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The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories. [ABSTRACT FROM AUTHOR]

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness. Author summary: Due to increasing utilization of high-throughput sequencing technologies, RNA sequences of many unknown viruses have been discovered in bats and other animal species. Research on the pathogenic potential of these viruses is hampered by incomplete viral genome sequences and difficulties in isolating infectious virus from the animal hosts. One example of these potentially zoonotic pathogens is Lloviu virus (LLOV), a filovirus which is closely related to Ebola virus. Here we applied molecular virological approaches, including minigenome assays, to complement the incomplete LLOV genome ends with sequences from related viruses and identify cis-acting elements required for LLOV replication and transcription that were missing in the published LLOV sequence. The resulting full-length clones were used to generate infectious recombinant LLOV. We used this virus for electron microscopic analyses, infection studies in human cells, host response analysis, and antiviral drug testing. Our results provide new insights into the pathogenic potential of LLOV and delineate a roadmap for studying uncultured viruses. [ABSTRACT FROM AUTHOR]

Reference 1 Hume AJ, Heiden B, Olejnik J, Suder EL, Ross S, Scoon WA, et al. (2022) Recombinant Lloviu virus as a tool to study viral replication and host responses. The x-axis label for panel D of Fig 5 is incorrect. [Extracted from the article]