Dania M Figueroa1, Eeva Kuisma2, M Jeremiah Matson1,3, Alain U Ondzie2, Trent Bushmaker1, Stephanie N Seifert1, Francine Ntoumi4, Beatriz Escudero-Pérez5,6, César Muñoz-Fontela5,6, Chris Walzer2,7, Sarah H Olson2, Cynthia Goma-Nkoua8, Jean-Vivien Mombouli8, Robert J Fischer1, Vincent J Munster9,10. 1. Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Hamilton, MT, USA. 2. Wildflife Conservation Society, Health Program, Bronx, NY, USA. 3. Marshall University, Joan C. Edwards School of Medicine, Huntington, WV, USA. 4. Fondation Congolaise pour la Recherche Médicale (FCRM), Brazzaville, Republic of Congo. 5. Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Strasse 74, 20359, Hamburg, Germany. 6. German Center for Infection Research (DZIF), Partner Site Hamburg, Bernhard Nocht Strasse 74, 20359, Hamburg, Germany. 7. Department of Interdisciplinary Life Sciences, Research Institute of Wildlife Ecology, University of Veterinary Medicine, Vienna, Austria. 8. Laboratoire National de Santé Publique, Brazzaville, Republic of the Congo. 9. Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, Hamilton, MT, USA. vincent.munster@nih.gov. 10. Rocky Mountain Laboratories, NIAID/NIH, 903S 4th Street, Hamilton, MT, 59840, USA. vincent.munster@nih.gov.
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. METHODS: The 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. RESULTS: The 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. CONCLUSION: We 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.
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. METHODS: The 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. RESULTS: The 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. CONCLUSION: We 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.
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