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.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

Precision cut lung slices: a novel versatile tool to examine host-pathogen interaction in the chicken lung

The avian respiratory tract is a common entry route for many pathogens and an important delivery route for vaccination in the poultry industry. Immune responses in the avian lung have mostly been studied in vivo due to the lack of robust, relevant in vitro and ex vivo models mimicking the microenvironment. Precision-cut lung slices (PCLS) have the major advantages of maintaining the 3-dimensional architecture of the lung and includes heterogeneous cell populations. PCLS have been obtained from a number of mammalian species and from chicken embryos. However, as the embryonic lung is physiologically undifferentiated and immunologically immature, it is less suitable to examine complex host-pathogen interactions including antimicrobial responses. Here we prepared PCLS from immunologically mature chicken lungs, tested different culture conditions, and found that serum supplementation has a detrimental effect on the quality of PCLS. Viable cells in PCLS remained present for ≥ 40 days, as determined by viability assays and sustained motility of fluorescent mononuclear phagocytic cells. The PCLS were responsive to lipopolysaccharide stimulation, which induced the release of nitric oxide, IL-1β, type I interferons and IL-10. Mononuclear phagocytes within the tissue maintained phagocytic activity, with live cell imaging capturing interactions with latex beads and an avian pathogenic Escherichia coli strain. Finally, the PCLS were also shown to be permissive to infection with low pathogenic avian influenza viruses. Taken together, immunologically mature chicken PCLS provide a suitable model to simulate live organ responsiveness and cell dynamics, which can be readily exploited to examine host-pathogen interactions and inflammatory responses

Acute paretic syndrome in juvenile White Leghorn chickens resembles late stages of acute inflammatory demyelinating polyneuropathies in humans

Background: Sudden limb paresis is a common problem in White Leghorn flocks, affecting about 1% of the chicken population before achievement of sexual maturity. Previously, a similar clinical syndrome has been reported as being caused by inflammatory demyelination of peripheral nerve fibres. Here, we investigated in detail the immunopathology of this paretic syndrome and its possible resemblance to human neuropathies. Methods: Neurologically affected chickens and control animals from one single flock underwent clinical and neuropathological examination. Peripheral nervous system (PNS) alterations were characterised using standard morphological techniques, including nerve fibre teasing and transmission electron microscopy. Infiltrating cells were phenotyped immunohistologically and quantified by flow cytometry. The cytokine expression pattern was assessed by quantitative real-time PCR (qRT-PCR). These investigations were accomplished by MHC genotyping and a PCR screen for Marek’s disease virus (MDV). Results: Spontaneous paresis of White Leghorns is caused by cell-mediated, inflammatory demyelination affecting multiple cranial and spinal nerves and nerve roots with a proximodistal tapering. Clinical manifestation coincides with the employment of humoral immune mechanisms, enrolling plasma cell recruitment, deposition of myelinbound IgG and antibody-dependent macrophageal myelin-stripping. Disease development was significantly linked to a 539 bp microsatellite in MHC locus LEI0258. An aetiological role for MDV was excluded. Conclusions: The paretic phase of avian inflammatory demyelinating polyradiculoneuritis immunobiologically resembles the late-acute disease stages of human acute inflammatory demyelinating polyneuropathy, and is characterised by a Th1-to-Th2 shift

The culture of primary duck endothelial cells for the study of avian influenza

Background: Endothelial cells play a major role in highly pathogenic avian influenza (HPAI) virus pathogenesis in gallinaceous poultry species (e.g. chicken, turkey and quail). Upon infection of gallinaceous poultry with HPAI viruses, endothelial cells throughout the body become rapidly infected, leading to systemic dissemination of the virus, disseminated intravascular coagulation, oedema and haemorrhaging. In contrast, the pathogenesis of HPAI viruses in most wild bird species (e.g. duck, goose and gull species) is not associated with endothelial tropism. Indeed, viral antigen is not found in the endothelial cells of most wild bird species following infection with HPAI viruses. This differential endothelial cell tropism in avian species is poorly understood, mainly due to the absence of appropriate cell culture systems. Results: Here, we describe the isolation and purification of primary duck endothelial cells from the aorta or bone marrow of Pekin duck embryos. Cells were differentiated in the presence of vascular endothelial growth factor and, if needed, enriched via fluorescent-activated cell sorting based on the uptake of acetylated low-density lipoprotein. The expression of von Willebrand factor, a key marker of endothelial cells, was confirmed by polymerase chain reaction. Monocultures of duck endothelial cells, either derived from the aorta or the bone marrow, were susceptible to infection with an H5N1 HPAI virus but to a much lesser extent than chicken endothelial cells. Conclusions: The methods described herein to isolate and purify duck endothelial cells from the aorta or bone marrow could also be applied to obtain microvascular endothelial cells from other tissues and organs, such as the lung or the intestine, and represent a valuable tool to study the pathogenesis of avian viruses

