Jpn. J. Infect. Dis., 59, 2006

Laboratory and Epidemiology Communications

Sequence Characteristics of HA Gene in Influenza Type A (H1N1) Virus
Isolated during the 2005 - 2006 Season in Aichi Prefecture, Japan
Mami Hata*, Masako Tsuzuki**, Kenji Sakae***, Hiroko Minagawa,Takashi Kimura1 and Yutaka Miyazaki1
Department of Microbiology, 1Aichi Prefectural Institute of Public Health, Nagoya 462-8576, Japan
Communicated by Ichiro Kurane(Accepted May 12, 2006)
identity among the isolates in cluster b was 99.1 - 100%,
without changes in the predicted amino acid sequences. A/New Caledonia/20/99, one of the 2005 - 2006 season vaccinestrains, also belonged to cluster b, and the Aichi isolates incluster b exhibited 98.1 - 98.4% nucleotide sequence identity
to this vaccine strain. In contrast, the isolates in cluster a were
genetically more distant from A/New Caledonia than were
those in cluster b, showing 97.4 - 97.5% identity.
A comparison of the predicted amino acid residues between
each of the Aichi isolates and A/New Caledonia/20/99 isshown in Fig. 2. Amino acid sequences were 100% identicalamong isolates within each of two clusters. Aichi/168/2005
and Aichi/21/2006 (isolated from the specimen collected on
January 13) are shown as representative strains of clusters band a, respectively, in Fig. 2. Amino acid numbering corresponds to the number of HA1 in the H3 subtype, as reported
previously (3). A total of 5 amino acid changes from A/New
Caledonia/20/99 were detected in the HA1 protein of Aichi/
168/2005-like viruses, including V169A, N190D, W255R,
Y256F, and V317A (not shown in Fig. 2). In contrast, Aichi/
21/2006-like viruses contained 11 predicted amino acid

In Aichi Prefecture, the first influenza outbreak of the 2005 2006 influenza season occurred from November to December
2005 in the northeast area (1). As of March 26, 2006, we
had obtained a total of 182 influenza virus isolates from therespiratory specimens collected through the surveillance program in Aichi Prefecture. Of these, 70 (39%) were influenza
AH1 virus, 106 (58%) were influenza AH3 virus, and 6 (3%)
were influenza B virus. The percentage of AH1 virus isolatesin Aichi was somewhat higher than that in the entire country(22%, Infectious Disease Surveillance Center, National Institute of Infectious Diseases [NIID], Tokyo, Japan. Available
online at <http://idsc.nih.go.jp/disease/influenza/inf-keiho/
YR05/flu12/jmap12.html>.). In the present report, we describe
antigenic and genetic aspects of AH1 isolates obtained in the2005 - 2006 season in Aichi Prefecture.
Throat swab specimens were collected from influenza
patients as sentinels in the influenza surveillance, and wereinoculated onto Madin-Darby Canine Kidney (MDCK) cells.
Influenza virus was identified by hemagglutination inhibition (HI) test using culture supernatant of infected MDCK
cells as the hemagglutinin and type-specific sera against
influenza viruses (provided from the NIID). Viral RNA wasextracted from culture supernatants with High Pure Vial
RNA Kit (Roche Applied Science, Penzberg, Germany). The
complete coding sequence of the HA1 gene was amplified
by One-step RT-PCR Kit (Invitrogen, Carlsbad, Calif., USA).
The primers used were 5´-gcaaaagcaggggaaaataa-3´ and 5´ttgatctgcagcatagccag-3´, which yield a 1,177-bp amplicon.
Direct sequencing of the amplified fragment was performed
with a Model-4200 automated DNA sequencer (Li-cor,Lincoln, Nebr., USA). The NA subtype of each isolate wasdetermined using type-specific primers (2). All 14 AH1 isolates were proved to be of the N1 subtype.
Out of the 70 AH1 isolates identified so far in the 2005 2006 season, we determined nucleotide sequences of the HA1gene of 14 isolates. The phylogenetic tree of the isolates isshown in Fig. 1. The isolates formed two clusters in the 2005 2006 season (Fig. 1). The isolates in the early season (1)are clustered into a group (cluster b in Fig. 1) including
Aichi/168/2005, the first isolate of the season in our institute(isolated from a specimen collected on November 17). The*Corresponding author: Mailing address: Department of Microbiology, Aichi Prefectural Institute of Public Health, Nagare 76, Tsujimachi, Kita-ku, Nagoya 462-8576, Japan. Tel: +81-52910-5674, Fax: +81-52-913-3641, E-mail: mami_hata@pref.
aichi.lg.jp
Present address: **Department of Planning and Information,
Aichi Prefectural Institute of Public Health.
***Aichi Food Hygiene Center, Ohbu 474-0036, Aichi.

