|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Dipartimento di Medicina Clinica e
Sperimentale, University of Padua, and the Dipartimento di Medicina
Interna, Cardioangiologia, Epatologia, Policlinico S. Orsola,
University of Bologna, Italy.
Chronic hepatitis C virus (HCV) infection has been associated with
development of mixed cryoglobulinemia type 2 (MC2), a
lymphoproliferative disorder characterized by B cell monoclonal
expansion and immunoglobulin M/k cryoprecipitable immunoglobulin
production. A short sequence (codons 384-410) of the HCV E2 protein,
which has the potential to promote B cell proliferation, was
investigated in 21 patients with HCV-related MC2 and in a control group
of 20 HCV carriers without MC2. In 6 of the 21 (29%) patients with
MC2, all the clones isolated from plasma, peripheral blood mononuclear
cells, and liver showed sequence length variation compared with the
hypervariable region 1 (HVR1) consensus sequence; 5 patients had an
insertion at codon 385, and 1 patient had a deletion at codon 384. Inserted residues at position 385 were different within and between
patients. No such mutations were observed in any of the HVR1 clones
from control patients without MC2, and the difference between the 2 groups was statistically significant (P = .02). Analysis
of 1345 HVR1 sequences obtained from GenBank strongly supported the
conclusion that the observed insertions and deletion represent a rare
event in HCV-infected patients, suggesting that they are significantly associated with MC2. The physical and chemical profiles of the 385 inserted residues detected in the MC2 patients were consistent with the
possibility that these mutations, which occurred in a region containing
immunodominant epitopes for neutralizing antibodies and binding sites
for B lymphocytes, may be selected by functional constraints for
interaction with host cells.
(Blood. 2001;98:2657-2663) The hepatitis C virus (HCV), besides being
the major cause of chronic hepatitis, cirrhosis, and hepatocellular
carcinoma in many parts of the world, has also been implicated in the
pathogenesis of mixed cryoglobulinemia type 2 (MC2).1-3
This is a lymphoproliferative disorder characterized by B cell
monoclonal expansion, leading to the production of large amounts of
circulatory, temperature-sensitive immunocomplexes that accumulate in
different tissues and cause the classical syndrome of arthralgia,
purpura, weakness, systemic vasculitis, and
glomerulonephritis.4-6 In some patients, further expansion
of the monoclonal B cell tissue mass into high-grade non-Hodgkin
lymphoma has been observed.7 In most published studies,
the prevalence of HCV infection in patients with MC2 ranged from 80%
to 90%, whereas reported prevalence of MC2 in HCV-infected patients
has been variable, with higher rates in Italy and southern Europe than
in northern Europe.3,8 How HCV promotes the
development of MC2 in a subgroup of chronically infected patients is
still unclear, but host and viral factors have been implicated.
It has been shown that the HLAB8-DR3 haplotype might confer
susceptibility to HCV-related MC2.9 As to the mechanisms
promoting B cell proliferation, these might involve direct infection
or, more likely, chronic, antigen-driven stimulation of B lymphocytes
by HCV. A recent study10 favors the latter hypothesis by
suggesting that the E2 envelope protein of HCV could be the chronic
antigenic stimulus for clonal selection and expansion of B cells. The
E2 protein of HCV contains the hypervariable region 1 (HVR1)-expressing immunodominant epitopes for B cells involved in
virus neutralization.11-15 Furthermore, it has been
reported that E2 can bind directly to B lymphocytes through interaction with the tetraspanin CD81, and such interaction has been suggested to
promote B cell proliferation by lowering their activation
threshold.16 Thus, the E2 protein has the potential for
multifunctional interaction with B lymphocytes. Still it remains
unclear why monoclonal expansion and proliferation leading to MC2
occurs only in a small subset of infected patients. As do other RNA
viruses, HCV replicates as a mixture of different, but closely related,
genomic variants in the form of virus quasi species.17
HVR1 has the highest heterogeneity within the HCV quasi species, and
this leads to the possibility that binding to and stimulation of B
cells might be favored by specific HVR1 sequences. To assess whether
variations in the sequence or conformation of the E2 protein could play
a role in the development of HCV-related MC2, we have here compared the
HVR1 sequence and quasi species in HCV chronic carriers with or without
MC2. In some patients the analysis was extended to HCV derived from
plasma, from peripheral blood mononuclear cells, and from liver
biopsies to evaluate possible compartmentalization of specific HCV sequences.