Herpesvirus Telomerase RNA(vTR)-Dependent Lymphoma Formation Does Not Require Interaction of vTR with Telomerase Reverse Transcriptase (TERT)

Telomerase is a ribonucleoprotein complex involved in the maintenance of telomeres, a protective structure at the distal ends of chromosomes. The enzyme complex contains two main components, telomerase reverse transcriptase (TERT), the catalytic subunit, and telomerase RNA (TR), which serves as a template for the addition of telomeric repeats (TTAGGG)n. Marek's disease virus (MDV), an oncogenic herpesvirus inducing fatal lymphoma in chickens, encodes a TR homologue, viral TR (vTR), which significantly contributes to MDV-induced lymphomagenesis. As recent studies have suggested that TRs possess functions independently of telomerase activity, we investigated if the tumor-promoting properties of MDV vTR are dependent on formation of a functional telomerase complex. The P6.1 stem-loop of TR is known to mediate TR-TERT complex formation and we show here that interaction of vTR with TERT and, consequently, telomerase activity was efficiently abrogated by the disruption of the vTR P6.1 stem-loop (P6.1mut). Recombinant MDV carrying the P6.1mut stem-loop mutation were generated and tested for their behavior in the natural host in vivo. In contrast to viruses lacking vTR, all animals infected with the P6.1mut viruses developed MDV-induced lymphomas, but onset of tumor formation was significantly delayed. P6.1mut viruses induced enhanced metastasis, indicating functionality of non-complexed vTR in tumor dissemination. We discovered that RPL22, a cellular factor involved in T-cell development and virus-induced transformation, directly interacts with wild-type and mutant vTR and is, consequently, relocalized to the nucleoplasm. Our study provides the first evidence that expression of TR, in this case encoded by a herpesvirus, is pro-oncogenic in the absence of telomerase activity

Herpesvirus Telomerase RNA (vTR) with a Mutated Template Sequence Abrogates Herpesvirus-Induced Lymphomagenesis

Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma

The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge

International audienceMammalian type I interferons (IFNα/β) are known to modulate inflammatory processes in addition to their antiviral properties. Indeed, virus-induced type I interferons regulate the mammalian phagocyte immune response to bacteria during superinfections. However, it remains unresolved whether type I IFNs similarly impact the chicken macrophage immune response. We first evidenced that IFNα and IFNβ act differently in terms of gene expression stimulation and activation of intracellular signaling pathways in chicken macrophages. Next, we showed that priming of chicken macrophages with IFNα increased bacteria uptake, boosted bacterial-induced ROS/NO production and led to an increased transcriptional expression or production of NOS2/NO, IL1B/IL-1β and notably IFNB/IFNβ. Neutralization of IFNβ during bacterial challenge limited IFNα-induced augmentation of the pro-inflammatory response. In conclusion, we demonstrated that type I IFNs differently regulate chicken macrophage functions and drive a pro-inflammatory response to bacterial challenge. These findings shed light on the diverse functions of type I IFNs in chicken macrophages

Doppelresonanzmessungen an in einer Penningfalle gespeicherten Europium-Ionen zur Bestimmung des Kern-g-Faktors

