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

Adam J Hume,Ferenc Jakab,Darrell N Kotton,Stephen Ross,Andrew Emili,Elke Mühlberger,Baylee Heiden,Esther Bullitt,Jacquelyn Turcinovic,Daniel Cifuentes,Volker Thiel,Mitchell R White,Joseph E Kaserman,John H Connor,Jessie Huang,Judith Olejnik,Maria Ericsson,Ellen L Suder,Andrew Wilson ,Gábor Endre Tóth ,Whitney A Scoon ,Tran Thi Nhu Thao ,Gábor Kemenesi ,Konstantinos–Dionysios Alysandratos ,Ryan Hekman

doi:10.1371/journal.ppat.1010268

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.

Highlights

Zoonotic viruses are a major public health threat
One example of these potentially zoonotic pathogens is Lloviu virus (LLOV), a filovirus which is closely related to Ebola virus
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

Summary

Introduction

Zoonotic viruses are a major public health threat. A single spillover event from an animal host into the human population can initiate deadly epidemics or even pandemics. Examples of bat-borne viruses that have been transmitted to humans, either directly or via intermediate hosts, causing multiple epidemics include Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Hendra virus, Nipah virus, and Marburg virus (MARV). For other viruses, such as Middle East Respiratory Syndrome coronavirus (MERSCoV), pandemic SARS-CoV-2, and Ebola virus (EBOV), there is strong evidence that bats might be the natural reservoir, these viruses have not yet been isolated from bats [1]. While some of these represent reemerging viruses that were already known to cause severe disease in humans, this list includes a number of bat-borne viruses that were either unknown or understudied prior to spillover into the human population

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