Phylogenetic analyses confirm the high prevalence of hepatitis C virus (HCV) type 4 in the Seine-Saint-Denis district (France) and indicate seven different HCV-4 subtypes linked to two different epidemiological patterns

Yoann Morice1, Dominique Roulot1,2, Véronique Grando2, Jérome Stirnemann1, Elyanne Gault1, Vincent Jeantils3, Michelle Bentata3, Bernard Jarrousse3, Olivier Lortholary3, Coralie Pallier4 and Paul Dény1

Laboratoire de Bactériologie, Virologie-Hygiène, Hôpital Avicenne, Equipe d’accueil Agents Transmissibles et Hôtes, Signalisation Cellulaire et Oncogenese, UFR Santé Médecine Biologie Humaine, Université Paris 13, Bobigny, France1
Services d’Hépatologie-Gastroentérologie, Réseau hépatite C Nord-Est Parisien, Hôpitaux Avicenne et Jean Verdier, UFR Santé Médecine Biologie Humaine, Université Paris 13, Bobigny et Bondy, France2
Services de Médecine Interne et de Maladies Infectieuses et Tropicales, Centre d’Information et de Soins de l’Immunodéficience Humaine du 93 (CISIH 93), Hôpitaux Avicenne et Jean Verdier, UFR Santé Médecine Biologie Humaine, Université Paris 13, Bobigny et Bondy, France3
Service de Microbiologie, Unité de Virologie, CHU de Bicêtre, Le Kremlin-Bicêtre, France4

Author for correspondence: Paul Dény. Fax +33 1 48 95 59 11. e-mail paul.deny{at}avc.ap-hop-paris.fr


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis C virus (HCV) has been classified into six clades as a result of high genetic variability. In the Seine-Saint-Denis district of north-east Paris, the prevalence of HCV-4, which usually infects populations from Africa or the Middle East, is twice as high as that recorded for the whole of continental France (10·2 versus 4·5%). Although the pathogenicity of HCV-4 remains unknown, resistance of HCV-4 to therapy appears to be similar to that observed for HCV-1. In order to characterize the epidemiology of HCV-4 in Paris, sequences of the non-structural 5B gene (332 bp) were obtained from 38 HCV-4-infected patients. Extensive phylogenetic analyses indicated seven different HCV-4 subtypes. Moreover, phylogenetic tree topologies clearly distinguished two epidemiological profiles. The first profile (52·6% of patients) reflects the intra-suburban emergence of two distinct HCV-4 subclades occurring mainly among intravenous drug users (65% of patients). The second profile shows six subclades [HCV-4a, -4f, -4h, -4k, -4a(B) and a new sequence] and accounts for patients from Africa (Egypt and sub-Saharan countries) who have unknown risk factors (77·8% of patients) and in whom no recent diffusion of HCV-4 is evident. This study indicates the high diversity of HCV-4 and the extension of HCV-4a and -4d subclades among drug users in France.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis C virus (HCV) is responsible for most parenteral non-A, non-B hepatitis virus infections. HCV infection is characterized by virus persistence leading to chronic infection in 85% of cases. It is estimated that 170 million individuals are infected with HCV worldwide. Aside from factors yet to be defined, the rate of progression of chronic hepatitis to cirrhosis is partly linked to the age of the patient at the time of infection, alcohol intake or to co-infection with human immunodeficiency virus (HIV). Cirrhosis may occur in 15 to 20% of infected patients thereby increasing the risk of developing hepatocellular carcinoma (Di Bisceglie, 2000 ).

This enveloped, positive-stranded RNA virus has a high genetic variability due to the lack of fidelity of the HCV RNA-dependent RNA polymerase, which is encoded by the non-structural 5B (NS5B) gene. It has been suggested that HCV has been present in the human population for hundreds, perhaps thousands, of years (Smith et al., 1997 ). Furthermore, by analysis of the branching pattern from phylogenetic trees, Holmes et al. (1996) have suggested that the transmission of HCV has increased dramatically at an exponentially growing state in the last 50 years. Finally, HCV variability within a patient gives rise to a number of phylogenetically related lineages referred to as quasispecies (Martell et al., 1992 ).

HCV taxonomy is based upon phenetic analyses of nucleotide sequence data. Pairwise sequence similarity applied to either partial or total genome sequences encouraged H. Tokita and co-workers to divide HCV into 11 distinct genotypes (Tokita et al., 1995 , 1996 , 1998 ). However, phylogenetic analyses with bootstrapping grouped HCV-10a together with HCV-3 and indicated that HCV-7, -8, -9 and -11a shared a common ancestor with HCV-6 (de Lamballerie et al., 1997 ; Simmonds et al., 1996 ). Therefore, results from all phylogenetic inference methods support the existence of six HCV clades (Robertson et al., 1998 ).