Patient characteristics
Polymerase chain reaction amplification and cloning of the
HVR1
Total RNA was resuspended in 10 µL diethylpyrocarbonate-treated distilled water. Reverse transcription (RT) was performed as described previously.18 Briefly, the RNA was incubated at 70°C for 5 minutes and then reverse-transcribed in a 25 µL reaction mixture containing 50 pM outer antisense primer, 3 mM MgCl2, 1 mM deoxynucleoside triphosphate (dNTP), 1 mM dithiothreitol, 75 mM KCl, 5 mM Tris-HCl (pH 8.3), 20 U RNase inhibitor (Pharmacia LKB, Piscataway, NJ), and 130 U Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD). The mixture was incubated at 37°C for 60 minutes and then at 95°C for 5 minutes. E2 HVR1 was amplified after RT by nested PCR. First-round PCR was performed as follows: 10 µL cDNA was added to 40 µL mixture containing 50 pM external sense primer, 1.5 mM MgCl2, 23.5 mM Tris-HCl (pH 8.3), 35.5 mM KCl, and 1.5 U Taq polymerase (Perkin-Elmer, Norwalk, CT). Nested "hot-start" PCR was performed as follows: the bottom mixture contained 2.0 mM MgCl2, 0.2 mM each dNTP, 10 mM Tris-HCl (pH 8.3), 15 mM KCl, and 50 pM each internal primer. A wax layer was used to separate the lower mixture from the top reaction mixture containing 40 mM Tris-HCl (pH 8.3), 60 mM KCl, 1.5 U Taq polymerase, and 1 µL first-round PCR product. For the amplification of a 175 nt fragment (nt 1428-nt 1603) of the E2-HVR1, the following 2 sets of primers were used: outer primers, AS 5'CATTGCAGTTCAGGGCCGTGCTA3' and S 5'GGTGCTCACTGGGGAGTCCT3'; inner primers, AS 5'TGCCAACTGCCATTGGTGTT3' and S 5'TCCATGGTGGGGAACTGGGC3'. For first- and second-round PCR, 30 cycles with the following conditions were used: 30 seconds at 94°C, 25 seconds at 55°C, and 30 seconds at 72°C. PCR products were purified (QIAQuik columns; Qiagen, Chatsworth, CA) and ligated into TA cloning vectors (TA cloning kit; Invitrogen, Leek, The Netherlands) according to the manufacturer's instructions. HVR1 sequencing For sequencing, 8 clones were isolated from the plasma of 21 patients with MC2 and 20 patients in the chronic hepatitis control group; in total, 328 clones were analyzed. Deduced amino acid (aa) sequences from all the clones were aligned to the HVR1 consensus sequence published by Puntoriero et al.19Cycle sequencing was performed using an ABI Prism Big Dye terminator ready reaction kit following the manufacturer's instructions (PerkinElmer Cetus, Emeryville, CA). The inner set of primers previously described was used to determine the nucleotide sequence of each strand. Electrophoresis of the sequencing products was performed by an ABI 310 automated DNA sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Phylogenetic analysis Sequence alignment was obtained using the Pileup program of the GCG package (Accelcrys, San Diego, CA). Aligned sequences of each patient were compared to the sequence of the clone used as probe and then shaded using the Publish program of the GCG Package.Phylogenetic trees were derived using the Treecon for Windows package (UIA, Antwerp, Belgium).20 Tree topologies were inferred by the neighbor-joining method with the Kimura 2-parameter distance matrices. Physical and chemical profiles of HVR1 sequences Prediction of relative residue accessibility and antigenicity values were calculated over a 7-aa window by the