Recombinant Lloviu virus as a tool to study viral replication and host responses

Abstract

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.

Isolation of infectious Lloviu virus from Schreiber’s bats in Hungary

“…The RNA FISH analysis was performed once. The probes were evaluated with recombinant LLOV and reliably detected positive and negative-sense recombinant LLOV RNA in infected cells 21 .…”

“…Although rLLOV is able to infect known target cells of EBOV, including human primary macrophages, the inflammatory response in human macrophages, a hallmark of Ebola virus disease, is not induced by rLLOV. This suggests that LLOV might be able to infect humans, but the infection might not lead to disease, similar to the nonpathogenic Reston virus 21 . Public health preparedness to filovirus zoonoses is highly related to the ecological attributes of wildlife hosts.…”

Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers

“…SARS-CoV-2 stocks (isolate USA_WA1/2020, kindly provided by CDC’s Principal Investigator Natalie Thornburg and the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA)) and EBOV-ZsGreen [ 13 ], were grown in Vero E6 cells cultured in DMEM supplemented with L-glutamine (200 mM), penicillin (50 U/mL), streptomycin (50 mg/mL), and 2% FBS. Virus titers were determined in Vero E6 cells by tissue culture infectious dose 50 (TCID 50 ) assay using the Spearman and Kärber algorithm.…”

“…The inactivation of EBOV-containing samples is strictly regulated in that all inactivation procedures must be validated prior to approval. For this study, we used recombinant EBOV (Mayinga isolate) expressing ZsGreen (EBOV-ZsG) [ 13 ]. EBOV-ZsG grows to high viral titers, as needed for this study, and can be easily visualized because cells infected with EBOV-ZsGreen fluoresce green.…”

Isolation and genome characterization of Lloviu virus from Italian Schreibers’ bent-winged bats

“…Studies with recombinant and wild-type LLOV isolates confirmed the susceptibility of human-derived cell lines and primary human macrophages to LLOV infection in vitro . Based on these data, LLOV is now considered as a potential zoonotic virus with unknown pathogenicity to humans and bats [3,5].…”