Die differentielle Hyperfeinanomalie beschreibt den Isotopieeffekt der magnetischen Hyperfeinwechselwirkung und stellt eine Testmöglichkeit für Kernmodelle dar. Nachdem in einer Reihe von Messungen die A-Faktoren der Hyperfeinwechselwirkung im Grundzustand von zwei stabilen und fünf instabilen Europium-Isotopen bestimmt wurden, sollen nun die Kern-g-Faktoren der gleichen Isotope bestimmt werden.Um die Kern-g-Faktoren zu bestimmen, wurden zunächst die Termschemata des Grundzustandesder stabilen Isotope 151,153Eu+ simuliert. Wegen der hohen Kern- und Hüllenspins I=5/2, J=4 ergeben sich 54 Zeemanzustände und ein komplexes optisches Spektrum.Etwa 10^6 Ionen der beiden stabilen Isotope 151,153Eu+ werden in einer Penningfalle beieinem Magnetfeld von ca. 1.5T gespeichert. Durch Puffergas und Anregung einer Seitenbandfrequenzder Ionenbewegung wird optisches Pumpen in metastabile D-Zustände bei gleichzeitiger Kühlung der Ionenwolke verhindert.Bei Verstimmung eines frequenzverdoppelten Ti:Sa-Lasers wurde die Ionenfluoreszenz aufgenommen. Durch die Simulation der optischen Spektren gelang es, einzelne Übergänge des Isotopes 151Eu+ zu identifizieren. Mit Laser/Mikrowellen-Doppelresonanzmessungen erhält man ein in erster Ordnung dopplerfreies Mikrowellenspektren eines Überganges zwischen zwei Zeemanzuständen im Grundzustand.Durch Messung der Übergangsfrequenzen von insgesamt fünf Delta mI=+1 Übergängen und Bestimmung des Magnetfeldes durch Messung der Zyklotronfrequenz gespeicherter Elektronen konnte der gI-Faktor des Isotopes151Eu zu 151gI = 1.37734(6) bestimmt werden.The differential hyperfine anomalie describes the isotopic effect of the magnetic hyperfine interactionand is a verification of nuclear models. Having determined the A-factors of hyperfine interaction for a series of two stable an five unstable Europium-isotopes the gI-factors of those isotopeswere to be measured.Simulation of the level schemes of 151,153Eu with high spins I=5/2, J=4 the results in 54 Zeeman-levelsper isotope an a complex optical spectrum. About 10^6 ions of 151,153Eu+ have been stored in a penning trap at a magnetic flux of 1,5T. By excitationof a sideband of the ion movement at a buffergas athmosphere optical pumping into metastableD-levels is prevented and the ion cloud is cooled.By detuning a frequency doubled Ti:Sa-laser the ionic fluorescence has been detected and by thehelp of the simulaation of the optical spectrum, some transitions of 151Eu+ could be determined.Withlaser-microwave double resonance the transition frequency of two Zeeman-levels can be measuredin first order doppler-free.Five transition frequencies of Delta mI transitions have been measured. With this data the gI-factorof 151Eu was determined to 151gI = 1.37734(6)

VetBioNet - Periodic Technical Report Part B (M36) - Public Summary

VetBioNet seeks to strengthen the European capacity and competence to meet the challenges of emerging infectious disease outbreaks by establishing a comprehensive network of European BSL3 infrastructures, academic institutes, industries and international organisations.To reach this overall objective, the Activities of VetBioNet are dedicated to:• Opening the VetBioNet infrastructure resources to external users by providing Transnational Access (TNA) to BSL3 animal experimental facilities and laboratories, technological platforms, and sample collections.• New scientific and technological developments.• Enhanced preparedness of the major European BSL3 research infrastructures that will allow a swift response to (re-)emerging epizootic and zoonotic threats.• Harmonization of Best Practices and a larger use of global standards in European BSL3 infrastructures.• Determining the social impact deriving from VetBioNet activities.• Establishing a sustainability plan that will allow VetBioNet to offer its services beyond the project duration.• Providing improved scientific and technological standards for the services offered by the VetBioNet infrastructures

VetBioNet - Periodic Technical Report Part B (M18) - Public Summary

VetBioNet seeks to strengthen the European capacity and competence to meet the challenges of emerging infectious disease outbreaks by establishing a comprehensive network of European BSL3 infrastructures, academic institutes, industries and international organisations. VetBioNet is dedicated to advance research on epizootic and zoonotic diseases and to promote related technological developments.To reach this overall objective, the Integrating Activities of the VetBioNet partners are dedicated to:• Opening the VetBioNet infrastructure resources to external users by providing Transnational Access to BSL3 animal experimental facilities and laboratories, technological platforms, and sample collections.• New scientific and technological developments.• Enhanced preparedness of the major European BSL3 research infrastructures that will allow a swift response to (re-)emerging epizootic and zoonotic threats.• Harmonization of Best Practices and a larger use of global standards in European BSL3 infrastructures.• Determining the social impact deriving from VetBioNet activities.• Establishing a sustainability plan that will allow VetBioNet to offer its services beyond the project duration.• Providing improved scientific and technological standards for the services offered by the VetBioNet infrastructures