Characterization of full-length hepatitis C virus sequences for subtypes 1e, 1h and 1l, and a novel variant revealed Cameroon as an area in origin for genotype 1

Abstract

In this study, we characterized the full-length genome sequences of seven hepatitis C virus (HCV) isolates belonging to genotype 1. These represent the first complete genomes for HCV subtypes 1e, 1h, 1l, plus one novel variant that qualifies for a new but unassigned subtype. The genomes were characterized using 19-22 overlapping fragments. Each was 9400-9439 nt long and contained a single ORF encoding 3019-3020 amino acids. All viruses were isolated in the sera of seven patients residing in, or originating from, Cameroon. Predicted amino acid sequences were inspected and unique patterns of variation were noted. Phylogenetic analysis using full-length sequences provided evidence for nine genotype 1 subtypes, four of which are described for the first time here. Subsequent phylogenetic analysis of 141 partial NS5B sequences further differentiated 13 subtypes (1a-1m) and six additional unclassified lineages within genotype 1. As a result of this study, there are now seven HCV genotype 1 subtypes (1a-1c, 1e, 1g, 1h, 1l) and two unclassified genotype 1 lineages with full-length genomes characterized. Further analysis of 228 genotype 1 sequences from the HCV database with known countries is consistent with an African origin for genotype 1, and with the hypothesis of subsequent dissemination of some subtypes to Asia, Europe and the Americas.

Fig. 1.

An ML phylogenetic tree estimated using full-length genomic sequences of HCV. Reference sequences from confirmed subtypes of genotypes 1, 2, 3, 4, 5, 6 and 7 are included, together with the seven new isolates from this study (black circles). Each genotype is denoted at the right-hand side of the tree. Within genotype 1, all subtypes and equivalents are also indicated. Isolates are named using the following format: subtype_sampling country_sampling date (if available)_isolate name_accession number. The symbol ‘?’ indicates that a subtype has not been assigned. Bootstrap supports are shown at internal branches and the scale bar represents 0.10 nt substitutions per site. Country codes: CA, Canada; CM, Cameroon; EG, Egypt; ES, Spain; HEC, 〈?〉; HK, 〈?〉; ID, Indonesia; IN, India; JP, 〈?〉; MD, 〈?〉; TH, 〈?〉; UK; US, USA; VN, Vietnam.

Fig. 2.

An ML phylogenetic tree estimated using 141 HCV-1 partial NS5B sequences (positions 8276–8615 in the H77 reference genome). Each subtype or subtype equivalent is denoted at the right-hand side of the tree. Isolates from Cameroon are represented with red branches, and isolates from Canada are represented with blue branches, while isolates from France are indicated with purple branches. The seven isolates determined in this study are indicated by black circles. A black square indicates an isolate previously classified as subtype 1a, but which in this study was shown to belong to subtype 1e. For other details, see legend of Fig. 1.

Fig. 3.

Distribution of genotype 1 isolates in different countries and in Cameroon. (a) Histogram showing the frequencies of subtype 1e, 1h and 1l in four countries (CM, Cameroon; CA, Canada; FR, France; VN, Vietnam; ?, unknown country). The number of isolates is shown above each column while the country of origin is indicated below. (b) Histogram showing the frequencies of genotype 1 subtypes sampled in Cameroon. The number of isolates is shown above each column.

Fig. 4.

Analysis of amino acid sequences in the E2 and NS5A regions. Amino acids are shown using standard IUPAC codes and bars indicate indels. Isolate names are listed to the left of the alignment while amino acid positions (relative to the H77 reference genome) are indicated above.