Determining the genotype of HCV isolates is crucial for epidemiological and clinical analyses (reviewed by Zein, 2000 ). First, the geographical distribution of different types and subtypes of HCV varies significantly. Some genotypes are distributed worldwide while others are found predominantly in specific geographical regions; for example, HCV-4 typically infects populations from Egypt (Angelico et al., 1997 ; Ray et al., 2000 ), Central Africa (Fretz et al., 1995 ; Xu et al., 1994 ) and the Middle East (Shemer-Avni et al., 1998 ; Shobokshi et al., 1999 ). Second, the efficiency of anti-HCV therapy is clearly linked to the infecting HCV genotype. A low response rate to therapy occurs in patients infected with either HCV-1 (Nousbaum et al., 1995 ) or -4 (el-Zayadi et al., 1996 ; Remy et al., 1998 ). In contrast, patients infected with either HCV-2 or -3 have a stronger response to either monotherapy (Martinot-Peignoux et al., 1998 ) or bitherapy (Poynard et al., 1998 ). Third, in France HCV-1b is associated with patients who had received a previous blood transfusion (Nousbaum et al., 1995 ), whereas HCV-3a is predominantly associated with intravenous drug users (IVDUs) (Pawlotsky et al., 1996 ). In addition, multivariate analyses by Angelico et al. (1997) showed that the high prevalence of HCV-4 in Egyptian patients was associated independently with an earlier parenteral treatment for either schistosomiasis or haematemesis, implying possible transmission of HCV during previous health care.

Since January 1996, anti-HCV-positive serum samples from patients from the Seine-Saint-Denis district of north-east Paris, France, have been prospectively tested against viral peptides that represent the six major HCV genotypes (HCV serotyping 1–6 assay HCO2, Murex) (Leruez-Ville et al., 1998 ). A high prevalence of HCV-4 infection was observed (10·2%), which is in contrast to what has been described previously in the French population (Castelain et al., 1997 ; Martinot-Peignoux et al., 1999 ). To confirm such a high prevalence and to further investigate HCV lineages, we combined phylogenetic inference methods with epidemiological information. Two factors could contribute to the appearance of HCV-4. On the one hand, there is a wide diversity of ethnic groups in our area, as 19% of the population originated from other countries, predominantly from Africa because of the historical relationships between France and Africa. On the other hand, the current major route of HCV transmission within large cities and suburbs of industrialized countries is the use of intravenous drugs, which is also the main cause of infection with HIV among AIDS cases in the Seine-Saint-Denis district (France).

In order to investigate the epidemiology of HCV-4 infections near Paris, 45 patients with suspected HCV-4 infection were studied. After optimizing the PCR amplification of the NS5B gene sequences, we obtained HCV sequences (332 bp) from all patients. We first investigated the phylogenetic lineage of the NS5B gene between positions 7938 and 8269 (numbered as in Choo et al., 1991 ), as this region is currently used for HCV classification and permits further classification of HCV-4 subtypes (Stuyver et al., 1994 ). For a better understanding of HCV-4 classification, the first 351 nucleotides of the E1 gene were also sequenced in a set of selected samples. The analyses presented here underline the wide diversity of HCV-4 subtypes in France and identify two distinct epidemiological profiles that account for the emergence of HCV-4: the first profile reflects the intra-suburban emergence of two distinct HCV-4 subtypes, mainly among IVDUs, whereas the second profile is linked to patients originating from Africa whose risk factors are unknown.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Patients and samples.
This study was carried out on samples from 45 HCV-infected patients: 44 patients were followed up at the Avicenne and Jean Verdier Hospitals (Seine-Saint-Denis district, France) and one child was followed up at Le Kremlin Bicêtre Hospital. Included in this study were 43 patients with antibodies against HCV-4 antigens (HCV serotyping 1–6 assay HCO2, Murex) and with an HCV-positive result for RNA present in sera (Amplicor HCV Monitor Test Kit, Roche). Two HCV-seropositive samples from children without any type-specific reactivity were also included in the study. One child (6 years old) was suspected to have been infected with HCV-4 through mother-to-infant transmission. The other (11 years old) was native of former Zaire, now called the Democratic Republic of the Congo (DRC), where HCV-4 infection has been described previously (Bukh et al., 1993 ). Eight HCV-infected patients with serological reactivity against HCV-1 (n=1), -2 (n=2), -3 (n=1), -5 (n=2) and -6 (n=2) were selected as patients infected with HCV-non-4.

