Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, January 2001, p. 62-73, Vol. 8, No. 1
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.1.62-73.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Diversity of Hepatitis C Virus Quasispecies
Evaluated by Denaturing Gradient Gel Electrophoresis
K. A.
Harris* and
C. G.
Teo
Hepatitis and Retrovirus Laboratory, Central
Public Health Laboratory, Public Health Laboratory Service, London
NW9 5HT, United Kingdom
Received 8 June 2000/Returned for modification 12 September
2000/Accepted 18 October 2000
 |
ABSTRACT |
Denaturing gradient gel electrophoresis (DGGE) was used to
study the diversity of hepatitis C virus (HCV) quasispecies.
Optimized DGGE running conditions were applied to screen for variations in sequences cloned from amplicons originating from the
nonstructural 5b (NS5b) gene of HCV in blood of hemophilia
patients, intravenous drug users, and blood donors
(five specimens from each study group, ca. 40 clones studied per
specimen). Clones identified by DGGE as unique were
sequenced. NS5b sequence entropy and mean genetic distance in
hemophiliacs did not differ significantly from those in the other
groups, pointing to a lack of correlation
between HCV diversity and the multiplicity of past HCV exposures.
DGGE was also applied to investigate variation
in the HCV envelope 2/hypervariable region 1 (E2/HVR-1) in serum
samples serially taken from two patients during the
seroconversion phase of HCV infection. E2/HVR-1 sequence entropy
changes were small and not correlated with rising
anti-HCV antibody levels, reflecting mutational changes not mediated
by antibody selection.
| |
INTRODUCTION |
Most studies assessing the diversity
of hepatitis C virus (HCV) quasispecies are conducted by amplifying
selected portions of the genome by PCR, isolating individual subgenomic
fragments by a cloning procedure, and then characterizing the
nucleotide sequence of each clone 15, 17, 20. Evaluating
the diversity of HCV quasispecies in clinical specimens often requires
the sequencing of a large number of clones. Less onerous procedures,
e.g., those that analyze single-strand conformation polymorphism and
heteroduplex mobility of PCR amplicons, have been described 11,
18, 26. We have developed an alternative procedure based on
denaturing gradient gel electrophoresis (DGGE) 10 that
permits intrahost HCV genetic diversity to be screened more comprehensively.
In the DGGE procedure, double-stranded (ds) DNA is electrophoresed
through an acrylamide gel containing a gradient of denaturant that
increases in the direction of electrophoresis. The DNA molecules melt
when they reach a part of the gel that is sufficiently denaturing. At
this position, denaturation starts to occur at melting domains of the
molecule. As electrophoresis proceeds, conditions become more
denaturing and more domains melt. Single-stranded domains, as they are
formed, retard the movement of the DNA through the gel matrix. Sequence
differences of as little as 1 base can dramatically alter the stability
of the melting domains. However, at positions of the gel where the
concentration of denaturants is high, DNA can become completely single
stranded and the migration is no longer dependent on sequence. To
prevent complete denaturation of ds DNA, a "GC-clamp" is attached
to one of the PCR primers, facilitating the detection of mutations
along the whole length of a DNA molecule 23. The
sensitivity of DGGE in distinguishing between mutations is highly
dependent on the quality of the gradient gels and the differences in
migratory positions of DNA molecules and is not necessarily related to
the number of nucleotide differences.
Nucleotide variation in three subgenomic regions, the 5' noncoding
region (5'NCR), the nonstructural 5b (NS5b) gene, and hypervariable region 1 of the envelope glycoprotein 2-coding (E2/HVR-1) region was
investigated. The 5'NCR is a highly conserved region of the genome.
Nucleotide variations therein permit genotypes to be assigned, allowing
inferences of how HCV evolves over long intervals (decades and
centuries) to be drawn 3. In this study, PCR clones
derived from 5'NCR were used to optimize DGGE running conditions and to determine if DGGE can discriminate between sequences with
single-nucleotide changes. The NS5b gene is a relatively variable
subgenomic region 1, 13, 14. It displays a diversity wide
enough to allow observation of how the HCV genome drifts over a
relatively short period (months and years) 25. In our
study, clones amplified from this region were used to test whether the
optimized DGGE conditions were adequate to screen a large array of PCR
clones bearing a variety of sequence changes and to investigate if its sequence diversity differs between HCV-infected people belonging to
different at-risk groups. E2/HVR1 is the most hypermutable locus of the
HCV genome 6. It exhibits the highest sequence diversity
of any region of the genome, encoding a 27-amino-acid peptide at the
amino terminus of the E2 gene against which the host antibody response
is targeted 28, 29. The DGGE procedure was applied to
E2/HVR-1 to study how the HCV quasispecies changes during the early
(seroconverting) phase of acute infection 2, 7, 16.
| |
MATERIALS AND METHODS |
Specimens.
For studies of variation in 5'NCR and the NS5b
region, we used sera from 15 HCV RNA-positive individuals referred to
our laboratory for confirmatory testing. Five serum samples were from
hemophilia patients, five were from injecting drug users (IDUs) and
five were from blood donors. All the individuals were known not to have
undergone antiviral therapy. The hemophilia patients had received
clotting-factor therapy before anti-HCV antibody screening became
routine in blood donor centres. To investigate E2/HVR-1 evolution in
the early, acute phase of infection, panels of serum samples each
serially taken from two plasma donors undergoing HCV seroconversion was
used. These specimens were purchased from Bioclinical Partners Inc.