double-prediction method of Antheprot21 (http://antheprot-pbil.ibcp.fr/ns_sommaire.html).Analysis of the HVR1 quasi species and comparison of plasma, peripheral blood mononuclear cells, and liver HVR1 The HVR1 quasi species in plasma, PBMC2s, and liver were studied and compared using the heteroduplex tracking assay (HTA) and the clonal frequency analysis (CFA) techniques. These methods, which have been described in detail elsewhere,18,22,23 allowed us to resolve on nondenaturing gel electrophoresis intra-sample sequence heterogeneity through the mismatches between a radiolabeled probe (from one cDNA clone representing the quasi-species major variant) and the target sequences (the heterogeneous PCR product in the HTA and the PCR product from individual cDNA clones in the CFA). Briefly, to generate a probe, the insert of one clone was amplified by PCR. After column purification, the PCR product was end-radiolabeled with T4 polynucleotide kinase (Gibco BRL) plus -32P] adenosine
triphosphate. Initially, quasi-species analysis was performed with the
HTA technique whereby the probe, which represented the quasi-species
major variant of the plasma sample, is hybridized to the heterogeneous
HVR1 PCR product from each specimen, and the different viral sequences
are then resolved by nondenaturing polyacrylamide gel electrophoresis.
Nucleotide changes between the probe and the target DNA led to the
formation of heteroduplex bands characterized by retarded mobility
during gel electrophoresis. Probe hybridized with its own unlabeled
sequence was used to indicate the homoduplex control. Shift control was
made by hybridizing a probe with a PCR product obtained from a
different patient. As previously demonstrated, the degree of shift
between heteroduplex bands and homoduplex bands was directly
proportional to the number of nucleotide changes between the probe and
the target DNA.
To analyze in detail the quasi-species composition of a patient, 20 independent clones per specimen were then examined by CFA using the same blood-derived, patient-specific probe obtained for the analysis of the heterogeneous PCR product. In 4 selected patients in the MC2 group, the nucleotide sequence was determined from at least 1 clone from each unique variant by CFA in plasma, PBMC2s, and liver biopsy sample. Statistical analysis To examine whether the percentages of patients with mutated HVR1 sequence were significantly different in the group with HCV-related MC2 and in the control group with HCV infection without MC2, a statistical test of independence using the Fisher exact test was applied to the data from the 2 groups.Nucleotide sequence accession number The GenBank accession numbers of the nucleotide sequences reported here are AJ406073 to AJ406149.
HVR1 sequences and quasi species in patients with mixed cryoglobulinemia HCV-RNA from plasma samples of the 21 patients with MC2 and of the 20 HCV carriers without MC2 was used for RT-PCR amplification, cloning, and sequencing of a 175-nt long fragment, including the 384 to 410 HVR1 sequence. For each patient 8 independent clones were studied, and the derived aa sequences were aligned and compared with the consensus HVR1 sequence described by Puntoriero et al.19As shown in Figure 1A, different degrees
of quasi-species complexity were identified in the patients with MC2.
