Laboratoire de Bactériologie, Virologie-Hygiène, Hôpital Avicenne, Equipe daccueil Agents Transmissibles et Hôtes, Signalisation Cellulaire et Oncogenese, UFR Santé Médecine Biologie Humaine, Université Paris 13, Bobigny, France1
Services dHépatologie-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 dInformation et de Soins de lImmunodéficience 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
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Abstract |
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Introduction |
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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 16 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.
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Methods |
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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 manufacturers 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 5464 °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 5846 °C) for 30 cycles. PCR products (389 bp) were purified from agarose gels using a QIAquick gel extraction kit (Qiagen), according to the manufacturers 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 AJ401094AJ401101; accession numbers of the NS5B gene sequences are AJ291245AJ291294).
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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).
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Results |
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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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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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Discussion |
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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-
plus ribavirin (el-Zayadi et al., 1999
) thereby contributing to the increase of treatment-resistant HCV in France.
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Acknowledgments |
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Received 26 October 2000;
accepted 11 January 2001.