1 Department of Medical Virology, Landspitali-University Hospital, University of Iceland, Reykjavik, Iceland
2 Department of Internal Medicine, Landspitali-University Hospital, University of Iceland, Reykjavik, Iceland
3 Department of Medical Microbiology, Malmö University Hospital, Lund University, SE-20502 Malmö, Sweden
Correspondence
Anders Widell
Anders.Widell{at}mikrobiol.mas.lu.se
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank accession numbers of the sequences reported in this paper are AY307450AY307770.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The transmission of HCV can occur with infectious doses of different sizes, which can confer a low infection rate, e.g. after accidental needle-stick (Arai et al., 1996; Suzuki et al., 1994
), sexual (Morisca et al., 2001
), perinatal (Rapicetta et al., 2000
) or nosocomial exposure (Esteban et al., 1996
; Ross et al., 2000
; Allander et al., 1995
; Widell et al., 1999
). Low-level contaminated blood products, such as non-solvent detergent treated Gammagard, were shown to have infected between 5 and 80 % of the recipients, depending upon the batch (Healey et al., 1996
; Widell et al., 1997
).
Quasispecies evolution of HCV has been reported after administration of anti-D immunoglobulin (McAllister et al., 1998; Casino et al., 1999
). These studies demonstrated that virus variants in the infectious source were relatively homogeneous, and distinct variants in HVR-1 were observed in each anti-D recipient, indicating evolution of the virus in that region. The changes observed were not random but rather strongly constrained, thus conserving the hydrophobicity pattern of HVR-1.
Recipients of blood components such as red cells, platelets and plasma, prior to screening for HCV antibodies among blood donors in the early 1990s (Alter & Houghton, 2000), probably received the largest infectious doses because of the large volume transferred at each transfusion. Thus, transfused patients theoretically should have the greatest potential for broad viral quasispecies. An extensive study by Farci et al. (2000)
focusing on early events after transfusion suggests that the magnitude of genetic diversity early after infection is important for the outcome of disease, i.e. the more progressive forms of disease have a larger genetic diversity compared with the resolving HCV cases. In a recent study of HCV from blood donors and their recipients (Lin et al., 2001
), the authors observed more extensive recipient sequence divergence with time, reaching the degree seen between unlinked subjects. These analyses included data obtained by direct sequencing of PCR products at one time-point per patient. Similar data have been reported recently by Cantaloube et al. (2003)
. However, extensive quasispecies data linking a single HCV-infected donor quasispecies to several transfused recipients are not available.
The aim of the present study was to assess the evolution of HCV quasispecies in one blood donor and several recipients infected from his blood components. We had access to frozen sera from 1993 from an HCV-infected blood donor and 13 of his viraemic recipients (Löve et al., 1995). These samples were analysed for viral heterogeneity in 1993 when the donor and the recipients were first found to be infected with HCV. The donor and six viraemic recipients still alive in 1998 were analysed again for quasispecies evolution. The study was done by extensive cloning and sequencing of the HCV HVR-1 region.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All samples were anonymously coded and unlinked to the patient source. The study was approved by the Ethical Committee of the National University Hospital of Iceland.
HCV diagnosis and RNA detection.
The initial virus diagnosis by detection of HCV antibodies was made with the Ortho second-generation ELISA (Ortho Diagnostic Systems) and confirmed with the second-generation recombinant immunoblot assay (RIBA; Chiron). The HCV diagnosis of the samples taken in 1998 was confirmed with third-generation ELISA and RIBA tests.
Processing and cloning of 1993 and 1998 samples was done completely independently and the guidelines recommended by Kwok & Higuchi (1989) were strictly adhered to to prevent contamination between samples and clones. The molecular studies were based on viral RNA, extracted from 100 µl serum by the method described by Chomczynski & Sacchi (1987)
. Extracted RNA was divided into three to four aliquots and stored in the presence of RNase inhibitor (Promega) at -70 °C.
For detection of viral RNA, a nested PCR directed to a region in the 5'-untranslated region was used (Widell et al., 1991). The detection limit was about 300 genomic copies ml-1.
Amplification and direct sequencing of HVR-1.
