Abstract
Early detection of Ebola virus spillover into wildlife is crucial for rapid response. We developed and validated a portable, cold-chain independent Ebola virus RT-qPCR assay.MethodsThe field syringe-based RNA extraction method was compared with a conventional laboratory-based spin-column RNA extraction method. Next, the qPCR efficiency and limit of detection of the assay was compared to standard laboratory-based reagents and equipment. The specificity of the assay was confirmed by testing against multiple Zaire Ebolavirus (EBOV) variants and other ebolavirus species. Lastly, swabs from an EBOV-infected non-human primate carcass, stored at environmental conditions mimicking central and west Africa, were analyzed to mimic in field conditions.ResultsThe syringe-based RNA extraction method performed comparably to a standard laboratory spin-column-based method. The developed assay was comparable in sensitivity and specificity to standard laboratory-based diagnostic assays. The assay specifically detected EBOV and not any of the other tested ebolavirus species, including Reston ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, and Tai Forrest ebolavirus. Notably, the assays limit of detection for EBOV isolates were all below 4 genome copies/μL. The assay was able to detect EBOV in oral, nasal, thoracic cavity, and conjunctiva swabs obtained from an infected non-human primate.ConclusionWe developed a field-based Ebolavirus assay which is comparable in sensitivity and specificity to laboratory-based assays. Currently, the assay is being incorporated into wildlife carcass surveillance in the Republic of the Congo and is being adapted for other infectious disease agents.
Highlights
Since its discovery in 1976, there have been 17 recorded Ebola virus (EBOV, species Zaire ebolavirus) outbreaks affecting humans in west and central Africa [1]
Postmortem sampling of EBOV-infected non-human primate We studied a cynomolgus macaque included as control in EBOV vaccine studies and euthanized because of signs of Ebola Virus Disease and viremia
The qPCR efficiency and limit of detection (LoD) between instruments was evaluated by testing the LightCycler 480 RNA Master Hydrolysis Probes (LC480) reagents on both the BM and the SmartCycler (Fig. 1b)
Summary
Since its discovery in 1976, there have been 17 recorded Ebola virus (EBOV, species Zaire ebolavirus) outbreaks affecting humans in west and central Africa [1]. EBOV can cause mortality events in several wildlife species including gorillas, chimpanzees, and duikers [2, 3]. Multiple EBOV zoonotic spillover events during the 2001–2003 Gabon and Republic of Congo (RoC) outbreaks resulted from direct contact with infected gorilla, chimpanzee, and duiker carcasses [2]. The ability to rapidly detect EBOV in wildlife carcasses has the potential to limit human contact with EBOV and prevent large-scale outbreaks. Early detection of mortality events due to EBOV in wildlife could be used to limit further spread and aid in the design of data-driven countermeasures (e.g. wildlife vaccination) [7]
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