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To enable control of African swine fever (ASF) in Eastern and Southern Africa, prototype live vaccine candidates were generated by targeted gene deletions from a Kenyan genotype IX ASF virus (ASFV). It was attempted to delete known nonessential genes involved in virulence (encoding TK, dUTPase, CD2v, 9GL), possibly essential genes (p12, pA104R, ribonucleotide reductase), and genes with widely unknown functions (pK145R). Isolation of the desired virus recombinants by plaque assays or limiting dilutions on a wild boar lung cell line (WSL-HP) was facilitated by substitutive reporter gene insertions encoding fluorescent proteins (GFP, DsRed), or the human membrane protein CD4. The latter protein permitted enrichment of recombinant virus particles by magnetic activated cell sorting (MACS). The isolated ASFV recombinants were characterized by PCR and sequencing of the mutated genome parts, and replication kinetics and virus spread in cell culture were investigated. Deletion of TK, CD2v, or pK145R had no detectable effect on in vitro growth of ASFV Kenya. Interestingly, virus mutants lacking the DNA binding protein pA104R which has been considered to be essential for DNA replication, also exhibited almost wild type-like growth properties.
In contrast, ASFV mutants lacking ribonucleotide reductase or p12 could not be purified to homogeneity on WSL-HP cells, indicating these proteins are essential for virus replication in cell culture. Therefore, trans-complementing cells lines stably expressing ASFV p12 have been prepared which can now be used for mutant virus purification. If this approach is successful the resulting defective mutant ASFV Kenya-p12 might be suitable as a safe “disabled in second cycle” (DISC) live vaccine in swine.
In a novel approach to improve reverse genetics of ASFV the CRISPR/Cas9 cell line WSL-gRp30 (Hübner et al., 2018a) was co-transfected with genomic DNA of ASFV-KenyaCD2vDsRed, sgRNA plasmids targeting K145R or 9GL, and GFP-expressing recombination plasmids for homology-directed repair. For booting up of the noninfectious virus genome the cells were infected with phylogenetically distant helper virus (genotype II ASFV Armenia, 84% identity) which was selectively inhibited on the used cell line. The desired double-fluorescent double-deletion mutants could be isolated after few plaque purification steps on selective WSL-gRp30 cells. Next generation sequence (NGS) analyses of reconstituted ASFV Kenya genomes showed that no unwanted recombination with the helper virus occurred, indicating that the method might be also suitable for booting of synthetic ASFV genomes cloned and mutagenized in E. coli or yeast.
The modified CRISPR/Cas9 system of S. pyogenes might be also usable for generation of ASFV resistant pigs. To evaluate this alternative control measure WSL cell clones stably expressing Cas9 nuclease and single or multiple sgRNAs against essential ASFV proteins were prepared and tested for their susceptibility to infection. Strain specific sgRNAs targeting the p30 gene of ASFV Kenya or Armenia selectively inhibited the respective viruses, and a p12 gene-specific sgRNA abrogated replication of both genotypes almost completely. Interestingly, coexpression of four ASFV-specific sgRNAs did not enhance virus inhibition, but might help to reduce the frequency of escape mutants which were occasionally isolated from the single sgRNA-expressing cells, and exhibited silent base substitutions or in-frame deletions within the target genes. First attempts to express the in vitro tested CRISPR/Cas9 constructs in transgenic pigs are in progress.
CRISPR/Cas9 supported rescue of a defective BAC clone of pseudorabies virus (PrV) vaccine strain Bartha (Hübner et al., 2018b) was used to develop putative vectored vaccines against ASFV. In the present study expression cassettes for the codon-optimized p12 and p54 genes of ASFV were successfully inserted into the PrV genome. The insertions did not significantly affect PrV recombination in cell culture, and the transgenes were expressed at similar levels as in ASFV-infected cells. It has to be tested whether coinfection with vector constructs for these and other immunogenic ASFV proteins is able to protect pigs against a lethal challenge.