Viral RNA was reverse transcribed and amplified in a single-tube reaction using primers encompassing the HVR-1 region of genotype 1a (nt 12901309 and 18731854 for the first PCR, 35 cycles) as described previously (Widell et al., 1997). The enzymes employed were AMV-derived reverse transcriptase (Promega) and AmpliTaq polymerase (Roche). Primers corresponding to nt 13001319 and 18701848 were used in the second, inner PCR (35 cycles), which also employed AmpliTaq. PCR products were detected by agarose gel electrophoresis and ethidium bromide staining and visualized with ultraviolet light. DNA was purified on 1·0 % agarose gels from which the nucleic acid bands were removed and purified by a gel-extraction method (QIAquick; Qiagen) using a microcentrifuge. Subsequently, for direct sequencing of amplimers, the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS (PE Biosystems) was used with either inner PCR primers or special sequencing primers (Widell et al., 1997
). Both strand products were then analysed on a gene sequencer (373 Sequenator ABI; PE Biosystems) and edited with software programs (Factura and Sequence Navigator; PE Biosystems). The present study is entirely based upon the 81 nucleotides of HVR-1. (In our GenBank submissions of isolate and clone sequences, the HVR-1 coding region as well as the flanking 27 upstream and 171 nucleotides downstream were included.)
Cloning and sequencing of HVR-1.
To study the genomic heterogeneity of HVR-1, PCR products from all viraemic samples were cloned and 820 clones picked and subsequently sequenced. For the cloning, a cloning vector, pBAD TOPO TA (Invitrogen), was used in competent cells. Each clone was cultured overnight and the plasmids were purified on a Jet Star plasmid mini-prep kit (Genomed) and cycle-sequenced bi-directionally using vector specific M13-20 forward and M13-reverse primers.
To assess the variability of the PCRs and cloning procedures, donor DE samples from 1993 and 1998 each underwent two independent complete reverse transcription and nested PCRs with subsequent independent cloning procedures, labelled A and B, respectively. A further independent extraction/RT-PCR labelled DE98C (without cloning) was done on the donor 1998 sample.
Phylogenetic analysis.
Sequences obtained either from PCR products or clones were aligned by BioEdit version 4.8.10 (T. A. Hall, University of North Carolina, NC, USA). Pairwise nucleotide p-distances (the proportion of nucleotide sites at which the two sequences are different) between isolates were calculated with the Kimura two-parameter model using the Molecular Evolution Genetic Analysis (MEGA) version 2.1 software (S. Kumar, K. Tamura, I. B. Jakobsen & M. Nei, 2001). MEGA 2.1 was also used to compare predicted p-distances regarding synonymous versus non-synonymous changes by the Kumar algorithm and to calculate predicted amino acid differences between clones. In addition, differences between clones in 1993 and 1998 in the same individual were also calculated.
Neighbour-joining trees based on nucleotide p-distances were displayed in circular form when necessary and otherwise in linear form. Robustness of trees was always assessed by bootstrap analysis (1000 resamplings) provided with the MEGA 2.1 software.
Statistical methods.
Distance and amino acid difference data were transferred via Microsoft Excel 2000 to Statistica 5.1 (StatSoft). Since the number of observed distances varied with the number of clones obtained in each person, we used post hoc comparisons by the Tukey honest significant difference test for unequal N. This test allows for several parallel significance calculations between patients with different numbers of observations. P values of less than 0·05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Although fewer clones (about 10 per recipient) were analysed in the recipients, it was evident that the clones of each recipient displayed a very limited quasispecies distribution, despite the fact that 0·35 years had elapsed from transfusion to the diagnosis of HCV infection. The most diverse were R12 and R14. The narrow pattern among recipients with nucleotide p-distances of 0·044 (R1), 0·006 (R2), 0·010 (R3), 0·005 (R4), 0·004 (R5), 0·013 (R7), 0·013 (R8), 0·003 (R9), 0·013 (R10), 0·012 (R11), 0·062 (R12), 0·012 (R13) and 0·037 (R14) was in clear contrast to donor DE whose mean nucleotide p-distances among clones was 0·130. All differences between the donor and each recipient were highly significant by post hoc testing (P<0·00003). In addition, R12 significantly differed (P<0·005) from all other recipients except R1 and R14. Recipient R1 was a special case since one clone (R0193clo10) was different from all his other clones and closely related, but not identical, to a sequence found in the donor 1998 sample (data not shown). Overall, quasispecies diversity in 1993 was not different in the two individuals (R1 and R5) whose HCV infection resolved spontaneously compared with those who remained chronically HCV infected.
