Development and evaluation of a panel of filovirus sequence capture probes for pathogen detection by next-generation sequencing.

Adeyemi Adesokan,Adrienne T Hall,Anna N Honko,Timothy D Minogue,Randal J Schoepp,Jean-Jacques Muyembe,Jens H Kuhn,Prime Mulembekani,Gustavo F Palacios,P Alexander Rolfe,Joseph N Fair,Jeffrey W Koehler

doi:10.1371/journal.pone.0107007

Abstract

A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance.

Highlights

Filoviruses are highly pathogenic viruses that can cause outbreaks with significant disease and high lethality
We developed a panel of pathogen-specific probes to detect multiple filoviruses including Ebola virus (EBOV), Sudan virus (SUDV), Reston virus (RESTV), Taı Forest virus (TAFV), Bundibugyo virus (BDBV), and the Marburg virus (MARV) variants Musoke, Ci67, and Angola
The nonhuman primate (NHP) experiment and procedures were approved by the USAMRIID Institutional Animal Care and Use Committee (IACUC) and was carried out in compliance with the regulations outlined in the USDA Animal Welfare Act (PHS Policy) and other Federal statutes and regulations relating to animals and experiments involving animals

Summary

Introduction

Filoviruses are highly pathogenic viruses that can cause outbreaks with significant disease and high lethality. Based on revised filovirus naming standards [1], the filoviruses are assigned to three different genera, Ebolavirus, Marburgvirus, and Cuevavirus. They have at least 30% sequence divergence from virus to virus and include Ebola virus (EBOV), Sudan virus (SUDV), Taı Forest virus (TAFV), Reston virus (RESTV), and Bundibugyo virus (BDBV). Recent outbreaks and newly discovered filoviruses have highlighted the geographic range and diversity of the filoviruses, including the identification of RESTV as a highly pathogenic virus of cynomolgus macaques [2] that leads to apparently asymptomatic infections in humans [3]. Additional filoviruses have recently been discovered, including BDBV in Uganda [4], and Lloviu virus (LLOV), the only member of the genus Cuevavirus, in Spain [5]

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