV-6A replication. Furthermore, we elucidated the suppressive role of interferon (IFN) signaling in virus reconstitution, highlighting the potential of JAK1/JAK2 inhibition via ruxolitinib (RUX) to improve viral yields. By systematically optimizing nucleofection and stimulation conditions, we developed a robust and reproducible methodology that significantly enhances virus reconstitution. Our refined method, validated across multiple experimental conditions, facilitates the generation of recombinant HHV-6A mutants, including a novel reporter virus for detailed studies of virus replication kinetics and gene functions. Collectively, these advances provide a powerful platform for mechanistic studies of HHV-6A biology and lay the groundwork for future investigations into herpesvirus latency, reactivation and antiviral strategies","Das Humane Herpesvirus 6A (HHV-6A) ist ein Betaherpesvirus, das aufgrund seiner ausgeprägten Latenzneigung und langsamen Replikation erhebliche Herausforderungen für die Forschung darstellt. Diese Doktorarbeit befasst sich mit dem entscheidenden Engpass der effizienten Rekonstitution rekombinanter HHV-6A-Viren aus bakteriellen, künstlichen Chromosomen (BAC-Konstrukten). Dieser Prozess wurde bislang durch geringe Transfektionseffizienzen und langwierige Virusvermehrung erschwert. Wir zeigten, dass eine Vorbehandlung mit Dimethylsulfoxid (DMSO) und Exonuklease V (ExoV) in Kombination mit einer optimierten Zellregeneration nach der Nukleofektion die Transfektionseffizienz auf bis zu 30 % steigern konnte. Wir identifizierten koordinierte Stimulationsstrategien, die Virusreplikation deutlich verbesserten. Insbesondere die Kombination des Hypoxie-Mimetikums IOX2 und Hydrocortison (HC) reduzierte die Rekonstitutionsdauer drastisch auf zwei Wochen, und lieferte den ersten Nachweis, dass die hypoxische Antwort eine Rolle bei der HHV-6A-Replikation spielt. Darüber hinaus konnten wir die hemmende Wirkung des Interferon (IFN)-Signalwegs bei der Virusrekonstitution aufklären und zeigen, dass eine Hemmung von JAK1/JAK2 durch Ruxolitinib (RUX) die Virusausbeute verbessert. Durch die systematische Optimierung der Nukleofektions- und Stimulationsbedingungen entwickelten wir eine robuste und reproduzierbare Methode, die Virusrekonstitution signifikant verbessert. Unsere verbesserte Methode, die unter verschiedenen experimentellen Bedingungen validiert wurde, ermöglicht die effiziente Herstellung rekombinanter HHV-6A-Viren, einschließlich eines neuartigen Reportervirus, für detaillierte Studien zur viralen Replikationskinetik und Genfunktionen. Insgesamt bieten diese Fortschritte eine leistungsfähige Plattform für mechanistische Studien zur HHV-6A-Biologie und bilden die Grundlage für zukünftige Untersuchungen zur Latenz, Reaktivierung und antiviralen Strategienentwicklung von Herpesviren."]},{"key":"dc:title","label":"Title","values":["Systematic Enhancement of Human Herpesvirus 6A Reconstitution: Bridging Technical Refinement and Host Determinants of Virus Replication"]}]}],"canonical_facts":{"dc:creator":["Reich, Jana"],"dc:date.issued":["2026"],"dc:description.abstract":["Human herpesvirus 6A (HHV-6A) is a betaherpesvirus that presents substantial challenges for research due to its strong propensity for latency and slow replication. This thesis addresses the critical bottleneck of efficient recombinant HHV-6A reconstitution from bacterial artificial chromosome (BAC) constructs, a process historically hampered by low transfection efficiencies and prolonged propagation duration. We demonstrated that pretreatment with dimethyl sulfoxide (DMSO) and exonuclease V (ExoV), coupled with refined post-nucleofection cell recovery, increased transfection efficiencies