In 1993, samples from recipients exposed to the components from the same donation differed in sequence clustering, such as R1 and R2 in contrast to R3; R4 in contrast to R5; R7 in contrast to R8; and R9 in contrast to R10. This patient grouping was in general supported by high bootstrap values. The opposite outcome was also observed where recipients were exposed to different blood donations obtained several months apart, for example R8 and R9 shared HVR-1 sequences and R10 and R11 had closely related HVR-1 sequences, despite years elapsing since transfusion and first diagnosis of HCV infection.
Donor DE 1998 sequences compared with the sequences of six recipients at follow-up in 1998
In concordance with the donor 1993 sequence diversity, the quasispecies in the donor DE 1998 sample was wider (mean nucleotide p-distance 0·172; P<0·0005) and contained several groups supported by high bootstrap values (Fig. 3). For the six recipients being followed up and included in the same dendrogram, quasispecies diversity was limited in R7, R8, R10 and R13 (0·0100·016) and somewhat broader in R11 (0·040; P<0·05) and R14 (0·086; P<0·001). R14 still showed a bimodal pattern of distribution, supported by high bootstrap values for each lineage. Despite original exposure to donor DE with broad quasispecies, no recipient changed to the clades of any other recipient.
To facilitate longitudinal comparisons over 5 years within the same person, individual neighbour-joining trees based on nucleotide p-distances between clones were calculated for DE, R7, R8, R10, R11, R13 and R14 (Fig. 4). For DE, several 1998 variants were related to 1993 precursors (upper half of tree), but several lineages were lost and a new cluster had appeared. In the recipients, the nucleotide evolution was much more restricted, in particular for R7, R8, R10 and R11. Recipients R13 and R14 changed more, leaving the R13 1998 quasispecies as relatively restricted while the bimodal pattern in R14 had evolved.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several main points of this study should be emphasized. First, and the key finding of our study, is that a substantial fraction (here four of six) of HCV-infected recipients, despite original exposure to a broad quasispecies, exhibited a narrow quasispecies pattern over at least a 5-year period of observation. In one case (R11) the recipient never developed HCV antibodies, whereas all the others did. This may suggest that the humoral immunity affects quasispecies evolution minimally. The lack of viral drift in several cases indicates that, despite an error prone viral RNA polymerase, viral variability and quasispecies development may be limited in a substantial number of patients and in this respect HCV infection may resemble GBV-C infection. Indeed several studies (Ross et al., 2000; Allain et al., 2000
) mention that a few patients show this conserved pattern, which is in contrast to the favoured view of a generally rapidly mutating HVR-1 region in HCV infection (Kato et al., 1994
). The few mutations that occurred during follow-up in recipients R7, R8, R10 and R11 were mainly synonymous. In contrast, donor DE and recipient R14 both showed increasingly broader quasispecies and predominance for non-synonymous substitutions, following the accepted view on HCV HVR-1 quasispecies evolution. Interestingly, R13, who was rather conserved both in 1993 and in 1998, showed a similar rate of amino acid changes.
Secondly, several recipients' variants in 1993 had a moderate range of nucleotide differences, despite onset of infection 0·35 years earlier. Most recipient isolates were closely related to one or more 1993 donor clones. Variants within each recipient in 1993 were similar and clustered closely together except in three cases. Recipient R1 had, when traced, a very recent onset of HCV infection and displayed one distantly related clone (clone DE93clo10) compared with others, as described above. Another exception was R14, who had a bimodal distribution of clones and continued to evolve along this pattern. Finally, R12, who was analysed 3 years after transfusion, showed a quasispecies pattern, which was at that time (1993) broader than R14, but R12 died before follow-up.
Thirdly, some recipients, despite being exposed to infectious material from the same donation, displayed quite different sequences, such as R1 and R2 compared with R3, R7 compared with R8, and R9 compared with R10. This finding was not linked to the type of blood component they had received and is difficult to understand. In recipients R4 and R5, also infected from a shared donation by donor DE, the sequences of the clones were more alike but grouped independently, which was supported by bootstrap analysis. Two of the recipients (R9 and R11) received donations on two occasions from the same donor. This did not seem to affect the diversity of viral variants.
Finally, a technical aspect. When the whole RNA extraction RT-PCR was repeated for the DE93 sample, the results were similar but not completely identical. New, different clones were detected, while some others were not detectable in the second RT-PCR/cloning run. This illustrates that the whole procedure from extraction through reverse transcription and PCR to cloning is subject to biological variation as well as competition between primer and different target sequences in vitro. When a full duplicate procedure was done on the donor 1998 sample, again there was variability between runs. The obvious conclusion is that results from PCR products must be interpreted with caution.