Fig. 1. Phylogenetic tree based on the nucleotide sequences of the HA1
region of the H1 genes. The tree was generated by the N-J method.
The Aichi isolates in the 2005 - 2006 season and reference strains (A/New Caledonia/20/99, A/Moscow/13/98) were analyzed. Two clusters
are indicated by a and b.

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Fig. 2. The predicted amino acid sequence of the HA1 region of the Aichi isolates compared with A/New Caledonia/20/99.
Amino acid numbering corresponds to H3 subtype, with additional amino acid residues present in the H1 subtype sequence
indicated by an asterisk. Residue substitutions are shown using one-letter amino acid code, and a dot (.) indicates aminoacid residues homologous to those of A/New Caledonia/20/99.
Table 1. Antigenic analysis of the AH1 isolateschanges from A/New Caledonia 20/99, and only three of themwere common with Aichi/168/2005-like viruses (V169A,N190D, W255R). The HA1 polypeptide has been proposed
to contain five antigenic sites, based on a study of the threedimensional structure (4). It has been proposed that a newdrift variant with epidemiologic importance generally had fouror more amino acid substitutions located in more than oneof the antigenic sites (5). Among the 11 amino acid changes
in the HA1 protein of Aichi/21/2006-like viruses, at least two(V133T and K144E) were located in site A, which forms
the center of the most obvious antibody-binding sites (4).
Moreover, K81R was in antigenic site Cb, which is located at
the bottom of the HA1 globular head (6,7). This substitution
also affects oligosaccharide attachment. These substitutions
in different antigenic sites could potentially alter antibodybinding properties of the viruses.
The antigenic analysis further confirmed the results of thesequence analysis. The antigenicity of the isolates was compared to that of A/New Caledonia/20/99, A/Moscow/13/98(H1N1), and A/New York/55/2004 (H3N2). The results of
the representative strains are shown in Table 1. Aichi/168/
2005-like viruses showed HI titers of 1:160 for anti-A/New
Caledonia serum (homologous titer 1:160), 1:10 for antiA/Moscow serum, and less than 1:10 for anti-A/New York
serum (data not shown). In contrast to Aichi/168/2005-likeviruses, Aichi/21/2006-like viruses showed a substantial
decrease in titers, with 1:40 for anti-A/New Caledonia serum.
The results indicated that the Aichi/168/2005-like viruses wereantigenically similar to A/New Caledonia, whereas Aichi/21/
2006-like viruses were antigenically distinct from A/NewCaledonia.
In the early stage of the 2005 - 2006 influenza season, fromNovember to December 2005, most viral isolates showedHI titers within a twofold difference from the homologous
titer for A/New Caledonia (1). However, about 40% of AH1
isolates showed fourfold lower HI titers to anti-A/New
Caledonia serum than homologous A/New Caledonia afterJanuary 2006. It is possible that these isolates with the lowered titers were Aichi/21/2006-like virus.
From the 2000 - 2001 to the 2005 - 2006 influenza seasons,Antigens

Antisera toA/New Caledonia/20/99 A/Moscow/13/98

Reference viruses
A/New Caledonia/20/99
A/Moscow/13/98

160––3202005 - 2006 season isolates
A/Aichi/168/2005
A/Aichi/17/2006

160
160

10104040<10<10A/Aichi/21/2006
A/Aichi/103/2006–, not tested.

the World Health Organization recommended A/New Caledonia/20/99 as the H1N1 vaccine component for both thesouthern and northern hemispheres. However, our findingemphasizes the importance of vigilance against the emergenceof influenza drift variants which may make the previously
recommended influenza vaccine ineffective.
Nucleotide sequence accession number: The nucleotide
sequences of the HA1 gene can be found in GenBank under
the indicated accession numbers as follows: A/Aichi/168/
2005, AB243744; A/Aichi/2/2006, AB255389; A/Aichi/11/
2006, AB255390; A/Aichi/17/2006, AB255391; A/Aichi/21/2006, AB255392; A/Aichi/46/2006, AB255393; A/Aichi/67/2006, AB255394; A/Aichi/103/2006, AB255395; A/Aichi/111/2006, AB255396; A/Aichi/132/2006, AB255397; and A/
Aichi/150/2006, AB255398.
This article appeared in part in the Infectious Agents Surveillance Report (IASR), vol. 27, p. 12, 2006.
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