1 Departamento de Ciencias Experimentales y de la Salud, Universitat Pompeu Fabra, Dr. Aiguader 80, 08003 Barcelona, Spain
2 Hepatología, Instituto de Enfermedades Digestivas, Hospital Clinic, 08036 Barcelona, Spain
3 Hospital Figueres, 17600 Girona, Spain
4 Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, 28040 Madrid, Spain
Correspondence
Juana Díez
juana.diez{at}upf.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the nucleotide sequences reported here are AY683898AY684043.
Present address: Department of Molecular Biology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia.
Present address: Departament de Biologia Estructural, Institut de Biologia Molecular de Barcelona (CSIC), Josep Samitier 1-5, 08028 Barcelona, Spain.
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INTRODUCTION |
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The mechanisms responsible for the high rate of viral persistence are thought to be the result of a complex hostvirus interaction early after infection (Racanelli & Rehermann, 2003). However, little is known about these early virus and host determinants because the acute phase of infection is often asymptomatic and thus most diagnoses are made during the chronic stage, i.e. months or years after the events that determined the clinical course of the infection. While HCV induces strong humoral and cellular immune responses, their roles in virus clearance or persistence have not been fully elucidated. Studies in humans and chimpanzees indicate that a robust intrahepatic CD4+ and CD8+ T-cell response during the first weeks after infection is associated with viral clearance (Cooper et al., 1999
; Major et al., 2004
; Thimme et al., 2002
). Also, antibodies may play a role here because an early antibody recognition of the hypervariable region 1 (HVR1) in the envelope E2 protein was correlated with virus clearance (Allander et al., 1997
), and infection of chimpanzees could be inhibited by a human hyperimmune serum against HVR1 (Farci et al., 1996
). In line with this are studies on the rate of HVR1 evolution that have suggested that the HVR1 region is under immune pressure exerted by neutralizing antibodies (Booth et al., 1998
; Kato et al., 1993
; Shimizu et al., 1994
; Weiner et al., 1992
). Nevertheless, by using infectious retroviral pseudotypes, a recent study has shown that neutralizing antibody responses early after infection do not seem to play a role in the resolution of an acute infection (Logvinoff et al., 2004
).
What is the strategy of HCV to survive and establish persistence? This question is still unresolved. One interesting aspect is that HCV seems to have a wide cell tropism and can infect not only hepatocytes but also cells of the immune system (Bain et al., 2001; Sung et al., 2003
). Another important feature is that HCV behaves in infected patients as a complex mixture of genetically distinct but closely related variants, termed quasispecies, which results in a high genetic variability and adaptability (Martell et al., 1992
; Mellor et al., 1995
; Simmonds, 1995
). The quasispecies distribution of RNA viruses may influence the transmission, the pathogenesis and the outcome of viral infections (Domingo et al., 2001
; Pawlotsky, 2003
). New variants are continuously generated during virus replication as a result of errors made by the viral RNA-dependent RNA polymerase, which lacks proof-reading activity. This genetic variability could give the virus an advantage in adapting to a changing host environment including availability of permissive cells, the presence of innate and adaptive immune responses and antiviral treatment.
The quasispecies nature of HCV may help to establish viral persistence by escaping the host immune surveillance (Chang et al., 1997; Christie et al., 1999
; Kao et al., 1995
; Kato et al., 1994
; Shimizu et al., 1994
; Tsai et al., 1998
; van Doorn et al., 1995
; Weiner et al., 1992
, 1995
). However, due to the difficulty in obtaining samples at early time points after infection, very limited information is available on HCV quasispecies at that stage (Farci et al., 2000
; Manzin et al., 1998
, 2000
; Ray et al., 1999
). Moreover, the above studies were conducted in patients usually infected with large quantities of virus from potentially heterogeneous sources, even belonging to different genotypes.
In the present study, we had the unique opportunity of analysing HCV quasispecies features as early as 11 weeks post-infection in samples from acutely infected patients that presented different disease outcomes (Bruguera et al., 2002). All patients were infected from a single common donor during a nosocomial episode, allowing us to assess the evolution of a single viral strain in patients in whom the infection either became persistent or was spontaneously resolved. The study was done by extensive cloning and sequencing of an HCV E1/E2 segment that included the HVR1 region.
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METHODS |
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PCR amplification.
