Positively selected amino acid sites in the entire coding region of
Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, 1111 Yata, Mishima-shi, Shizuoka-ken 411-8540, Japan
Received 6 December 2000; received in revised form 10 April 2001; accepted 26 July 2001
To predict the amino acid sites important for the clearance of hepatitis C virus (HCV) subtype 1b in vivo, positively selected amino acid
sites were detected by analyzing the sequence data collected from the international DNA databank. The rate of nonsynonymous substitutionsper nonsynonymous site was compared with that of synonymous substitutions per synonymous site for each codon site in the entire codingregion. As a result, 13 out of 3010 amino acid sites were found to be positively selected. Among the 13 positively selected amino acid sites,eight were located in the structural proteins and five were in the nonstructural proteins. Moreover, eight were located in B-cell epitopes andtwo were in T-cell epitopes. These observations suggest that both the antibody and the cytotoxic T lymphocyte are involved in the clearanceof HCV subtype 1b in vivo. These positively selected amino acid sites represent candidate vaccination targets for HCV subtype 1b. q 2001Elsevier Science B.V. All rights reserved.
Keywords: Hepatitis C virus; Synonymous substitution; Nonsynonymous substitution; Positive selection; Epitope; Vaccine
et al., 1998). In the polyprotein of HCV, a region includingthe N-terminal 27–31 amino acid sites in E2 is known to be
Hepatitis C virus (HCV), the sole member of the genus
the most variable and is called hypervariable region 1
Hepacivirus in the family Flaviviridae (van Regenmortel et
(HVR1; Hijikata et al., 1991; Weiner et al., 1991). HVR1
al., 2000), is an enveloped, non-segmented, single-stranded,
has been proposed as a major target of the immune response
and positive-sense RNA virus (Choo et al., 1989). The
(Weiner et al., 1992; Farci et al., 1996; Zibert et al., 1997a).
genome of HCV is approximately 9.5 kilobases long, encod-
More than 50% of humans infected with HCV establish
ing a polyprotein of approximately 3000 amino acids (Kato
chronic hepatitis, which may progress to cirrhosis and hepa-
et al., 1990). The polyprotein is co- and post-translationally
tocellular carcinoma (Alter et al., 1992). Interferon alpha
cleaved into core protein (C), envelope glycoprotein 1 (E1),
and ribavirin are used for treatment of HCV infection.
E2, p7, nonstructural protein 2 (NS2), NS3, NS4A, NS4B,
However, they are not highly effective, especially for
NS5A, and NS5B in order from its N-terminus by the cellu-
HCV subtype 1b (Davis et al., 1998; McHutchison et al.,
lar signalase and the viral proteinases (Grakoui et al., 1993).
1998). Therefore, development of effective therapies and
The genomic sequences from different HCV isolates are
vaccines against HCV is an urgent subject worldwide.
highly divergent (Kato et al., 1989). According to the phylo-
For developing effective vaccines against HCV, it is
genetic analysis, HCV has been classified into six clades, in
important to identify epitopes involved in the clearance of
which various numbers of subtypes are included (Robertson
HCV in vivo, because they may be the candidate vaccinationtargets. Although it may be difficult to identify thoseepitopes experimentally because of the lack of an efficient
Abbreviations: C, core protein; DDBJ, DNA databank of Japan; E, envel-
in vitro cell culture system and an in vivo animal model
ope glycoprotein; HCV, hepatitis C virus; HVR, hypervariable region; NS,nonstructural protein; P, probability; c
system for proliferation of HCV (Blight et al., 2000), they
tions per codon; cN, number of nonsynonymous substitutions per codon; dS,
may be identified as the positively selected amino acid sites.
number of synonymous substitutions per synonymous site; dN, number of
This is because the amino acid mutations at those epitopes
nonsynonymous substitutions per nonsynonymous site; sS, number of
may provide selective advantage to the mutants by allowing
synonymous sites per codon; sN, number of nonsynonymous sites per codon
them to escape from the immune response (Endo et al.,
* Corresponding author. Tel.: 181-559-81-6847; fax: 181-559-81-6848.
E-mail address: [email protected] (T. Gojobori).