{blacksquare} RNA purification, reverse transcription, PCR and sequencing.
RNA was extracted from 250 µl of serum with 750 µl of TRIzol (Life Technologies), according to the manufacturer’s protocols. The RNA pellet was resuspended in 50 µl of diethyl pyrocarbonate-treated water containing 40 U of RNasin (Promega) and stored at -80 °C. Extracted RNA (5 µl) was reverse transcribed for 45 min at 42 °C using 0·5 mM of each dNTP, 10 pmol of random hexamers, 10 mM DTT, 1x first strand buffer, 25 U RNasin and 100 U MoMLV reverse transcriptase superscript II (Life Technologies) in a final volume of 25 µl. Samples were heated for 5 min at 95 °C. To avoid selective PCR amplification or contamination due to nested-PCR, single-step PCR amplification was carried out with degenerate primers (Table 1). PCR was performed in 80 µl aliquots containing 5 µl of cDNA, 20 pmol of sense and antisense primers, 0·2 mM of each dNTP, 1x Expand High Fidelity buffer and 2·6 U Expand High Fidelity PCR system enzyme mix (Boehringer Mannheim). After a denaturation step at 94 °C (1·5 min), DNA was amplified in a GeneAmp PCR system 2400 (Perkin Elmer). The following cycling parameters were used for NS5B amplification: denaturation at 94 °C (30 s), annealing at 54–64 °C (45 s) and elongation at 72 °C (1 min). During the first five cycles, the annealing temperature was 64 °C (or 60 °C for selected samples); this was then reduced by 0·3 °C per cycle for 30 cycles (touch-down PCR). The last five cycles were performed at 54 °C (or 50 °C for selected samples). After 40 cycles, a final extension cycle was carried out at 72 °C (4 min). For E1 amplification, different primers (Table 1) were used in a similar touch-down approach (annealing temperature 58–46 °C) for 30 cycles. PCR products (389 bp) were purified from agarose gels using a QIAquick gel extraction kit (Qiagen), according to the manufacturer’s protocols. Purified DNA NS5B fragments were directly sequenced on both strands using an ABI Prism 377 automated sequencer (Perkin Elmer) with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer). Amplified E1 products were cloned into the pCRII vector (Invitrogen) and two clones were sequenced on both strands. All new sequences are labelled FrSSD (French Seine-Saint-Denis) followed by the number of the sample. All sequences have been submitted to EMBL (accession numbers of the E1 gene sequences are AJ401094–AJ401101; accession numbers of the NS5B gene sequences are AJ291245–AJ291294).


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Table 1. Sequence of primers used for PCR amplification of the NS5B and the E1 genes of HCV

 
{blacksquare} Sequence alignment and phylogenetic analyses.
Viral sequences were aligned using CLUSTAL W software, version 1.8 (Thompson et al., 1994 ). Neighbour-joining (NJ) analyses were performed using DNADIST and NEIGHBOR in the PHYLIP package, version 3.572 (Felsenstein, 1989 ), with pairwise distances estimated using Kimura two-parameter distances. We analysed the robustness of different branches by bootstrapping [103 replicates (programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE in the PHYLIP package)]. Maximum likelihood (ML) analyses were performed using PAUP 4.0b3 (Swofford, 1998 ), with most parameters estimated from the data. Due to computational limits imposed by the ML calculations, the number of sequences analysed was limited to all HCV-4 sequences that were available both in the E1 and in the NS5B region (n=19) and bootstrapping was performed on 100 replicates. All maximum parsimony (MP) analyses were carried out using PAUP 4.0b3 with heuristic searches. All characters were weighted equally and left unordered. We estimated the reliability of the various inferred clades by bootstrapping (103 replicates, heuristic search achieved by stepwise addition). All trees were visualized with TreeView 1.5 (Page, 1996 ).

