Heterogeneity of hepatitis C virus genotype 4 strains circulating in south-western France

Florence Nicot1, Florence Legrand-Abravanel1, Karine Sandres-Saune1, Anne Boulestin1, Martine Dubois1, Laurent Alric2, Jean-Pierre Vinel3, Christophe Pasquier1 and Jacques Izopet1

1 Service de Virologie, Hôpital Purpan, EA2046-IFR30, CHU Toulouse, 31059 Toulouse Cedex, France
2 Service de Médecine Interne, Hôpital Purpan, EA2046-IFR30, CHU Toulouse, 31059 Toulouse Cedex, France
3 Service de Gastro-entérologie, Hôpital Purpan, EA2046-IFR30, CHU Toulouse, 31059 Toulouse Cedex, France

Correspondence
Jacques Izopet
izopet{at}cict.fr


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis C virus (HCV) is a major cause of liver disease. Knowledge of HCV variability is crucial for clinical and epidemiological analysis. HCV genotype 4 (HCV-4) has become increasingly prevalent in European countries, including France, in recent years. The present study investigates the heterogeneity of HCV-4 in south-western France by phylogenetic analysis of NS5B sequences from 166 patients. The E2 region of 90 strains was also analysed. Genotype 4 accounts for 7·4 % of HCV infections in this area. Analysis of the NS5B region revealed 12 subtypes and the NS5B and E2 phylogeny data were congruent, except for one strain. The epidemiological data indicated two main groups of patients. One included intravenous drug users (IVDUs) of French origin, who were infected by homogeneous strains of subtypes 4a or 4d. The second group comprised non-IVDU patients who were infected with heterogeneous strains. This group was subdivided into patients of French origin, who were infected with eight subtypes, and patients from non-European countries (Central Africa or the Middle East), who were mainly infected with 4f, 4k, 4r and other subtypes; they showed the greatest genetic heterogeneity. This study of a large cohort of patients shows the great diversity of HCV-4 strains, and that these subtypes have spread differently.

The GenBank/EMBL/DDBJ accession numbers for the NS5B sequences reported in this paper are AY743050–AY743215 and those for the E2 sequences are AY742960–AY743049.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
More than 170 million people throughout the world are infected with hepatitis C virus (HCV). Persistence of the virus in the liver leads to chronic hepatitis in 70 % of infected patients, which can progress to cirrhosis and liver cancer (Poynard et al., 2003; WHO, 1997).

This single-stranded RNA virus is genetically extremely heterogeneous because of its rapid replication and the poor fidelity of its RNA-dependent RNA polymerase, encoded by the NS5B region. Studies on the taxonomy of HCV based upon phylogenetic analyses of nucleotide sequences indicate that there are six HCV genotypes (1–6) (Robertson et al., 1998; Simmonds, 1999), each of which may have various subtypes. The virus population in an individual is defined as quasispecies (Martell et al., 1992). Several methods are used for genotyping, based on analysis of portions of the genome (5'NC, NS5B, core, E1). Phylogenetic analysis of the NS5B region is commonly used for subtyping (Salemi & Vandamme, 2002), but little is known of the suitability of the E2 region (Sandres et al., 2000).

Knowledge of HCV variability is crucial for clinical and epidemiological analyses. Prediction of sustained virological response and choice of treatment duration depend on genotype (Poynard et al., 2003; Zein, 2000). Subtyping has yet to be shown to be relevant to treatment, but this information could be useful in epidemiological investigations (Pybus et al., 2001). Genotypes differ in both their geographical distribution and mode of transmission (Pybus et al., 2001). HCV strains 1, 2, 3 and 4 are encountered worldwide, whereas genotypes 5 and 6 are restricted to well-defined areas. Genotypes 1a and 3a mainly infect patients who have been infected via intravenous drug use (IVDU), whereas genotypes 1b and 2 infect patients who have received contaminated blood transfusions. Several recent studies carried out in Europe have indicated changes in genotype distribution and have underlined the increasing prevalence of HCV genotype 4 (HCV-4) (Matera et al., 2002; Sánchez-Quijano et al., 1997; Schröter et al., 2002; van Asten et al., 2004). This genotype is endemic to Central Africa (Ndjomou et al., 2003; Xu et al., 1994) and the Middle East (Chamberlain et al., 1997), including Egypt (Angelico et al., 1997; Ray et al., 2000), but is spreading into European countries, mainly among intravenous drug users (IVDUs) (van Asten et al., 2004). Previous studies on small groups of patients in north and south-east France found an increased occurrence of HCV-4 (Morice et al., 2001; Tamalet et al., 2003).