1996; Fitch et al., 1997; Nielsen and Yang, 1998; Suzuki
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 1 9 ( 0 1 ) 0 0 6 4 0 - 0
Y. Suzuki, T. Gojobori / Gene 276 (2001) 83–87
and Gojobori, 1999; Yamaguchi-Kabata and Gojobori,
CLUSTAL W (Thompson et al., 1994). We then extracted
sequences which did not contain any gaps within each
Positive selection operating at the amino acid sequence
coding region in the pairwise alignment. The subtype 1b
level can be detected by comparing the rate of nonsynon-
sequences were identified by reconstructing a phylogenetic
ymous substitution per nonsynonymous site (rN) with that of
tree for each coding region. The numbers of nucleotide
synonymous substitution per synonymous site (rS). A higher
sequences collected for C, E1, E2, NS2, NS3, NS4,
value of rN than rS is an indicator of positive selection,
NS5A, and NS5B were 129, 135, 59, 74, 68, 70, 69, and
whereas the inverse is an indicator of negative selection
61, respectively. These sequences originated largely from
(Hughes and Nei, 1988, 1989). By comparing rN and rS at
unrelated patients and only a few were from the same
single codon sites, it is possible to detect positive selection
patients. In this paper, the amino acid positions are
at single amino acid sites (Fitch et al., 1997; Nielsen and
Yang, 1998; Suzuki and Gojobori, 1999; Yamaguchi-Kabata and Gojobori, 2000).
In this study, we identified positively selected amino acid
sites in the entire coding region of HCV subtype 1b by using
A multiple alignment was made for each coding region by
the method of Suzuki and Gojobori (1999) to predict the
using CLUSTAL W. The positively selected amino acid
amino acid sites important for the clearance of HCV subtype
sites were identified by using the method of Suzuki and
Gojobori (1999). Briefly, a phylogenetic tree was recon-structed by the neighbor-joining method (Saitou and Nei,1987) using the number of synonymous substitutions (Nei
and Gojobori, 1986). The ancestral sequence was inferred ateach node in the phylogenetic tree by using the maximum
parsimony method (Hartigan, 1973). Then, the averagenumbers of synonymous (sS) and nonsynonymous (sN)
In this study, we focused on HCV subtype 1b because of
sites and the total numbers of synonymous (cS) and nonsy-
its clinical importance and a large number of sequence data
nonymous (cN) substitutions throughout the phylogenetic
deposited in the international DNA databank. Note that a
tree were estimated for each codon site. A probability (P)
relatively large number of sequences are required for detect-
of obtaining the observed or more biased numbers of synon-
ing positively selected amino acid sites by using the method
ymous and nonsynonymous substitutions was computed for
of Suzuki and Gojobori (1999). Actually, the numbers of
each codon site, assuming a binomial distribution. In the
sequences for another subtype were too small to detect posi-
computation, sS/(sS 1 sN) and sN/(sS 1 sN) were used as the
tively selected amino acid sites in the entire coding region.
probabilities of the occurrences of synonymous and nonsy-
For detecting positively selected amino acid sites in the
nonymous substitutions, respectively. The significance level
entire coding region of HCV subtype 1b, the simplest way is
was set at 5%. A significantly larger value of cN than cS was
to analyze only sequence data which contain the entire
considered as an indicator of positive selection, whereas the
coding region. However, the number of such sequences
inverse was considered as an indicator of negative selection.