In 4 patients (patients 6, 9, 14, and 21), the quasi species was highly
homogeneous, and only 1 major HVR1 variant was detected in all clones;
in the remaining 17 patients, 2 to 5 variants were identified. In 6 patients (patients 1 to 6), the HVR1 aa sequences had different lengths than the consensus sequence. In 5 of them (patients 1 to 5), a single
aa insertion was detected at position 385, and it was found in all the
clones analyzed. Although the insertion site was identical in all 5 patients and in all clones from each patient, the inserted residue
often varied among clones. It was the same within the whole quasi
species in patients 3 and 4 only, in whom it showed a T385 and a D385
insertion, respectively. In patient 6, a 1-aa deletion at position 384 was detected in all 8 clones analyzed. In the remaining 15 patients
with MC2 (patients 7 to 21), no such aa insertion or deletion was
detected in any of the clones analyzed. The results obtained in 5 patients with an insertion at aa 385 and in those of patient 6 with a
deletion at aa 384 were fully confirmed with a second experiment of PCR
amplification, cloning, and sequencing, starting from a second batch of
the same plasma sample (data not shown). Phylogenetic analysis of the
obtained HVR1 sequences was then performed to exclude the possibility
of false results from PCR contamination. By using the neighbor-joining method, a phylogenetic tree was constructed for all 57 unique aa
sequences obtained from the 21 patients with MC2. As shown in Figure
2, sequences from each clustered together
and were well confined within subtrees, as expected from
epidemiologically unrelated patients. Furthermore, clusters of
sequences from the 6 patients with aa insertions or deletions were
intermingled with clusters of nonmutated sequences, thus indicating the
lack of a unique lineage for sequences with aa alterations in HVR1,
distinct from all the other sequences detected. In agreement with these
molecular profiles, there was no evidence of possible epidemiologic
relationship among the 6 patients with mutated sequences in whom
demographic data, geographic origin, and risk factors were considered.
HVR1 sequences and quasi species in patients with no mixed cryoglobulinemia No aa insertions or deletions within the HVR1 sequence were detected when 160 clones obtained from the 20 patients without MC2 were sequenced (Figure 1B). The frequency of sequence length modification was, therefore, significantly higher in patients with MC2 (6 of 21; 29%) than in patients without MC2 (0 of 20; P = .02). We also investigated the frequency of HVR1 sequence length alterations in a much higher number of sequences obtained from the GenBank database. Among the 1345 HVR1 (codons 384-410) sequences found, only 27 (2%) harbored 1 to 4 insertions, and in all but one the insertion occurred at codon 385. Only 7 sequences (0.5%) had 1 or 2 aa deletions. These results indicate that aa insertions or deletions within the 27 residues of the HVR1 sequence, as detected in our patients with MC2, is a rare finding in HCV-infected patients and is significantly associated with MC2.Physical and chemical profiles of HVR1 sequences The insertions at 385 detected within the HVR1 sequences in patients with MC2 were either hydrophilic (H and D in patient 1, R in patient 2, T in patient 3, D in patient 4, and E in patient 5) or were characterized by high turn propensity (P in patient 1, G in patient 2, and G in patient 5) and, therefore, appeared able to confer conformational restriction to this portion of the protein. Furthermore, insertion was located just before T386, a polar aa that might have an important structural and functional role given that it is highly conserved among sequences of HCV. Interestingly, T386 was also maintained in the sequence with the aa deletion found in patient 6. Recently, an HCV envelope protein E2 model was proposed, based on aa similarities with the envelope protein E of tick-borne encephalitis virus.24 According to this model, the insertion 385 we detected in patients with MC2 occurred in a highly exposed portion of the E2 protein that was the target of the immunoresponse and was in close steric contiguity with the CD81 binding site. According to these data, the changes detected in our patients with MC2 could affect antigenicity and binding properties of the HVR1 sequence. Indeed, the prediction of relative residue accessibility and antigenicity values, calculated over a 7-aa window by the double-prediction method of Antheprot, indicated that these values were increased at codon 385 in the HVR1 sequence of patients with MC2 compared with the consensus sequence and with those derived from patients without MC2 (mean relative residue accessibility and antigenicity values, 68.3 and 40.0, respectively).Heteroduplex gel shift assay and sequencing analysis of HVR1 quasi species in different compartments To investigate whether the 385 insertion, when detected in plasma, was also present in other compartments, detailed HVR1 quasi-species analysis was performed in plasma, PBMC2s, and liver biopsy specimens from 2 patients with MC2 (patients 3 and 4) using a combination of 2 techniques heteroduplex tracking assay and clonal frequency analysis.
The same analysis was then performed in 2 other patients with MC2, but
not in patients with mutations in plasma HVR1 sequences (patients 11 and 12), to verify the presence, if any, of circulating variants with
the 385 insertion in PBMC2s or liver biopsy samples.
