Complete Genome Sequence of a Hepatitis E Virus Genotype 1e Strain from an Outbreak in Nigeria, 2017

Hepatitis E virus genotype 1 (HEV-1) is associated with large epidemics. Notably, HEV subtype 1e (HEV-1e) has caused HEV outbreaks in sub-Saharan Africa.

H epatitis E virus (HEV) is the most common causative agent of acute viral hepatitis.
Eight HEV genotypes (HEV-1 to HEV-8) have been described, of which five are well recognized as human pathogens (1). HEV-1 and HEV-2 are waterborne, transmitted through fecal-oral routes, and are responsible for large HEV outbreaks in resourcelimited countries. HEV-3 and HEV-4 are linked to zoonotic transmission and cause sporadic infections in industrialized countries. It has been reported that HEV subtype 1e (HEV-1e) was responsible for a large outbreak in sub-Saharan Africa (Chad). The T3 strain (GenBank accession no. AY204877) was obtained from an infected person from France during an outbreak in Chad in 1983 and represents the only complete genome sequence of HEV-1e available (2). We report the second full-length genome sequence of an HEV-1e strain (NG/17-0503) from a recent outbreak in Nigeria in 2017. The virus was isolated from a 35-year-old resident of the Mobbar local government area of Borno State, Nigeria. The patient's serum tested positive for HEV antibodies using Wantai HEV IgM rapid test and Wantai HEV IgM enzyme-linked immunosorbent assay (ELISA; Sanbio, The Netherlands).
Viral RNA was extracted from serum using the QIAamp viral RNA minikit with the QIAcube BioRobot workstation (Qiagen, Hilden, Germany), followed by cDNA synthesis using SuperScript first-strand synthesis (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Real-time reverse transcription-PCR (RT-PCR) targeting the HEV open reading frames (ORF) 2 and 3 showed viremic HEV infection, with a viral load of 2.22 ϫ 10Eϩ6 IU/ml. Sequence analysis of partial ORF1 (307 bp) and ORF2 (401 bp) implemented using the BLASTn search engine (https://blast.ncbi.nlm.nih.gov) indicated that NG/17-0503 had highest nucleotide identity with the T3 strain. Real-time RT-PCR and consensus nested RT-PCR were conducted as previously described (3). The complete viral genome was amplified in fragments using the Kapa HiFi HotStart ReadyMix PCR kit (Roche, Mannheim, Germany) with HEV-1 universal and NG/17-0503 genomespecific primers ( Table 1). The 5= and 3= ends were amplified using 5= and 3= rapid amplification of cDNA ends (Roche). All HEV amplicons were sequenced using Sanger sequencing with a BigDye Terminator 3.1 kit (Thermo Fisher, USA) in sense and antisense directions. The whole-genome sequence was assembled and analyzed using Geneious software 10.0.5 (Biomatters Ltd., Auckland, New Zealand) (4). Phylogenetic analyses were performed with the MEGA 7.0.26 software (5).
The complete genome of NG/17-0503 is 7,284 nucleotides in length, excluding the poly(A) tail, with a GϩC content of 57.6%. The genome contains three ORFs (ORF1, 1,693 amino acids; ORF2, 660 amino acids; ORF3, 123 amino acids) encoding the HEV viral proteins. NG/17-0503 shared the highest identity of 94.2% with the T3 strain from Chad, followed by 88.7% with an HEV-1d Moroccan strain (GenBank accession no. MH918640). Sequences were aligned using the MAFFT algorithm (6). Phylogenetically, NG/17-0503 and T3 formed a separate subcluster within HEV-1 (Fig. 1). In conclusion, we identified and characterized the full-length genome of an HEV-1e strain circulating during an outbreak in Nigeria in 2017. Sequence and phylogenetic analyses showed that NG/17-0503 is the second full-length HEV-1e genome available to date. Since the Chad HEV-1e strain from 1983 was from a neighboring country of Nigeria, the detection of NG/17-0503 is strongly suggestive of local transmission of this endemic virus over decades.
Data availability. The complete genome sequence of NG/17-0503 has been deposited in the GenBank database under the accession no. MH918640.