{blacksquare} List of reference sequences.
Reference sequences used for comparison with those obtained in this study were retrieved from databases and classified by subtypes. Sequences used for comparison of the E1 gene are as follows: HCV-4a, ED43 (Y11604) and HEMA51 (D45193); HCV-4b, Z1 (L16677); HCV-4c, B203 (L39284), GB116 (L29601), GB215 (L29604), GB358 (L29606) and NL81 (L39314); HCV-4d, DK13 (L16656) and NL52 (L39310); HCV-4e, CAM600 (L29587), CAM736 (L38323) and GB809 (L29624); HCV-4f, CAMG22 (L29592), CAMG27 (L29597) and FR12 (L38332); HCV-4g, GB549 (L29620); HCV-4h, GB438 (L29610); HCV-4i, CAR4/1205 (L36439); HCV-4k, B14 (L39282) and CAR4/901 (L36440); HCV-4a(B), GB809.4 (L29628), Z4 (L16652) and 1196E1-4 (D43677); and unspecified HCV subtype 4, Z7 (L16653). Sequences used for comparison of the NS5B gene are as follows: HCV-1a, HCV-1 (M62321) and HC-J1 (D10749); HCV-1b, HC-J4 (D13558) and HCV-BK (M58335); HCV-1c, HC-G9 (D14853); HCV-2a, HC-J6 (D00944); HCV-2b, HC-J8 (D10988); HCV-2c, BEBE1 (D50409); HCV-3a, NZL1 (D17763) and HCV-K (D28917); HCV-3b, TrKj (D49374); HCV-10a, JK049 (D63821); HCV-4a, ED43 (Y11604), HEMA51 (D45194), SD001 (D86533), SD003 (D86535), SD004 (D86536), SD016 (D86540), SD024 (D86541) and SD033 (D86542); HCV-4c, GB116 (L29602), GB215 (L29605), GB358 (L29607), GB48 (L29614), FR9 (L38376) and NL81 (L44603); HCV-4d, SD006 (D86537) and SD008 (D86538); HCV-4e, CAM600 (L29590) and GB809.3.1 (L29626); HCV-4f, CAMG22 (L29593) and FR12 (L38370); HCV-4g, GB549 (L29618); HCV-4h, GB438 (L29611); HCV-4i, CAR4/1205 (L36437); HCV-4j, CAR1/501 (L36438); HCV-4k, B14 (L44597); HCV-4l, SD002 (D86534) and SD015 (D86539); HCV-4m, SD035 (D86543); unspecified HCV subtype 4, EG81 (L78841); HCV-5a, SA13 (AF064490) and EUH1480 (Y13184); HCV-6a, EUHK2 (Y12083); HCV-6b, Th580 (D84262); HCV-7b, VN235 (D84263); HCV-8b, VN405 (D84264); HCV-9a, VN004 (D84265); and HCV-11a, JK046 (D63822).


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
PCR amplification results and NS5B phylogenetic studies of FrSSD HCV sequences
We studied 43 adults with antibodies reactive against HCV-4 and two children with clinical indications suggestive of a HCV-4 infection. Eight HCV-infected patients were considered as controls because they had serological reactivity against peptides corresponding to HCV-1, -2, -3, -5 and -6. Among these controls, at least one NS5B sequence for each of HCV-1 (FrSSD27), -2 (FrSSD101), -3 (FrSSD26) and -5 (FrSSD104) was obtained. PCR amplification of the two samples from patients with an anti-HCV-6 serological reactivity failed. After optimization, PCR amplification was efficient enough to allow direct sequencing of both DNA strands, except for two samples which were cloned. Forty-six HCV NS5B gene sequences (332 bp) were obtained from 45 patients with a suspected HCV-4 infection. Two sequences (FrSSD55 and FrSSD56) were obtained within a 4 month interval from a single treated patient. Sequence alignment showed neither an insertion nor a deletion in this region. We inferred the phylogenetic relationships of the 46 FrSSD sequences together with the four control sequences and the 21 reference sequences representing the six (Robertson et al., 1998 ) to 11 (Tokita et al., 1998 ) major HCV types that were retrieved from databases.

Phylogenetic trees obtained using NJ and MP analyses (Fig. 1) indicate that NS5B sequences from samples positive for antibodies against HCV-4 antigens did not always group within a clade. A well-defined clade clearly separate from clades 1, 2, 3, 5 and 6 comprised 39 FrSSD sequences plus the reference sequence HCV-4a (Chamberlain et al., 1997 ; Simmonds et al., 1993a ). This HCV-4 clade is supported by high bootstrap values (BV) of 100 and 97·9 (103 replicates, NJ and MP analyses, respectively). This result indicates that among the patients studied, 36 out of 43 (83·7%) with antibodies that were reactive against HCV-4 antigens were infected with a virus strain from the HCV-4 lineage. The other seven patients with antibodies that were reactive against HCV-4 non-structural protein 4 peptides were found to be currently infected with a virus strain from another HCV lineage. FrSSD45 and FrSSD93 sequences cluster with HCV-3a sequences and FrSSD34, FrSSD49, FrSSD54, FrSSD88 and FrSSD98 sequences cluster within HCV-1 sequences (Fig. 1, star-tagged samples). In each of these cases, the BV (>=83·3) confirms these affiliations. To investigate why the type-specific serological reactivity of these seven patients did not correlate with the characterized virus strain, two new serological typing assays (versions HCO2 and HCO3) were carried out on serum from the same date (Table 2). The new version of the assay, HCO3, allowed us to correlate both the serological type and the genotype for one sample (FrSSD88, HCV-1/1b). A persistently positive reactivity against HCV-4 was evident only for FrSSD54 (HCV-1a), while most results were controlled as non-reactive. Using phylogenetic analyses, we also demonstrated HCV-4 infection of the two samples from children (FrSSD97 and FrSSD136). In conclusion, we were able to obtain and analyse 39 HCV-4 NS5B sequences from 38 HCV-infected patients living in the suburbs of Paris.



