Download Variation of hepatitis C virus following serial transmission: multiple

Journal
of General Virology (1999), 80, 717–725. Printed in Great Britain
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Variation of hepatitis C virus following serial transmission :
multiple mechanisms of diversification of the hypervariable
region and evidence for convergent genome evolution
Carmela Casino,1 Jane McAllister,1 Fiona Davidson,2 Joan Power,3 Emer Lawlor,3 Peng Lee Yap,2
Peter Simmonds1 and Donald B. Smith1
1
Department of Medical Microbiology, University of Edinburgh, Medical School, Teviot Place, Edinburgh EH8 9AG, UK
Edinburgh and South East Scotland Blood Transfusion Service, Lauriston Place, Edinburgh EH3 9HB, UK
3
Irish Blood Transfusion Board, St Finbarr’s Hospital, Cork and Pelican House, Dublin, Ireland
2
We have studied the evolution of hepatitis C virus (HCV) from a common source following serial
transmission from contaminated batches of anti-D immunoglobulin. Six secondary recipients were
each infected with virus from identifiable primary recipients of HCV-contaminated anti-D
immunoglobulin. Phylogenetic analysis of virus E1/E2 gene sequences [including the hypervariable
region (HVR)] and part of NS5B confirmed their common origin, but failed to reproduce the known
epidemiological relationships between pairs of viruses, probably because of the frequent
occurrence of convergent substitutions at both synonymous and nonsynonymous sites. There was
no evidence that the rate at which the HCV genome evolves is affected by transmission events.
Three different mechanisms appear to have been involved in generating variation of the
hypervariable region ; nucleotide substitution, insertion/deletion of nucleotide triplets at the
E1/E2 boundary and insertion of a duplicated segment replacing almost the entire HVR. These
observations have important implications for the phylogenetic analysis of HCV sequences from
epidemiologically linked isolates.
Introduction
The ability of hepatitis C virus to establish a persistent
infection following transmission to a new host is generally
thought to be related to continued evolution of the virus
envelope glycoproteins E1 and E2. Attention has focused
particularly on a 25–30 amino acid hypervariable region (HVR)
at the NH terminus of the E2 protein that induces the
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production of specific antibodies that may be neutralizing in
vivo (Farci et al., 1996) and may be capable of blocking virus
attachment to cells in vitro (Zibert et al., 1995). Previous work
has identified both invariant and variable positions within the
HVR ; amino acid replacements at some variable sites are
limited to a small number of residues with similar chemical
Author for correspondence : Donald Smith.
Fax j44 131 650 6531. e-mail Donald.B.Smith!ed.ac.uk
The GenBank accession numbers for the nucleotide sequences
reported here are AF119717–AF119768, AF056764–AF056785 and
AF056795–AF056812.
0001-5972 # 1999 SGM
properties, while at other sites almost every residue can occur
(Sekiya et al., 1994 ; Sherman et al., 1996 ; McAllister et al.,
1998). Detailed comparisons of the HVR sequences in different
individuals infected from a common infectious source has
provided evidence for the operation of selection against amino
acid substitution at some positions, and positive selection for
amino acid replacement at others (McAllister et al., 1998).
However, most previous studies of the evolution of the
HVR have been confined to longitudinal studies of acutely or
persistently infected individuals, or in a few cases to individuals
infected from an identifiable source (Weiner et al., 1993 ; Hohne
et al., 1994 ; Esteban et al., 1996). To date, there is no
information about the effect of multiple transmission events on
virus evolution in humans, and it is possible that very different
patterns of variation might be observed under these circumstances. For example, continued selection of antigenically
distinct variants of the HVR might drive the virus population
away from a fitness optimum, and subsequent transmission to
a new host might result in selection for less divergent variants,
as observed previously for human immunodeficiency virus
(Zhang et al., 1993) and during experimental transmission of
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C. Casino and others
HCV in chimpanzees (Kojima et al., 1994). Transmission of
virus to an immunologically naı$ ve host might also result in a
slowing in the rate of virus evolution if immunological
selection is a major force in driving virus evolution. These
types of effect have implications for the study of the rate of
virus evolution, the extrapolation of short-term rates of
sequence change as a way of reconstructing the history of virus
infections, and in the interpretation of phylogenetic relationships between viruses in different infected individuals where
transmission is suspected.