was relatively small. Thus, we divided the entire coding
The number of synonymous substitutions per synonymous
region of HCV subtype 1b into eight regions encoding C,
site (dS) and that of nonsynonymous substitutions per nonsy-
E1, E2, NS2, NS3, NS4, NS5A, and NS5B and analyzed
nonymous site (dN) were estimated by cS/sS and cN/sN,
each region separately. p7 and E2 were combined as E2, and
NS4A and NS4B were combined as NS4, because p7 (63amino acid sites) and NS4A (54 amino acid sites) were tooshort to analyze positively selected amino acid sites. The
numbers of amino acid sites for C, E1, E2, NS2, NS3, NS4,NS5A, and NS5B were 191, 192, 426, 217, 631, 315, 447,
The results for identifying positively selected amino acid
sites in the entire coding region of HCV subtype 1b are
A total of 7262 entries which included the successive
summarized in Fig. 1. dS exceeded dN at most of the
terms ‘hepatitis C virus’ in their organism names were
amino acid sites (2426/3010, 80.60%), and negative selec-
collected from the international DNA databank (DDBJ
tion was detected at more than half (1560/3010, 51.83%) of
release 40). These entries included all clades and subtypes
all amino acid sites. The HCV polyprotein contains many B-
of HCV. To collect nucleotide sequences for each coding
cell and T-cell epitopes. For example, nearly the entire
region of HCV subtype 1b, we defined HCV-JS (Accession
coding region of E1 and E2, and NS3 have been reported
number: D85516; Tanaka et al., 1995) as a reference
as B-cell and T-cell epitopes, respectively (Zibert et al.,
sequence for HCV subtype 1b and made 7261 pairwise
1997a, 1999; Tabatabai et al., 1999). However, the amino
alignments, each of which consisted of HCV-JS and one
acid sites involved in the clearance of HCV subtype 1b in
of the other sequences, by using the computer program
vivo may be limited, because most of the sites in the entire
Y. Suzuki, T. Gojobori / Gene 276 (2001) 83–87
Fig. 1. Distribution of the value of (1 2 P) in the entire coding region of HCV subtype 1b. The entire coding region is divided into eight coding regions (C, E1,E2, NS2, NS3, NS4, NS5A, and NS5B). The abscissa indicates the amino acid positions and the ordinate indicates the value of (1 2 P) for each amino acid site. When dN is larger than dS, the value is indicated above the abscissa, whereas in the opposite situation, the value is indicated below the abscissa. Dotted linesindicate the 5% significance level. A filled rectangle in E2 indicates the position of HVR1.
coding region, including E1, E2, and NS3, are negatively
Table 1Functions of positively selected amino acid sites in the entire coding region
Indeed, dN exceeded dS at only 265 amino acid sites
(8.80%), and positive selection was detected at only 13
amino acid sites (0.43%) (Fig. 1). Among the 13 positively
selected amino acid sites, eight were located in the structural
proteins (C, E1, and E2) and five were in the nonstructural
proteins (NS2, NS3, NS4, NS5A, and NS5B). Since the
structural proteins occupy only 26.9% (809/3010) of the
HCV polyprotein, positively selected amino acid sites
seemed to be located more densely in the structural proteins
than in the nonstructural proteins (P ¼ 0:009).
Table 1 summarizes the functions of positively selected
amino acid sites for HCV subtype 1b. Most of the sites are
located in B-cell (Zibert et al., 1997a, 1999; Jolivet-
Reynaud et al., 1998; Pereboeva et al., 1998, 2000; Nakano
et al., 1999) and T-cell (Tabatabai et al., 1999; Wang and
Eckels, 1999) epitopes, suggesting that both antibodies and
cytotoxic T lymphocytes (CTLs) are involved in the clear-
ance of HCV subtype 1b in vivo. However, a larger numberof positively selected amino acid sites were located in B-cell
a The amino acid position is numbered according to HCV-JS.
epitopes than in T-cell epitopes. This is probably because
Y. Suzuki, T. Gojobori / Gene 276 (2001) 83–87
the recognition of T-cell epitopes is restricted by the haplo-
type of human leukocyte antigen, whereas that of B-cellepitopes is not. The sequence data used in this study were
Allain, J.-P., Dong, Y., Vandamme, A.-M., Moulton, V., Salemi, M., 2000.
collected from the international DNA databank, which
Evolutionary rate and genetic drift of hepatitis C virus are not correlatedwith the host immune response: studies of infected donor-recipient
included HCV subtype 1b sequences largely from unrelated
clusters. J. Virol. 74, 2541–2549.
patients. Therefore, the positive selection may be more effi-
Alter, M.J., Margolis, H.S., Krawczynski, K., Judson, F.N., Mares, A.,
ciently detected in B-cell epitopes than in T-cell epitopes in
Alexander, W.J., Hu, P.Y., Miller, J.K., Gerber, M.A., Sampliner,
this study. This is consistent with the above observation that
R.E., Meeks, E.L., Beach, M.J., 1992. The natural history of commu-
positively selected amino acid sites are more densely
nity-acquired hepatitis C in the United States. N. Engl. J. Med. 327,
located in the structural proteins than in the nonstructural
Blight, K.J., Kolykhalov, A.A., Rice, C.M., 2000. Efficient initiation of
proteins, because B-cell epitopes are mainly located in
HCV RNA replication in cell culture. Science 290, 1972–1974.
structural proteins. Since most positively selected amino
Bush, R.M., Bender, C.A., Subbarao, K., Cox, N.J., 1999. Predicting the
acid sites were located in B-cell and T-cell epitopes,
evolution of human influenza A. Science 286, 1921–1925.
amino acid positions 345, 827, 2719, and 2968 may also
Choo, Q.-L., Kuo, G., Ralston, R., Weiner, A.J., Overby, L.R., Bradley,
be parts of B-cell and T-cell epitopes.