(Franklin, Mass.) (catalogue numbers HCV6211 and HCV6214).
PCR amplification and cloning.
RNA was extracted from 100 µl of sera or plasma using the Amplicor HCV specimen preparation kit
(Roche Diagnostic Systems, Branchburg, Calif.). The final pellet was
resuspended in 50 µl of nuclease-free water. Extracted RNA (22.2 µl) was reverse transcribed in 5 mM MgCl2-1 mM each
deoxynucleoside triphosphate (dNTP)-3.3 µM random hexamers-0.34 U
of RNasin (Promega, Madison, Wis.)-5 U of Moloney murine leukemia
virus reverse transcriptase (Life Technologies, Paisley, United Kingdom).
Amplification from the 5'NCR was carried out in 50-µl reaction
volumes each containing 10 µl of cDNA solution, 1.5 mM
MgCl2, 1 mM each dNTP, 25 pmol of sense primer 126 (5'GTGGTCTGCGGAACCGG), 25 pmol of antisense primer 299 (5'GGGCACTCGCAAGCACCC) 12, and 0.7 U of EXPAND
high-fidelity polymerase (Roche Molecular Biochemicals, Lewes, United
Kingdom). The reaction mixtures were heated to 94°C for 30 s,
and this was followed by 35 cycles of 94°C for 30 s, 65°C for
40 s, and 72°C for 50 s and a final cycle of 30 s at 72°C. This primer pair amplified a 174-bp fragment between genomic positions 
199 and 26.
Amplification from the NS5b gene was carried out in 50-µl reaction
mixtures each containing 10 µl of cDNA, 0.7 U of EXPAND
high-fidelity
polymerase, 25 pmol of sense primer 1204 (5'GGAGGGGCGGAATACCTGGTCATAGCCTCCGTGAA),
25 pmol of
antisense primer 1203 (5'ATGGGGTTCTCGTATGATACCCGCTGCTTTGACTC),
and 1 mM each dNTP in Opti-prime PCR buffer 4 (Stratagene, La
Jolla, Calif.). The reaction mixtures were heated to 94°C for
30 s, and this was followed by 35 cycles of 94°C for 30 s, 54°C
for 40 s, and 72°C for 50 s and a final cycle of 30 s
at 72°C.
The sequence amplified is a 401-bp fragment between
positions
7903 and
8309.
Amplification of the E2/HVR-1 region was carried out in 50-µl
reaction mixtures containing 10 µl of cDNA in 5 mM MgCl
2,
1
mM each dNTP, 25 pmol of sense primer HVR-S2
(5'TACTACTCCATGGTGGGAGACTGGGC)
27, 25 pmol of
antisense primer HVR-A2 (5'GATGTGCCAGCTGCCATTGG),
and 0.7 U
of EXPAND high-fidelity polymerase. The reaction mixtures
were heated
to 94°C for 1 min, and this was followed by 30 cycles
of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. These primers
amplify a
189-bp product between positions 1082 and 1271 of the
HCV genome
4.
PCR products were cloned using the TOPO TA cloning kit (Invitrogen BV,
De Schelp, The Netherlands). Colonies with inserts
from the three
subgenomic regions were picked and amplified directly
using PCR
conditions described above. The number of cycles was
reduced to 20 for
amplification of the 5'NCR and E2/HVR-1 sequences.
A GC clamp sequence
(5'CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG)
was
incorporated in the 5' end of the forward primer sequences
(HVR-S2,
sense 126, and sense 1204) during PCR to prevent complete
denaturation
of ds DNA during DGGE
23.
DGGE analysis.
The denaturing-gradient polyacrylamide gel
was prepared as follows. Acrylamide solutions were prepared as two
stock solutions. For 5'NCR and NS5b analysis, one solution contained
30% denaturants consisting of 12% polyacrylamide (supplied as
Protogel by National Diagnostics, Hull, United Kingdom), 12%
formamide, and 2.1 M urea in 0.6× Tris-acetate-EDTA (TAE) buffer, and
the second solution contained 70% denaturants consisting of 12%
polyacrylamide, 28% formamide, and 4.9 M urea in 0.6× TAE buffer. For
analysis of the E2/HVR-1, these solutions were diluted down to 10 and
65% denaturants, respectively, using an acrylamide solution that
contained no denaturants. The gradient gel was formed by transferring,
using a gravity-driven gradient maker (GRI, Braintree, United Kingdom), 30 ml of each gel solution between the glass plates of the Ingeny Phor
U2 vertical electrophoresis apparatus (GRI). This apparatus allows two
gels to be made for each electrophoresis run. Wells in each gel were
formed with a 48-well square-toothed comb, after which the gels were
allowed to polymerize for 1 h. PCR products (3 to 5 µl of each)
were mixed with 2 to 4 µl of loading dye and applied to the gel.