For characterization of the generated ASFV mutants and PrV vector constructs, monospecific antisera against several ASFV gene products (p11.5, p12, p54, pK145R, p285L) were prepared by immunization of rabbits with bacterial GST fusion proteins. The anti-p12 serum showed only weak and strain-specific reactions with the ASFV Kenya protein, but was nevertheless useful for identification of p12-expressing PrV recombinants and WSL cell lines. All other sera showed satisfying reactions in Western blot and mostly immunofluorescence analyses, and allowed i.a. precise localization of the pK145R and p285L proteins in ASFV-infected cells and virions (Hübner et al., 2019).
As the animal-to-human interface becomes increasingly narrow, transmission events of zoonotic pathogens between animals and humans become more and more probable. While SARS-CoV-2 already accomplished a spillover infection to humans and is responsible for the current pandemic, the bat H9N2 IAV with so far unknown zoonotic potential was only recently discovered. In order to identify I) the role and potential of a newly discovered, potentially pre-pandemic virus, such as the bat H9N2, or II) possible future prevailing virus mutant variants of an already known pandemic virus, such as SARS-CoV-2, it is important to characterize these emerging viruses in vivo as soon and as good as possible.
The first objective in this dissertation (Publications I and II) therefore deals with the characterization of bat H9N2 and the estimation of its zoonotic or even pandemic potential.
In Publication I, a general susceptibility of directly inoculated Egyptian fruit bats to bat H9N2 was confirmed by successful seroconversion, although exhibiting only moderate viral shedding. All three contact animals remained seronegative, though one contact bat showed slight lesions in the histopathological analysis.
Publication II further addressed the question of the zoonotic potential of this virus. Inoculation of day-old turkey hatchlings demonstrated moderate susceptibility to bat H9N2 infection with a measurable seroconversion, while day-old chicken hatchlings were not susceptible to bat H9N2. Ferrets proved to be highly susceptible to bat H9N2 with high viral shedding, a transmission efficiency rate of 100% to direct contact animals at 2 days post contact, but with only minimal clinical signs. Importantly, the virus demonstrated the ability to evade the MxA-restriction factor and to replicate efficiently in human lung tissue explants. Furthermore, seasonal IAV- and standard IAV-vaccines showed no cross reactivity against the bat-N2 protein in humans. Therefore, further research on such viruses is urgently needed in order to prevent a renewed pandemic situation in the future as caused by SARS-CoV-2.
The second objective in this dissertation dealt with the identification and characterization of emerging SARS-CoV-2 Variants of Concern (VOCs).
Therefore, in Publication III, competitive infection experiments were performed using the Syrian golden hamster, the ferret, and transgenic mouse models (K18-hACE2 and hACE2-KI). These studies revealed replicative and transmissive predominance of Alpha VOC over Beta VOC, but not over SARS-CoV-2 WT in the hamster model, although Beta VOC substantially replicated in the lungs of donor animals. In contrast, the Alpha VOC had an unambiguous replication and transmission advantage over WT SARS-CoV-2 in the ferret and both mouse models. A recombinant SARS-CoV-2 WT-SAlpha virus helped to assign the fitness advantage of this variant particularly to the spike protein-associated mutations.
In Publication IV, in vitro results inferred an early replicative fitness advantage of Omicron BA.1 over Delta VOC, although the opposite was observed in competitively inoculated hamsters, ferrets and naive hACE2-KI mice. In addition, Publication IV demonstrated a disadvantage in transmission for the VOC Omicron BA.1 over the Delta VOC and a lack of susceptibility of ferrets after a single infection with the VOC Omicron BA.1. An mRNA vaccination of K18-hACE2 mice caused a drastic reduction of infectious virus particles in organ material following an infection with a recombinant SARS-CoV-2 WT-SDelta, but not when challenged with the SARS-CoV-2 SOmicron BA.1 clone.