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Fig. 1. Phylogenetic analyses of the NS5B gene (positions 7938–8269) from HCV genomes (FrSSD) characterized within the Seine-Saint-Denis district, France, and 21 reference sequences. The tree was reconstructed using pairwise distances estimated with both Kimura two-parameter distances and NJ algorithms and artificially rooted using HCV-1 sequences as the outgroup. Similar topology was obtained using MP heuristic analysis. For each type (HCV-1 to -6), BV (%) are indicated above and below the branches for 103 NJ and 103 MP replicates, respectively. Samples differing between serological typing and genotyping results are indicated by an asterisk. For each reference sequence, the proposed subtype is indicated in parenthesis. Accession numbers are given in Methods. Thick branches correspond to the six major clades. Scale represents the percentage substitution that is expected per site.

 

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Table 2. Study of the specificity of HCV-typing results for seven samples, each having initial anti-HCV-4-positive reactivity and discordant genotyping results

 
Variability and classification of FrSSD HCV-4 sequences
In order to evaluate the genetic variability of the HCV-4 NS5B gene, various phylogenetic inferences were applied to the 39 FrSSD and 29 reference HCV-4 sequences (see Methods). Typical results for MP analyses are shown for all 68 sequences in Fig. 2(a). A similar branching pattern was observed using NJ reconstruction from distance data (see BV in Fig. 2a). The 34 FrSSD sequences that are scattered among the five previously characterized NS5B subtypes include HCV-4a (Fujimura et al., 1996 ; Chamberlain et al., 1997 ; Tokita et al., 1998 ), -4d (Tokita et al., 1998 ), -4f, -4h and -4k (Stuyver et al., 1994 , 1995 ). In contrast, four sequences (FrSSD120, FrSSD136, FrSSD162 and FrSSD173) formed a monophyletic clade that included no NS5B reference sequence. Two of these viral genomes were sequenced in the E1 gene. NJ and MP analyses of the first 351 nucleotides of the E1 gene revealed that FrSSD120 and FrSSD136 were grouped into a particular clade that included three reference E1 sequences: Z4, 1196E1-4 and GB809.4 (103 replicates, NJ BV=100 and MP BV=99·6) (Fig. 2b). This subtype was characterized by sequencing the E1 gene of the Z4 isolate and was initially referred to as HCV-4a by Bukh et al. (1993) . However, in both the NS5B and the E1 regions, this isolate is distantly related to a complete HCV-4a reference sequence (isolate ED43) (Chamberlain et al., 1997 ). The Z4 isolate was therefore assigned to the HCV-4a(B) subtype (Fig. 2b).



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Fig. 2. Result of MP (and NJ) analyses for (a) the NS5B (329 bp; positions 7941–8269) and (b) the E1 (351 bp; positions 574–924) genes using HCV-1 sequences as the outgroup (see Methods). BV (%) are indicated above and below the branches for 103 NJ and 103 MP replicates, respectively (NO: branch not observed). Vertical bars indicate virus sequences or subtypes characterized within the Seine-Saint-Denis district. Other HCV-4 subtypes are indicated above their respective branches. The NS5B cladogram is a strict consensus of 28200 trees obtained through MP heuristic analysis on 39 FrSSD HCV-4 genomes and 29 HCV-4 reference sequences. The E1 cladogram is a strict consensus of eight trees obtained through MP heuristic analysis of eight HCV-4 FrSSD sequences and 25 reference sequences. Note that two distinct HCV-4a subtypes have been described previously. To specify such differences, the label 4a(B) was used for the E1 sequence Z4 from Bukh et al. (1993) .