HCV-RNA was extracted from serum by the Trizol method following the instructions of the manufacturer (Invitrogen). Specific amplification of HCV-RNA was carried out with primers described previously (Gimenez-Barcons et al., 2001) as follows: cDNA was synthesized from viral RNA using 1·5 µM reverse primer HV2 and 0·45 U AMV reverse transcriptase µl1 in 20 µl final volume. Ten microlitres of this cDNA was used as a template for PCR amplification using the forward primer HV1 and the reverse primer HV2 at 0·5 µM each, in a final volume of 50 µl. One tenth of the external PCR product was used in a second PCR amplification using the internal primers HV3 and HV4. The resulting fragment of 388 nt comprised an E1/E2 segment (position 14431784 in genotype 1b HCV consensus sequence; Los Alamos National Laboratory HCV database) that includes the hypervariable region 1 (HVR1). PCR conditions were as follows: first 5 min denaturation step at 95 °C, then, 35 cycles consisting of denaturation for 30 s at 95 °C, annealing for 30 s at 45 °C and extension for 1 min at 72 °C, followed by a final extension for 10 min at 72 °C. All reactions were carried out using the Expand High Fidelity PCR system (Roche) with 1·8 mM MgCl2 final concentration.
HCV-quasispecies analysis.
At least two different PCR amplification reactions were done from each cDNA and combined to diminish molecular bottleneck during clonal analysis. These PCR products were purified from agarose gels and incubated for 30 min at 72 °C with Taq polymerase (EcoTaq; Ecogen) to introduce single overhanging A's at the 3' ends. The product of this reaction was cloned with the pGEM-T vector system (Promega) following manufacturer's instructions. A mean of 16 recombinant clones from each sample (range from 10 to 24) were sequenced using standard dideoxy sequencing with fluorescence labelled nucleotides (Perkin-Elmer Applied Biosystems). Although the amount of RNA used for the amplification reactions could not be quantified before use, the observed higher genetic complexities in the serum from patients with low viral load strongly argues against a molecular bottleneck effect in sample preparation. Sequences were aligned using CLUSTAL W (Thompson et al., 1994). The features of intrapatient HCV populations were defined and calculated as follows. The genetic distance is defined as the number of nucleotide differences between two sequences and was calculated by Kimura two-parameters (Kimura, 1980
). Distances between amino acid sequences are given as Hamming distances. The mean of genetic distances and Hamming distances is the mean of the values taken for all sequence pairs derived from a single sample. Both, the genetic distance and the number of synonymous and non-synonymous changes per synonymous and non-synonymous sites were calculated using the MEGA 2 program (Kumar et al., 2001
). The mutation frequency is the number of different mutations found relative to the number of nucleotides sequenced; it is calculated by dividing the number of different mutations found in a set of genomes (compared to the consensus nucleotide sequence of the same set) by the total number of nucleotides sequenced. The Shannon entropy (Sn) is a measure of the proportion of identical sequences in a mutant distribution. The possible values of Sn range from zero (when all genomes are identical) to one (when all genomes differ from one another). Sn was calculated following the formula: Sn=
i[(pixln pi)/ln n], in which pi is the frequency of each sequence in the mutant spectrum and n is the total number of sequences compared (Volkenstein, 1994
). The statistical significance of comparisons among patient samples was analysed with a Student's t-test. P values less than 0·05 were considered significant.
Nucleotide sequence accession numbers.
The nucleotide sequences reported here have been submitted to GenBank under the accession numbers AY683898AY684043.
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RESULTS |
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Quasispecies analysis reveals a correlation of high HCV load with the presence of a predominant viral form
The quasispecies nature of HCV has been related to important aspects of virus evolution and pathogenesis. To get an insight into the actual complexity of the intrapatient HCV quasispecies from this nosocomial outbreak and to analyse possible correlates with the clinical outcome of the infection, viral populations were determined for all seven patients early after infection. The above-mentioned amplified E1/E2 HCV-RNA segment was cloned into a bacterial vector. A mean of 16 clones (range from 10 to 24) per sample were sequenced and analysed. Fig. 2 shows a schematic representation of the quasispecies composition for each sample. In the high viral-load group, the major form ranged from 54 to 66 % of all nucleotide sequences while in the low viral-load group, it ranged from 23 to 25 %. Parallel observations were made from the analysis of the corresponding predicted amino acid sequence. The major deduced amino acid form ranged from 62 to 89 % and from 29 to 41 % in the high and low viral-load groups, respectively. These differences were statistically significant with P-values <0·0007 and <0·0004 for nucleotide and amino acid values, respectively. In patients S7 and S9, two major forms with an approximately equal frequency co-existed in the quasispecies. Taken together, these results indicated that grouping the patients according to viral load was reflected in the structure of the quasispecies.
