﻿JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2005, p. 6130­6132 Vol. 43, No. 12
0095-1137/05/$08.000 doi:10.1128/JCM.43.12.6130­6132.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Molecular Evolution of Human Influenza A/H3N2 Virus in Asia
and Europe from 2001 to 2003
X. Sherry Chi, Trentice V. Bolar, Ping Zhao, John S. Tam,
Ruth Rappaport, and Sheau-Mei Cheng*
Applied Immunology and Microbiology Division, Wyeth Vaccines Research, Pearl River, New York 10965
Received 9 May 2005/Returned for modification 15 August 2005/Accepted 20 September 2005
Hemagglutinin sequences of 146 human influenza A/H3N2 strains identified in respiratory specimens from
Asia and Europe during the 2001-2003 influenza seasons were analyzed by DNA sequencing. Our results
suggest that four amino acid substitutions, L25I, H75Q, H155T, and Q156H, led to the antigenic conversion
of the previously predominant A/Panama/2007/99-like strains to the more recent A/Fujian/411/2002-like
strains.
Influenza virus genomes are well known to undergo anti-
genic drifts that enable escape from preexisting immunity and
potentially cause epidemics in humans (10). Monitoring anti-
genic variations in circulating influenza viruses is crucial for
anticipating epidemics and for vaccine design.
The surface glycoprotein hemagglutinin (HA) is the major sur-
face antigen of influenza viruses, against which neutralizing anti-
bodies are elicited during virus infection and vaccination (12). HA
is synthesized as a single polypeptide that is subsequently cleaved
into two polypeptides, HA1 and HA2. The HA1 polypeptide
mutates more frequently than HA2 and plays a crucial role in
natural selection (13, 14). Five antigenic sites (A to E) on the
three-dimensional structure of the HA protein of A/Aichi/2/68
(H3N2) have been proposed to be the antibody-binding sites of
the protein (13, 14). About one-third of the HA1 amino acids lie
on or near these five antigenic sites, although the importance of
these amino acid positions is not clear (6). Recently, several mod-
els have been proposed to predict the antigenic evolution of the
A/H3N2 viruses (6, 8, 9, 11).
A/Panama/2007/99-like viruses have been circulating world-
wide since 1999, which predates the emergence of A/Fujian/
411/2002-like viruses. The appearance of A/Fujian/411/2002-
like viruses prompted a change in the selection of vaccine
components for the Southern Hemisphere in 2003 and for the
Southern and Northern Hemispheres in 2004. In this report,
we utilized a high-throughput sequencing method to determine
the nucleotide sequence of the HA1 gene segments of influ-
enza virus in nasal swabs collected from infected children aged
6 months and older during the 2001-2003 influenza seasons.
The specimens were divided into the following five groups,
based on the date of collection (Table 1): groups 1, 3, and 5
were from Asian countries, including Bangladesh, Singapore,
Malaysia, and South Korea, and groups 2 and 4 were from
European countries, including the United Kingdom, Scotland,
Finland, The Netherlands, Poland, and Italy. Viral RNA was
extracted from influenza viruses propagated once in MDCK
cells as previously described (4). HA1 fragments correspond-
ing to nucleotides 37 to 1154 were amplified by reverse tran-
scription-PCR using oligonucleotides 5-CTATCATTGCTT
TGAGCTAC (primer 1) and 5-ATCTGCTGCTTGTCCTGT
GC (primer 2). The PCR products were purified and subjected
to sequencing on a genetic analyzer (ABI PRISM 3100; Ap-
plied Biosystems, Foster City, CA) with the above-mentioned
oligonucleotides and two additional oligonucleotides, 5-ATG
CCAAACAATGACAAATT (primer 3) and 5-TGTTTG
GCATAGTCACGTTC (primer 4), except for group 3 speci-
mens, for which only primers 1 and 4 were used for sequencing.
Multiple alignments of the sequences were performed by the
ClustalV method, and the phylogenetic tree was constructed by
the neighbor-joining method using MegAlign version 5.03
(DNASTAR) as previously described (4).
