Background
Viral hepatitis presents a major health problem throughout the world [
1]. Hepatitis C virus (HCV) is the most important agent of chronic or acute parenterally transmitted viral hepatitis [
2]. The virus is able to establish persistent infections in more than 90% of infected people and cause quiescent, progressive and asymptomatic infections for several years [
3,
4]. Its replication is characterized by high rate of genetic mutation, as a result of lack of fidelity of the RNA-dependent RNA polymerase encoded by the non-structural 5B (NS5B) gene [
5‐
7]. High genetic mutation of the HCV genome results in rapid generation of viral variants. Even within an infected individual HCV variants emerge having genomes that are either very closely related, referred to as quasispecies, or genetically distinct, referred to as genotypes or subtypes [
8‐
10].
Previously, HCV was classified into six phylogenetically distinct genotypes numbered 1–6 [
11,
12]. Subsequently, comparison of sequences derived from various geographical locations have shown that a large number of HCV variants exist [
13,
14]. Recently, analysis of over 1300 (near-)complete HCV genome sequences available on public databases using consensus criteria classified HCV into seven confirmed genotypes (numbered 1 to 7) and 67 subtypes; and identified 21 additional complete coding region sequences of unassigned subtype [
15]. Molecular epidemiology studies have shown that these HCV genotypes and subtypes are differently distributed in different regions of the world, and certain genotypes predominate in certain regions [
16,
17].
Africa is reported as the World Health Organization (WHO) region with the highest HCV prevalence. The average prevalence estimated for sub-Saharan Africa is approximately 3.0% [
18‐
20]; and there exist extensive diversity within genotypes in Central and West African countries [
21‐
24]. Importantly, genotypes 1 and 2 are suggested to be endemic in West Africa [
25,
26] where the present study country, Ghana, is located. A recent comprehensive, systematic review by Akosua Agyeman and colleagues [
27] estimated the prevalence of chronic HCV infection among Ghanaian blood donors as 2.6%. Other studies also confirmed the predominance of genotype 2 HCV [
22,
28]. However, the prevailing HCV subtypes in the country are yet to be completely determined. Recently, a Bayesian phylogeographic analysis using discrete trait model showed Ghana as the most likely geographic region for the origin of HCV genotype 2 [
29]. Candotti and colleagues previously analysed the E1/E2 and NS5 regions; and, noting the challenge in distinguishing genotype 2 subtypes, have described the Ghanaian HCV sequences as diverse. Notwithstanding, other studies have found the core encoding region suitable and ideal for HCV classification at the subtype level [
30,
31]. This study therefore aimed to describe the genetic diversity of prevailing HCV genotypes in healthy individuals in Accra, Ghana, based on analysis of the core encoding region. Analysis showed that the core sequences of the HCV isolates detected were complex and phylogenetically distinct from other HCV 2 subtypes. This study thus reports that the core gene of Ghanaian HCV isolates may not be suitable for distinguishing HCV genotype 2 subtypes.
Methods
Samples
This study was cross-sectional and experimental. It was carried out on 200 consenting voluntary blood donors sampled through a national blood donation exercise in Accra. Random samples were collected in four millilitres EDTA tubes (Vacutainer Systems, Frankh Lakes, N. J., USA). Within 2 h of drawing blood, plasma was separated after centrifugation and immediately frozen at –70 °C until used for serological testing and virus RNA extraction.
HCV antibodies testing and confirmation
Plasma from donor specimens were serologically screened and confirmed for the presence of anti-HCV antibodies. Initial screening was done with SERODIA-HCV particle agglutination (PA) assay (FUJIREBIO INC., Tokyo, Japan). All sera, both PA reactive and nonreactive were retested with Murex anti-HCV version 4.0 Enzyme-linked Immunosorbent Assay (ELISA) (Murex Biotech SA Ltd., Kyalami, South Africa). All assays were performed strictly according to the manufacturers’ instructions. HCV RNA extraction and amplification were performed on sera that were reactive by either PA or anti-HCV EIA; and PCR-confirmed specimens were genotyped.