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For the high viral-load patients S1 and S8, we obtained sera from a later time point and were able to amplify the respective HCV region. The comparable quasispecies analysis revealed a slight increase in Sn over time while the mutation frequencies, distances and ratio of synonymous/non-synonymous substitutions did not show consistency (Fig. 2 and Table 2
). Importantly, despite the ongoing HCV replication in both patients for about 20 weeks, the quasispecies characteristics of the high viral-load group were maintained.
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DISCUSSION |
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The infection history of our patients is particularly interesting because (i) all 10 patients involved in the outbreak were infected on the same day from a single-seropositive person, (ii) samples were available at an early time point after infection, and (iii) the infection dose was lower than in previously studied transmissions via blood transfusion. This latter aspect was inferred by two observations, first the infection source was a vial of heparin presumably contaminated by a needle bearing HCV-positive blood and second, the low HCV load in both patients that were the possible transmitters (Bruguera et al., 2002). This kind of low dose HCV transmissions are expected to be representative of most new infections as blood screening programmes have drastically reduced the risks of the high dose transmissions via blood transfusions.
It is interesting to note that from all the 10 HCV-infected patients, two had cleared the infection. Although these are very low numbers, this frequency is what one would expect from any HCV infection: a chronic carrier state develops in about 7085 % of cases whereas a spontaneous virus clearance is observed in 1530 %. This, together with the observation that the infecting virus strain was very homogeneous, would suggest that host factors contribute more to the infection outcome than the particular virus variant. Indeed this hypothesis fits well to studies in which chimpanzees infected with the same clonal HCV sequence presented different disease outcomes (Major et al., 2004). However to substantiate this point, studies with larger cohorts and different HCV genotypes are warranted.
Previous reports have suggested a correlation between HCV persistence and either a higher quasispecies complexity or a sequential increasing complexity early after infection (Farci et al., 2000; Ray et al., 1999
). These studies were conducted with injecting drug users (Ray et al., 1999
) or post-transfused patients (Farci & Purcell, 2000
) infected with unrelated HCV strains, which even belonged to different genotypes. Such a correlation was explained by a more effective immune response in patients who resolved the infection. In such a patient group, the immune pressure was thought to progressively clear virus variants from the quasispecies thus resulting in the decline of complexity (Farci & Purcell, 2000
). In contrast, in our present study, we observed that the quasispecies with a lower complexity were always related to persistent infection (patients S1, S3, S8 and S11). In addition, the quasispecies with the higher complexity related either to persistent infection (patient S10) or to spontaneous HCV clearance (patients S7 and S9) demonstrating that the complexity per se is not a reliable predictor for the outcome of an HCV infection. A possible explanation for the observed differences between our observations and previous reports could be because of different genetic heterogeneity, genotypes and size of the inoculum. In any case, future studies of early post-infection samples should help to clarify this matter.
How might the correlation of the existence of a dominant HCV form within the quasispecies with the high viral load be rationalized? Suppose that a clonal virus transmission event will start an infection. Then, variants will accumulate inevitably because of the high error rate of the virus replication machinery. Since the mutations will spread across the genome, the created quasispecies will mainly consist of minor forms. However, mutants that might overcome intrapatients selective constraints will gain selective advantage and expand relative to the other variants. These mutants will subsequently become the major forms in the virus population. If this simplistic view is correct, then a successfully selected mutant will directly lead to a high viral load and a more homogeneous quasispecies. Indeed, this is what has been suggested in quasispecies models (Eigen & Biebricher, 1988). Furthermore, overcoming the initial intrapatient barrier will increase the chance to develop a chronic infection. In fact, this is the case in our limited cohort.
In conclusion, while this study on HCV quasispecies complexity did not result in a correlate with the clinical outcome of the infection, it revealed an interesting feature of basic HCV evolution. HCV populations in high viral-load patients seemed to harbour a major viral variant possibly as a result of overcoming intrapatients selective constraints. It would be interesting to understand the underlying process, be it immune-mediated selection or host-cell adaptation. The recently developed method to mimic the natural infection by HCV pseudotypes should help to shed light on this issue in the future (Hsu et al., 2003; Matsuura et al., 2001
).
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ACKNOWLEDGEMENTS |
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Received 3 August 2004;
accepted 25 August 2004.
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