A total of 13 amino acid changes were found in the HA1
protein of the reference vaccine strain, A/Fujian/411/2002,
compared to that of the A/Panama/2007/99 strain, including
changes at positions 21, 25, 50, 75, 83, 131, 155, 156, 183, 186,
202, 222, and 225. These changes were allocated to the HA1
sequences for the five groups of specimens; the results are
summarized in Table 1. For phylogenetic analysis, 25 HA1
sequences (five from each of the five groups) were selected
from 146 specimens in an attempt to represent the varieties
within each group. The sequences formed clusters (Fig. 1) that
corresponded to different stages of antigenic drift. We at-
tempted to establish a pattern by further examining the HA1
sequences of all 146 specimens with respect to these 13 amino
acid changes. Figure 2 shows the frequencies of individual
amino acid changes within each group. The HA1 sequences in
groups 1 and 2 all exhibited amino acid changes S21P, L183H,
and S186G, regardless of origin (Asia or Europe), suggesting
that these three changes were inherited from earlier drifts. Six
additional changes (R50G, E83K, A131T, V202I, W222R, and
G225D) were detected in group 1 sequences, five of which (all
but A131T) were also observed in group 2. More changes were
detected in specimens in later groups. Nearly half (47%) of
the sequences from group 3 exhibited 12 of the 13 changes
found in the HA1 sequence of A/Fujian/411/2002, including
the three amino acid substitutions L25I, H75Q, and H155T,
which were also found in group 4 at a frequency of 43%. The
* Corresponding author. Mailing address: Applied Immunology and
Microbiology Division, Wyeth Vaccines Research, 401 N. Middletown
Rd., Building 180, Pearl River, NY 10965. Phone: (845) 602-5115. Fax:
(845) 602-5296. E-mail: chengsm@wyeth.com.
6130
occurrence of these three substitutions increased signifi-
cantly, to 100%, in group 5. Moreover, 39% of group 4
(European) viruses and 70% of group 5 (Asian) viruses also
exhibited an additional amino acid substitution, Q156H,
which appears to be critical for the antigenic evolution of
the viruses from A/Panama/2007/99-like to A/Fujian/411/
2002-like, as reported previously (3).
A hallmark of influenza virus is its ability to undergo rapid
antigenic variation (14). New variants are usually subject to
intense selective pressure from existing human immunity
gained from exposure to their viral ancestors (5). In this
study, we investigated the antigenic drift of the HA1 se-
quence of the human influenza A/H3N2 virus during the
2001-2003 influenza seasons. This study confirms a progres-
sive antigenic drift from predominantly A/Panama/2007/99-
like strains to A/Fujian/411/2002-like strains in our A/H3N2
influenza specimens during the 2001-2003 seasons. Among
the 13 amino acid differences between the HA1 protein of
A/Fujian/411/2002 and that of A/Panama/2007/99, many
were detected before March 2002 in both Asia and Europe.
Changes were more frequent and advanced in Asia since
only 1 out of 17 specimens in group 2 exhibited amino acid
changes other than S21P, L183H, and S186G (Fig. 2). Three
amino acid changes--L25I, H75Q, and H155T--were found
later in the year 2002 but prior to the emergence of the key
amino acid change Q156H. These three substitutions were
not found separately from each other and appeared to occur
at the same time, suggesting that these three amino acid
changes arose from a single genetic event. Although most of
the viruses examined during this period were genetically closer to
A/Fujian/411/2002 than to A/Panama/2007/99 based on their
HA1 sequences, it was not until the emergence of the last amino
acid change, Q156H, that the antigenic property of the viruses
completely shifted to that of A/Fujian/411/2002. In addition, 39%
of the European specimens collected from November 2002 to
May 2003 contained A/Fujian/411/2002-like viruses, based on
the HA1 sequence data. This percentage is significantly
higher than that previously reported by Paget et al. (7),
based mostly on classical serology assays. However, it is not
clear whether the difference is due to sampling or to the
sensitivity of the assay method. The sharp rise of the A/
Fujian/411/2002-like viruses in the population coincides with
FIG. 1. Phylogenetic analyses of influenza A/H3N2 viruses based on 320 amino acid sequences of HA1. Twenty-five HA1 sequences (five from
each of the five groups) were selected from 146 specimens in an attempt to represent the varieties within the same group. The changes in the
deduced amino acid sequences were compared with the sequence of A/Panama/2007/99. Only the amino acid changes corresponding to the
sequence of A/Fujian/411/2002 are shown in square brackets. Bold letters indicate the reference vaccine strains. C1, C2, C3, C4, and C5 correspond
to groups 1, 2, 3, 4 and 5, respectively. The group numbers are followed by hyphens and then the isolate numbers. AA, amino acid.
