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

PLoS One. 2014 Sep 10;9(9):e107007. doi: 10.1371/journal.pone.0107007. eCollection 2014.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • DNA Probes / chemical synthesis*
  • DNA, Complementary / genetics
  • Democratic Republic of the Congo
  • Ebolavirus / genetics*
  • Ebolavirus / isolation & purification
  • Filoviridae / classification
  • Filoviridae / genetics*
  • Filoviridae / isolation & purification
  • Hemorrhagic Fever, Ebola / diagnosis
  • Hemorrhagic Fever, Ebola / veterinary*
  • Hemorrhagic Fever, Ebola / virology
  • Humans
  • Macaca mulatta
  • Polymerase Chain Reaction / methods*
  • RNA, Viral / genetics*
  • RNA, Viral / isolation & purification
  • Sequence Analysis, DNA

Substances

  • DNA Probes
  • DNA, Complementary
  • RNA, Viral

Grants and funding

This project was funded by the Defense Threat Reduction Agency (DTRA; http://dtra.mil) under grant numbers CB3630 and CB2999. Reference materials, including aliquots of diagnostic samples, were obtained through a collaboration with National Institute of Biomedical Research (French acronym INRB) in Kinshasa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.