Electrophoresis was carried out in 0.6× TAE buffer at 60°C and 100 V
for 16 h. Running buffer was continuously circulated to maintain a
constant temperature throughout the electrophoresis tank. The gels were
stained with SYBR Green I (Flowgen; Lichfield, Staffordshire, United
Kingdom), and bands were visualized by UV transillumination.
Sequence analysis.
Following DGGE, the migration positions
of the various PCR products were first inspected. For cloned products,
the migration position of the dominant clone was identified. The PCR
product from a representative dominant clone was then chosen for
sequencing, together with products that yielded migration positions
different from the dominant clone. PCR products were purified by
electrophoresis through 2% agarose followed by excision of the
appropriate band. DNA was recovered using the Igenie DNA extraction kit
(Immunogen, Sunderland, United Kingdom). The 5'NCR products were
sequenced with the ABI Prism DNA sequencing kit (PE Applied
Biosciences, Warrington, United Kingdom), and electrophoresis was
carried out using the ABI 373 automated sequencer. The NS5b products
were sequenced using ABI prism Big Dye terminator sequencing kit, which particularly facilitated characterization of the sequence of the entire
length of the amplicon in both the forward and reverse directions.
Sequences were aligned and compared using the CLUSTAL V algorithm in
the MEGALIGN program of the LASERGENE package (DNASTAR
Inc, Madison,
Wis.). Phylogenetic analysis was also carried out
using DNADIST and
FITCH from the PHYLIP suite of programs and
TREEVIEW to generate
dendrograms
9.
Statistical analysis.
Sequence data from the NS5b segment
formed the basis for the determination of HCV genetic diversity.
Shannon entropy (Sn) values were calculated as a measure of genetic
complexity. They incorporate the number of sequence variants, the
frequency of each variant, and the total number of variants analyzed
21
where
N = total number of sequences and
pi = frequency of each sequence. Sn values can
range from 0 (when 1 variant is present)
to 1 (where each variant
occurs once). The data were first verified
to be normal by
Shapiro-Wilks testing. Student's
t testing of
mean values
and variance ratio testing of standard deviations
were then applied.
Genetic diversity was calculated as the average
genetic distance
between species, using the CLUSTAL V algorithm.
The statistical
significance of differences in Sn and genetic
distance values between
groups was evaluated using the Kruskal-Wallis
test.
Nucleotide sequence accession numbers.
Sequences were
deposited in GenBank under accession numbers AF282631 to AF282674
and AY003921 to AY004035.
| |
RESULTS |
Optimizing DGGE running conditions using 5'NCR amplicons.
Two
patients chronically infected with HCV were used for the optimization
of the DGGE technique. A fragment of the HCV 5'NCR was amplified, by
PCR, from the sera of these patients and then cloned. PCR products
derived from the resulting colonies had a GC clamp attached and were
then analyzed by DGGE. In addition, 5'NCR PCR products from a further
eight patients were used in the optimization process. These were not
cloned. Two of the colony PCR products from one patient, CHC001,
differed by a 1-nucleotide (nt) substitution from each other and from
the dominant clones. The sequences of colony products derived from the
other patient, CHC002, were all identical. These products were then
analyzed by DGGE using a 0 to 80% denaturant gradient. Initial
electrophoresis conditions were 200 V for 6 h at 60°C. Figure
1A shows the final migratory positions of
the two sets of colony PCR products attained under these DGGE
conditions (panels a and b; the two products with different sequences
are indicated by asterisks in panel a). Thus, while these DGGE
conditions reproducibly led to uniform migration of DNA sharing the
same sequence, they failed to distinguish DNA products bearing 1-nt
changes. Panel c of Fig. 1A shows that there was little difference in
the migratory positions reached in noncloned amplicons derived from the
5'NCR of HCV carried by the eight individuals.
View larger version (71K):
[in this window]
[in a new window]
|
FIG. 1.
DGGE gel migratory positions of colony PCR products
amplified from the 5'NCR of HCV in sera of two patients with chronic
hepatitis C, CHC001 and CHC002 (panels a and b, respectively) and 5'NCR
PCR products directly amplified from eight different HCV carriers
(panel c). Sequences of two of the colony products from CHC001
(indicated by asterisks in panel a) differ by 1 nt between each other
and the dominant clone; sequences of the colony products from CHC002
are all identical. (A) Positions attained after electrophoresis through
a 0 to 80% denaturant gradient. (B) Positions attained after
electrophoresis through a 30 to 70% gradient.
|
|
Narrower gradients were empirically tested using the same panel of
colony and noncloned PCR products to determine the gradient
that would
result in the clear resolution of unique variants.
The best result was
achieved using a 30 to 70% gradient. Figure
1B shows the migratory
positions of these PCR products through
such a gradient. Cloned
sequences bearing 1-nt substitutions (panel
a) and noncloned sequences
from different HCV carriers could then
be discriminated (panel c).
Increasing the electrophoresis time
for the DGGE procedure to 16 h
(overnight) and reducing the voltage
to 100 V did not alter
discrimination (data not shown). Forty
colony 5'NCR products derived
from five hemophiliacs, five IDUs,
and five blood donors were
systematically studied using the overnight
running conditions. For each
set of clones, clones whose reamplified
DNA products resulted in DGGE
bands migrating to positions different
from each other were subjected
to DNA sequencing analyses. In
all cases, products of 5'NCR-derived
clones yielding minority
DGGE banding positions were found to carry
sequences differing
from the majority sequence (data not
shown).
