Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Präventions- und Therapiestrategien gegen Orthopockenviren

Trotz der Ausrottung des hochpathogenen Variola Virus nach einer global angelegten Kampagne der WHO besteht das Restrisiko einer kriminellen Freisetzung des Pocken-Erregers, der dann auf eine inzwischen immunologisch weitgehend ungeschützte Bevölkerung treffen würde. Seit der Einstellung der flächendeckenden Impfungen steigt zudem die Gefahr von zoonotischen Erkrankungen durch Kuhpocken-, Affenpocken- und Vaccinia Viren. Daher werden weiterhin Impfstoffe sowie Therapeutika entwickelt und in Tiermodellen auf ihre Sicherheit und Wirksamkeit geprüft. In der vorliegenden Arbeit wurden sowohl Therapieansätze als auch Immunisierungsstrategien in Tiermodellen untersucht. In einem ersten Ansatz wurden durch die Immunisierung von Legehennen sowohl mit aktiven als auch mit inaktivierten Orthopockenviren spezifische IgY generiert. Die immuntherapeutische Wirksamkeit dieser qualitativ hochwertigen Antikörper wurde in einem Orthopockenvirus/Maus-Modell getestet, bei dem die Infektion mit Vaccinia Virus Western Reserve auf intranasalem Weg erfolgte. Die IgY wurden zu verschiedenen Zeitpunkten vor bzw. nach der Infektion ebenfalls intranasal verabreicht. Die Resultate dieser Studie belegen, dass es möglich ist, OPVspezifische IgY prophylaktisch und therapeutisch gegen OPV-Infektionen einzusetzen. In einem zweiten Ansatz wurde der Impfstoff IMVAMUNE auf seine Wirksamkeit im kürzlich etablierten Calpox Virus/Weißbüschelaffen-Modell untersucht. Diese weitgehend replikationsdefiziente Lebendvakzine der dritten Generation zeigt ein gutes Sicherheitsprofil und hat bereits von der FDA den sogenannten Fast Track- Status für ein beschleunigtes Zulassungsverfahren erhalten [1]. Die hier vorgestellten Ergebnisse zeigen, dass der Impfstoff die Tiere nicht vor einer letalen Dosis Calpox Virus schützt. Dabei spiegelte in diesem Modell die schwache Antikörperinduktion die ungenügende Schutzwirkung besser wieder als bei anderen Studien in nichthumanen Primatenmodellen, bei denen verglichen mit traditionellen Vakzinen die Antikörperantwort durch IMVAMUNE zwar ähnlich stark ausfällt, die Schutzwirkung aber schwächer ist, was den Einsatz von IMVAMUNE beim Menschen als Prävakzine als die sicherste Anwendungsform nahelegt.Despite of the eradication of the highly pathogenic variola virus as a consequence of the WHO’s global campaign there remains a risk of a criminal release of the smallpox pathogen which would encounter an immunologically almost unprotected human population by now. Since the cessation of the global immunization, the threat of zoonotic infections by cowpox viruses, monkeypox viruses and vaccinia viruses has increased. For this reason, vaccines and therapeutics are still being developed and their safety and efficacy are being evaluated. This study entails the investigation of both therapeutic approaches and immunization strategies in animal models. In a first approach orthopoxvirus-specific IgY were generated by the immunization of laying hens with infectious as well as inactivated orthopoxviruses. The immunotherapeutic efficacy of these high-quality antibodies was tested in an orthopoxvirus/mouse model, in which the infection with vaccinia virus western reserve was performed by the intranasal route. IgY were administered intranasally at different time points before and accordingly after infection. The results of this study demonstrate the possibility to apply OPV-specific IgY prophylactic and therapeutic against OPV-infections. In a second approach the efficacy of the vaccine IMVAMUNE was evaluated in the recently established calpox virus/marmoset model. This widely replication deficient live vaccine of the third generation reveals an extensive safety profile and received the FDA’s fast track status for an accelerated approval procedure. The results presented here demonstrate that the vaccine does not protect the animals against a lethal dose of calpox virus. In this model the observed weak induction of antibodies better reflected the insufficient protection than other studies working with non-human primate models. In the latter, compared to traditional vaccines, the antibody induction by IMVAMUNE is also strong but the protection is weaker suggesting the safest application of IMVAMUNE in humans would be as a prevaccine

External Quality Assessment (EQA) for Molecular Diagnostics of Zika Virus: Experiences from an International EQA Programme, 2016–2018