As for limitations of this study, the potential infidelity of the Taq polymerase used has been a matter of debate (Smith et al., 1997) and obviously we cannot exclude the presence of some sporadic mutations' caused in vitro. However, our main finding was of the strong and constant clustering of viral variants in most recipients.
In conclusion, our study, which may be the most extensive quasispecies study based on a single blood donor and many viraemic recipients of whom several were studied 5 years later, showed that limited quasispecies had been established when the retrospective study was done and that such a steady state can be maintained for long periods. This is partially in contrast with the common view of a rapidly evolving HVR-1 in the HCV and should be evaluated further. The question of the role of viral variants in the evolution of the HCV still remains to be answered, as well as their significance in the pathogenesis of disease.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allander, T., Gruber, A., Naghavi, M., Beyene, A., Söderström, T., Björkholm, M., Grillner, L. & Persson, M. A. A. (1995). Frequent patient-to-patient transmission of hepatitis C virus in a haematology ward. Lancet 345, 603607.[Medline]
Alter, H. J. & Houghton, M. (2000). Clinical Medical Research Award. Hepatitis C virus and eliminating post-transfusion hepatitis. Nat Med 6, 10821086.[CrossRef][Medline]
Arai, Y., Noda, K., Enomoto, N., Arai, K., Yamada, Y., Suzuki, K. & Yoshihara, H. (1996). A prospective study of hepatitis C virus infection after needlestick accidents. Liver 16, 331334.[Medline]
Cantaloube, J.-F., Biagini, P., Attoui, H., Gallian, P., de Micco, P. & de Lamballiere, X. (2003). Evolution of hepatitis C virus in blood donors and their respective recipients. J Gen Virol 84, 441446.
Casino, C., McAllister, J., Davidson, F., Power, J., Lawlor, E., Yap, P. L., Simmonds, P. & Smith, D. B. (1999). Variation of hepatitis C virus following serial transmission: multiple mechanisms of diversification of the hypervariable region and evidence for convergent genome evolution. J Gen Virol 80, 717725.[Abstract]
Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanatephenolchloroform extraction. Anal Biochem 162, 156159.[CrossRef][Medline]
Esteban, J. I., Gomez, J., Martell, M. & 7 other authors (1996). Transmission of hepatitis C virus by a cardiac surgeon. N Engl J Med 334, 555560.
Farci, P., Shimoda, A., Coiana, A. & 9 other authors (2000). The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 288, 339344.
Forns, X., Purcell, R. H. & Bukh, J. (1999). Quasispecies in viral persistence and pathogenesis of hepatitis C virus. Trends Microbiol 7, 402410.[CrossRef][Medline]
Healey, C. J., Sabharwal, N. K., Daub, J., Davidson, F., Yap, P. L., Fleming, K. A., Chapman, R. W., Simmonds, P. & Chapel, H. (1996). Outbreak of acute hepatitis C following the use of anti-hepatitis C virus-screened intravenous immunoglobulin therapy. Gastroenterology 110, 11201126.[Medline]
Holland, J., Spindler, K., Horodysky, F., Graban, E., Nichol, S. & VandePol, S. (1982). Rapid evolution of RNA genomes. Science 215, 15771585.[Medline]
Kato, N., Ootsuyama, Y., Sekiya, H., Ohkoshi, S., Nakazawa, T., Hijikata, M. & Shimotohno, K. (1994). Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection. J Virol 68, 47764784.[Abstract]
Kwok, S. & Higuchi, R. (1989). Avoiding false positives with PCR. Nature 339, 237238.[CrossRef][Medline]
Lin, H. J., Seeff, L. B., Barbosa, L. & Hollinger, F. B. (2001). Occurrence of identical hypervariable region 1 sequences of hepatitis C virus in transfusion recipients and their respective blood donors: divergence over time. Hepatology 34, 424429.[CrossRef][Medline]
Löve, A., Rydbeck, R., Kristensson, K., Örvell, C. & Norrby, E. (1985). Hemagglutininneuraminidase glycoprotein as a determinant of pathogenicity in mumps virus hamster encephalitis. Analysis of mutants selected with monoclonal antibodies. J Virol 53, 6774.[Medline]
Löve, A., Smáradóttir, A., Thorsteinsson, S. B., Stanzeit, B. & Widell, A. (1995). Hepatitis C virus genotypes among blood donors and their recipients in Iceland determined by the polymerase chain reaction. Vox Sang 69, 1822.[Medline]
McAllister, J., Casino, C., Davidson, F., Power, J., Lawlor, E., Yap, P. L., Simmonds, P. & Smith, D. B. (1998). Long-term evolution of the hypervariable region of hepatitis C virus in a common-source-infected cohort. J Virol 72, 48934905.
