﻿RESEARCH Open Access
A two-year survey of the oseltamivir-resistant
influenza A(H1N1) virus in Yamagata, Japan and
the clinical effectiveness of oseltamivir
and zanamivir
Yoko Matsuzaki1*
, Katsumi Mizuta2
, Yoko Aoki2
, Asuka Suto2
, Chieko Abiko2
, Kanako Sanjoh3
, Kanetsu Sugawara4
,
Emi Takashita4,5
, Tsutomu Itagaki6
, Yuriko Katsushima7
, Makoto Ujike5
, Masatsugu Obuchi5
, Takato Odagiri5
,
Masato Tashiro5
Abstract
Background: Oseltamivir is the preferred antiviral drug for influenza, but oseltamivir-resistant A(H1N1) viruses have
circulated worldwide since the 2007-2008 influenza season. We aimed to determine the rate of oseltamivir
resistance among A(H1N1) isolates from Yamagata, Japan, to compare the virological characteristics between
isolates from the 2007-2008 and 2008-2009 seasons, and to evaluate the clinical effectiveness of oseltamivir.
Results: Oseltamivir resistance, determined by detecting the H275Y mutation in the neuraminidase (NA) gene, was
observed in 2.5% (2 of 79) and 100% (77 of 77) of isolates from the 2007-2008 and 2008-2009 seasons, respectively.
Antigenic analysis suggested that antigenically different variants of A(H1N1) viruses circulated in the 2008-2009
season. Growth testing demonstrated that the ability of the 2008-2009 isolates to replicate in MDCK cells was
similar to those of the oseltamivir-susceptible isolates from the 2007-2008 season. A phylogenetic analysis revealed
that two oseltamivir-resistant viruses isolated in the 2007-2008 season were closely related to other oseltamivir-
susceptible viruses in Yamagata but were different from oseltamivir-resistant viruses isolated in Europe and North
America in the 2007-2008 season. The oseltamivir-resistant viruses isolated in Japan in the 2008-2009 season were
phylogenetically similar to oseltamivir-resistant isolates from Europe and North America during the 2007-2008
season. Furthermore, the median duration of fever after the start of oseltamivir treatment was significantly longer
in oseltamivir-resistant cases (2 days; range 1-6 days) than in oseltamivir-susceptible cases (1.5 days: range 1-2 days)
(P = 0.0356).
Conclusion: Oseltamivir-resistant A(H1N1) isolates from Yamagata in the 2007-2008 season might have acquired
resistance through the use of oseltamivir, and the 2008-2009 oseltamivir-resistant isolates might have been
introduced into Japan and circulated throughout the country. Influenza surveillance to monitor oseltamivir-
resistance would aid clinicians in determining an effective antiviral treatment strategy.
Background
During the 2007-2008 season, increased levels of resis-
tance to oseltamivir among influenza A (H1N1) viruses
were reported in Europe and North America [1-6], and
oseltamivir-resistant viruses were also detected in the
southern hemisphere [7,8]. The frequency of oseltamivir-
resistance in A(H1N1) isolates was highest (67%) in Nor-
way [9]. During the same season in Japan, it is estimated
that up to 2.6% of all influenza A(H1N1) isolates were
resistant to oseltamivir [10]. It was reported that some of
the resistant viruses found in Japan during the 2007-2008
season were not phylogenetically related to those found in
Europe and that these resistant isolates from Japan
emerged independently in Japan [11,12]. Further, during
the 2008-2009 season, the A(H1N1) virus was prominent
* Correspondence: matuzaki@med.id.yamagata-u.ac.jp
1
Course of Clinical Nursing, Yamagata University Faculty of Medicine,
Yamagata 990-9585, Japan
Matsuzaki et al. Virology Journal 2010, 7:53
http://www.virologyj.com/content/7/1/53
© 2010 Matsuzaki et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
in influenza outbreaks in Japan, and national surveillance
showed that 99.6% of A(H1N1) isolates had the histidine-
to-tyrosine substitution at residue 275 (H275Y) of the
neuraminidase (NA) gene; this mutation is associated with
oseltamivir resistance [13]. Oseltamivir is widely used in
clinical settings in Japan. Therefore, an increase in oselta-
mivir-resistant influenza viruses is an important problem
that is likely to influence the treatment strategy for influ-
enza virus infections.