D.W., Houghton, M., 1989. Isolation of a cDNA clone derived from ablood-borne non-A, non-B hepatitis genome. Science 244, 359–362.
The positively selected amino acid sites in B-cell and T-
Davis, G.L., Esteban-Mur, R., Rustgi, V., Hoefs, J., Gordon, S.C., Trepo,
cell epitopes may be the vaccination targets against HCV
C., Shiffman, M.L., Zeuzem, S., Craxi, A., Ling, M.-H., Albrecht, J.,
subtype 1b, because these sites should be highly immuno-
1998. Interferon alfa-2b alone or in combination with ribavirin for the
genic and involved in the clearance of HCV subtype 1b in
treatment of relapse of chronic hepatitis C. N. Engl. J. Med. 339, 1493–
vivo. However, it should be noted that these sites are often
highly variable, so that HCV may escape from the immune
Endo, T., Ikeo, K., Gojobori, T., 1996. Large-scale search for genes on
which positive selection may operate. Mol. Biol. Evol. 13, 685–690.
response by producing antigenic mutants (Nowak et al.,
Farci, P., Shimoda, A., Wong, D., Cabezon, T., De Gioannis, D., Strazzera,
1991; Weiner et al., 1992; Farci et al., 2000). This may be
A., Shimizu, Y., Shapiro, M., Alter, H.J., Purcell, R.H., 1996. Preven-
facilitated by a high evolutionary rate of HCV (Ina et al.,
tion of hepatitis C virus infection in chimpanzees by hyperimmune
1994; Smith et al., 1997; Allain et al., 2000; Suzuki et al.,
serum against the hypervariable region 1 of the envelope 2 protein.
2000) and shifting immunodominance of the immune
Proc. Natl. Acad. Sci. USA 93, 15394–15399.
Farci, P., Shimoda, A., Coiana, A., Diaz, G., Peddis, G., Melpolder, J.C.,
response (Nowak et al., 1995). Nevertheless, it has been
Strazzera, A., Chien, D.Y., Munoz, S.J., Balestrieri, A., Purcell, R.H.,
reported that if the immune response is sufficiently strong
Alter, H.J., 2000. The outcome of acute hepatitis C predicted by the
in an acute infection, HCV is unable to escape from
evolution of the viral quasispecies. Science 288, 339–344.
the immune response even if it is directed against highly
Fitch, W.M., Bush, R.M., Bender, C.A., Cox, N.J., 1997. Long term trends
variable epitopes (Missale et al., 1996; Zibert et al.,
in the evolution of H(3) HA1 human influenza type A. Proc. Natl. Acad.
1997a,b; Farci et al., 2000). Moreover, the composite
Grakoui, A., Wychowski, C., Lin, C., Feinstone, S.M., Rice, C.M., 1993.
vaccines containing different amino acid residues, particu-
Expression and identification of hepatitis C virus polyprotein cleavage
larly predicted future amino acid residues, at positively
products. J. Virol. 67, 1385–1395.
selected amino acid sites may be useful for preventing
Hartigan, J.A., 1973. Minimum mutation fits to a given tree. Biometrics 29,
proliferation of escape mutants (Bush et al., 1999). Since
both antibodies and CTLs seem to be involved in the
Hijikata, M., Kato, N., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S., Shimo-
tohno, K., 1991. Hypervariable regions in the putative glycoprotein of
clearance of HCV subtype 1b, it may be more effective to
hepatitis C virus. Biochem. Biophys. Res. Commun. 175, 220–228.
use both B-cell and T-cell epitopes as the vaccination
Hughes, A.L., Nei, M., 1988. Pattern of nucleotide substitution at major
histocompatibility complex class I loci reveals overdominant selection.