The G+C content of a DNA fragment predicts the denaturing conditions
under which it will be completely retarded in the gel
matrix. We
predicted that, having a lower G+C content than the
5'NCR, both the
NS5b and E2/HVR-1 fragments would be resolved
in a lower concentration
of denaturants. This allowed us to analyze
NS5b-derived clones on the
upper half of the gel used to analyze
5'NCR-derived clones. For the
E2/HVR-1 colony products, a gel
with a denaturing gradient of 10 to
65% was used. In initial DGGE
runs, five or six of the dominant colony
products derived from
both the NS5b and E2/HVR-1 were sequenced to
confirm that minor
variants could indeed be detected. In later runs,
two or three
dominant colony products were
sequenced.
DGGE and sequence analysis of NS5b-amplified clones from
individuals belonging to different risk groups.
Intrahost NS5b
variation in the hemophilia patients, IDUs, and blood donors (five from
each group) was then studied using DGGE conditions optimized for the
analysis of 5'NCR clones. As with the study of 5'NCR variation, every
product of each individual screened by DGGE to yield a banding position
different from the majority position was processed for DNA sequencing.
Figure 2 illustrates representative DGGE bands obtained and the sequence changes that accounted for the different positions in clones from a blood donor (BD259) (Fig. 2A) and a hemophilia patient (H858) (Fig. 2B). PCR products whose sequences differed by as little as 1 nt
regularly migrated to different positions. Figure
3 displays dendrograms illustrating NS5b sequence diversity in individuals from the three study groups. Sequences from each individual tended to cluster tightly,
segregated away from clusters of sequences from other individuals in
the same genotype. This tight clustering is the result of nucleotide
substitutions occurring at one to three sites along different positions
of the NS5b amplicon in the minority variants (the complete sequence
data are in GenBank). Only in two individuals (BD268 and BD424) was a
single substitution observed at the same site in a small proportion of
minority variants (BD268 III and V, and BD424 V and VII); such an
occurrence leads to positioning of these minority variants away from
the main cluster (Fig. 3A).
View larger version (149K):
[in this window]
[in a new window]
|
FIG. 2.
The upper panels show DGGE gel migratory positions of
colony PCR products amplified from the HCV NS5b subgenomic region from
serum of blood donor BD259 (A) and hemophilia patient H858 (B). The
lower panels show a comparison of nucleotide sequences characterized
after unique sequences were screened by the DGGE procedure. Colony
products with unique sequences are assigned different Roman numerals at
the top of the upper panels and to the left of the lower panels.
Numbers in parentheses in the lower panels denote the number of clones
with the unique sequences.
|
|
View larger version (65K):
[in this window]
[in a new window]
|
FIG. 3.
Unrooted tree showing the diversity of NS5b sequences
cloned from serum blood donors (A), IDUs (B), and hemophilia patients
(C). Sequences from study individuals are shown in plain type; Roman
numerals in parentheses denote unique sequences found within each
specimen. Representative sequences obtained from the GenBank database
are shown in bold type. The number and line at the bottom denote the
proportion of nucleotides substituted for a given horizontal branch
length. Dendrograms were produced using DNADIST, FITCH, and TREEVIEW in
the PHYLIP suite of programs.
|
|
Table
1 summarizes the entropy and
genetic diversity values derived from NS5b sequences. Although the
median Sn and GD values
in the blood donor group are higher than those
in the other two
groups, the differences between the groups were not
significant.
Comparison of the variant NS5b sequences identified from
each
sample with sequences deposited in GenBank showed five of the
individuals to be infected by type 1a, six to be infected by 1b,
one to
be infected by 2a, one to be infected by 2b, and two to
be infected by
3a. None were infected with more than one genotype
or subtype.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Comparison of median values and ranges for HCV NS5b
entropy and genetic distance in three at-risk groups
|
|
Analysis of changes in the E2/HVR-1 region during HCV
seroconversion.
DGGE analysis allowed the changes in E2/HVR-1
sequences over a short period in two seroconverting individuals, SC1
and SC2, to be evaluated rapidly. Figure
4 shows the distribution
of E2/HVR-1 sequences from each patient at six time points, and Fig.
5 documents the base
changes that led to variation in DGGE banding positions.
View larger version (73K):
[in this window]
[in a new window]
|
FIG. 4.
(A) DGGE profiles of colony PCR products amplified from
the HCV E2/HVR-1 subgenomic region obtained from serially collected
sera from acutely infected patient SC1. (B) Comparison of E2/HVR-1
unique sequences from SC1. Unique sequences and their corresponding
DGGE profiles are assigned different Roman numerals. Dots represent
sequence identity, and dashes represent deletions.
|
|
View larger version (77K):
[in this window]
[in a new window]
|
FIG. 5.