We have investigated these questions by studying virus
E1\E2 sequences from six HCV-infected individuals for whom
the source of infection could be traced to a different individual
each of whom had received anti-D immunoglobulin in Ireland
in 1977. One of the donors to the plasma pools used to
manufacture batches of anti-D used in 1977 was acutely
infected with HCV genotype 1b (Power et al., 1994).
Retrospective studies have established that batches of this antiD were PCR-positive and infectious (Lawlor et al., 1999), and
that virus from different anti-D recipients was of genotype 1b
and closely related to that present in archived batches of antiD immunoglobulin and the single implicated donor (Power et
al., 1995 ; Smith et al., 1997 b ; McAllister et al., 1998). By
studying virus sequences in secondary recipients of these
HCV-infected anti-D recipients we have therefore been able to
investigate the effect of virus transmission on the rate and
characteristics of virus evolution.
Methods
Sera. Plasma and serum samples were obtained in 1994 from HCVinfected individuals identified through targeted lookback by the Irish
Blood Transfusion Service Board as having received blood from or being
a member of the family of a woman who had received an infectious batch
of anti-D immunoglobulin in 1977. All individuals were PCR-positive for
the virus 5h non-coding region (5hNCR) and were infected with HCV
subtype 1b as assessed by RFLP analysis of the 5hNCR (Davidson et al.,
1995) and\or by phylogenetic analysis of the E1 and NS5B regions
(Smith et al., 1997 b).
PCR. Virus RNA was extracted from 0n1 ml of plasma or serum by
incubation with proteinase K–polyadenylic acid–SDS as reported
previously (Jarvis et al., 1994). Synthesis of cDNA was carried out using
5 µl extracted RNA and 10 units of avian myeloblastosis virus reverse
transcriptase (Promega) in 20 µl buffer containing 50 mM Tris–HCl
pH 8n0, 5 mM MgCl , 5 mM DTT, 50 mM KCl, 0n05 µg\µl BSA, 15 %
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DMSO, 600 µM each of dGTP, dATP, dTTP and dCTP, 1n5 µM primer
and 10 units RNasin (Promega). A fragment containing the COOH
terminus of E1 and an NH terminal region of E2 was amplified using the
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primers 2174 (5h TTCATCCAYGTRCASCCRAACCA 3h, antisense,
positions 1986–2006 numbered from the AUG initiation codon) and
2173 (5h CAYCGNATGGCNTGGGAYATGATG 3h, sense, positions
1287–1310) for the first round of PCR, and primers 8914 (5h
CGGGATCCGGGTGCTCACTGGGGAGTCCTGGCGGGC 3h, sense,
positions 1389–1415 incorporating a BamHI site) and 2070 (5h
GGAATTCGTGAARCARTACACYGGRCCRCANAC 3h, antisense,
positions 1845–1870 incorporating an EcoRI site) for the second round of
HBI
PCR. Optimized conditions for PCR amplification of the HVR utilized
5 µl cDNA and 30 cycles, each consisting of 0n6 min at 94 mC, 0n7 min at
50 mC, 1n5 min at 72 mC and a final extension period of 6n5 min at 72 mC.
Reactions were carried out with 0n4 units Taq DNA polymerase
(Promega) in 50 µl buffer containing 50 mM KCl, 10 mM Tris–HCl
pH 9n0, Triton X-100, 1n5 mM MgCl , 30 µM each of dGTP, dATP,
#
dTTP, dCTP and 0n25 µM of each of the outer primers. For the second
round of PCR, 1 µl of PCR product was transferred to a fresh tube
containing 100 µl reaction mix and subjected to 30 cycles of PCR as
before.