In conclusion, we identified 13 positively selected amino
acid sites in the entire coding region of HCV subtype 1b.
Hughes, A.L., Nei, M., 1989. Nucleotide substitution at major histocompat-
ibility complex class II loci: evidence for overdominant selection. Proc.
These sites may be candidate vaccination targets against
Ina, Y., Mizokami, M., Ohba, K., Gojobori, T., 1994. Reduction of synon-
ymous substitutions in the core protein gene of hepatitis C virus. J. Mol. Evol. 38, 50–56.
Jolivet-Reynaud, C., Dalbon, P., Viola, F., Yvon, S., Paranhos-Baccala, G.,
Piga, N., Bridon, L., Trabaud, M.A., Battail, N., Sibai, G., Jolivet, M.,1998. HCV core immunodominant region analysis using mouse mono-
The authors thank Dr Allison Wyndham at the National
clonal antibodies and human sera: characterization of major epitopes
Institute of Genetics, Japan, for providing valuable
useful for antigen detection. J. Med. Virol. 56, 300–309.
comments on an earlier version of the manuscript. We are
Kato, N., Ohkoshi, S., Shimotohno, K., 1989. Japanese isolates of the non-
grateful to two anonymous reviewers for suggestions to
A, non-B hepatitis viral genome show sequence variations from the
improve the manuscript. This work was supported, in part,
original isolate in the U.S.A. Proc. Jpn. Acad. 65, 219–223.
Kato, N., Hijikata, M., Ootsuyama, Y., Nakagawa, M., Ohkoshi, S., Sugi-
by grants from the Ministry of Education, Culture, Sports,
mura, T., Shimotohno, K., 1990. Molecular cloning of the human hepa-
Science, and Technology, Japan. Y.S. is supported by the
titis C virus genome from Japanese patients with non-A, non-B
JSPS Research Fellowships for Young Scientists.
hepatitis. Proc. Natl. Acad. Sci. USA 87, 9524–9528.
Y. Suzuki, T. Gojobori / Gene 276 (2001) 83–87
McHutchison, J.G., Gordon, S.C., Schiff, E.R., Shiffman, M.L., Lee, W.M.,
Tabatabai, N.M., Bian, T.-H., Rice, C.M., Yoshizawa, K., Gill, J., Eckels,
Rustgi, V.K., Goodman, Z.D., Ling, M.-H., Cort, S., Albrecht, J.K.,
D.D., 1999. Functionally distinct T-cell epitopes within the hepatitis C
1998. Interferon alfa-2b alone or in combination with ribavirin as initial
virus non-structural 3 protein. Hum. Immunol. 60, 105–115.
treatment for chronic hepatitis C. N. Engl. J. Med. 339, 1485–1492.
Tanaka, T., Kato, N., Cho, M.-J., Shimotohno, K., 1995. A novel sequence
Missale, G., Bertoni, R., Lamonaca, V., Valli, A., Massari, M., Mori, C.,
found at the 30 terminus of hepatitis C virus genome. Biochem.
Rumi, M.G., Houghton, M., Fiaccadori, F., Ferrari, C., 1996. Different
Biophys. Res. Commun. 215, 744–749.
clinical behaviors of acute hepatitis C virus infection are associated
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improv-
with different vigor of the anti-viral cell-mediated immune response.
ing the sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight matrix
Nakano, I., Fukuda, Y., Katano, Y., Hayakawa, T., 1999. Conformational
choice. Nucleic Acids Res. 22, 4673–4680.
epitopes detected by cross-reactive antibodies to envelope 2 glycopro-
van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Carstens, E.B.,
tein of the hepatitis C virus. J. Infect. Dis. 180, 1328–1333.
Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J.,
Nei, M., Gojobori, T., 1986. Simple methods for estimating the numbers of
Pringle, C.R., Wickner, R.B., 2000. Virus Taxonomy. Academic Press,
synonymous and nonsynonymous substitutions. Mol. Biol. Evol. 3,
Wang, H., Eckels, D.D., 1999. Mutations in immunodominant T cell
Nielsen, R., Yang, Z., 1998. Likelihood models for detecting positively
epitopes derived from the nonstructural 3 protein of hepatitis C virus
selected amino acid sites and applications to the HIV-1 envelope
have the potential for generating escape variants that may have impor-
tant consequences for T cell recognition. J. Immunol. 162, 4177–4183.