RNA preparation and PCR amplification
Total RNA was extracted from 100 μl of plasma by SepaGene RNA extraction kit (Sanko Junyaku Co. Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Extracted RNA were vacuum-dried and resuspended in 40 μl of diethyl pyrocarbonate (DEPC)-treated water (DEPC was by SIGMA Chemical Co., Steinheim, Germany); and stored at –70 °C until use. Extracted RNA was reverse-transcribed with Moloney murine leukemia virus (M-MuLV) reverse transcriptase and cDNA amplification was by AmpliTaq Gold DNA polymerase with primers located in the highly conserved 5′-untranslated region (5’UTR) as described below.
HCV RNA detection
RT-PCR was performed by a one-step method using Ready-To-Go RT-PCR bead system (Amersham Pharmacia Biotech Inc., N. J, USA) optimized for first strand cDNA synthesis and PCR. The RT-PCR reaction was carried out in a final volume of 50 μl containing 10 units of RNAguard, 100 units of M-MuLV reverse transcriptase, 200 μM of each deoxynucleotide triphosphate (dNTP), 2 units of AmpliT
aq Gold DNA polymerase and 1.5 mM MgCl
2 in RNase-free water, to which 0.65 μl of 10 μM each HCV-specific outer primer and 5 μl of RNA template were added. Globin mRNA was used as HCV positive control. Previously described primers, HCV 19 (GCGACACTCCACCATAGAT) and HCV 20 (GCTCATGGTGCACGGTCTA [
32] were used for the RT-PCR. These yielded a fragment of 329 bp. Reverse transcription was achieved by incubating the mixture at 42 °C for 30 min, followed by AmpliT
aq Gold DNA polymerase activation step at 95 °C for 5 min. First round PCR comprised 40 cycles with the following parameters: denaturation at 94 °C for 30 s, annealing at 50 °C for 45 s, and extension at 72 °C for 1 min. The reverse transcription and amplification reactions were done using Perkin-Elmer GeneAmp PCR System 2400 (Norwalk CT, USA).
Second round PCR was carried out using Ready-To-Go PCR reaction system. Bead in the reaction tube was dissolved in 22.3 μl RNase-free water; 0.35 μl each of the pair of inner primers HCV 21/HCV22 [
32] and 2 μl of first round PCR product were added. When brought to a final volume of 25 μl, the reaction mixture contained 1.5 units of T
aq DNA Polymerase, 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl, 1.5 mM MgCl
2 and 200 μM of each dNTP. Nested PCR conditions comprised preheating at 94 °C for 2 mins, followed by two 20-cycle rounds of PCR. Conditions for the first 20 cycles of amplification comprised denaturation at 94 °C for 30 s, annealing at 53 °C for 45 s and extension at 72 °C for 1 min. Conditions for the second 20 cycles round of amplification comprised 94 °C for 30 s, 55 °C for 45 s and 72 °C for 1 min. The expected product size was 268 bp. PCR products were evaluated by electrophoresis in 2% agarose gel in 1X Tris-Acetic Ethylenediaminetetraacetic acid (1X TAE). Tris base (Tris[hydroxymethyl]aminomethane), EDTA and ethidium bromide were obtained from SIGMA Chemical Co., Steinheim, Germany; agarose was from Invitrogen, Life Technologies, Paisley, Scotland.