This dissertation includes numerous, comprehensive experimental studies that are generally important for the characterization of emerging, potentially pre-pandemic viruses and may provide crucial information about the future dominance of certain virus variants in an ongoing pandemic. Here, the need for the use of a variety of animal models becomes apparent. By characterizing and classifying potentially zoonotic strains, these methods will help to better prepare for potentially upcoming pandemics and, in the case of a zoonotic or even pandemic event, to better detect and understand the circulating strains and their evolution.
Rekombinante Vektorvakzinen, basierend auf Viren der Newcastle-Krankheit (NDV) haben sich als kostengünstig, schnell herstellbar und sicher für die Applikation bei Geflügel und Säugetieren erwiesen. Im Rahmen dieser Arbeit wurden zwei rekombinante Vakzineviren, die das lentogene NDV Clone 30 als Vektor nutzen, für die Prävention der hochpathogenen aviären Influenza (HPAI) bei Hühnerküken, die maternale aviäre Influenzavirus-spezifische Antikörper (AIV-MDA) aufweisen, und die Pest der kleinen Wiederkäuer (PPR) bei Ziegen evaluiert.
Während bekannt ist, dass rekombinante NDV/AIV-H5-Vakzineviren in spezifisch pathogenfreien Eintagsküken eine protektive Immunantwort induzieren, ist diese bei Küken in HPAI-Endemiegebieten meist durch vorhandene AIV-MDA negativ beeinflusst. Durch die Generierung einer NDV-Rekombinanten (rNDVsolH5_H5), die das HA des HPAIV H5N1 von zwei individuellen Fremdgenen exprimiert, konnte eine Überexpression des H5 und in Folge der okulonasalen Immunisierung von zwei- und drei-Wochen-alten AIV-MDA+-Küken die Überwindung der AIV-MDA und die Bildung von wirtseigenen AIV-H5-spezifischen Antikörpern erreicht werden. Die in Folge einer HPAIV H5N1-Belastungsinfektion vermittelten Protektionsraten beliefen sich auf 85 % bei zwei-Wochen-alten und auf 100 % bei drei-Wochen-alten Hühnern, deren Ausscheidung des virulenten Wildtypvirus im Vergleich zu Kontrolltieren signifikant reduziert war. Auch wenn das rekombinante Impfvirus in ein-Wochen-alten AIV-MDA+-Küken replizieren konnte, weist die Schutzrate von 40 % darauf hin, dass die Immunisierung sehr junger AIV-MDA+-Küken nicht zu empfehlen ist.
Die subkutane Immunisierung von Ziegen mit dem rekombinanten Vektorvirus rNDV_HKur, das das Hämagglutinin des Morbillivirus der kleinen Wiederkäuer (small ruminant morbillivirus, PPRV) exprimiert, schützte diese vor einer virulenten Wildtypvirus-Infektion. Nach zweimaliger Applikation wurden, wie nach der Impfung mit einer atttenuierten PPRV-Lebendvakzine, weder Erkrankungszeichen beobachtet noch eine hämatogene Streuung und nur geringgradige Replikation bzw. Ausscheidung des PPR-Wildtypvirus nachgewiesen, was auf die Bildung PPRV-neutralisierender Antikörper nach der Immunisierung zurückzuführen ist. Somit konnte gezeigt werden, dass sich das rekombinante NDV/PPRV-H für die Prävention der PPR bei Ziegen eignet. Mit NDV als Vektorvirus konnten nachweislich die Anforderungen sowohl bezüglich der DIVA-Applikation als auch einer hohen Thermostabilität erreicht werden.
Um NDV als Vektorvirus gezielt zu verändern bzw. für dessen unterschiedliche Anwendungen zu verbessern, ist es von Vorteil die Funktion der einzelnen viralen Proteine zu kennen. Mit dem erstmaligen Nachweis der Expression des W-Proteins können nun mit Hilfe des generierten W-spezifischen Peptidantiserums weitere in-vitro- und in-vivo-Analysen erfolgen, um dessen Funktion im viralen Replikationszyklus näher zu untersuchen.