 
Most of the FrSSD NS5B sequences (n=15) belonged to a group that included the HCV-4a ED43 isolate. However, this topology was not supported with high BV (103 replicates, NJ BV=64·4 and MP BV=37·2). Interestingly, the NL81 isolate, which is now classified as HCV-4c (Stuyver et al., 1995 ), is also included in this group. Furthermore, eight FrSSD NS5B sequences from this group have a high nucleotide sequence similarity (mean distance estimated through Kimura two-parameter, 0·0296) and, together with another HCV-4c isolate (FR9) (Stuyver et al., 1995 ), seem to share a possible common ancestor (NJ BV=72·8 and MP BV=63·8) (Fig. 2a). To further characterize the affiliation of these isolates, the E1 gene of two samples (FrSSD25 and FrSSD77) was sequenced and analysed with other reference sequences, including NL81. Phylogenetic analyses of the E1 sequences clearly indicate that FrSSD25, FrSSD77, NL81, HEMA51 and ED43 are monophyletic and that this topology is supported by bootstrapping approaches (103 replicates, NJ BV=99·9 and MP BV=98·8). Branch topologies of the reference HCV-4c sequences (GB116, GB215 and GB358) obtained in both the E1 and the NS5B regions (NJ BV=99·3/99·7 and MP BV=85·2/97·8, respectively) also argue for the existence of a specific HCV-4c clade (Fig. 2). Furthermore, ML analyses (data not shown) and NJ reconstruction (Fig. 3) for subsets of both the E1 and the NS5B gene sequences, which combine the available HCV-4 sequences characterized in the same regions, confirm that HCV-4a and -4c are more convincingly separated into two distinct lineages when the E1 sequences are analysed. In summary, these data might suggest that NL81 and, by extension, FR9, are more likely to share a more recent common ancestor with HCV-4a than with HCV-4c sequences.



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Fig. 3. NJ trees for (a) the E1 and (b) the NS5B genes corresponding to HCV-4 FrSSD genomes and reference sequences. Scale represents the percentage substitution that is expected per site (note the difference of scale between the E1 and the NS5B genes). NJ trees are artificially rooted using the HCV-4i subtype sequence as an outgroup. BV (%) are indicated in parentheses for 103 NJ, 103 MP and 102 ML replicates, respectively. Note that FrSSD158 might represent the prototype for a new HCV-4 subtype.

 
Among the 20 remaining sequences, 12 were associated with the reference sequences SD006 and SD008 (Tokita et al., 1998 ), with BV>=96·5 in both the NJ and the MP NS5B analyses (Fig. 2a). These 12 sequences were assigned to HCV-4d. The respective affiliation of seven sequences to HCV-4f (FrSSD36, FrSSD125, FrSSD159 and FrSSD160), -4h (FrSSD35 and FrSSD61) and -4k (FrSSD174) was supported by BV>=87·5 in both the NJ and the MP approaches (see values in Fig. 2a). One sequence (FrSSD158) that is distantly related to other subtypes (mean distance 0·1826) is evident (Fig. 2a). Sequencing of the E1 gene confirmed that this viral genome did not cluster with any of the HCV-4 sequences characterized previously (Fig. 2b).

Phylogenetic analyses and epidemiological data
Epidemiological data for each of the 38 HCV-4-infected patients were collected. We focused on the geographical origin of each patient, previous travel destinations and on the suspected route of HCV transmission, for example, IVDU, blood transfusion or mother-to-infant transmission (Table 3). Interestingly, two opposite topologies were obtained through phylogenetic tree reconstructions.


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Table 3. Distribution of HCV-4 subtypes among 38 HCV-infected patients with regard to age, sex, birthplace and risk factors

 
Phylogenetic analyses (Fig. 4) clearly distinguished two groups of sequences each forming a tight, well-defined, clade. The first group (NJ BV=98·7 and MP BV=88·4) comprises eight (out of 15) sequences belonging to the HCV-4a subtype, whereas the second group (NJ BV=100 and MP BV=98·9) comprises 12 sequences belonging to the HCV-4d subtype. Pairwise distances of the sequences inside each of these two groups are low (Table 4



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Fig. 4. Superimposition of phylogenetic results from 39 HCV-4 FrSSD NS5B sequences and epidemiological data (birthplace and risk factors) from infected patients living in the Seine-Saint-Denis district. This phylogenetic tree was reconstructed using the NJ approach on the data set that includes only the FrSSD sequences (indicated with the number of the sample). BV (%) are indicated for 103 NJ and 103 MP replicates. Geographical origin of the patients and suspected routes of transmission are indicated. The emergence of two distinct subclades (HCV-4a and -4d) among French HCV-infected patients is evident. Note the difference of risk factors between the French and the African groups.

 

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Table 4. Pairwise distances of sequences from HCV-4-infected patients living in the Seine-Saint-Denis area

 
On the other hand, sequences from patients who were born in Africa are much more heterogeneous (Fig. 4) and distantly related (Table 4). All seven viral sequences from the six patients who were born in Egypt cluster into the HCV-4a subtype. Our analyses indicate that the HCV-4a subtype viruses that were characterized among patients infected in France originated from the Egyptian clade. Other sequences corresponded to viruses from patients who were born in sub-Saharan countries (Burundi, Cameroon, DRC, Gabon and the Republic of the Congo), with the exception of a French patient who had lived for a long time in several sub-Saharan countries (Table 3). As shown previously (Fig. 2), these highly heterogeneous sequences did not form a well-defined HCV clade and were scattered into subtypes HCV-4a(B), -4f, -4h and -4k, except for the FrSSD158 sequence representing a prototype of a possible new subtype.