HCV genotyping
HCV PCR positive samples were genotyped by type-specific PCR using primers that selectively amplify different genotypes as described by Ohno et al. [
33]. Briefly, the core region of the HCV genome was reverse-transcribed, and two rounds of PCR were performed. PCR amplification was done using Ready-To-Go RT-PCR. RT-PCR reaction bead containing M-MuLV reverse transcriptase. The bead was dissolved in 40.5 μl of RNase-free water; 2.5 μl of 1 μg/ul random hexamer pd.(N)
6 (GIBCO BRL, Gaithersburg, Md.) and 5 μl RNA template was added. RNA reverse-transcription was achieved by incubating the reaction mixture at 42 °C for 25 mins and heating at 95 °C for 5 mins. First round PCR utilized primers Sc2 and Ac2 [
33], and T
aq DNA Polymerase. One microliter of each 2.5 μM primer was added to the RT reaction tube. Amplification conditions included pre-incubation at 94 °C for 1 min, followed by 40 cycles with the following parameters: a preliminary 20 cycles amplification through denaturation at 94 °C for 1 min, annealing at 45 °C for 1 min and extension at 72 °C for 1 min; followed by additional 20 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min.
Two different second-round PCRs were performed for each sample by the method of Ohno et al. [
33] modified for use in a Ready-To-Go PCR bead system. Briefly, two different primer mixtures were prepared per sample - one containing S7, S2a, G1b, G2a, G2b and G3b primers (Mix 1) and another containing S7, G1a, G3a, G4, G5a and G6a primers (Mix 2). The total reaction volume was 25 μl and made up of 18.5 μl RNase-free water, 6 μl of 2.5 μM PCR primer Mix 1 or Mix 2 and 0.5 ul of first-round PCR amplicon as template. Amplification was performed by preheating at 94 °C for 1 min, followed by 30 cycles of reaction. Each cycle comprised denaturation at 94 °C for 1 min, annealing at 62 °C for 45 s and extension at 72 °C for 1 min. Ten microliters of the second-round PCR product was evaluated in 2% agarose gel electrophoresis. HCV genotype was determined by identifying genotype-specific DNA bands in gel by their expected sizes based on primers used [
33].
Cloning and sequencing
To verify HCV genotype and determine possible inter- and intra-patient subtype differences, HCV core amplicons (approximately 429 bp) were further analysed. A semi-nested PCR was performed with Sc2 and Ac2 as first-round primers and S7 and Ac2 as second-round primers [
33] . The PCR conditions were as described for genotyping above. PCR products were purified from 2% agarose gel using QIAquick gel purification protocol (Qiagen Ltd., Germany) according to the manufacturer’s instructions. Purified amplicons were cloned directly into pCR 2.1-TOPO plasmid vector (~ 3.9 kb) and used to transform chemically competent
Escherichia coli. Positive clones were detected through purification by Miniprep protocol (Qiagen Ltd.) and digestion with
Eco RI. LB Agar, LB Broth Base, pCR 2.1 TOPO vector and
Escherichia coli were obtained from Invitrogen, Life Technologies, Paisley, Scotland; and
Eco RI was from Roche Diagnostics GmbH., Mannheim, Germany.
For each isolate, at least two clones were sequenced on both strands using BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). Sequencing products were purified by ethanol precipitation protocol. Electrophoresis and data acquisition were done on an automated ABI PRISM 310 genetic analyser (Applied Biosystems). Consensus nucleotide sequences obtained from the isolates were used in phylogenetic analysis.
Phylogenetic relationships and evolutionary analyses
Nearly complete core coding sequence (420 nt) corresponding to positions 342–761 of H77 reference sequence [accession number AF009606] was analysed. Representative sequences from the 7 different HCV clades were selected from the 2015 updated alignment of HCV genotypes and subtypes provided by the International Committee on Taxonomy of Viruses (ICTV), available at
https://hcv.lanl.gov/content/sequence/NEWALIGN/align.html/ (the ICTV website). More genotype 2 sequences from Ghana were included for analysis to clarify phylogenetic relationships. Reference and test viral core sequences were first aligned using CLUSTAL W program and edited using BioEdit version 7.2.5 [
34]. Subsequently, multiple sequence alignment was performed by using MUSCLE program implemented in Molecular Evolutionary Genetics Analysis (MEGA) software [
35] (
megasoftware.net/index.html); and evolutionary distances were determined based on the maximum likelihood model. Phylogenetic trees with all branch lengths drawn to scale were constructed by neighbour-joining algorithm modelled with Kimura-2 parameter method set for 1000 bootstrap replicates; and values above 700 (70%) were considered to support branching clusters. All phylogenetic relationships and molecular evolutionary analyses were conducted using MEGA version 7.