Sequence analysis. PCR products and the plasmid pUC18 were
cleaved with both BamHI and EcoRI (Promega) and purified from 0n8 %
low melting point agarose (Gibco BRL). Ligations were performed using
50 ng vector, 300 ng of PCR insert and 5 units of T4 DNA ligase
(Promega), and were incubated at room temperature overnight. Ligation
reactions were transformed into competent TG1 cells and plated on Luria
agar plates containing 200 µg\ml ampicillin, 10 µg\ml X-Gal and 200 µM
IPTG. Plasmid DNA was extracted from transformants producing white
colonies by alkali denaturation and sequenced by standard methods using
the Sequenase 2.0 enzyme (Amersham) and the primers DBS6 (5h
CACTGGGGAGTCCTGGCGGGC 3h, positions 1395–1415), S6645 (5h
TGCCARCTNCCRTTGGTRTT 3h, positions 1583–1603) and pUC
reverse primer (5h CAGGAAACAGCTATGAC 3h). Nucleotide
sequences were entered, aligned and checked using Simmonic 2000 (P.
Simmonds).
Phylogenetic analysis. Phylogenetic analysis was carried out
using the Molecular Evolutionary Phylogenetic Analysis (MEGA)
version 1.02 package (Kumar et al., 1993). Evolutionary distances were
calculated using the Kimura 2-parameter method (all sites) or the
Jukes–Cantor correction (synonymous sites). Mean distances between
groups of sequences were computed from distance matrices using the
program Dst strp (P. Simmonds).
Results
Study group
Six different HCV-infected individuals (SR8, SR7, SR11,
SR14, SR13 and SR6) were identified whose likely source of
infection was from an individual (R1, R5, R10, R12, R15 and
R21 respectively) who had received an infectious batch of antiD immunoglobulin in 1977 (Table 1). Three of the secondary
recipients (SR6, SR7 and SR8) were infected following blood
transfusion of blood from individuals subsequently identified
as HCV-infected recipients of 1977 anti-D immunoglobulin.
Two secondary recipients (SR11 and SR14) are children of
HCV-infected recipients of 1977 anti-D who may have been
infected before or shortly after birth. Some studies of vertical
transmission of HCV have first detected HCV RNA 2–6
months after birth (Ohto et al., 1994 ; Zanetti et al., 1995), but
there is also evidence that transmission can occur before birth
(Weiner et al., 1993). Alternatively, it is possible that these
transmissions occurred through parenteral exposure later in
life. The remaining secondary recipient (SR13) is a relative of
an anti-D recipient, and the timing and route of transmission
are unknown. Serum samples obtained in 1994 were analysed
for each of these individuals.
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Table 1. Primary and secondary recipients of anti-D immunoglobulin
Secondary
recipient
Age in
1994
Sex
Anti-D
recipient
SR7
SR8
SR11
SR13
SR14
82
70
11
42
17
M
F
M
F
F
R5
R1
R10
R15
R12
SR6
42
F
R21
Year of
anti-D
exposure
1977
1977
1977
1977
1977,
1978
1977
Fig. 1. Evolutionary distances between epidemiologically linked pairs of
sequences. The mean Jukes–Cantor distance between virus E1/E2
sequences from secondary recipients and their respective implicated
primary anti-D recipients (A) and between sequences from different
primary anti-D recipients (B) are plotted on the y-axis.
Convergent evolution of the E1/E2 genes
Phylogenetic analysis of virus E1\E2 sequences revealed
that all six secondary recipients were infected with an HCV
of subtype 1b virus which was closely related to the virus
present in infectious batches of anti-D immunoglobulin
manufactured in 1977 and in primary recipients of these
batches. However, a surprising finding was that virus sequences
in secondary recipients were no more closely related to virus
sequences from the anti-D recipient implicated as their source
of infection than were two different recipients of anti-D
immunoglobulin (Fig. 1). This finding is unexpected since virus
sequences present in the infectious batch were relatively
homogeneous (Smith et al., 1997 b ; McAllister et al., 1998) and
so virus from a secondary recipient and an implicated primary
recipient should share a more recent common ancestor than
that shared by different primary recipients. Although the pair
R5 and SR7 had a relatively low pairwise distance, consistent
with the relatively recent time of transmission (1988), the pair
R1 and SR8 with the most extreme pairwise distance had
Means of
transmission
Transfusion
Transfusion
Vertical?
Horizontal?
Vertical?
Time of
transmission
1988
1988
1984?
1977
1978?