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In this study, we focused on HCV-4 infections because we had observed previously that HCV-4 was highly prevalent in the Seine-Saint-Denis area (Leruez-Ville et al., 1998 ). Since 1996, among the 1171 patients living in this district who had typable anti-HCV antibodies, the sera of 122 patients (10·2%) were found to be reactive against HCV-4 peptides (unpublished results). Furthermore, using the new 1–6 serotyping HCO3 assay (Murex), we found recently that 17% of the serum samples that had been previously non-typable using the earlier HCO2 assay version had reactivity against HCV-4 (E. Gault, unpublished). In the present study, we demonstrated that specific serological typing results identifying HCV-4 infection correlate with the affiliation of viral genomes into the HCV-4 clade in 36 out of the 43 (83·7%) cases examined. Therefore, all these results confirm the ‘corrected’ prevalence of HCV-4 in our area to be at least 8·7%, i.e. twice as high as the previously reported prevalence of HCV-4 observed among the French population (4·5 and 4·1%) (Castelain et al., 1997 ; Martinot-Peignoux et al., 1999 ). The characterization of various HCV-4 subtypes and the high prevalence of HCV-4 among patients in our area might be explained (i) by immigration of patients who were most probably infected with HCV-4 in Africa and (ii) by the emergence of two distinct HCV-4 subclades in France among IVDUs.

By studying 38 HCV-4-infected patients, we could clearly ascertain that two distinct epidemiologies co-exist. Firstly, whichever phylogenetic analysis approach was used, phylogenetic tree topologies were similar and indicated that, in our area, patients born or infected in France were infected with either HCV-4a or -4d. HCV transmission is mostly associated with IVDUs (65%) or blood transfusion patients (20%) and, more rarely, due to mother-to-infant transmission (5%). Among each group of French HCV-infected patients, HCV-4a and -4d represented 52·6% of all of the HCV-4 viruses described in our study. Among the ‘acquired-in-France’ HCV-4a and -4d viruses, low virus genetic distances (Fig. 4) argue for relatively recent ancestors of both HCV-4a and -4d. Indeed, if we postulate that the duration of HCV infection might be back-dated to the dates of either the first use of intravenous drugs or the first blood transfusion, which are available for a few French patients, it can be suggested that such virus intra-subtype diversity occurred within the last 25 years. Finally, these data strongly suggest an introduction of these two distinct HCV-4 subtypes on two separate occasions and their spread to date among IVDUs. In France, it has also been reported recently (Le Pogam et al., 1998 ) that HCV-4d viruses have probably been transmitted from patient-to-patient in a haemodialysis unit through nosocomial infection.

A second and opposite epidemiological profile concerns African patients. All Egyptian patients examined were infected with HCV-4a. A high prevalence of this subtype has already been described in Egypt (Angelico et al., 1997 ; Rapicetta et al., 1998 ; Ray et al., 2000 ). Therefore, these patients probably acquired their infection in their own country. A relationship between HCV and parenteral anti-schistosomiasis treatment has been reported (Darwish et al., 1993 ; Hibbs et al., 1993 ) and this could contribute to explaining such a high prevalence of HCV-4 in Egypt. Moreover, genetic distances, regardless of the two samples (FrSSD55 and FrSSD56) obtained from the same patient following treatment with interferon (IFN) plus amantadine or ribavirin, are higher among Egyptian HCV-4a subtypes than among French HCV-4a subtypes (Table 4). This could reflect an HCV-4a genetic variability that is seen for the whole of Egypt (Ray et al., 2000 ) but which is concentrated in our area and argues against the recent diffusion of a single HCV-4a strain among Egyptian patients living in France.

In our study, risk factors for HCV infection for sub-Saharan patients remain mostly undetermined (75%). HCV-4 genomes that were characterized from patients originating from Burundi, Cameroon, DRC, Gabon or the Republic of the Congo are highly heterogeneous. Indeed, phylogenetic inference approaches underline this wide diversity by showing different NS5B subtypes, i.e. HCV-4f, -4h, -4k, -4a(B) and a new sequence (FrSSD158) (Figs 2a and 4). Many other subtypes (HCV-4c, -4e, -4g, -4i and -4j) have been characterized previously using NS5B phylogenetic studies among patients who also live in sub-Saharan African countries (Cameroon, Central African Republic and Gabon) (Stuyver et al., 1994 , 1995 ). An ancient endemic presence of HCV-4 in these countries could explain this wide genetic diversity. Indeed, considering virus evolution among recipients of contaminated anti-D immunoglobulins (Smith et al., 1997 ), it has been suggested that diversification among HCV types might have occurred between 6 and 15 hundred years ago. This estimation might sustain such a hypothesis. However, it remains intriguing that no HCV-4 infections are evident among descendants of African people deported to Martinique, a Caribbean island, during the last four centuries [R. Césaire (Laboratoire Virologie, Hôpital de Fort-de-France, Martinique, France), personal communication]. Therefore, it is likely that outbreaks of HCV infection arose in African countries during the 20th century (Holmes et al., 1996 ; Ray et al., 2000 ).