We have now investigated the variation in HCV-4 strains in a large cohort of patients in the Midi-Pyrénées area of south-west France. The NS5B region of the HCV-4 strains was characterized by phylogenetic analysis and a second region of the genome, E2, was also investigated in a subset of these strains. Epidemiological data were used to determine the spread of the different subtypes.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Patients.
HCV strains in 2239 patients infected with HCV living in the Midi-Pyrénées area, France, were examined between March 2000 and May 2003. They were genotyped by sequencing the NS5B region and subjected to phylogenetic analysis (Sandres-Sauné et al., 2003) in the virology laboratory of Toulouse University Hospital. In total, 166 subjects were infected with genotype 4. The E2 region of the virus was also characterized in a subset of 90 patients. Data on patient age, gender, risk factors for HCV acquisition and virus load were collected.

HCV RNA quantification.
HCV RNA in sera was measured by quantitative RT-PCR [Cobas Monitor HCV 2.0 (Roche Diagnostics)] according to the manufacturer's instructions. The detection limit was 600 IU ml–1.

Sequencing and phylogenetic analysis of HCV NS5B.
HCV RNA was extracted from 140 µl serum by using the QIAamp viral RNA method (Qiagen). Aliquots (10 µl) of extracted RNA were amplified by RT-PCR using a Qiagen one-step RT-PCR kit. The mix contained 3·5 mM MgCl2, 10 U ribonuclease inhibitor (Invitrogen), 2 U uracil–DNA glycosylase (Roche) and 200 nM primers Pr3 and Pr2 (Sandres-Sauné et al., 2003). A 391 bp segment was amplified. A hemi-nested PCR was performed for 19·9 % of HCV RNA samples, because amplification was insufficient after the first round of PCR. The first step was carried out with primers Pr3 and Pr4 and the second step with Pr3 and Pr5 (Sandres-Sauné et al., 2003). The segment obtained was 382 bp. HCV PCR products were analysed by electrophoresis through 2 % agarose gel and ethidium bromide staining.

HCV PCR products were then purified on columns (QIAquick PCR purification kit) and sequenced with the PCR primers that were used for amplification. Cycle sequencing was done by using fluorescent dye-terminator technology (BigDye Terminator V3.1; Applera) with AmpliTaq DNA polymerase and an ABI PRISM 3100 sequencer (Applera). Sequence chromographs were analysed by using SEQUENCE ANALYSIS and SEQUENCE NAVIGATOR software.

Phylogenetic analyses compared the NS5B sequences with 264 reference sequences in GenBank, including 27 NS5B regions from complete genomes, to determine genotypes. The NS5B regions of the HCV genomes of the 166 patients infected with genotype 4 were then sequenced to determine subtypes by comparing them with reference sequences of genotype 4. Primers were removed from sequences (360 bp) before analysis. Nucleotide sequences were aligned with CLUSTAL_X 1.83 software (Thompson et al., 1997). The evolutionary distances between NS5B sequences were calculated with MEGA 2.1 (Kumar et al., 2001) with the Kimura two-parameter model. The mean pairwise distance is the mean of all genetic distances calculated on pairwise sequences. Phylogenetic trees were created by the neighbour-joining (NJ) method with the approach implemented in MEGA: firstly, with reference sequences to determine the subtype of each strain and, secondly, only with strains of patients. The reproducibility of the branching pattern was determined by bootstrap analysis (1000 replicates). Subtypes were determined when bootstrap values (BVs) were >70 %. Values of >70 % of 1000 replicates are considered as supporting the grouping as, in general, a BV of 70 % corresponds to a confidence value of 95 % (Hillis & Bull, 1993; Robertson et al., 1998). Phylogenetic trees were drawn by using the TreeView 1.66 program.

