New Cell Lines Derived from European Tick Species

Tick cell lines are important tools for research on ticks and the pathogens they transmit. Here, we report the establishment of ten new cell lines from European ticks of the genera Argas, Dermacentor, Hyalomma, Ixodes and Rhipicephalus originating from Germany and Spain. For each cell line, the method used to generate the primary culture, a morphological description of the cells and species confirmation by sequencing of the partial 16S rRNA gene are presented. Further molecular analysis of the two new Ixodes ricinus cell lines and three existing cell lines of the same species revealed genetic variation between cell lines derived from ticks collected in the same or nearby locations. Collectively, these new cell lines will support research into a wide range of viral, bacterial and protozoal tick-borne diseases prevalent in Europe.</jats:p

African Swine Fever Virus – Exploring Virus Evolution and Vector Dynamics

African swine fever virus (ASFV) is one of the most threatening animal viruses which has dramatically expanded its distribution range within the last years. ASFV was first described and is endemic in sub-Saharan Africa where it is transmitted in a sylvatic cycle between indigenous suids and Ornithodoros soft ticks. Therefore, ASFV is the only known DNA-arbovirus and, in addition to that, the only member of the genus Asfivirus within the family Asfarviridae. Being highly infectious to domestic pigs and wild boar, the virus was introduced into Georgia in 2007 and has subsequently spread throughout eastern Europe reaching the European Union in 2014. Despite almost 100 years of intensive research and the occurrence of African swine fever (ASF) on four continents including Europe, many aspects of its epidemiology, vector dynamics and virus evolution are unknown. In our study, first evidence is presented on endogenous ASFV-like (EASFL)- elements which are integrated into the genome of ASFV natural vectors, O. moubata soft ticks. Through a series of experiments including next-generation sequencing, infection experiments, phylogenetic and BEAST analyses as well as PCR-screening, evidence is provided that these elements belong to an ancestral ASFV strain that might have existed 50,000 to 30,000 years BCE. Further results suggest that the EASFL-elements are involved in protecting ticks against ASFV infection and might belong to a generalised tick defence mechanism. In order to evaluate factors influencing ASFV epidemiology in eastern Europe, experiments were conducted on possible indigenous vector species and circulating virus isolates. In the absence of the natural tick vector, blow fly larvae were considered as possible mechanical vectors involved in ASFV transmission and persistence. Results are presented that even after feeding on highly infectious wild boar tissue, fly larvae and pupae showed no contamination with infectious virus. On the contrary, the maggots appeared to have inactivated the virus in the organ tissue through their salivary secretions. Further experiments conducted on an ASFV-strain isolated from northeastern Estonia resulted in the first report of an ASFV-strain with attenuated phenotype isolated in Eastern Europe. Results from NGS-analyses provided evidence for a major genome reorganisation in that strain that included a large deletion and a duplication of multiple ASFV genes. Taken together, this study provides novel insights into the epidemiology of ASF and evolution of ASFV one of the major threats to animal health worldwide and therefore does not only contribute significantly to basic research but possibly also to specific knowledge necessary for future disease management.Das Virus der Afrikanischen Schweinepest (ASPV) ist einer der gefährlichsten Tierseuchenerreger weltweit. ASPV wurde 1921 erstmalig in Afrika (Kenia) beschrieben und ist bis heute in Afrika endemisch, wo es vor allem südlich der Sahara in einem sylvatischen Zyklus zwischen Warzenschweinen und Lederzecken der Gattung Ornithodoros zirkuliert. Daher ist ASPV, als einziger derzeit bekannter Vertreter seiner Gattung (Asfivirus) und Familie (Asfarviridae), das einzige bekannte Virus mit einem DNA-Genom, das von Arthropoden übertragen wird. ASPV, hochpathogen für Haus- und Wildschweine, wurde 2007 von Afrika nach Georgien eingetragen. Seitdem breitet sich das Virus unkontrolliert in Osteuropa aus und hat 2014 auch die östlichen Mitgliedstaaten der Europäischen Union (Estland, Lettland, Litauen und Polen) erreicht. Trotz fast 100 Jahren intensiver Forschung und dem Vorkommen auf vier Kontinenten, existiert weder ein Impfstoff noch Behandlungsmöglichkeiten und viele Aspekte der Epidemiologie, Vektordynamik und Virusevolution von ASPV sind weiterhin unklar. In dieser Arbeit werden erstmalig endogene, ASPV-ähnliche Elemente (EASFL-Elemente) im Genom des biologischen Zeckenvektors, Ornithodoros moubata, beschrieben. Durch Sequenzierungen und Metagenomanalysen, sowie phylogenetische, BEAST und Real-Time PCR-Analysen konnte gezeigt werden, dass diese Elemente zu einem ASPV-ähnlichen Virus gehören, das vor etwa 30.000-50.000 Jahren in das Genom der Zecke integriert worden sein könnte. Daten aus Infektionsexperimenten mit verschiedenen Ornithodoros-Spezies legen weiterhin nahe, dass die EASFL-Elemente eine zentrale Rolle bei der Abwehr einer viralen Infektion in der Zecke spielen könnten. Im Weiteren beschäftigt sich diese Arbeit mit der Untersuchung verschiedener Faktoren, die die Epidemiologie von ASPV in Osteuropa beeinflussen können. Hierzu wurden zunächst Fliegenlarven als mögliche mechanische Vektoren und Reservoir für ASPV untersucht. Im Ergebnis konnte hier gezeigt werden, dass Fliegenlarven, die sich über mehrere Tage von stark virushaltigem Gewebe ernährten, nicht mit infektiösem Virus kontaminiert waren. Zusätzlich wurde gezeigt, dass die Larven das Virus im Organgewebe, vermutlich durch die Sekretion von Enzym-haltigen Speichel, effektiv inaktivieren. In weiteren Experimenten wurde ein estnisches ASPV-Isolat, das im Feld und in Infektionsexperimenten mit Haus- und Wildschweinen eine abgeschwächte Virulenz zeigte, mittels Vollgenomsequenzierung genetisch charakterisiert. Hierdurch konnte gezeigt werden, dass dieses Isolat sowohl eine große Deletion als auch eine Duplikation im viralen Genom aufweist, die als mögliche Ursache für die abgeschwächte Virulenz in Betracht gezogen werden muss. Zusammenfassend ermöglichen die Ergebnisse dieser Arbeit neue Einblicke in die Evolution und Epidemiologie von ASPV, einem der gefährlichsten viralen Tierseuchenerreger, der mittlerweile zu einer Gefahr für Schweinepopulationen weltweit geworden ist