HVR1 of these 4 patients was amplified by RT-PCR starting from paired samples of plasma, liver biopsy specimens, and PBMC2s. PCR products were then used to characterize the quasi species in the 3 different compartments by heteroduplex gel shift analysis. Patient-specific radiolabeled probes from the plasma major variant were
generated and initially hybridized with the unlabeled heterogeneous PCR
products from plasma, PBMC2s, and liver biopsy samples. HTA allowed
rapid determination of the quasi-species composition in the 3 samples
from each patient regarding number of variants and their relative
genetic distances. Results of HTA are illustrated in Figure
3: in each panel, the heteroduplex shift patterns corresponding to the quasi species from plasma, liver, and
PBMC2s of each patient are reported. In each patient the migration profile of the HVR1 variants was identical in the different samples, indicating the presence of the same major variants. To more precisely assess quasi-species composition in terms of complexity (number of
variants with a distinct shift pattern) and relative abundance of each
detected variant, 20 clones from each compartment were then analyzed by
CFA. Because it is sensitive enough to discriminate among variants that
differ by only a few nucleotide substitutions, CFA allowed the
resolution of details in the quasi-species composition. Results of this
analysis are reported in Figure 4A for
patients 3 and 4 and in Figure 4B for patients 11 and 12.
In patient 3, the shift profile of clones derived from plasma, liver,
and PBMC2s were identical In patient 11, the gel shift patterns revealed the presence of a similar HCV population in plasma, liver, and PBMC2s. In this case, the restricted number of clones with distinct patterns and the slight mobility differences observed between clones was indicative of a tight genetic relation among variants within and between the 3 compartments. Similarly, in patient 12 the quasi species in plasma, liver, and PBMC2 samples was homogeneous and was primarily represented by variants with highly similar migration patterns. Sequences from clones with unique gel shift profiles on CFA from these 4 patients were determined. In each case, the deduced aa sequences from plasma, PBMCs, and liver were then aligned, and the results were consistent with the presence of identical sequences in the 3 compartments previously observed by CFA.
Mixed cryoglobulinemia type 2 has been clearly associated with
chronic HCV infection, but how the virus promotes B-cell clonal expansion remains undefined. In this study we have identified a
mutation of HVR1 of the E2 protein in a significant proportion of
patients with MC2. This mutation consisted of a change in HVR1 sequence
length, highly conserved among all HCV genotypes and composed of 27 aa.
Five patients with mutated sequence length had 1 residue insertion at
codon 385, and one patient had 1 residue deletion at codon 384. Interestingly, 100% of the variants isolated from each of these
patients had the mutation, independently on whether HCV had been
derived from plasma, PBMC2s, or liver. Thus, though we cannot exclude
the presence of nonmutated HVR1 as a minor variant, HVR1 sequence
length alterations were predominant in 6 of 21 of our patients with
MC2. Whether the mutated HVR1 sequences found in patients with MC2 were
the major variant at the time of contamination or emerged and expanded
during chronic infection is a matter of speculation. Our findings were
consistent with both possibilities. The high prevalence of mutated HCV
(each of the 8 clones within the same quasi species) found in patients with MC2 The pathogenic role of HCV in the development of MC2 has been associated with the ability of the virus to induce strong, protracted B-cell clone proliferation. This might be the consequence of chronic antigenic stimulation by HCV antigens acting directly on B cells or of involvement in the immunocomplexes that elicit the IgM monoclonal response. After studying immunoglobulin antigen receptor variable region genes, De Re et al10 suggest that the HCV E2 protein may indeed represent one of the chronic stimuli driving B-cell selection and clonal expansion. On the other hand, Pileri et al16 show that the E2 protein can also bind directly to B lymphocytes through interaction with the cellular receptor CD81 and that such interaction, which might occur through cellular receptors also involved in virus entry, has been suggested to promote B-cell proliferation by lowering the activation threshold. Although in these studies the discrete domains of E2 representing the epitopes for neutralizing antibodies and B-cell binding sites have not been identified, data suggest that the HVR1 could be directly involved in interaction with and in stimulation of B cells. Several studies show that HVR1, including the N-terminus of HVR1, does contain immunodominant epitopes for neutralizing antibodies.11-15 According to a recently proposed model of the HCV envelope protein E2, the 385 insertion we detected in patients with MC2 would occur in a highly exposed portion of the E2 protein that is the target of the immunoresponse and is in close steric contiguity with the CD81 binding site.24 We suggest that selection and expression of the mutated HVR1 sequence seen in patients with MC2 could reflect a replicative advantage resulting from optimized fitness with cell receptors or coreceptors involved in virus binding and entry. These receptors or coreceptors could also be responsible for triggering B-cell proliferation by the mutated HCV, thus explaining the association with MC2. Further studies on the binding and stimulatory activity on B lymphocytes of the identified HVR1 sequences are warranted to prove or reject this hypothesis.