Genotype 4 reference sequences used in this study were taken from the database http://hcv.lanl.gov/content/hcv-db/ and classified by subtype (Table 1).


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Table 1. Reference sequences used in the NS5B tree

Abbreviations: AR, Argentina; BE, Belgium; BU, Burundi; CA, Canada; CAR, Central African region; CG, Congo; CM, Cameroon; EG, Egypt; FR, France; GB, Gabon; MQ, Martinique.

 
Sequencing and phylogenetic analysis of HCV E2.
HCV RNA was extracted from 140 µl serum by using the QIAamp viral RNA method (Qiagen) and aliquots (10 µl) were amplified by RT-PCR using a QIAgen one-step RT-PCR kit. The mix contained 2·5 mM MgCl2, 10 U ribonuclease inhibitor (Invitrogen) and 300 nM primers [external sense, KS2, and external antisense, KS1 (Table 2)]. The thermal profile was: 30 min reverse transcription at 50 °C, 15 min denaturation at 95 °C, 50 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, followed by 10 min at 72 °C. A 394 bp segment of the E2 gene was amplified. Nested RT-PCR was carried out on 2 µl of the first PCR products if amplification was insufficient, with 2·5 U AmpliTaq DNA polymerase (Applera) and 300 nM primers [nested sense, KS4, and nested antisense, KS3 (Table 1)]. The nested PCR thermal profile was: 95 °C for 7 min, 30 cycles at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s and a final elongation at 72 °C for 10 min. A 212 bp segment of the E2 region was amplified.


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Table 2. PCR primers for E2 amplification

 
Phylogenetic analysis was done as above for the NS5B region. The sequence of the E2 gene was analysed by using 174 bp fragments. The single subtype 4a reference (GenBank no. Y11604) in the database http://hcv.lanl.gov/content/hcv-db/ was used (Simmonds, 1995).

Statistical analysis.
Continuous variables were analysed by Student's unpaired t-test. Categorical variables were tested by the {chi}2 test. P values of <0·05 were considered to be significant.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Phylogenetic analysis of the NS5B region
Genotype 4 accounted for 7·4 % (n=166) of the strains from patients infected with HCV in the Midi-Pyrénées area, whilst genotype 1 accounted for 58·1 %, genotype 2 for 13·1 %, genotype 3 for 20 %, genotype 5 for 1·3 % and genotype 6 for 0·1 %.

The sequences of genotype 4 were of two main subtypes: 44 % were of subtype 4a and 34 % were of subtype 4d (Table 3). The other sequences were of subtypes 4k (5 %), 4f (4 %), 4r (4 %), 4t (1·5 %), 4c (1 %), 4h (1 %), 4i (1 %), 4n (1 %), 4o (1 %) and 4p (1 %). The subtype was not determined in 1·5 % of cases; these sequences were called 4U. The distribution of strains is illustrated by the NS5B tree (Fig. 1). The mean pairwise distance of the NS5B sequences was 0·1289±0·0109 substitutions per site (range, 0·0031–0·3028). Clade 4a was made up of a homogeneous group of sequences and divergent sequences (G4MP003, G4MP006, G4MP028). Strains classified as subtype 4a formed a cluster with strains of subtype 4c. Clade 4c had a high BV that allowed strains in clade 4a to be differentiated from those in clade 4c. Clades 4d, 4f, 4h, 4i, 4k, 4n, 4o, 4p, 4r and 4t were well-defined clades, supported by BVs of >70 %. The three 4U sequences were distributed throughout the tree.


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Table 3. HCV-4 strains with country of origin and subtypes

Abbreviations: CAR, Central Africa region; CF, Central African Republic; CG, Congo; CM, Cameroon; EG, Egypt; FR, France; GB, Gabon; RW, Rwanda; SA, Saudi Arabia; SY, Syria; ZA, Zaire.

 


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Fig. 1. Phylogenetic analysis of the NS5B gene of HCV genomes from the Midi-Pyrénées area. BVs for 1000 replicates are indicated below branches. Subtypes were determined when sequences that we studied and reference sequences clustered together with a BV of >70 %.