Transfusion
1984
exactly the same time of transmission. Similarly, except for the
pair R5 and SR7, phylogenetic analysis of sequences revealed
no evidence for groupings within the anti-D recipient cohort
between secondary recipients and their respective implicated
anti-D recipients (data not shown). This was true even if the
HVR was excluded from the analysis, or if comparisons were
limited to nonsynonymous or synonymous sites (Fig. 2).
This lack of grouping of epidemiologically linked sequences
may be due to the frequent occurrence of convergent
substitutions within the region studied. Within the 18 amino
acid COOH-terminal region of E1 studied here there were four
polymorphic sites where the same substitution was observed
in different primary or secondary anti-D recipients. However,
each of these substitutions sometimes occurred in a secondary
recipient without being observed in the corresponding implicated primary anti-D recipient. Another three substitutions
occurred in a single sequence from the primary and secondary
recipient pairs described here, but occurred amongst published
subtype 1b sequences, again consistent with convergent
substitution. Finally, four substitutions were unique to the
sequences reported here and may represent artefacts introduced during PCR amplification since this number of sporadic
amino acid substitutions would be expected amongst cloned
PCR products if the error rate of Taq DNA polymerase was
2i10−& per nucleotide copied, at the lower end of published
estimates (Smith et al., 1997 a).
There was also evidence for convergent evolution in the
region of E2 following the HVR, since the majority of
substitutions that segregated within an infected individual or
that differed between an anti-D recipient and their associated
secondary recipient also occurred amongst other anti-D
recipients or amongst epidemiologically unrelated subtype 1b
sequences. Of the 27 positions within this region that were
variable amongst unrelated subtype 1b sequences, 23 were
also polymorphic amongst the anti-D cohort, usually with the
same amino acid replacements. Fifteen of these positions were
discordant between the clones of a secondary recipient and
those of the implicated primary anti-D recipient, but there was
no consistent association between these polymorphisms in
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Fig. 2. For legend see facing page.
HCA
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HCV hypervariable region evolution
different recipients. None of these positions were polymorphic
amongst 24 clones derived from virus isolated from archived
batches of 1977 anti-D immunoglobulin (McAllister et al.,
1998).
Surprisingly, there was also evidence that many synonymous substitutions were convergent. In the region of
E1\E2 flanking the HVR, six synonymous substitutions were
confined to a particular anti-D recipient\secondary recipient
pair, but at 51 positions the same synonymous substitution
was observed in different anti-D recipients. Several of these
substitutions occurred in virus sequences from a secondary
recipient and one or more primary anti-D recipients, but not in
virus from their implicated primary anti-D recipient. This
segregation of substitutions is unlikely to be due to heterogeneity in the infectious source of anti-D since only three of
the polymorphic positions were variable amongst cloned
sequences from archived batches of 1977 anti-D immunoglobulin, and there was no association between polymorphisms
in different anti-D recipients or secondary recipients.
Variation of the HVR
The consensus HVR sequences of the six secondary
recipients differed from each other at an average of 11n9 amino
acid positions, similar to the difference between different antiD recipients (mean 9n8) or between epidemiologically unrelated
subtype 1b sequences (mean 13n2) (McAllister et al., 1998).
Variation between the five pairs of primary and secondary
recipients (Fig. 3) was less than this for all pairs (3, 6, 7 and 9
differences, mean 6n25) except for sequences from R1 and SR8
that differed at 17 out of 27 positions because of a genome
rearrangement (see below). The extent of variability between
different virus sequences within individual secondary recipients
was limited to five or fewer positions in three individuals, but
14 or more positions were variable amongst the viruses cocirculating in the remaining three individuals (Fig. 3). This
compares with the previous observation that only three out of
seventeen primary anti-D recipients were infected with more
than one HVR variant (McAllister et al., 1998). This difference
is surprising given the shorter duration of infection in
secondary recipients (mean of less than 10n1 years compared
with 17 years for anti-D recipients). There was no simple
relationship between the route of infection and the degree of
variability within individual secondary recipients since only a
single variant was observed in one (SR7) of the three
individuals who became infected following a blood transfusion
from an infected anti-D recipient and would have acquired a
large quantity of virus at transmission (Fig. 3).