Taking a Promising Vaccine Candidate Further: Efficacy of ASFV-G-&Delta;MGF after Intramuscular Vaccination of Domestic Pigs and Oral Vaccination of Wild Boar

African swine fever (ASF) is a pandemic threat to the global pig industry and wild suids. A safe and efficacious vaccine could monumentally assist in disease eradication. In the past years, promising live attenuated vaccine candidates emerged in proof-of-concept experiments, among which was &ldquo;ASFV-G-&#8710;MGF&rdquo;. In our study, we tested the vaccine candidate in three animal experiments intramuscularly in domestic pigs and orally in wild boar. Further, a macrophage-grown vaccine virus and a virus grown on permanent cells could be employed. Irrespective of the production system of the vaccine virus, a two-dose intramuscular immunization could induce close-to-sterile immunity with full clinical protection against challenge infection. After oral immunization, 50% of the vaccinees seroconverted and all responders were completely protected against subsequent challenge. All nonresponders developed ASF upon challenge with two acute lethal infections and two mild and transient courses. The latter results show a lower efficiency after oral administration that would have to be taken into consideration when designing vaccination-based control measures. Overall, our findings confirm that &ldquo;ASFV-G-&#8710;MGF&rdquo; is a most promising vaccine candidate that could find its way into well-organized and controlled immunization campaigns. Further research is needed to characterize safety aspects and define possible improvements of oral efficiency

Assessment of African swine fever vaccine candidate ASFV-G-∆MGF in a reversion to virulence study

Abstract African swine fever (ASF) has gained panzootic dimensions and commercial vaccines are still unavailable. Recently, a series of live attenuated vaccines has raised hope for an efficacious and safe vaccine, among them “ASFV-G-∆MGF”. We tested the latter in an in vivo reversion to virulence study in accordance with International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products guidelines. Upon forced animal passaging, a virus variant emerged that was associated with transient fever and an increased replication and shedding. However, all animals were healthy upon completion of the study and reversion to significant virulence was not observed. The genomic changes did not affect the recombination site but involved deletions and reorganizations in the terminal regions of the genome. Thus, our study underscores that in-depth safety characterization is needed for live ASF vaccines. For this particular candidate, additional studies should target long-term effects and transmission characteristics before thorough benefit-risk analysis can be carried out

Characterization of a Novel African Swine Fever Virus p72 Genotype II from Nigeria

African swine fever (ASF) is a high-consequence transboundary hemorrhagic fever of swine. It continues to spread across the globe causing socio-economic issues and threatening food security and biodiversity. In 2020, Nigeria reported a major ASF outbreak, killing close to half a million pigs. Based on the partial sequences of the genes B646L (p72) and E183L (p54), the virus responsible for the outbreak was identified as an African swine fever virus (ASFV) p72 genotype II. Here, we report further characterization of ASFV RV502, one of the isolates obtained during the outbreak. The whole genome sequence of this virus revealed a deletion of 6535 bp between the nucleotide positions 11,760–18,295 of the genome, and an apparent reverse complement duplication of the 5′ end of the genome at the 3′ end. Phylogenetically, ASFV RV502 clustered together with ASFV MAL/19/Karonga and ASFV Tanzania/Rukwa/2017/1 suggesting that the virus responsible for the 2020 outbreak in Nigeria has a South-eastern African origin