(A) DGGE profiles of colony PCR products amplified from
the HCV E2/HVR-1 subgenomic region obtained from serially collected
sera from acutely infected patient SC2. (B) Comparison of E2/HVR-1
unique sequences from SC2. Dots represent sequence identity, and dashes
represent deletions.
|
|
For SC1, DGGE showed that a single variant (I) was exclusively carried
by the first sample (day 0) (Fig.
4A). Over the seroconversion
period,
variant I remained dominant, while most of the minority
variants (II to
X, XI, and XII) appeared only once and variant
X appeared twice (both
on day 43). Sequence analysis revealed
point deletions in variants III
and VII and longer deletions in
variants V and XI (Fig.
4B). All these
deletions predict the translation
of truncated envelope protein
products (data in GenBank). In addition,
point substitutions were found
in variants III and VII and in
variants without any deletions (II, IV,
VI, VIII, IX, and XII)
(Fig.
4B). Substitutions in variants VIII, IX,
and XII were synonymous,
with the amino acid sequence specified being
identical to that
specified by variant I while substitutions in
variants II, IV,
and X were nonsynonymous (data in GenBank). The small
number of
nucleotide substitutions precluded analysis of variation in
synonymous/nonsynonymous
and transition/transversion ratios over
time.
E2/HVR-1 entropy values obtained for the samples from SC1 at each time
point were low, ranging from 0 (day 0) to 0.153 (day
43). No trends
were observed as entropy values changed with the
changes in circulating
viral load, serum alanine aminotransferase
(ALT) level, and anti-HCV
level (Fig.
6A).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 6.
Changes in HCV viral load, ALT level, anti-HCV antibody
level, and HCV E2/HVR-1 sequence entropy (Sn) in patients SC1 (A) and
SC2 (B). Viral load and antibody level data were as provided by the
distributor of the study samples. OD, optical density.
|
|
In contrast to SC1, DGGE showed the presence of three variants (I to
III) in the first sample from SC2 (Fig.
5A). Sequence
analysis of the
clones showed that while the minority variants
II and III differed by 1 nt, both these variants were substantially
different from variant I
(Fig.
5B). Over the seroconversion period,
variant I remained dominant,
and minority variants other than
II and III appeared (Fig.
4B). Of the
minority variants, only
two (variants II and XIII) re-emerged: variant
II on days 23,
49 and 56 and variant XIII on days 32, 49 and 56. The
rest of
the minority variants appeared only once. Figure
5B also shows
that the various sequences may be segregated into three groups
according to nucleotide motifs: variants II and III, those clustering
with variant I (variants IV, V, VI, VII, X, XI, XII, XV, and XVI),
and
those clustering with variant XIII (variants VIII, IX, XIV,
and XVII).
Phylogenetic analysis confirmed the grouping of variant
sequences to
the three clusters (data not shown). The deletion
variants (V, X, and
XI), all mutants of variant I, also specify
the translation of
truncated products (data in
GenBank).
Despite the presence of clusters, little variation in diversity was
seen in SC2 over the seroconversion period, with the entropy
values
varying from 0.03 (day 10) to 0.188 (day 56). There was
also no trend
in the entropy changes as the viral load and ALT
and anti-HCV levels
changed (Fig.
6B).
| |
DISCUSSION |
This study, using PCR clones derived from three regions of the HCV
genome 5'NCR, NS5b, and E2/HVR-1, which possessed different degrees of
nucleotide sequence variability, shows that the DGGE approach is
applicable to the investigation of HCV genetic diversity at the
intrahost level. Sequence differences in a relatively large number of
clones from a subgenomic fragment (up to 48 per gel, two gels per run)
can be screened during a single procedure. No radioisotopes are needed.
Clones yielding differing gel migratory positions are rapidly
identified, to be processed further for nucleotide sequencing analyses
if required. We regularly observed that single-nucleotide variations in
the amplicons led to differences in the migratory positions reached.
Convenient overnight runs were made possible by carrying out
electrophoresis at reduced voltage. A long electrophoresis run can be
tolerated because once GC-clamped DNA fragments travel to positions in
the gel gradient where all but the clamp region has denatured, they do
not migrate further through the gel matrix.
The assessment of quasispecies diversity, as opposed to complexity,
requires sequencing procedures to be carried out. These are
particularly necessary to define sequence changes in the minority variants. The diversity and complexity of the NS5b region characterized from blood samples from hemophiliacs, IDUs, and blood donors were evaluated using DGGE combined with sequencing of PCR clones derived from this region. Entropy (a measure of complexity) and mean genetic distance (a measure of diversity) are both indices of how rapidly HCV
mutates and of the extent of multiple HCV carriage. We found that mean
values of the two measures were not significantly different between
groups. Assuming that the NS5b subgenomic region mutates at about the
same rate among individuals in the three groups, the lack of
significance in the mean entropy and genetic distance values suggests
equal likelihood of carrying multiple HCV variants. That blood donors
should have the same extent of sequence diversity as hemophiliacs and
IDUs is surprising, considering that they are thought to be less
exposed to multiple HCV transmission events. It may be that the
multiple HCV transmission events hypothesized to occur in IDUs are, for
various reasons, not associated with an increase in genetic complexity.