Submitted February 26, 2001; accepted July 3, 2001.
Supported by a grant (MM06104479-007) from the 40% MURST, Italy.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Alfredo Alberti, Department of Clinical and Experimental Medicine, University of Padua, Via Giustiniani, 2 Padua 35128, Italy; e-mail: labepvir{at}unipd.it.
1. Alter MJ, Margolis HS, Krawczynski K, et al. The natural history of community-acquired hepatitis C in the United States: the Sentinel Counties Chronic non-A, non-B Hepatitis Study Team. N Engl J Med. 1992;327:1899-1905[Abstract]. 2. Agnello V, Chung RT, Kaplan LM. A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med. 1992;327:14901495. 3. Misiani R, Bellavita P, Fenili D, et al. Hepatitis C infection in patients with essential mixed cryoglobulinemia. Ann Int Med. 1992;117:573-577. 4. Perl A, Gorevic PD, Ryan DH, et al. Clonal B cell expansion in patients with essential mixed cryoglobulinemia [abstract]. Clin Exp Immunol. 1989;76:54[Medline] [Order article via Infotrieve]. 5. Monteverde A, Sabattini E, Poggi S, et al. Bone marrow findings further support the hypothesis that essential mixed cryoglobulinemia type II is characterized by a monoclonal B-cell proliferation. Leuk Lymphoma. 1995;20:119-124[Medline] [Order article via Infotrieve]. 6. Franzin F, Efremov DG, Pozzato G, Tulissi P, Batista F, Burrone OR. Clonal B-cell expansion in peripheral blood of HCV-infected patients. Br J Haematol. 1995;90:548-552[Medline] [Order article via Infotrieve].
7.
Silvestri F, Pipan C, Barillari G, et al.
Prevalence of hepatitis C virus infection in patients with lymphoproliferative disorders.
Blood.
1996;87:4296-4301 8. Ferri C, Greco F, Longobardo G, et al. Association between hepatitis C virus and mixed cryoglobulinemia. Clin Exp Rheumatol. 1991;9:621-624[Medline] [Order article via Infotrieve].
9.
Lenzi M, Frisoni M, Mantovani V, et al.
Haplotype HLA-B8-DR3 confers susceptibility to hepatitis C virus-related mixed cryoglobulinemia.
Blood.
1998;91:2062-2066
10.
De Re V, De Vita S, Marzotto A, et al.
Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin's lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia.
Blood.
2000;96:3578-3584
11.
Weiner AJ, Geysen HM, Christopherson C, et al.
Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infection.
Proc Natl Acad Sci U S A.
1992;89:3468-3472
12.
Farci P, Schimoda A, Wong D, et al.
Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein.
Proc Natl Acad Sci U S A.
1996;93:15394-15399 13. Shimizu YK, Igarashi H, Kiyohara T, et al. A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures. Virology. 1996;223:409-412[CrossRef][Medline] [Order article via Infotrieve]. 14. Zibert A, Schreier E, Roggendorf M. Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment. Virology. 1995;208:653-661[CrossRef][Medline] [Order article via Infotrieve]. 15. Zibert A, Kraas W, Meisel H, Jung G, Roggendorf M. Epitope mapping of antibodies directed against hypervariable region 1 in acute self-limiting and chronic infections due to hepatitis C virus. J Virol. 1997;71:4123-4127[Abstract].
