BACKGROUND: Filoviruses, including Ebola and Marburg viruses, cause severe hemorrhagic fever in humans and primates. These viruses pose significant threats to public health, making rapid and sensitive detection critical for controlling outbreaks. We developed and validated a hemi-nested generic PanFilo assay to detect all Ebola virus species, Marburg viruses, and recently discovered bat filoviruses. This assay was deployed to 15 European laboratories and evaluated through testing of eight non-infectious samples. OBJECTIVES: Laboratories were asked to determine the detection limit of positive controls and test all samples using the assay provided. The deployed assay enables direct Nanopore sequencing of PCR products, by using tagged primers during the second round of PCR. Sequencing of the samples was carried out on a voluntary basis. RESULTS: Multicenter validation revealed a 95 % limit of detection of 5309 RNA copies/µL for Ebola, 10,273 copies/µL for Marburg, and 2145 copies/µL for Mengla virus. In an implementation quality assessment, 93.3 % (84/90) of samples containing filovirus RNA were correctly identified and 100 % (30/30) of filovirus-negative samples were correctly identified. Thirteen laboratories sequenced PCR products, with nine identifying all positive samples correctly. CONCLUSION: The assay enables rapid and reliable detection of filoviruses, with sequencing capabilities for identifying both known and novel variants. This assay might be used for detection during the initial phase of an emerging filovirus outbreak, before a specific assay has been developed. However, our distribution across 15 laboratories revealed variability challenges due to reagents, human performance, and sequencing capacity, emphasizing the need for more training and standardization.

Centre for Infectious Disease Control National Institute for Public Health and the Environment Bilthoven the Netherlands

Charité Universitätsmedizin Berlin corporate member of Freie Universität Berlin and Humboldt Universität zu Berlin Institute of Virology Berlin Germany

Charité Universitätsmedizin Berlin corporate member of Freie Universität Berlin and Humboldt Universität zu Berlin Institute of Virology Berlin Germany; German Center for Infection Research associated partner Charité Berlin Germany

Charité Universitätsmedizin Berlin corporate member of Freie Universität Berlin and Humboldt Universität zu Berlin Institute of Virology Berlin Germany; German Center for Infection Research associated partner Charité Berlin Germany; Labor Berlin Charité Vivantes GmbH Berlin Germany

Croatian Institute of Public Health Zagreb Croatia

Department of Genetics and Microbiology Faculty of Science BIOCEV Charles University Praha Czech Republic

Department of Microbiology Immunology and Transplantation Rega Institute KU Leuven Leuven Belgium

Department of Microbiology Institute of Biomedicine and Translational Medicine Faculty of Medicine University of Tartu Tartu Estonia

Department of Molecular Medicine University of Padova Padova Italy; Microbiology and Virology Unit Padova University Hospital Padova Italy

Department of Viroscience Erasmus MC Rotterdam the Netherlands

Emergency Response and Biopreparedness Unit Department of Infectious Diseases National Institute of Health Doutor Ricardo Jorge Lisbon Portugal

Helsinki University and Helsinki University Hospital HUS Diagnostic Center Helsinki Finland

Institute of Diagnostic Virology Friedrich Loeffler Institute Greifswald Insel Riems Germany

Laboratory of Microbiology School of Medicine Aristotle University of Thessaloniki Thessaloniki Greece

Malopolska Centre of Biotechnology Jagiellonian University Gronostajowa 7A 30 387 Krakow Poland

National Reference Laboratory for Arbovirus Unit of Arbo Hanta and Emerging viruses Department of Infectious diseases National Health Institute of Italy Rome Italy

Unite des Virus Emergents France

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