Ebola Virus Epidemiology and Evolution in Nigeria

Folarin, Onikepe A.; Ehichioya, Deborah; Schaffner, Stephen F.; Winnicki, Sarah M.; Wohl, Shirlee; Eromon, Philomena; West, Kendra L.; Gladden-Young, Adrianne; Oyejide, Nicholas E.; Matranga, Christian B.; Deme, Awa Bineta; James, Ayorinde; Tomkins-Tinch, Christopher; Onyewurunwa, Kenneth; Ladner, Jason T.; Palacios, Gustavo; Nosamiefan, Iguosadolo; Andersen, Kristian G.; Omilabu, Sunday; Park, Daniel J.; Yozwiak, Nathan L.; Nasidi, Abdusallam; Garry, Robert F.; Tomori, Oyewale; Sabeti, Pardis C.; Happi, Christian T.

Samples from patients with suspected EVD were shipped both to the virology laboratory at Lagos University Teaching Hospital for diagnostics and to the African Center of Excellence for Genomics of Infectious Diseases (ACEGID) at RUN for diagnostics and sequencing. Whole-blood samples shipped to RUN were inactivated with AVL buffer (Qiagen) or TRIzol LS reagent (Life Technologies) in a 4:1 ratio, both according to the manufacturer's protocol. Inactivated samples were stored in a −20°C freezer. AVL buffer and TRIzol LS reagent have been used extensively in virus inactivation including for EBOV [2] [3] [4] [5] [6] [7] . Samples inactivated in AVL buffer were extracted using the QIAamp Viral RNA Mini Kit extraction protocol (Qiagen), according to the manufacturer's protocol. Samples inactivated in TRIzol reagent were extracted using chloroform modified with an AVL buffer inactivation and QIAamp Viral RNA Mini Kit extraction protocol. Following this modified protocol, 140 µL of chloroform was added to 1 mL of a TRIzol-inactivated sample. After vortex and centrifugation, 200 µL of the aqueous phase was transferred to a tube with 700 µL of AVL buffer without carrier RNA added. The sample was then processed according to the manufacturer's protocol for extraction, using the QIAamp Viral RNA Mini Kit. Extracted RNA samples were divided into aliquots for sequencing at both RUN and the Broad Institute of MIT and Harvard. Samples destined for the Broad Institute were shipped on dry ice and subsequently stored at −80°C.