Nowak, M.A., Anderson, R.M., McLean, A.R., Wolfs, T.F., Goudsmit, J.,
Weiner, A.J., Brauer, M.J., Rosenblatt, J., Richman, K.H., Tung, J., Craw-
May, R.M., 1991. Antigenic diversity thresholds and the development
ford, K., Bonino, F., Saracco, G., Choo, Q.-L., Houghton, M., Han, J.H.,
1991. Variable and hypervariable domains are found in the regions of
Nowak, M.A., May, R.M., Phillips, R.E., Rowland-Jones, S., Lalloo, D.G.,
HCV corresponding to the Flavivirus envelope and NS1 proteins and
McAdam, S., Klenerman, P., Koppe, B., Sigmund, K., Bangham,
the Pestivirus envelope glycoproteins. Virology 180, 842–848.
C.R.M., McMichael, A.J., 1995. Antigenic oscillations and shifting
Weiner, A.J., Geysen, H.M., Christopherson, C., Hall, J.E., Mason, T.J.,
immunodominance in HIV-1 infections. Nature 375, 606–611.
Saracco, G., Bonino, F., Crawford, K., Marion, C.D., Crawford, K.A.,
Pereboeva, L.A., Pereboev, A.V., Morris, G.E., 1998. Identification of anti-
Brunetto, M., Barr, P.J., Miyamura, T., McHutchinson, J., Houghton,
genic sites on three hepatitis C virus proteins using phage-displayed
M., 1992. Evidence for immune selection of hepatitis C virus (HCV)
peptide libraries. J. Med. Virol. 56, 105–111.
putative envelope glycoprotein variants: potential role in chronic HCV
Pereboeva, L.A., Pereboev, A.V., Wang, L.F., Morris, G.E., 2000. Hepatitis
infections. Proc. Natl. Acad. Sci. USA 89, 3468–3472.
C epitopes from phage-displayed cDNA libraries and improved diag-
Yamaguchi-Kabata, Y., Gojobori, T., 2000. Reevaluation of amino acid
nosis with a chimeric antigen. J. Med. Virol. 60, 144–151.
variability of the human immunodeficiency virus type 1 gp120 envelope
Robertson, B., Myers, G., Howard, C., Brettin, T., Bukh, J., Gaschen, B.,
glycoprotein and prediction of new discontinuous epitopes. J. Virol. 74,
Gojobori, T., Maertens, G., Mizokami, M., Nainan, O., Netesov, S.,
Nishioka, K., Shin-i, T., Simmonds, P., Smith, D., Stuyver, L., Weiner,
Zibert, A., Kraas, W., Meisel, H., Jung, G., Roggendorf, M., 1997a. Epitope
A., 1998. Classification, nomenclature, and database development forhepatitis C virus (HCV) and related viruses: proposals for standardiza-
mapping of antibodies directed against hypervariable region 1 in acute
tion. Arch. Virol. 143, 2493–2503.
self-limiting and chronic infections due to hepatitis C virus. J. Virol. 71,
Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
Zibert, A., Meisel, H., Kraas, W., Schulz, A., Jung, G., Roggendorf, M.,
Smith, D.B., Pathirana, S., Davidson, F., Lawlor, E., Power, J., Yap, P.L.,
1997b. Early antibody response against hypervariable region 1 is asso-
Simmonds, P., 1997. The origin of hepatitis C virus genotypes. J. Gen.
ciated with acute self-limiting infections of hepatitis C virus. Hepatol-
Suzuki, Y., Gojobori, T., 1999. A method for detecting positive selection at
Zibert, A., Kraas, W., Ross, R.S., Meisel, H., Lechner, S., Jung, G.,
single amino acid sites. Mol. Biol. Evol. 16, 1315–1328.
Roggendorf, M., 1999. Immunodominant B-cell domains of hepatitis
Suzuki, Y., Yamaguchi-Kabata, Y., Gojobori, T., 2000. Nucleotide substi-
C virus envelope proteins E1 and E2 identified during early and late
tution rates of HIV-1. AIDS Rev. 2, 39–47.
time points of infection. J. Hepatol. 30, 177–184.
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