To clarify sequence relationships, similarity plotting and bootscanning were performed using SimPlot software version 3.5.1 [
36] with window and step sizes of 300 and 20 nucleotides respectively. Representative sequences of all genotype 2 subtypes so far described, and other genotype 2 sequence from Ghana, were used as references in the SimPlot analyses.
Sequence repository
All sequences have been deposited with DNA Databank of Japan (DDBJ) under accession numbers LC271214 to LC271217).
List of reference sequences
Reference sequences retrieved and used for comparison with those obtained in this study are listed by genotype as follows: genotype 1: GenBank nos. HQ537007, AF009606, D90208, D14853, KJ439768; genotype 2: JF735116, D00944, AB047639, HQ639944, AB031663, AB030907, AB661382, D50409, D10988, JF735114, JF735120, KC844042, DQ155561, HM777359, AB031663, JF735111, FN666429, JF735115, KC197238 and JF735112, KJ642629 (GH02), KJ642628 (GH03), KJ642625 (GH06), KJ642623 (GH08); genotype 3: JF735124, D17763 and KJ470619; genotype 4: FJ025854, Y11604, FJ462435, FJ462436 and DQ418786; genotype 5: AF064490 and Y13184; genotype 6: DQ278893, D84262 and Y12083; and genotype 7: EF108306
Discussion
This study investigated HCV infections and characterized viral isolates in healthy Ghanaians in Accra, the capital of Ghana. Serological screening using two standard conventional assays showed high discrepancy. Confirmation by PCR revealed low level HCV viraemia in the studied population. A previous study reported frequent recovery and low viral load in Ghanaian HCV infections [
22]. Other reports also show that HCV infection in West and Central Africans rarely progresses to chronic liver disease as compared to what occurs in America, Europe and China [
37‐
39]. Also, antibody responses to HCV are usually directed against the viral envelope proteins - most likely those derived from the hypervariable region 1 of the E2 region [
40]. Therefore, extensive sequence variation of the infecting HCV strain could potentially have impact on serodiagnosis when conventional screening assays are used. As such, suitability of the assays deserves regular evaluation. Also, low viral load may explain low level viraemia detected in this study. However, the extensive polymorphism observed in HCV genome, even in conserved regions including the core, as seen in the Ghanaian samples, may give false negative results due to non-complementarity with the primer sequence used; or even a non-specific annealing of primers, which could lead to production of more than one band that may be interpreted as mixed infections. These underscore the importance of molecular characterization of HCV in different geographical areas to inform a rational design and interpretation of HCV diagnostics.
To distinguish HCV subtypes, primer-specific PCR procedure was initially applied and that classified HCV strains into subtypes under genotype 2. Sequence-based characterization by phylogenetic analysis, however, showed an identical clustering of the Ghanaian isolates under genotype 2, but the isolates exhibited high genetic variability and appeared distinct from previously described subtypes of HCV 2. Similar unique clustering pattern was observed with some previously described HCV core sequences from Ghana. Further detailed exploration of sequence patterns of the isolates by similarity plotting and bootscanning showed that the sequences were complex, having mosaic patterns of various subtypes in diverse forms. This complexity might explain their unique phylogenetic clustering and inability to assign subtypes. Thus, based on the findings of this study, the core gene sequences are not suitable for assigning subtypes of Ghanaian HCV. This adds to previous attempts at subtyping that proved unsuccessful [
22,
28]. These collectively highlight the dominance of genotype 2, identical sequence clustering, extensive genetic diversity along the entire HCV genome that gives rise to complex sequence patterns and hence difficulty to distinguish subtypes of Ghanaian HCV sequences.
