A 40-year-old Liberian man, who had been living in Elwa, Monrovia, travelled by air through Accra (Ghana) and Lome (Togo) to Lagos, Nigeria. Upon arrival at Lagos International Airport on 20 July 2014, he collapsed and was taken to a private hospital in the Obalende area of Lagos, Nigeria (Figure 1), where he was admitted. He presented with high fever, lymphadenopathy and sore throat and was treated for malaria and typhoid fever until he later developed diarrhoea, vomiting and microscopic haematuria. The culmination of the clinical presentation, the patient's non-response to treatment for malaria and typhoid, as well as his epidemiological link to Liberia, raised the suspicion for EVD. On 22 July 2014, blood samples in EDTA tubes and urine samples were collected and sent for viral investigations to the CMUL-LUTH Virology Unit Laboratory. Aliquots of the specimens were sent in parallel to the African Center of Excellence for Genomics of Infectious Diseases (ACEGID) at Redeemers University in Ogun State, Nigeria.
No ethics approval was required for the diagnosis of an infection of great public health importance and which was part of the normal course of management of any infected patient.
Due to the fact that no BSL-3 or BSL-4 facilities were available to handle the patient's specimens, all samples received were handled in a BSL-2 facility with extreme care and in line with US Centers for Disease Control and Prevention (CDC) safety guidelines. Personal protective equipment (PPE) was worn in accordance with the 'donning and doffing of PPE' protocol set out by the CDC. Sample carrier boxes were sprayed with 10% hypochlorite solution on a disposable laboratory absorbent mat in a biosafety class IIA cabinet. Specimen containers were removed from the carrier box and wrapped immediately with a laboratory paper towel soaked in 10% hypochlorite in order to disinfect any possible spillage around the specimen container. An identical process was used for removal of samples from the container box. Outer gloves were sprayed with 10% hypochlorite solution every time hands were withdrawn from the safety cabinet and then disposed of in double-jacketed bio-hazard waste bags if they were soiled during sample handling or at the end of the procedure. Aliquots of specimens used for viral assays were immediately inactivated in a guanidinium-thiocyanate lysis buffer for viral nucleic acid extraction (Qiagen, Germantown, Maryland, United States).
For viral RNA extraction, neat (undiluted), 1:10 and 1:100 dilutions in sterile phosphate-buffered saline specimens of both blood and urine were processed using a Qiagen RNA extraction kit (Qiagen, Germantown, Maryland, United States). Segments of the L-gene of the Ebola virus, the S-gene of the Lassa virus and the 3' non-coding region of the Dengue fever virus were amplified in singleplex PCRs using the primers listed in Table 1. Separate master mixes for each queried virus were prepared and cycled as described in the AmbionAgPath-ID One-Step RT-PCR kit (Applied Biosystems, Foster City, California, United States) protocol. Subsequently, PCR amplicons were subjected to 1.5% agarose gel electrophoresis with 1X SYBR  Safe DNA gel staining dye (Invitrogen, Carlsbad, California, United States) at 120V for 30 minutes and images were taken with a BioDocAnalyze 2.0 (Biometra, Goettingen, Germany).
PCR amplicons on the agarose gel were purified using an agarose gel extraction kit (Jena Bioscience, Jena, Germany). Purified non-infectious PCR products, packaged and transported using triple-level packaging, were sequenced using the Filo A2.3 primer on the Sanger dideoxy sequencing technology platform with an Applied Biosystems 3130xl Genetic Analyser at Jena Bioscience in Jena, Germany. A sequence trace file was used for BLAST analysis in GenBank. The RNA-dependent RNA polymerase (L) gene, partial Coding sequence (CDS) for EBOV/Hsap/NGA/2014/LIB-NIG 01072014 was deposited in the National Center for Biotechnology Information (NCBI) with the accession number KM251803.1. A set of 30 different Filovirus genomes were selected from NCBI's GenBank. The FASTA format of the L-gene region of all the selected genomes was downloaded and aligned with the KM251803.1 sequence using the MUSCLE tool of MEGA-6 software (Figure 2). The Tamura 3 parameter (T92) was found to be the best maximum likelihood model to infer the phylogenetic tree. A discrete Gamma distribution was used to model evolutionary rate differences among sites (+G, parameter = 0.8048) and the rate variation model allowed for some sites to be evolutionarily invariable ([+I], 36.3524% sites). The reliability of each node on the phylogenetic tree was tested by bootstrapping with 1000 replicates.
Investigations carried out on viral RNA purified from the blood and urine specimens of the patient revealed the presence of the expected amplicon size (300 bp) for Filoviruses (Figure 2). However, no signals were observed for Lassa or Dengue fever viruses. The filovirus amplicons were still detected in blood specimens with logscale dilutions of 1 (1:10) and 2 (1:100), whereas in urine samples the signal was no longer detectable at a log 3 dilution.
Phylogenetic analysis of the L-gene partial CDS indicated that the virus evolved from the Guinea and Mano River isolates of the 2014 outbreak with a bootstraping value of 87% (Figure 3). Other isolates represented on the phylogenetic tree confirmed the evolutionary relationships between the different filoviruses.