1 Laboratoire de Biologie Clinique, HIA Val de Grâce, 74 boulevard de Port Royal, 75230 Paris cédex 05, France
2 Service de Maladies Infectieuses, CHU de Batna, Batna, Algeria
3 Unité de Recherche "Epidémiologie et Sciences de l'Information", INSERM U444, Faculté de Médecine de Saint Antoine, 27, rue de Chaligny, 75571 Paris cédex 12, France
Correspondence
Elisabeth Nicand
rt{at}filnet.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper are AY568357AY568365, AY596462 and AY596463.
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INTRODUCTION |
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Although HEV strains belong to a single serotype, they display considerable genetic diversity according to the time and place of isolation. Different classification systems for HEV isolates have been proposed. According to the systems, they have been divided into four major genotypes (1 to 4) or at least nine groups (Schlauder & Mushahwar, 2001). These taxonomic systems are based on the full-length sequences of representative HEV strains isolated from epidemics (Aye et al., 1992
; Huang et al., 1992
; Tsarev et al., 1992
) or sporadic HEV isolates (Schlauder et al., 1999
; Takahashi et al., 2002). Partial sequencing of HEV genotype 1 strains revealed genetic changes with sporadic acute hepatitis E and inter-epidemic polymorphism (Shrestha et al., 2004
), but few studies have looked at the genetic diversity of HEV isolates within one epidemic. In India, analysis of the replicase gene of 17 HEV strains isolated from seven epidemics that occurred over a 17-year period (19761993), each epidemic being represented by one to five HEV isolates, showed only minor inter-epidemic genetic variation and no intra-epidemic diversity (Arankalle et al., 1999
). Analysis of ORF1 (replicase gene) and ORF2 (capsid gene) sequences of HEV stool isolates from three Indian epidemics that occurred over a 9-year period, each epidemic being represented by five HEV isolates, showed clear inter-epidemic diversity (3 % for ORF1 and 4 % for ORF2) and unexpected inter-patient variation within the same epidemic, suggesting either the co-circulation of closely related HEV strains or the emergence of quasispecies (Aggarwal et al., 1999
).
Quasispecies is a level of genomic diversity that characterizes RNA viruses in which the non-proofreading polymerase leads to error-prone replication, resulting in better environmental adaptability and capacity for rapid evolution during passage from host to host (Schneider & Roossinck, 2001). It has been described mainly in persistent virus infections, such as those due to human immunodeficiency virus type 1 and hepatitis C virus, during which virus populations develop a high degree of sequence variation within each infected individual (Sanchez-Palomino et al., 1996
). It is less common in acute self-limited virus diseases such as those caused by dengue virus (Wang et al., 2002
) or hepatitis A virus (Sanchez et al., 2003
).
To assess the genetic heterogeneity of the HEV strains involved in an outbreak, we carried out a retrospective analysis of both inter- and intra-patient diversity on 23 serum samples collected during a water-borne outbreak that occurred in 19861987 in eastern Algeria. Our results provide the first proof of the quasispecies nature of epidemic HEV.
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METHODS |
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HEV genome amplification.
Total RNA was extracted from the 69 serum samples using the QIAamp virus RNA mini kit (Qiagen) according to the manufacturer's instructions. Based on the sequence of the Burmese strain, oligonucleotides were designed to target the HEV ORF1 spanning nt 44124910 (replicase gene) and ORF2 nt 66537100 (capsid gene). Nested PCR was performed as described elsewhere (Tam et al., 1991). Briefly, the RT-PCR step was performed in a GeneAmp 2400 thermocycler using the Titan One Tube RT (Boehringer Mannheim) as follows. After incubation at 50 °C for 45 min, the first PCR amplification was carried out at 94 °C 7 min, 45 °C 45 s, 68 °C 45 s for one cycle; 92 °C 30 s, 45 °C 45 s, 68 °C 45 s for 39 cycles. Nested PCR was performed using 5 µl of the PCR product with Pfu polymerase (Stratagene) and the following conditions: 94 °C 7 min, 50 °C 45 s, 72 °C 45 s for one cycle and 92 °C 30 s, 50 °C 30 s, 72 °C 45 s for 39 cycles. Amplification products were analysed by conventional agarose gel electrophoresis. The specificity of amplimers was controlled using internal digoxigenin end-labelled oligonucleotide probes (Buisson et al., 2000
).
Molecular typing of the epidemic strain
Restriction endonuclease analysis.
HEV genotyping was carried out on ORF1 and ORF2 nested PCR products by endonuclease restriction analysis as described previously (Gouvea et al., 1998; Buisson et al., 2000
). Briefly, it was possible to distinguish between genotypes 1 and 2 by digestion of the ORF1 PCR product with SmaI and the ORF2 product with KpnI and NotI. The ORF2 product was also digested with BsmI to distinguish African genotype 2 strains from the Mexico prototype (Buisson et al., 2000
).