In addition to this, the epidemiology of HCV in blood donors is poorly
understood 5. Seropositive blood donors may thus be as
susceptible to HCV multiple transmission as are IDUs through a variety
of unknown routes. A recent study has identified intravenous drug use
in a substantial proportion of British anti-HCV-positive blood donors
19.
That HCV genetic complexity and diversity in hemophiliacs do not differ
significantly from those in the other two groups is also unexpected.
HCV-infected hemophilia patients are exposed to HCV from having
undergone therapy with clotting-factor concentrates prepared from blood
donors who were not yet screened for anti-HCV. Before routine screening
of blood donors for anti-HCV came into effect, hemophiliacs had been
particularly prone to multiple HCV transmissions. This arose not only
because of the frequency of clotting-factor infusions but also because
each concentrate was derived from plasma fractions that originated from
very large pools of blood donors 24. Despite the
vulnerability of hemophiliacs to infection by multiple HCV variants, we
found no evidence of mixed HCV genotype infection, neither did we find
higher NS5b entropy or genetic distance values in this group compared
to the IDU and blood donor groups. Moreover, sequencing of NS5b clones derived from the hemophilia patients shows that in all five patients, variation in sequences of the minority variants involved
single-nucleotide substitutions from the majority variant, accounting
for the tight clustering of sequences seen in Fig. 4C. These data are
consistent with quasispecies evolution from a single HCV founder strain
and again point to the rarity of multiple HCV carriage in hemophiliacs. Such an occurrence may be due to resistance of the already infected host to HCV reinfection, which may in turn be due to failure of the
newly transmitted strains to supplant the strain that had persisted
after having first established infection. However, it is possible that
mixed infections are in fact common but that one subtype prevails and
the other becomes undetectable. It would be feasible to apply the DGGE
procedure to larger numbers of blood specimens to confirm the absence
or paucity of multiple infection in hemophilia patients. This
confirmation would imply that vaccination programs employing live,
attenuated HCV vaccines might be more effective in preventing
primary HCV infections than inactivated, subunit, or epitope
vaccines would be.
HCV quasispecies evolution in the pre- and early seroconversion stage
of HCV infection has been studied, and the role of immune selection is
being clarified 8, 15. We note that in one of the study
patients (SC1), HCV viremia had peaked prior to the rise in ALT and
antiviral antibody levels, while in the other (SC2), viremia was
already declining at the time of the rise in ALT and antibody levels
(Fig. 6). Between our two study patients, there were also differences
in HCV complexity and diversity in the circulation before and after the
appearance of antibody. In SC1, there was no variation in the E2/HVR-1
region in the earliest samples, and the variants that subsequently
emerged possessed changes (deletions and substitutions) suggesting that
these and the dominant variant belong to one monophyletic group. By
contrast, the HCV infecting SC2 belonged to at least three monophyletic groups. Such a disparity in the complexity of HCV in specimens at the
acute stage of infection has been noted in a previous study of three
patients with acute hepatitis C 15. Variation in HCV complexity and diversity in early specimens may be due to the absence
(as exemplified in SC1) or presence (as exemplified in SC2) of multiple
strains in the inoculum, to differences in the way hepatocytes permit
the replication of particular strains 22, or to whether
effective humoral or cellular responses have been triggered
8.
Investigation of the two seroconverting patients by the combined
DGGE-sequencing protocol also revealed that despite the difference in
the patients with regard to carriage of single or multiple strains,
there was no substantial difference in their rates of diversification.
The highest E2/HVR-1 entropy values reached in each patient did not
even attain the highest values of the relatively conserved NS5b region
of the 15 individuals studied earlier (Table 1). There was also no
observable trend to the changes in entropy as antibody levels increased
(Fig. 6). Furthermore, for SC1 and SC2, nucleotide substitutions within
HVR-1 appeared as frequently as those outside it (Fig. 4B and 5B).
Overall, the E2/HVR-1 findings agree with observations on naive
chimpanzees experimentally infected with HCV, in which a paucity of mutations in HVR-1 during the acute phase of infection
has also been noted 28. They also agree with data from a
recent study of human patients undergoing acute HCV infection, in
whom the diversity of and number of sequence variants in HVR-1 between the first PCR-positive and the pre-seroconversion blood
specimens were not substantially changed 8.
In SC1 and SC2, the low frequency of the E2/HVR-1 mutations and the
poor correlation of these mutations with anti-HCV changes suggest that
at the early stage of HCV infection, mutations arise stochastically and
are not antibody mediated. It is noted that various reports of
mutations in HVR-1 which ascribe them to neutralizing pressure from
anti-HCV 29, 30 relate only to studies involving the use
of antibody preparations from blood collected from patients with
chronic, not acute, hepatitis C. However, a study of HVR variation in
acutely infected patients showed that in some individuals E2/HVR-1
changes may be immune mediated 8. Such individuals tended
to progress to chronic infection. The paucity of E2/HVR-1 changes in
early infection tended to be associated with resolving infection; SC1
and SC2 may thus belong to the latter category.