Morsica, G., Sitia, G., Bernardi, M. T., Tambussi, G., Novati, R., de Bona, A., Gianotti, N. & Lazzarin, A. (2001). Acute self-limiting hepatitis C after possible sexual exposure: sequence analysis of the E-2 region of the infected patient and sexual partner. Scandinavian J Infect Dis 33, 116120.[CrossRef][Medline]
Okuda, M., Hino, K., Korenaga, M., Yamaguchi, Y., Katoh, Y. & Okita, K. (1999). Differences in hypervariable region 1 quasispecies of hepatitis C virus in human serum, peripheral blood mononuclear cells, and liver. Hepatology 29, 217222.[Medline]
Pawlotsky, J. M., Germanidis, G., Frainais, P. O., Bouvier, M., Soulier, A., Pellerin, M. & Dhumeaux, D. (1999). Evolution of the hepatitis C virus second envelope protein hypervariable region in chronically infected patients receiving alpha interferon therapy. J Virol 73, 64906499.
Rapicetta, M., Argentini, C., Spada, E., Dettori, S., Riccardi, M. P. & Toti, M. (2000). Molecular evolution of HCV genotype 2c persistent infection following mother-to-infant transmission. Arch Virol 145, 965977.[CrossRef][Medline]
Ross, R. S., Viazov, S., Gross, T., Hofmann, F., Seipp, H. M. & Roggendorf, M. (2000). Transmission of hepatitis C virus from a patient to an anesthesiology assistant to five patients. N Engl J Med 343, 18511854.
Simmonds, P., Holmes, E. C., Cha, T. A. & 7 other authors (1993). Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol 74, 23912399.[Abstract]
Smith, D. B., McAllister, J., Casino, C. & Simmonds, P. (1997). Virus quasispecies: making a mountain out of a molehill? J Gen Virol 78, 15111519.
Soler, M., Pellerin, M., Malnou, C. E., Dhumeaux, D., Kean, K. M. & Pawlotsky, J. M. (2002). Quasispecies heterogeneity and constraints on the evolution of the 5' noncoding region of hepatitis C virus (HCV): relationship with HCV resistance to interferon-alpha therapy. Virology 298, 160173.[CrossRef][Medline]
Steinhauer, D. A. & Holland, J. J. (1987). Rapid evolution of RNA viruses. Annu Rev Microbiol 41, 409433.[CrossRef][Medline]
Suzuki, K., Mizokami, M., Lau, J. Y., Mizoguchi, N., Kato, K., Mizuno, Y., Sodeyama, T., Kiyosawa, K. & Gojobori, T. (1994). Confirmation of hepatitis C virus transmission through needlestick accidents by molecular evolutionary analysis. J Infect Dis 170, 15751578.[Medline]
Tokita, H., Okamoto, H., Iizuka, H., Kishimoto, J., Tsuda, F., Miyakawa, Y. & Mayumi, M. (1998). The entire nucleotide sequences of three hepatitis C virus isolates in genetic groups 79 and comparison with those in the other eight genetic groups. J Gen Virol 79, 18471857.[Abstract]
Widell, A., Månsson, A. S., Sundström, G., Hansson, B. G. & Nordenfelt, E. (1991). Hepatitis C virus RNA in blood donor sera detected by the polymerase chain reaction: comparison with supplementary hepatitis C antibody assays. J Med Virol 35, 253258.[Medline]
Widell, A., Zhang, Y. Y., Andersson-Gäre, B. & Hammarström, L. (1997). At least three hepatitis C virus strains implicated in Swedish and Danish patients with intravenous immunoglobulin-associated hepatitis C. Transfusion 37, 313320.[CrossRef][Medline]
Widell, A., Christensson, B., Wiebe, T., Schalén, C., Hansson, H. B., Allander, T. & Persson, M. A. A. (1999). Epidemiologic and molecular investigation of outbreaks of hepatitis C virus infection on a pediatric oncology service. Ann Intern Med 130, 130134.
Received 19 June 2003;
accepted 7 October 2003.