To assess the genetic heterogeneity of HEV isolates involved in this epidemic, the 448 bp ORF2 PCR fragment was subjected to RFLP analysis. Crude PCR products were digested in a final volume of 20 µl with 1·5 U Fnu4HI (New England Biolabs) for 2 h at 37 °C. Digested products were initially analysed on a 3 % agarose gel (2 % LE agarose; Promega; 1 % high resolution agarose; Sigma). Digested amplimers (6 µl) were loaded on a precast polyacrylamide gel and subjected to electrophoresis (as recommended by the supplier) in a GenPhor electrophoresis unit (GeneGel Excel 12.5/2; Amersham Biosciences). Separated products were silver stained (PlusOne silver staining kit; Amersham Biosciences). RFLP profiles were analysed using 1D Image analysis software (Kodak Digital).
The same procedure was used on the following cloning products. To study the diversity of the virus population, 12 isolates [seven randomly selected isolates with the most common RFLP profile (A) and five isolates displaying the other two RFLP profiles (B and C)] were cloned as described below.
Cloning and sequence analysis.
To assess inter-patient diversity, the ORF2 products amplified from 23 of 69 samples were purified on Microspin 200-HR columns (Amersham Biosciences) and sequenced in both directions with the DTCS sequencing kit (Beckman Coulter) by using an automated DNA sequencer (CEQ 8000; Beckman Coulter).
Intra-patient diversity was evaluated by analysing the 12 isolates described previously. Firstly, amplified ORF2 products were treated with Taq polymerase (Promega) (30 min, 72 °C) resulting in DNA 3' A ends then purified and cloned into pCR-Script SK(+) (Stratagene) or into pGEM-T (Rapid ligation kit; Promega) as recommended by the manufacturers. After transformation, 100 clones of each isolate were screened by PCR using ORF2 primers. Then, the ORF2 PCR products obtained from these 100 independent clones were digested with Fnu4HI, yielding the RFLP profiles.
For each of the 12 isolates, to investigate the diversity of RFLP profiles, PCR products of three clones characterized by the most common RFLP profiles and all clones with the less common RFLP profiles were purified with the Wizard miniprep kit (Promega), and sequenced twice. Sequencing was performed on both strands using PCR or M13 consensus primers and the DTCS sequencing kit. DNA sequences were aligned by using the CLUSTAL X software. Comparison of nucleotide sequences generated a consensus sequence for each profile, resulting in the variants named Tanef 86/87 A to Tanef 86/87 K. Evolutionary distances were determined by the Kimura two-parameter method using the DNADIST program from the PHYLIP 3.5 package. Evidence of the phylogenetic grouping was assessed by the bootstrap method (1000 replicates). Phylogenetic trees were created with the Macintosh version of the TREEVIEW program. The full-length genome sequences used as references in this study are listed in Fig. 3.
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RESULTS |
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Molecular characterization of Tanefdour 86/87 HEV isolates
Restriction endonuclease analysis of the 23 ORF2 PCR products revealed a KpnI site at nt 164 in genotype 1 strains (Burma strain). An additional KpnI site at position 147 was demonstrated in Tanefdour 86/87 HEV strain but was not present in the Burmese strain and in the closely related Algerian isolate named Mostaganem-80. Unlike the Burmese strain, neither the Tanefdour 86/87 HEV isolates nor Mostaganem-80 were digested by BsmI. SmaI at the specific site nt 397 in genotype 2 digested no ORF1 products of Tanefdour 86/87 HEV isolates.
To assess inter-patient genetic diversity, RFLP analysis was performed on the 448 bp ORF2 PCR products. Treatment with Fnu4HI revealed a large number of RFLP profiles among the Tanefdour 86/87 epidemic strain (Fig. 1). The 23 isolates could be segregated into three RFLP profiles, named from A to C. The most common profile A consisted of three bands of 120, 137 and 237 bp and grouped 18 isolates. The profile B with four bands (69, 120, 137 and 187 bp) and the profile C with five bands (78, 85, 110, 225 and 237 bp) gathered three and two isolates, respectively. Comparison of the nucleotide sequences of the ORF2 products from 23 HEV isolates confirmed that the Tanefdour 86/87 epidemic strain belonged to genotype 1. The ORF2 sequences from 18 of 23 isolates exhibited 99·7 to 100 % identity; those of the other five isolates exhibited 98·7 to 99 % identity.