up to 30%. We identified coordinated stimulation strategies that markedly enhanced virus replication. Notably, combining the hypoxia-mimetic IOX2 and hydrocortisone (HC) drastically reduced reconstitution time to two weeks, providing the first evidence that the hypoxic response plays a role in HHV-6A replication. Furthermore, we elucidated the suppressive role of interferon (IFN) signaling in virus reconstitution, highlighting the potential of JAK1/JAK2 inhibition via ruxolitinib (RUX) to improve viral yields. By systematically optimizing nucleofection and stimulation conditions, we developed a robust and reproducible methodology that significantly enhances virus reconstitution. Our refined method, validated across multiple experimental conditions, facilitates the generation of recombinant HHV-6A mutants, including a novel reporter virus for detailed studies of virus replication kinetics and gene functions. Collectively, these advances provide a powerful platform for mechanistic studies of HHV-6A biology and lay the groundwork for future investigations into herpesvirus latency, reactivation and antiviral strategies","Das Humane Herpesvirus 6A (HHV-6A) ist ein Betaherpesvirus, das aufgrund seiner ausgeprägten Latenzneigung und langsamen Replikation erhebliche Herausforderungen für die Forschung darstellt. Diese Doktorarbeit befasst sich mit dem entscheidenden Engpass der effizienten Rekonstitution rekombinanter HHV-6A-Viren aus bakteriellen, künstlichen Chromosomen (BAC-Konstrukten). Dieser Prozess wurde bislang durch geringe Transfektionseffizienzen und langwierige Virusvermehrung erschwert. Wir zeigten, dass eine Vorbehandlung mit Dimethylsulfoxid (DMSO) und Exonuklease V (ExoV) in Kombination mit einer optimierten Zellregeneration nach der Nukleofektion die Transfektionseffizienz auf bis zu 30 % steigern konnte. Wir identifizierten koordinierte Stimulationsstrategien, die Virusreplikation deutlich verbesserten. Insbesondere die Kombination des Hypoxie-Mimetikums IOX2 und Hydrocortison (HC) reduzierte die Rekonstitutionsdauer drastisch auf zwei Wochen, und lieferte den ersten Nachweis, dass die hypoxische Antwort eine Rolle bei der HHV-6A-Replikation spielt. Darüber hinaus konnten wir die hemmende Wirkung des Interferon (IFN)-Signalwegs bei der Virusrekonstitution aufklären und zeigen, dass eine Hemmung von JAK1/JAK2 durch Ruxolitinib (RUX) die Virusausbeute verbessert. Durch die systematische Optimierung der Nukleofektions- und Stimulationsbedingungen entwickelten wir eine robuste und reproduzierbare Methode, die Virusrekonstitution signifikant verbessert. Unsere verbesserte Methode, die unter verschiedenen experimentellen Bedingungen validiert wurde, ermöglicht die effiziente Herstellung rekombinanter HHV-6A-Viren, einschließlich eines neuartigen Reportervirus, für detaillierte Studien zur viralen Replikationskinetik und Genfunktionen. Insgesamt bieten diese Fortschritte eine leistungsfähige Plattform für mechanistische Studien zur HHV-6A-Biologie und bilden die Grundlage für zukünftige Untersuchungen zur Latenz, Reaktivierung und antiviralen Strategienentwicklung von Herpesviren."],"dc:identifier.uri":["https://refubium.fu-berlin.de/handle/fub188/52394","http://dx.doi.org/10.17169/refubium-52124"],"dc:language":["eng"],"dc:rights.uri":["https://www.fu-berlin.de/sites/ub/publizieren/refubium/rechtliches/index.html"],"dc:subject":["human herpesvirus 6","viral replication","reconstitution","stimuli","enhancers","laboratory methods"],"dc:title":["Systematic Enhancement of Human Herpesvirus 6A Reconstitution: Bridging Technical Refinement and Host Determinants of Virus Replication"],"dc:type":["doc-type:doctoralThesis"]},"updated_at":"2026-08-21T16:44:52Z"}