No evidence for resetting
In order to test the possibility that there is selection for
reversion of substitutions upon transmission to a naı$ ve host,
we compared the distance between virus sequences from
secondary recipients with those infecting anti-D recipients.
Viruses in these two groups are equally separated from their
common ancestor present in infectious batches of anti-D
immunoglobulin manufactured in 1977, but virus from secondary recipients has undergone an additional transmission
event. There was no significant difference between primary
and secondary recipients in the distance to virus sequences
from archived batches of anti-D, regardless of whether or not
the HVR was included in the analysis or whether amino acid,
nucleotide or only synonymous differences were considered
(data not shown). Similarly, for individual donor–recipient
pairs, virus from secondary recipients was just as divergent
from the 1977 sequence as was virus from the implicated antiD recipient. No difference in the extent of virus diversity
within an individual was observed between primary and
secondary recipients as groups or as individual pairs, or
between the secondary recipients infected as a result of blood
transfusion compared with those infected by other routes.
Hence, there is no discernible effect of secondary transmission
on the rate of virus evolution.
Generation of diversity in the HVR through duplication
Comparison of HVR sequences between anti-D recipients,
their secondary recipients and sequences detected in infectious
batches of anti-D immunoglobulin revealed evidence for three
different types of mechanism by which diversity of the HVR
was generated. First, individual anti-D recipients and secondary
recipients each contained different HVR sequences (Fig. 3) but
these sequences conformed to the restricted amino acid
diversity previously documented for different HVR positions
(Sekiya et al., 1994 ; Sherman et al., 1996 ; McAllister et al.,
1998). Within infected individuals HVR amino acid sequences
also differed between different clones at a small number of
positions, or in some cases more drastically although this
variation was generally less than that between variants
infecting different individuals. The majority of these differences
occurred in more than one clone, and so are likely to represent
polymorphisms in the virus population. Other substitutions
observed in only a single cloned sequence are likely to
represent artefacts introduced during PCR amplification since
their frequency was equivalent to an error rate of Taq DNA
polymerase of 5i10−&, within the range of published
estimates (Smith et al., 1997 a).
Fig. 2. Phylogenetic tree of synonymous sites in the region flanking the HVR. Nucleotide distances at synonymous sites
between E1/E2 sequences excluding the HVR were calculated using the Jukes–Cantor correction, and used to construct a
neighbour-joining tree. Numbers indicate the bootstrap support (100 replicates) for particular groupings. Sequences from
individuals with a known risk of transmission are linked by double lines. Sequences of four epidemiologically unrelated subtype
1b isolates were used as outgroups.
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Fig. 3. HVR amino acid sequences in anti-D recipients and paired secondary recipients. HVR sequences of individual clones
from different individuals are shown with ‘ . ’, indicating identity to the consensus sequence found in infectious batches of anti-D
immunoglobulin manufactured in 1977 (top line). Unique substitutions not otherwise observed amongst these or other
members of the anti-D recipient cohort (McAllister et al., 1998) and likely to represent misincorporation by Taq DNA
HCC
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Fig. 4. Structure of the HVR region in virus from SR8. The nucleotide sequence and predicted HVR amino acid sequence of
clone 5 from SR8. Amino acid positions that are strongly conserved amongst anti-D recipients and epidemiologically unrelated
subtype 1b sequences are indicated by asterisks. Below are shown the nucleotide differences and amino acid substitutions
observed amongst eight other clones from SR8. Nucleotides in bold are those found in both copies of the 33 nucleotide repeat
amongst the variants detected in SR8. The bottom line gives the consensus nucleotide and amino acid sequence of the HVR
from R1, the implicated blood transfusion donor to SR8.
A second type of variation was observed in a variant from
SR6 in which an additional codon (GGG) was present at the
extreme 5h end of the HVR (Fig. 3). Another variant contained
a two nucleotide insertion, but translation of the remainder of
the polyprotein would have become out of frame. Both of
these variants shared several substitutions with other variants
detected in SR6, consistent with a relatively recent origin,
although it was not possible to investigate the diversity in the
implicated donor. Single HVR sequences from SR6 and R10
contained nucleotide deletions that affected the reading frame
downstream of the HVR. These may be derived from defective
viruses or may represent deletions introduced during PCR
amplification.