16.
Pileri P, Uematsu Y, Campagnoli S, et al.
Binding of hepatitis C virus to CD81.
Science.
1998;282:938-941
17.
Martell M, Esteban JI, Quer J, et al.
Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasi-species nature of HCV genome distribution.
J Virol.
1992;66:3225-3229
18.
Gerotto M, Sullivan DG, Polyak SJ, et al.
Effect of retreatment with interferon alone or interferon plus ribavirin on hepatitis C virus quasi-species diversification in nonresponder patients with chronic hepatitis C.
J Virol.
1999;73:7241-7247 19. Puntoriero G, Meola A, Lahm A, et al. Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants. EMBO J. 1998;17:3521-3533[CrossRef][Medline] [Order article via Infotrieve]. 20. Van de Peer Y, De Wachter R. TREECON for Windows: A software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Application Biosci. 1994;10:569-570. 21. Geourjon C, Deleage G. Antheprot 2.0: a three-dimensional module fully coupled with protein sequence analysis methods. J Mol Graph. 1995;13:209-212[CrossRef][Medline] [Order article via Infotrieve].
22.
Wilson JJ, Polyak SJ, Day TD, Gretch DR.
Characterization of simple and complex hepatitis C virus quasi-species by heteroduplex gel shift analysis: correlation with nucleotide sequencing.
J Gen Virol.
1995;76:1763-1771 23. Gretch DR, Polyak SJ, Wilson JJ, Carithers RL, Perkins JD, Corey L. Tracking hepatitis C virus quasi-species major and minor variants in symptomatic and asymptomatic liver transplant recipients. J Virol. 1996;70:7622-7631[Abstract]. 24. Yagnik AT, Lahm A, Meola A, et al. A model for the hepatitis C virus envelope glycoprotein E2. Proteins. 2000;15:355-366. 25. Casino C, McAllister J, Davidson F, et al. Variation of hepatitis C virus following serial transmission: multiple mechanisms of diversification of the hypervariable region and evidences for convergent genome evolution. J Gen Virol. 1999;80:717-725[Abstract]. 26. Sobolev BN, Poroikov W, Olenina LV, Kolesanova EF, Archakov Al. Comparative analysis of amino acid sequences from envelope proteins isolated from different hepatitis C virus variants: possible role of conservative and variable regions. J Viral Hepat. 2000;7:368-374[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. Torres-Puente, J. M. Cuevas, N. Jimenez-Hernandez, M. A. Bracho, I. Garcia-Robles, F. Carnicer, J. del Olmo, E. Ortega, A. Moya, and F. Gonzalez-Candelas Contribution of insertions and deletions to the variability of hepatitis C virus populations J. Gen. Virol., August 1, 2007; 88(8): 2198 - 2203. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Bianchettin, C. Bonaccini, R. Oliva, A. Tramontano, A. Cividini, M. Casato, G. Merlini, E. Silini, and M. U. Mondelli Analysis of Hepatitis C Virus Hypervariable Region 1 Sequence from Cryoglobulinemic Patients and Associated Controls J. Virol., May 1, 2007; 81(9): 4564 - 4571. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Zehender, C. De Maddalena, F. Bernini, E. Ebranati, G. Monti, P. Pioltelli, and M. Galli Compartmentalization of Hepatitis C Virus Quasispecies in Blood Mononuclear Cells of Patients with Mixed Cryoglobulinemic Syndrome J. Virol., July 15, 2005; 79(14): 9145 - 9156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. P. Hofmann, E. Herrmann, B. Kronenberger, C. Merkwirth, C. Welsch, T. Lengauer, S. Zeuzem, and C. Sarrazin Association of HCV-related mixed cryoglobulinemia with specific mutational pattern of the HCV E2 protein and CD81 expression on peripheral B lymphocytes Blood, August 15, 2004; 104(4): 1228 - 1229. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