Quality Control for Molecular Diagnostics (QCMD), an international provider for External Quality Assessment (EQA) programmes, has introduced a programme for molecular diagnostics of Zika virus (ZIKV) in 2016, which has been continuously offered to interested laboratories since that time. The EQA schemes provided from 2016 to 2018 revealed that 86.7% (92/106), 82.4% (89/108), and 88.2% (90/102) of the participating laboratories reported correct results for all samples, respectively in 2016, 2017, and 2018. The review of results indicated a need for improvement concerning analytical sensitivity and specificity of the test methods. Comparison with the outcomes of other EQA initiatives briefly summarized here show that continuous quality assurance is important to improve laboratory performance and to increase preparedness with reliable diagnostic assays for effective patient management, infection and outbreak control.Peer Reviewe

Inactivation and Removal of Chikungunya Virus and Mayaro Virus from Plasma-derived Medicinal Products

Background: Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related members of the Semliki Forest complex within the genus alphavirus and are transmitted by arthropods, causing acute febrile illness in humans. CHIKV has spread to almost all continents, whereas autochthonous MAYV infections have been reported in South America and in the Caribbean. Nevertheless, there was concern about potential spread of MAYV to other regions similar to CHIKV in the past. The risk for transmission of emerging viruses by blood transfusion and the safety of plasma-derived medicinal products (PDMPs) are constant concerns. The manufacturing processes of PDMPs include procedures to inactivate/remove viruses. Methods: In this study, we investigated the reduction of MAYV and CHIKV by heat inactivation in various matrices, solvent/detergent treatment and nanofiltration. Results: Unexpectedly, MAYV was significantly more resistant to heat and solvent/detergent treatment compared to CHIKV. However, being similar in size, both MAYV and CHIKV were removed below the detection limit by 35 nm virus filters. Conclusions: The inactivation profiles of different alphavirus members vary considerably, even within the Semliki Forest Complex. However, robust dedicated viral inactivation/removal procedures commonly used in the plasma product industry are effective in inactivating or removing MAYV and CHIKV

Establishment of an Alphavirus-Specific Neutralization Assay to Distinguish Infections with Different Members of the Semliki Forest complex

Background: Alphaviruses are transmitted by arthropod vectors and can be found worldwide. Alphaviruses of the Semliki Forest complex such as chikungunya virus (CHIKV), Mayaro virus (MAYV) or Ross River virus (RRV) cause acute febrile illness and long-lasting arthralgia in humans, which cannot be clinically discriminated from a dengue virus or Zika virus infection. Alphaviruses utilize a diverse array of mosquito vectors for transmission and spread. For instance, adaptation of CHIKV to transmission by Aedes albopictus has increased its spread and resulted in large outbreaks in the Indian Ocean islands. For many alphaviruses commercial diagnostic tests are not available or show cross-reactivity among alphaviruses. Climate change and globalization will increase the spread of alphaviruses and monitoring of infections is necessary and requires virus-specific methods. Method: We established an alphavirus neutralization assay in a 384-well format by using pseudotyped lentiviral vectors. Results: MAYV-specific reactivity could be discriminated from CHIKV reactivity. Human plasma from blood donors infected with RRV could be clearly identified and did not cross-react with other alphaviruses. Conclusion: This safe and easy to use multiplex assay allows the discrimination of alphavirus-specific reactivity within a single assay and has potential for epidemiological surveillance. It might also be useful for the development of a pan-alphavirus vaccine

Epigenetic silencing of the c-fms locus during B-lymphopoiesis occurs in discrete steps and is reversible

The murine c-fms (Csf1r) gene encodes the macrophage colony-stimulating factor receptor, which is essential for macrophage development. It is expressed at a low level in haematopoietic stem cells and is switched off in all non-macrophage cell types. To examine the role of chromatin structure in this process we studied epigenetic silencing of c-fms during B-lymphopoiesis. c-fms chromatin in stem cells and multipotent progenitors is in the active conformation and bound by transcription factors. A similar result was obtained with specified common myeloid and lymphoid progenitor cells. In developing B cells, c-fms chromatin is silenced in distinct steps, whereby first the binding of transcription factors and RNA expression is lost, followed by a loss of nuclease accessibility. Interestingly, regions of de novo DNA methylation in B cells overlap with an intronic antisense transcription unit that is differently regulated during lymphopoiesis. However, even at mature B cell stages, c-fms chromatin is still in a poised conformation and c-fms expression can be re-activated by conditional deletion of the transcription factor Pax5