In a broader context, similar genetic variability and unique clustering pattern of type 2 HCV strains have been observed in neighbouring West African countries including Benin, Burkina Faso, and Cote D’Ivoire [
25,
41]. Reports have also indicated that West African HCV sequences, irrespective of their geographical origin, rarely cluster with subtypes 2a to c [
26,
42]. This may reflect an ongoing high rate of sequence variation in the sub-region. This may shed light on the complex nature of the Ghanaian HCV sequences described in this study. There is therefore the need for a primer update of the 5’UTR region to improve performance of the RT-PCR assay. Furthermore, primer-specific PCR-based methods that can be useful for subtyping Ghanaian HCV need to be designed and validated using information derived from Ghanaian HCV sequence profiles.
In general, high genetic variation, effective viral clearance and/or slow progression to chronicity, as commonly reported in West African HCV infections, may implicate strong immune pressure and/or effective response to HCV infection. Cellular immune responses appear to play a role in protecting against HCV infection; and possible targets for HCV-specific cytotoxic T lymphocytes (CTL) recognition epitopes have been identified within the conserved core and the highly variable E2 regions [
43]. It is known that host immunogenetic factor, such as the human leukocyte antigen (HLA) system, which presents viral peptides for CTL-mediated immune response, play an important role in influencing pathogen diversity [
44‐
47]. In fact, some HLAs have been described that associate with either clearance or persistence of HCV infection [
48]. About a decade ago Chuang et al. [
49] studied Ghanaian blood donors to investigate the role of cellular immune responses and host genetics in the high rate of recovery from HCV infection in West Africa. The study by Chuang and colleagues hypothesized that the dominance of genotype 2 HCV strains and an efficient contribution of HLA-B*57 may constitute important explanatory factor. It could therefore be that complexity in viral sequences as observed in this study might result from viral adaptation driven by strong HLA-mediated immune pressure on HCV. Thus, HCV heterogeneity may serve as a means of escaping CTL-mediated immunity [
43,
50]. This remains to be completely defined.
Of note, HLA association with outcome of HCV infection appears to be population-specific [
49,
51‐
53]; and immune response also appears to be HLA type-specific [
54,
55], but data on these are scarce. Therefore, the seeming dynamic nature of HCV molecular evolution in Ghana, and perhaps the West African sub-region, makes it necessary to comprehensively explore the nature of immune response and the role of host genetic factors in viral diversity as well as outcome of HCV infection.
The study had some limitations. First, the number of isolates obtained for the study was low. Besides, the samples were collected only from Accra, the capital. Few samples analysed - not including samples from other regions of Ghana; and unavailability of appreciable number of HCV core sequences from neighbouring West African countries for phylogenetic comparison are some of the limitations of this study. Thus, the findings of this study may not suggest that other Ghanaian HCV-2 isolates have the same complex sequence profile. Second, specificity of the primers used for HCV detection might not be high enough. Even though published primers that had been optimized and proven to be highly specific were chosen for the test, over time, several HCV variants have evolved that might render the primers nonspecific. This could be a cause for false negative results, hence the need for primer update, especially, of the 5’UTR region to increase performance of the RT-PCR assay. Third, sequence-based HCV genotype data were obtained through direct nucleotide sequencing, which might not be sensitive enough for detecting minority HCV variants that could be obscured by the wild-type strains. Future studies should aim at initially screening a much larger number of samples, and possibly from different regions of Ghana, in order to obtain a more significant number of positive cases that can describe the genetic diversity of prevailing HCV genotypes in Ghana. Also, the use of new technology such as ultra-deep sequencing, which has higher capacity than direct sequencing in detecting both minority pathogen populations and presence of dual or multiple infections, would be very useful in analysing HCV especially in a region where the virus appears to be endemic and several subtypes may be in circulation. Nevertheless, considering the in-depth analysis conducted, and the fact that the findings of this study are largely in agreement with, and sheds light on, those reported from previous studies in other populations of Ghana and West Africa at large, it does not seem the above-stated limitations could significantly impact the results from which the study conclusions were drawn.
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