Development and Evaluation of a Panel of Filovirus Sequence Capture Probes for Pathogen Detection by Next-Generation Sequencing

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.

Enhanced virome sequencing using targeted sequence capture

“…Targeted sequence capture has been used extensively to assess the human exome, as well as specific gene targets (Lovett et al 1991;Albert et al 2007;Hodges et al 2007;Okou et al 2007). Sequence capture has also been applied to the study of specific viruses (Depledge et al 2011;Duncavage et al 2011;Koehler et al 2014). Our aim was to develop a comprehensive viral targeted sequence capture panel that could be used to (1) assess all viruses known to infect vertebrate cells and (2) detect divergent viruses.…”

Neglected filoviruses

“…Diagnostic tests for BDBV infection are currently limited to various pan-filovirus reverse transcriptase-polymerase chain reaction (RT-PCR) or quantitative reverse transcriptase-PCR (qRT-PCR) protocols (Lu et al 2015) and BDBV-specific sequence capture probes for next-generation sequencing (Koehler et al 2014). A handful of BDBV-specific monoclonal or polyclonal murine, rabbit or crab-eating macaque antibodies (Ou et al 2011;Holtsberg et al 2015;Keck et al 2015;Wang et al 2015), BDBV …”

“…No reports on medical countermeasures against RESTV infection are available, most likely due to the perception that RESTV is not an imminent threat to humans. Diagnosis of RESTV infection is possible via pan-filovirus RT-PCR or qRT-PCR protocols (Lu et al 2015) and RESTV-specific sequence capture probes for next-generation sequencing (Koehler et al 2014). Several serological or nucleic-acid based RESTVspecific diagnostic systems have been described (Kalter et al 1995;Ksiazek et al 1999;Niikura et al 2001;Ikegami et al 2002bIkegami et al , 2003aOu et al 2011).…”

“…Minigenomes and reverse genetics systems have yet to be established for TAFV. TAFV detection is possible via panfilovirus RT-PCR (Lu et al 2015) and TAFV-specific sequence capture probes for next-generation sequencing (Koehler et al 2014). Very few TAFV-specific antibodies have been described (Ou et al 2011;Furuyama et al 2016), including ebolavirus cross-reactive llama single-domain antibodies (Sherwood and Hayhurst 2013).…”

Next-Generation Sequencing for Pathogen Detection and Identification

“…Similarly, Koehler et al reported on a panel of Filovirus capture probes used in conjunction with MiSeq sequencing to enable detection of Ebola and Marburg viruses. Their method utilized DxSeq™ probes, which are singlestranded DNA probes of which both ends bind to target sequences followed by filling in of the intervening captured sequence by polymerase and subsequent circularization (Koehler et al, 2014;McCarthy, 2011). Despite the fact that a pre-amplification step was performed prior to sequence capture and subsequent MiSeq sequencing, real-time PCR was still more sensitive for detection than sequencing in this case.…”