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Nucleotide substitutions in the Tanef 86/87 A to Tanef 86/87 K variants resulted in substantial amino acid diversity. The amino acid sequences deduced from the nucleotide sequences revealed an alanine to valine substitution at codon 585 of the capsid gene in Tanef 86/87 D, J and K variants because of a single base transition of a cytosine by a thymidine. For the Tanef 86/87 H variant, the replacement of a thymidine by a cytosine resulted in the replacement of a tyrosine by a histidine at codon 562 (Fig. 4).
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DISCUSSION |
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An epidemiological survey of the jaundice epidemic that occurred in 198687 in the Algerian village of Tanefdour clearly demonstrated that springs were the source of HEV infections (Siriwardana et al., 2003). Therefore, any of the HEV isolates from the 23 PCR-positive patients belonging to genotype 1 could be considered as a representative strain, the so-called Tanefdour 86/87 strain. Nevertheless, a slight inter-patient heterogeneity was revealed by RFLP, which divided the 23 isolates into three separate profiles (A, B and C). Moreover, RFLP analysis of 100 clones from each of 12 patients revealed intra-patient heterogeneity, with eight additional profiles (D to K) accounting for 05 % of the clones from each patient.
Nevertheless, the range of inter-patient and intra-patient nucleotide identity (96·799·9 %) reveals overall sequence homogeneity, suggesting that a unique HEV strain, comprising multiple closely related variants, spread in the Tanefdour population.
No previous studies on HEV genome heterogeneity have included such a large cluster of HEV isolates, collected from one epidemic within an 8-week period. The inter- and intra-patient genetic diversity revealed by molecular analysis of ORF2, with major and minor variants, is consistent with a quasispecies structure of HEV epidemic strains according to the criteria for RNA viruses (Domingo et al., 1985; Martell et al., 1992
). As a consequence of the high genetic plasticity of RNA viruses due to the lack of proofreading activity of their polymerase, the quasispecies structure describes an equilibrium status between variants in a replicating virus population. This diversity confers an advantage for survival and evolution as documented for human, animal and plant viruses (Domingo et al., 1998
; Schneider & Roossinck, 2001
). In two extensively studied human models (acquired immunodeficiency syndrome and hepatitis C), the quasispecies diversity is generally correlated with an apparent slow or fast progression of the disease and appears to be a significant pathogenicity factor. The genomic variability of RNA viruses involved in self-limiting acute infections is currently being explored in situations other than chronic virus infections. Defective viruses with a quasispecies nature have been underlined for dengue virus type 3 from six patients in Taiwan (Wang et al., 2002
). Likewise, the hepatitis A virus appears to replicate as a complex distribution of mutants despite displaying conserved antigenic specificity (Sanchez et al., 2003
).
To date, no similar studies have been carried out on HEV. Our data, based on a large cluster of isolates from the Tanefdour epidemic, seems to confirm the quasispecies nature of HEV. Minor variants from serum samples represented no more than 5 % of the overall virus population. It is difficult to detect these mutants by direct sequencing of PCR products or by studying a few randomly selected clones. Sequence variability is not uniformly distributed in the HEV genome. Some regions within ORF1, such as the hypervariable region, are highly polymorphic whereas the amino- and carboxy-terminal ends of the capsid gene are well conserved. Antigen mapping identified immunoreactive domains in ORF2, with a major neutralization epitope located between aa 578 and 607 (Khudyakov et al., 1999; Meng et al., 2001
; Schofield et al., 2000
). HEV strains from different origins or genotypes have been cross-neutralized with antibodies spanning a 166 aa epitope (Meng et al., 2001
). This region is located in the 3' end of ORF2 studied here. In some variants, such as Tanef 86/87 D, a single mutation modified a restriction enzyme site resulting in an amino acid substitution. In other variants, the amino acid substitutions could only be deduced from sequence data. Therefore, despite the strong immune response elicited after HEV infection, some minor virus variants within ORF2 epitopes may escape neutralization. In this study, it is clear that high serum levels of anti-HEV antibodies were associated with the persistence of HEV viraemia at the time of sampling.
Besides genetic diversity referred to as population cloud size, the quasispecies is characterized by a dynamic evolution under selective pressures such as immunological response (Schneider & Roossinck, 2001). As patients were sampled within a short period of time during the acute stage of HEV infection, it is unlikely that intra-host selective pressure led to the emergence of a major variant in each patient. The nature of the selective pressure remains to be determined. In some adults and the elderly, immune memory from past exposure to HEV could have been reactivated by Tanefdour 86/87 strain, resulting in the selection of clones less sensitive to the neutralizing effect of long-term anti-HEV antibodies.
Given the strong predominance of three RFLP profiles, especially profile A, differences in fitness between HEV variants should be considered to be a result of gradual evolution of virus subpopulations. Unfortunately, sequential samples were not collected from patients during the course of the Tanefdour epidemic.
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ACKNOWLEDGEMENTS |
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Received 3 May 2004;
accepted 21 July 2004.
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