Phylogenetic analyses on sequences of various lengths, carried out mainly for the E1 and the NS5B genes, have contributed to the characterization of at least 15 different HCV-4 subtypes (Bukh et al., 1993 ; Stuyver et al., 1994 , 1995 ; Mellor et al., 1995 ; Rapicetta et al., 1998 ; Ray et al., 2000 ). However, because subtype affiliation sometimes depends upon only one characterized region, the accumulation of new data might contribute to some confusion among HCV-4 subtype classification. Concerning the existence of two HCV-4a subtypes, the first NS5B-characterized HCV-4a isolate (Simmonds et al., 1993a , b ) was from an Egyptian patient, whereas the first E1-characterized HCV-4a isolate (Z4) (Bukh et al., 1993 ) was from a DRC patient. To clarify this ambiguity, we used, in this study, the 4a label for sequences including the first HCV-4 complete genome and the 4a(B) label for those sequences clustering with the DRC Z4 isolate. Furthermore, and as recommended by Robertson et al. (1998) , we sequenced two distinct HCV genomic regions (E1 and NS5B) for ambiguous viral sequences that appeared to form a new subtype through NS5B phylogenetic evolutionary analyses. By studying both the topologies and the branch robustness of the E1 phylogenetic trees, we could unambiguously confirm that the sequences FrSSD120, FrSSD136, FrSSD162 and FrSSD173 were closely related to the Z4 isolate, which was characterized previously (Fig. 2). In our study, three out of four patients originating from DRC are infected with HCV-4a(B).

By studying the phylogenetic relationships from available HCV-4 subtypes (21 viruses) sequenced in both the E1 (351 bp) and the NS5B (329 bp) genes, we confirm the existence of at least 11 HCV-4 subtypes (Fig. 4). In particular, HCV-4a(B), -4d and a new sequence (FrSSD158), which might be proposed to be the prototype for a new HCV-4 subtype, are now characterized in these two genes.

In conclusion, we observed a wide diversity of African HCV-4 subtypes in our environment. Indeed, Seine-Saint-Denis HCV-4 isolates clustered among seven subtypes, i.e. HCV-4a, -4d, -4f, -4h, -4k, -4a(B) and FrSSD158. We could clearly assess the risk factors for HCV diffusion in infected French patients (IVDUs, 65% of cases). In contrast, HCV-4 did not seem to diffuse from person-to-person among African immigrants studied. It should be noted that HCV-4 emergence in France (this study and in Martinot-Peignoux et al., 1999 ) is also studied in the present day in some specific areas of other European countries (van Doorn et al., 1995 ; Sanchez-Quijano et al., 1997 ; Spada et al., 1998 ). Thus epidemiological studies are needed to enforce specific prevention procedures. Unfortunately, it appears clear that European IVDUs are at risk of HCV-4 infection (HCV-4a or -4d in our environment). The incidence of HCV infections with the new HCV-4 subtype in IVDUs has to be followed carefully. In contrast to infection with HCV-3a, patients infected with HCV-4 probably react poorly following treatment with IFN, similar to those patients infected with HCV-1. Furthermore, as suggested by preliminary data, these patients may react poorly when treated with IFN-{alpha} plus ribavirin (el-Zayadi et al., 1999 ) thereby contributing to the increase of treatment-resistant HCV in France.


   Acknowledgments
 
We thank M. Beaugrand, L. Guillevin, M. Robineau, J. C. Trinchet, P. Cohen, N. Ganne, F. Rouges, H. Touitou and P. Berlureau for helping to collect epidemiological data. We thank M. Milinkovitch and C. Goujon for helpful discussions and criticisms, especially for phylogenetic aspects. We are grateful to R. Césaire for giving us unpublished information from Martinique and B. Rodgers who gave us the opportunity to use the Murex 1–6 HCV serotyping Assay HCO3. Y.M. is supported by a grant from the French Ministry of Research. This work was supported by ‘le Réseau Fondamental VHC’ (French Ministry of Research) and by the ‘Association Nationale de Recherches sur le Sida’ (ANRS). Presented in part at the European Virology Meeting (2000) held in Glasgow (17–21 September, 2000).


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 26 October 2000; accepted 11 January 2001.