 
Phylogenetic analysis of the E2 region
The E2 gene was also analysed in a subset of 90 strains. Because subtypes 4a and 4d were most prevalent, 31 strains of subtype 4a and 29 strains of subtype 4d were selected randomly. They were analysed together with all strains of the other subtypes that were amplifiable (six of subtype 4r, two of 4c, four of 4f, eight of 4k, two of 4o, three of 4t and two of 4U, and one each of subtypes 4h, 4i and 4p).

The resulting phylogenetic tree (Fig. 2) showed a mean pairwise distance for E2 sequences of 0·3605±0·0327 substitutions per site (range, 0·0066–0·7071), which was greater than that of the NS5B sequences (P<0·001). Seven clades (A–G) were identified. Clade A had a moderate BV (59 %). All but one clade A sequence was of subtype 4a in the NS5B region. One strain (G4MP132) had been subtyped as 4d in the NS5B region. Clade E was composed of strains that were 4k in the NS5B region, clade B had 4c strains, clade C had 4d strains, clade D had 4f strains, clade F contained 4r strains and clade G contained 4t strains (Table 4). Several E2 sequences were dispersed throughout the tree, including two sequences that formed NS5B clade 4o, one belonging to NS5B clade 4h, one to NS5B clade 4i, one to NS5B clade 4p and two undetermined sequences (4U).



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Fig. 2. NJ tree for the E2 gene corresponding to HCV-4 genomes and the 4a reference sequence (GenBank accession no. Y11604). BVs for 1000 replicates are indicated below branches. Clades were determined when sequences clustered with a BV of >70 %.

 

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Table 4. HCV typing obtained by analysis of the NS5B and E2 sequence

 
Epidemiological characteristics
Demographic data were collected for each patient infected with HCV-4 (63 women, 103 men; mean age, 41·1±8·6 years). The mean serum HCV load was 5·5±0·4 log RNA copies ml–1, without any significant difference between subtypes. Of these, 51 (17 women and 34 men, 30 %) were also infected with human immunodeficiency virus (HIV). Patients with both HIV and HCV were mainly infected with subtype 4a (45 %) and 4d (45 %) strains; a few patients were infected with subtype 4f, 4k, 4n and 4r strains.

The epidemiological data for 140 patients revealed two main groups. The first was composed of patients who were infected via IVDU (n=83). Their mean age was 40 years. All but one were infected with subtypes 4a (55 %) or 4d (44 %) and all but one were of French origin. Only one IVDU patient was infected with a subtype 4o strain (G4MP162). Thirty-six patients were also infected with HIV (43·4 %); they were infected with subtypes 4a or 4d in similar proportions. The mean pairwise distances of the NS5B sequences for this group was 0·1070±0·0720 substitutions per site.

The second group was a population of non-IVDUs (n=57). They had acquired their HCV infection by transfusion, nosocomial, interfamilial or undetermined routes. This population was divided into two subgroups: patients of French origin and patients from non-European countries. The French patients (n=28; mean age, 41 years) were infected with eight subtypes [4a (46 %), 4r (4 %), 4c (7 %), 4d (32 %), 4k (4 %), 4o (4 %), 4p (4 %) or 4t (4 %)]; 25 % of them were also infected with HIV. Patients from non-European countries (n=29; mean age, 45 years) were older than the IVDUs (P<0·02). They included patients from Central African countries and four from the Middle East (G4MP002, G4MP003, G4MP028 and G4MP031; Table 3). Patients from Africa were infected with very varied subtypes [4f (28 %), 4k (28 %), 4r (12 %), 4U (12 %) and by 4h, 4i, 4o, 4p and 4t], whereas patients from the Middle East were all infected with subtype 4a. No patient was infected with strains of subtypes 4c or 4d. Only three (9 %) non-European patients were also infected with HIV. Patients infected with 4U strains were from Cameroon (G4MP159 and G4MP166) and the Central African Republic (G4MP164).

Mean pairwise distances for the non-European patients (0·1643±0·0450 substitutions per site) were greater than those for the French non-IVDUs (0·1340±0·072 substitutions per site) and the IVDUs (0·1070±0·0720 substitutions per site) (Fig. 3).