In summary, DGGE facilitates rapid and reliable assessment of HCV
quasispecies diversity. Combined with nucleotide-sequencing procedures,
it provided evidence that HCV genetic complexity and diversity in blood
donors and IDUs did not differ significantly from those in hemophilia
patients. This observation led us to hypothesize that multiple HCV
infection is uncommon because persistent infection by one HCV strain
prevents the establishment of infection by subsequently introduced
strains. We also found that few mutations emerged during the
seroconversion period of HCV infection of the two patients studied,
suggesting that genetic drifts occurring at this stage of infection are
not immune-mediated.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Microbiology, Great Ormond Street Hospital for Sick Children NHS Trust, Great Ormond St., London WC1N 3JH, United Kingdom.
| |
REFERENCES |
| 1.
|
Behrens, S. E.,
L. Tomei, and R. De Francesco.
1996.
Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus.
EMBO J.
15:12-22[Medline].
|
| 2.
|
Bukh, J.,
R. H. Miller, and R. H. Purcell.
1995.
Genetic heterogeneity of hepatitis C virus: quasispecies and genotypes.
Semin. Liver Dis.
15:41-63[Medline].
|
| 3.
|
Bukh, J.,
R. H. Purcell, and R. H. Miller.
1992.
Sequence analysis of the 5' noncoding region of hepatitis C virus.
Proc. Natl. Acad. Sci. USA
89:4942-4946[Abstract/Free Full Text].
|
| 4.
|
Choo, Q. L.,
K. H. Richman,
J. H. Han,
K. Berger,
C. Lee,
C. Dong,
C. Gallegos,
D. Coit,
R. Medina-Selby, and P. J. Barr.
1991.
Genetic organization and diversity of the hepatitis C virus.
Proc. Natl. Acad. Sci. USA
88:2451-2455[Abstract/Free Full Text].
|
| 5.
|
Dike, A. E.,
J. M. Christie,
J. B. Kurtz, and C. G. Teo.
1998.
Hepatitis C in blood transfusion recipients identified at the Oxford Blood Centre in the national HCV look-back programme.
Transfus. Med.
8:87-95[CrossRef][Medline].
|
| 6.
|
Domingo, E.,
E. Martinez-Salas,
F. Sobrino,
J. C. de la Torre,
A. Portela,
J. Ortin,
C. Lopez-Galindez,
P. Perez-Brena,
N. Villanueva,
R. Najera, et al.
1985.
The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance a review.
Gene
40:1-8[CrossRef][Medline].
|
| 7.
|
Farci, P.,
J. Bukh, and R. H. Purcell.
1997.
The quasispecies of hepatitis C virus and the host immune response.
Springer Semin. Immunopathol.
19:5-26[CrossRef][Medline].
|
| 8.
|
Farci, P.,
A. Shimoda,
A. Coiana,
G. Diaz,
G. Peddis,
J. C. Melpolder,
A. Strazzera,
D. Y. Chien,
S. J. Munoz,
A. Balestrieri,
R. H. Purcell, and H. J. Alter.
2000.
The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.
Science
288:339-344[Abstract/Free Full Text].
|
| 9.
|
Felsenstein, J.
1993.
PHYLIP (Phylogeny Inference Package 3.5c).
Department of Genetics, University of Washington, Seattle.
|
| 10.
|
Fodde, R., and M. Losekoot.
1994.
Mutation detection by denaturing gradient gel electrophoresis (DGGE).
Hum. Mutat.
3:83-94[CrossRef][Medline].
|
| 11.
|
Lee, J. H.,
T. Stripf,
W. K. Roth, and S. Zeuzem.
1997.
Non-isotopic detection of hepatitis C virus quasispecies by single strand conformation polymorphism.
J. Med. Virol.
53:245-251[CrossRef][Medline].
|
| 12.
|
Lin, H. J.,
N. Shi,
M. Mizokami, and F. B. Hollinger.
1992.
Polymerase chain reaction assay for hepatitis C virus RNA using a single tube for reverse transcription and serial rounds of amplification with nested primer pairs.
J. Med. Virol.
38:220-225[Medline].
|
| 13.
|
Lohmann, V.,
F. Korner,
U. Herian, and R. Bartenschlager.
1997.
Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity.
J. Virol.
71:8416-8428[Abstract].
|
| 14.
|
Lohmann, V.,
A. Roos,
F. Korner,
J. O. Koch, and R. Bartenschlager.
1998.
Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymerase of the hepatitis C virus.
Virology
249:108-118[CrossRef][Medline].
|
| 15.
|
Manzin, A.,
L. Solforosi,
E. Petrelli,
G. Macarri,
G. Tosone,
M. Piazza, and M. Clementi.
1998.
Evolution of hypervariable region 1 of hepatitis C virus in primary infection.
J. Virol.
72:6271-6276[Abstract/Free Full Text].
|
| 16.
|
Martell, M.,
J. I. Esteban,
J. Quer,
J. Genesca,
A. Weiner,
R. Esteban,
J. Guardia, and J. Gomez.
1992.
Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution.
J. Virol.
66:3225-3229[Abstract/Free Full Text].
|
| 17.
|
McAllister, J.,
C. Casino,
F. Davidson,
J. Power,
E. Lawlor,
P. L. Yap,
P. Simmonds, and D. B. Smith.
1998.
Long-term evolution of the hypervariable region of hepatitis C virus in a common-source-infected cohort.