TABLE 1. Changes in the deduced amino acid sequences of the HA1 protein of influenza A/H3N2 virusesa
Group Region Period (mo/yr)
No. of
specimens
No. of amino
acid changes
detectedb
Changes in amino acid residuesb
1 Asia 11/2001 to 03/2002 16 9 S21P, R50G, E83K, A131T, L183H, S186G, V202I, W222R, G225D
2 Europe 11/2001 to 03/2002 17 8 Same as group 1 but without A131T
3 Asia 04/2002 to 08/2002 58 12 Same as group 1 plus L25I, H75Q, and H155T
4 Europe 11/2002 to 05/2003 28 13 Same as group 1 plus L25I, H75Q, H155T, and Q156H
5 Asia 11/2002 to 05/2003 27 13 Same as group 1 plus L25I, H75Q, H155T, and Q156H
a
Compared with the HA1 sequence of A/Panama/2007/99.
b
Only changes corresponding to those in A/Fujian/411/2002 are included.
VOL. 43, 2005 NOTES 6131
the emergence of the key amino acid change Q156H. This
particular amino acid located near antigenic site B is con-
sidered 1 of the 18 amino acids in the HA1 protein under
positive selection and exhibiting a rapid rate of replacement
(2). It is likely that these changes altered the antigenic
property of the virus and facilitated the spread of A/Fujian/
411/2002-like viruses in Asia and Europe as well as in other
parts of the world such as South Africa (1).
The HA1 polypeptide has been proposed to contain five
antigenic sites, based on a study of its three-dimensional struc-
ture (13). Among the 13 amino acid changes in the HA1
protein of A/Fujian/411/2002 compared to that of A/Panama/
2007/99, at least 7 of them were located in one of the five
antigenic sites: R50G (site C), H75Q and E83K (site E),
A131T (site A), and H155T, Q156H, and S186G (site B) (6). In
addition, the L183H change is involved in sialic acid receptor
binding (12). However, not all amino acid changes in the HA1
sequence were sustained. The majority (94%) of HA1 se-
quences of European specimens from group 2 exhibited the
changes A106V and N144D, which are not found in A/Fujian/
411/2002, and the frequency of these changes declined to 50 to
60% in later European specimens (group 4). Similarly, about
30 to 40% of HA1 sequences in Asian specimens found in
groups 1 and 3 also exhibited these two changes. However,
none of the specimens in group 5 exhibited A106V, and less
than 10% exhibited N144D.
Nucleotide sequence accession numbers. The nucleotide se-
quences of the HA1 gene can be found in GenBank under
the indicated accession numbers: strain A/Panama/2007/99,
ISDNCDA001; strain A/Fujian/411/2002, ISDN38157; 146
clinical specimens, DQ179382 to DQ179527.
We thank Giuseppe Palladino for providing wild-type influenza ref-
erence strains and Fenglan Li and Wafa Al-Rimawi for technical
assistance.
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FIG. 2. Prevalence of genetic variations in the HA1 sequences of influenza A/H3N2 viruses. The percentages of HA1 sequences exhibiting the
13 amino acid changes reflect the differences in the A/Fujian/411/2002 strain relative to A/Panama/2007/99.
6132 NOTES J. CLIN. MICROBIOL.
