Core encoding sequences of Hepatitis C virus in Ghanaian blood donors are predominantly mosaics of different genotype 2 strains and cannot distinguish subtypes

Abstract

Background: Distribution of Hepatitis C virus (HCV) genotypes varies significantly worldwide. Genomic diversity between genotypes has implications for treatment, vaccine development and optimal design of HCV diagnostic assays. Molecular characterization of HCV in different geographical areas is therefore very essential for management and public health control of HCV infection. This study investigated the molecular epidemiology and characteristics of HCV genotypes in healthy individuals in Accra, Ghana.

Methods: An experimental study was carried out on blood samples obtained from voluntary blood donors. Two hundred samples were initially screened for HCV antibodies and infection was confirmed by RNA detection through RT-PCR of the 5'-untranslated region (5'UTR). The core gene sequences were analysed for HCV genotype determination by genotype-specific PCR; and then by cloning and direct sequencing followed by phylogenetic analysis. The sequences were further analysed in detail by similarity plotting.

Results: Molecular diagnosis confirmed the presence of HCV RNA in 2 out of 200 (1%) blood donors. Initial genotyping by genotype-specific PCR identified all two infections as subtypes 2a and 2b of genotype 2. Extensive evolutionary and genetic analyses indicated two epidemiological profiles. First, phylogenetic tree topologies clearly showed that, collectively, the core sequences of the Ghanaian HCV isolates belong to a single, distinct genetic group within HCV genotype 2 cluster, with high genetic similarity and rapid sequence variation in a single individual. Second, the sequences are mosaics comprising 2e and other genotype 2 subtype fragments. The analyses underscore a unique and complex HCV genotype 2 core sequence profile of the Ghanaian isolates.

Conclusions: Analysis of HCV core encoding sequences from Ghanaian blood donors in Accra confirmed predominance of genotype 2 HCV among healthy individuals. However, the isolates could not be classified into subtypes, possibly due to their complex sequence pattern that might suggest high mutability of the prevailing genotype. The core region of Ghanaian HCV therefore may not be suitable for distinguishing subtypes. These findings extend those from previous studies and thus underscore the need to search for subtype-informative region of Ghanaian HCV to elucidate the genetic diversity and factors determining outcome of HCV infections in Ghana.

Keywords: Blood donors; Core gene; Genotype; Ghana; HCV; Molecular epidemiology; Seroprevalence.

Fig. 1

Electrophoretic mobility patterns of PCR products from HCV detection, genotyping and (pre)cloning reactions In all gels first lane displays the molecular weight marker - 100 bp in ‘A’ and ‘B’ and 1 kb in ‘C’ and ‘D’. (a) HCV RNA detection in plasma samples. Lane 2 shows cDNA band (268 bp) of globin mRNA used as HCV positive control. Lanes 4 and 5 show 268 bp product indicating presence of HCV RNA in corresponding samples, HCV 152 and HCV 173. (b) Primer-specific genotyping of HCV 152 (lane 2) and HCV 173 (lane 3) showing migration positions of HCV genotypes 2a (190 bp) and 2b (337 bp). Two DNA bands with sizes corresponding to genotypes 2a and 2b observed in sample from donor labelled HCV 152 indicated this donor had dual genotype infection. (c) Semi-nested PCR amplification products (429 bp) from positive samples (HCV 152 and 173) purified for cloning and sequencing. (d) Five products [(HCV 173–1, 173–2, HCV 152–4, 152–5 and 152–8 of Eco RI digested rDNA (4.35 kb) clones purified from E. coli cultures. Positive clones showed two bands of sizes 3.9 kb and 429 bp corresponding to the vector and insert cDNA respectively

Fig. 2

Evolutionary relationship analyses of Ghanaian HCV isolates Phylogenetic analyses of HCV core-encoding region, performed by comparison of Ghanaian isolates with representative nucleotide sequences from all seven clades. The evolutionary history was inferred using neighbour-joining method; and the evolutionary distances were computed using Kimura 2-parameter method. Bootstrap scores were calculated from 1000 replicate analyses and values exceeding 700 (70%) are shown at tree nodes. The tree displays Ghanaian HCV isolates clustered under genotype 2, which are represented by coloured circles (blue); and subtype reference isolates, represented by the subtype (summarized to display only genotype, in the case of 1, 3, 4, 5 and 6) and isolate name. The scale represents number of nucleotide substitutions per site. HCV genotype 7a isolate EF108306 was used as outgroup. Evolutionary analyses were conducted in MEGA7. The branch leading to the group of Ghanaian isolates is 100% supported. The analyses show that the Ghanaian HCV isolates belong to single, unique phylogenetic group within HCV genotype 2 cluster. GH - Ghana

Fig. 3

SimPlot analyses of core gene sequences of four Ghanaian HCV isolates. In each of the figure units (a), (b), (c) and (d), upper panel shows sequence similarity plots (SimPlot analysis) and lower panel displays bootscan analyses results. Each analysis was performed by comparing sequence relationships of test viral sequences to representatives of reference genotype 2 and its subtypes 2a, 2b, 2c, 2d, 2e, 2f, 2i, 2 k, 2 m, 2q, 2r, 2 t and 2u retrieved from the International Committee on Taxonomy of Viruses (ICTV) website (https://hcv.lanl.gov/content/sequence/NEWALIGN/align.html/). Similarity plotting and bootscan analyses were performed in SimPlot version 3.5.1 with parameters set at simple consensus sequences; and with window and step sizes of 300 and 20 nucleotides respectively. The Y-axis represents percentage of sequence similarity to the corresponding subtype in the SimPlot analysis. The bootscan panel displays plots of bootstrap values (percentage permuted trees) calculated from multiple genome alignment of test viral sequences with reference subtype sequences. Bootscanning was performed using neighbour-joining method with Kimura-2 parameter modelling for 100 replicates. For all panels the x axis indicates nucleotide positions along the alignment (with gaps removed from the alignment). Reference sequences used are colour-coded and listed on the right of each plot. The analyses show the presence of a unique and complex HCV genotype 2 sequence profile of the Ghanaian isolates (core gene) comprising a 2e fragment (red colour-coded) of about 80 nucleotides from position 455 to 534 of the H77 reference sequence (GenBank accession number AF009606) and other variable fragments belonging to subtypes 2a, 2 k, 2q, 2r, 2u and/or 2