Finally, variants present in SR8 also contained an additional
codon at the NH terminus of the HVR and had HVR amino
#
acid sequences that were strikingly divergent from those from
the implicated transfusion donor R1 (Fig. 3). One of the
sequences from SR8 (clone 5) had a distinct HVR sequence that
contained an imperfect direct repeat of 33 nucleotides (18
matches) (Fig. 4). The first six nucleotides of the repeat encode
the amino acids Trp.Gly which are uncommon amongst published HCV sequences (McAllister et al., 1998) either at
position 1 or at positions 11 and 12 where they are specified by
the second copy. The repeated segment may derive from the
HVR of an ancestor of the SR8 variants since there is a strong
similarity between codons 13–20 of SR8 variants (excluding
clone 5) and codons 12–20 of R1 variants (excluding codon
15). This suggests a process in which a duplication event
occurred in a variant containing Trp.Gly at positions 11\12
and with a deletion at codon 15, followed by deletion of
codons 1–10 of the HVR. Subsequent evolution of the HVR
might then have led to the accumulation of substitutions in the
COOH terminus of the HVR in the lineage represented by
clone 5, and by the accumulation of substitutions in the NH
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terminus of the HVR and in the flanking regions of the other
main lineage.
Discussion
This study of serial transmission of HCV from a defined
source to primary and secondary recipients has failed to
uncover any effect of multiple transmission events on the rate
or characteristics of virus evolution. Three of the secondary
transmissions studied here involved blood transfusion and the
secondary recipient would have been infected with large
quantities of infectious virus. For the other three transmission
events the route and timing is uncertain, but the dose is likely
to have been much smaller than following transfusion of
infected blood. The lack of any consistent differences between
the extent of virus evolution in these different secondary
recipients argues that it may be valid to extrapolate the rate of
evolution observed over single transmission events (Smith et
al., 1997 b) to longer periods of time during which multiple
transmissions to different individuals will occur.
Reconstruction of epidemiological relationships within
a cohort
An important finding of this study is that the detailed
epidemiological relationships between viruses from anti-D
recipients and secondary recipients could not be adequately
reproduced by phylogenetic analysis of the E1\E2 region (Fig.
2). Although we were able to confirm that all primary and
polymerase during PCR amplification are indicated in bold type. Single nucleotide deletions are indicated by ‘ F ’, ambiguities by
‘ ? ’ and gaps introduced to maintain alignments by ‘ - ’. The number of clones with each sequence is indicated together with the
number of amino positions that are variable within virus sequences from an individual and the number that are variable between
viruses from the primary (R) and secondary recipient (SR).
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secondary anti-D recipients were infected with closely related
viruses of subtype 1b, the only consistent sub-grouping
observed within this cohort was for R5 and SR7 who became
infected following a blood transfusion in 1988. Even this
grouping was poorly supported if analysis was confined to
synonymous sites in the region flanking the HVR. This
deficiency may be partly due to the relative similarity (mean
evolutionary distance of 0n121) of virus sequences infecting
different anti-D recipients compared with that between
epidemiologically unrelated subtype 1b isolates (mean evolutionary distance of 0n23). However, in addition many of the
changes observed in virus sequences from both primary and
secondary recipients of virus from 1977 anti-D immunoglobulin were convergent. This was true not only for amino
acid replacements within the HVR as previously described
(McAllister et al., 1998), but also for both nonsynonymous and
synonymous substitutions in E1 and E2 flanking the HVR.
Similar observations were made when a less variable 222
nucleotide sequence from the virus NS5B region was analysed
(data not shown). All of the sequences shared a common
branch with the sequence of virus present in an infectious batch
of anti-D immunoglobulin, but there was no subgrouping of
sequences from donor–recipient pairs. Amongst the five pairs
of NS5B sequences, there were one nonsynonymous and
eleven synonymous substitutions, none of which were shared
by a donor–recipient pair. Two of the synonymous substitutions were observed in two or more different pairs of
sequences but not in ten independent sequences obtained from
an infectious batch (Smith et al., 1997 b). Since both these
substitutions and all those represented by a single sequence
also occurred in one or more epidemiologically unlinked HCV
subtype 1b sequences, these observations are consistent with
the occurrence of convergent synonymous substitution.