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Fig. 3. Mean pairwise distances (±SD) of NS5B sequences from HCV-4-infected patients living in the Midi-Pyrénées area, according to the epidemiological data.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This is the first study of a large cohort of patients to investigate the genetic diversity of HCV-4 in Europe. We find that genotype 4 is relatively widespread (7·4 %) in south-west France, with a total of 12 subtypes (4a, 4c, 4d, 4f, 4h, 4i, 4k, 4n, 4o, 4p, 4r and 4t) identified by phylogenetic analysis of the NS5B region. Analysis of the E2 region gave results that were congruent with those for the NS5B region. Finally, links between the epidemiological data and variations in genotype 4 reveal three populations infected with HCV-4, each with different subtypes, risk factors and subject origins (France, Africa or the Middle East).

Analysis of the phylogenetic relationships of the HCV-4 sequences in the NS5B and E2 regions indicate that this genotype is very heterogeneous. Mean pairwise distances of the E2 sequences were greater than those of the NS5B sequences, indicating that the E2 region of HCV-4 is more variable than the NS5B region, which is in line with several studies on other HCV genotypes (Duffy et al., 2002; Martell et al., 1992; McAllister et al., 1998). The topologies of the NS5B and E2 trees are congruent: sequences of one subtype were grouped in the same clades in both NS5B and E2 trees, except for three sequences, and BVs were similar. The discordance between the phylogenetic analyses of the two regions for strain G4MP132 is probably due to this patient having a mixed infection or a recombinant virus (Colina et al., 2004; Kalinina et al., 2002). The same holds for strains G4MP162 and G4MP163, which form clade 4o in the NS5B tree, but are dispersed in the E2 tree. Despite the lack of reference sequences for the E2 region, our data show that the E2 region discriminates well between subtypes.

HCV-4 is less common (7·4 %) in south-west France than in the Paris area (10·5 %) (Morice et al., 2001) or around Marseille (10·7 %) (Tamalet et al., 2003). Nevertheless, it is above the national average in 1999 (4·5 %) (Martinot-Peignoux et al., 1999). This may well be due to the greater numbers of immigrants in the Paris and Marseille areas.

The distribution of genotype 4 subtypes in south-west France shows great genetic diversity and the predominance of two subtypes, 4a and 4d. We identified two epidemiological profiles. Patients infected via IVDU were all infected with subtype 4a or 4d and were of French origin. Previous studies conducted in France have described IVDU as the main risk factor for patients infected with 4a and 4d (Martial et al., 2004; Morice et al., 2001). Those IVDU patients who were also infected with HIV were infected with subtypes 4a and 4d in similar proportions. Our data do not agree with those of a study conducted in other European countries (van Asten et al., 2004), where IVDUs co-infected with both HCV-4 and HIV were mainly infected with subtype 4a. This may well be because there were few genotype 4 strains (n=12) in the study by van Asten et al. (2004). This indicates that these strains have spread only recently in France. The second epidemiological profile on non-IVDU patients identified two subgroups of different geographical origins: French patients and patients from Africa or the Middle East. All of the patients in this group infected with HCV-4a were from the Middle East. Patients from Africa were infected with strains 4f, 4k, 4h, 4i, 4o, 4p, 4r, 4t or 4U. These results are in agreement with those of a study conducted in Seine-Saint-Denis (Morice et al., 2001).

Strains infecting non-IVDUs were more heterogeneous than those of IVDUs. Non-European, non-IVDU patients had the most heterogeneous sequences. The presence of so many divergent and co-existing lineages of genotype 4 indicates that this genotype has been in Central African countries for a long time. Non-European patients were probably infected in their home countries, but we have no data on dates of infection or immigration.

This study of a large cohort of infected patients shows the wide diversity of HCV-4 subtypes in the Midi-Pyrénées area, with the presence of 12 subtypes. French patients infected via IVDU are most likely to be infected with homogeneous subtypes 4a or 4d, whereas non-IVDU patients are infected with 11 different subtypes, with the subtypes of patients from African countries being 4f, 4k, 4h, 4i, 4o, 4p, 4r, 4t and 4U. IVDU is the main route of HCV-4 propagation in France.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 30 June 2004; accepted 17 September 2004.



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