J. Virol.
72:4893-4905[Abstract/Free Full Text].
|
| 18.
|
Moribe, T.,
N. Hayashi,
Y. Kanazawa,
E. Mita,
H. Fusamoto,
M. Negi,
T. Kaneshige,
H. Igimi,
T. Kamada, and K. Uchida.
1995.
Hepatitis C viral complexity detected by single-strand conformation polymorphism and response to interferon therapy.
Gastroenterology
108:789-795[CrossRef][Medline].
|
| 19.
|
Murphy, E. L.,
S. M. Bryzman,
S. A. Glynn,
D. I. Ameti,
R. A. Thomson,
A. E. Williams,
C. C. Nass,
H. E. Ownby,
G. B. Schreiber,
F. Kong,
K. R. Neal, and G. J. Nemo.
2000.
Risk factors for hepatitis C virus infection in United States blood donors. NHLBI Retrovirus Epidemiology Donor Study (REDS).
Hepatology
31:756-762[CrossRef][Medline].
|
| 20.
|
Ni, Y. H.,
M. H. Chang,
P. J. Chen,
H. H. Lin, and H. Y. Hsu.
1997.
Evolution of hepatitis C virus quasispecies in mothers and infants infected through mother-to-infant transmission.
J. Hepatol.
26:967-974[CrossRef][Medline].
|
| 21.
|
Pawlotsky, J. M.,
G. Germanidis,
A. U. Neumann,
M. Pellerin,
P. O. Frainais, and D. Dhumeaux.
1998.
Interferon resistance of hepatitis C virus genotype 1b: relationship to nonstructural 5A gene quasispecies mutations.
J. Virol.
72:2795-2805[Abstract/Free Full Text].
|
| 22.
|
Rumin, S.,
P. Berthillon,
E. Tanaka,
K. Kiyosawa,
M. A. Trabaud,
T. Bizollon,
C. Gouillat,
P. Gripon,
C. Guguen-Guillouzo,
G. Inchauspe, and C. Trepo.
1999.
Dynamic analysis of hepatitis C virus replication and quasispecies selection in long-term cultures of adult human hepatocytes infected in vitro.
J. Gen. Virol.
80:3007-3018[Abstract/Free Full Text].
|
| 23.
|
Sheffield, V. C.,
D. R. Cox,
L. S. Lerman, and R. M. Myers.
1989.
Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes.
Proc. Natl. Acad. Sci. USA
86:232-236[Abstract/Free Full Text].
|
| 24.
|
Simmonds, P.,
L. Q. Zhang,
H. G. Watson,
S. Rebus,
E. D. Ferguson,
P. Balfe,
G. H. Leadbetter,
P. L. Yap,
J. F. Peutherer, and C. A. Ludlam.
1990.
Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users.
Lancet
336:1469-1472[CrossRef][Medline].
|
| 25.
|
Smith, D. B., and P. Simmonds.
1997.
Review: molecular epidemiology of hepatitis C virus.
J. Gastroenterol. Hepatol.
12:522-527[Medline].
|
| 26.
|
Sullivan, D. G.,
J. J. Wilson,
R. L. J. Carithers,
J. D. Perkins, and D. R. Gretch.
1998.
Multigene tracking of hepatitis C virus quasispecies after liver transplantation: correlation of genetic diversification in the envelope region with asymptomatic or mild disease patterns.
J. Virol.
72:10036-10043[Abstract/Free Full Text].
|
| 27.
|
Toyoda, H.,
Y. Fukuda,
I. Nakano,
Y. Katano,
T. Takayama,
T. Kumada,
Nakano,
J. Takamatsu,
H. Saito, and T. Hayakawa.
1998.
Quasispecies nature of hepatitis C virus (HCV) in patients with chronic hepatitis C with mixed HCV subtypes.
J. Med. Virol.
54:80-85[CrossRef][Medline].
|
| 28.
|
van Doorn, L. J.,
I. Capriles,
G. Maertens,
R. DeLeys,
K. Murray,
T. Kos,
H. Schellekens, and W. Quint.
1995.
Sequence evolution of the hypervariable region in the putative envelope region E2/NS1 of hepatitis C virus is correlated with specific humoral immune responses.
J. Virol.
69:773-778[Abstract].
|
| 29.
|
Weiner, A. J.,
H. M. Geysen,
C. Christopherson,
J. E. Hall,
T. J. Mason,
G. Saracco,
F. Bonino,
K. Crawford,
C. D. Marion, and K. A. Crawford.
1992.
Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections.
Proc. Natl. Acad. Sci. USA
89:3468-3472[Abstract/Free Full Text].
|
| 30.
|
Zibert, A.,
E. Schreier, and M. Roggendorf.
1995.
Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment.
Virology
208:653-661[CrossRef][Medline].
|
Clinical and Diagnostic Laboratory Immunology, January 2001, p. 62-73, Vol. 8, No. 1
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.1.62-73.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Top
Abstract
Introduction
![<UP>Sn = </UP>[<UP>&Sgr;</UP>i(p<SUB>i</SUB><UP> ln </UP>p<SUB>i</SUB>]/<UP>ln </UP>N](/content/vol8/issue1/fulltext/62/img001.gif)