These findings imply that the investigation of epidemiological relationships within a cohort of individuals infected
directly or indirectly from a common source may require the
phylogenetic analysis of much longer genomic regions than
the variable (NS5B, 222 nucleotides) and hypervariable (E2,
363 nucleotides) regions considered here. This may also be the
case even when comparisons are made immediately after a
transmission event, since groupings of multiple E1\E2 virus
sequences from different anti-D recipients were not always
supported by bootstrap analysis despite their having had 17
years of independent evolution (Fig. 2), (McAllister et al.,
1998).
Multiple mechanisms for the generation of diversity in
the HVR
This detailed study of the evolution of the HVR of E2 from
a defined source reveals that diversification of the HVR can
occur through at least three different mechanisms. The most
familiar of these is nucleotide substitution at those positions in
HCE
the HVR at which amino acid replacements are tolerated. The
second mechanism is through the insertion of additional
nucleotides at the junction of E1 and E2, as observed in two
variants in SR6. Variation in this region has occasionally been
reported with insertion or deletion of 1–4 codons (Abe et al.,
1992 ; Kato et al., 1992 ; Hohne et al., 1994). In only one of these
cases is it clear that variants have been produced by insertion
of nucleotides into an otherwise unaltered HVR sequence
(Hohne et al., 1994), and a similar process seems to have
generated the variants observed in SR6 since additional G
residues occur within a sequence otherwise very similar to that
present in 1977 anti-D immunoglobulin. There are only 12
sites of insertion or deletion when the coding regions of 37
complete genomes of HCV are compared, most of which have
strong associations with particular virus types or subtypes.
Since there is evidence that virus types and subtypes diverged
at least several hundred years ago (Smith et al., 1997 b), the
repeated observation of insertion and deletion of nucleotides
at the junction of E1 and E2 during relatively short-term
longitudinal studies suggests that nucleotide rearrangements
may be relatively common in this part of the virus genome.
One way in which this could occur might be through stalling
of the RNA polymerase because of a conserved RNA
secondary structure, but we have been unable to detect such a
structure in the regions of the genome flanking the HVR.
The nucleotide sequence of virus from SR8 provides
evidence for a previously undescribed third mechanism
through which diversity of the HVR can be generated.
Comparison of virus sequences from SR8 and R1, the
implicated donor, suggests a COOH-terminal region of the
HVR was duplicated and replaced the NH -terminal region
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resulting in an unrelated HVR sequence with one extra amino
acid residue. Duplication events have not previously been
described for HCV, although they have been implicated in the
pathogenesis of a pestivirus, bovine viral diarrhoea virus
(Tautz et al., 1996).
Although the HVR of HCV is the most variable part of the
virus genome, it is also striking that this region is subject to
strong constraints on the ways in which it can change. Some
positions within the HVR are confined to particular amino acid
residues, or to a small selection of residues that share particular
chemical characteristics (McAllister et al., 1998). In addition,
there appears to be strong selection against changes in the size
of the HVR since the HVR is the same length in all six
genotypes of HCV with the exception of type 6 which for both
sequences currently available has a deletion of one residue
(Chamberlain et al., 1997). Such selection might explain why
virus variants with lengthened HVR sequences have often
been found to co-circulate with variants with various types of
HVR deletion (Kato et al., 1992 ; Hohne et al., 1994). Together
these features suggest that the HVR has an important function
in the replication of HCV, and that this function depends on
something more than its ability to be structurally flexible
(Taniguchi et al., 1993).
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HCV hypervariable region evolution
C. C. was supported by a study fellowship from the University of
Rome ‘ La Sapienza’, J. McA. and D. B. S. are supported by a grant from the
Wellcome Trust and P. S. is a Darwin Fellow.
Ohto, H., Terazawa, S., Sasaki, N., Hino, K., Ishiwata, C., Kako, M.,
Ujiie, N., Endo, C., Matsui, A., Okamoto, H., Mishiro, S., Kojima, M.,
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Received 21 September 1998 ; Accepted 18 November 1998
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