The purposes of this study were to investigate the per-
centage of A(H1N1) isolates from Yamagata Prefecture
during the 2007-2008 and 2008-2009 seasons that had
the H275Y mutation in the NA gene and to compare the
virological characteristics between the A(H1N1) viruses
isolated in those seasons. Additionally, we evaluated the
clinical effectiveness of oseltamivir and zanamivir against
oseltamivir-resistant A(H1N1) virus infections.
Results
The percentage of influenza A(H1N1) virus isolates with
the H275Y mutation
A total of 156 isolates from the Yamagata prefecture
obtained between December 2007 and March 2008
(2007-2008 isolates) and between December 2008 and
March 2009 (2008-2009 isolates) were sequenced for the
identification of the H275Y mutation in the NA gene.
The sequencing results demonstrated that 2.5% of the
2007-2008 isolates and 100% of 2008-2009 isolates had
the H275Y mutation associated with oseltamivir resis-
tance (Table 1).
The NA inhibition assay against oseltamivir and zanamivir
Seven isolates were tested for susceptibility to the NA
inhibitors oseltamivir and zanamivir. Two 2007-2008
isolates and two 2008-2009 isolates with the H275Y
mutation showed 234- to 1,968-fold reductions in sus-
ceptibility to oseltamivir when compared with three
2007-2008 isolates without the H275Y mutation (Table
2). However, the H275Y mutation had no impact on the
susceptibility to zanamivir.
Antigenic analysis
The antigenic analysis was performed by hemagglutina-
tion inhibition (HI) tests for reactivity with post-
infection ferret antisera against two A(H1N1) vaccine
strains (A/Solomon Islands/3/2006 [2007-2008 vaccine
strain] and A/Brisbane/59/2007 [2008-2009 vaccine
strain]) (Table 3). A/Yamagata/66/2008 and A/Yama-
gata/68/2008, the oseltamivir-resistant 2007-2008 iso-
lates, were antigenically similar to the other oseltamivir-
susceptible 2007-2008 isolates. However, oseltamivir-
resistant 2008-2009 isolates except A/Yamagata/45/2009
showed a fourfold decrease in the HI titer compared
with oseltamivir-resistant 2007-2008 isolates, indicating
that antigenically different variants of oseltamivir-
Table 1 Influenza A(H1N1) virus resistance to oseltamivir
in Yamagata, Japan
Season Total
tested
Number (%) of oseltamivir-resistant
isolates
neuraminidase H275Y mutation
Dec 2007-Mar
2008
79 2 (2.5)
Dec 2008-Mar
2009
77 77 (100)
Table 2 Inhibition of the enzyme activity of the A(H1N1)
isolates in Yamagata in the NA inhibition assay
Viruses Amino Acid at
position 275
in the NA
gene
IC50 values in the NA
inhibition assay (nM)
Oseltamivir Zanamivir
2007-2008 isolates
A/Yamagata/1/2008 Histidine 0.08 0.52
A/Yamagata/66/2008 Tyrosine 50.16 0.45
A/Yamagata/67/2008 Histidine 0.03 0.22
A/Yamagata/68/2008 Tyrosine 39.71 0.55
A/Yamagata/69/2008 Histidine 0.17 0.78
2008-2009 isolates
A/Yamagata/126/2008 Tyrosine 59.04 0.50
A/Yamagata/128/2008 Tyrosine 47.59 0.40
Table 3 Antigenic analysis of the A(H1N1) isolates in
Yamagata by the HI test
HI titer of post-infection ferret
sera to:
Viruses A/Solomon Islands/
3/2006
A/Brisbane/
59/2007
A(H1N1) vaccine strains
A/Solomon Islands/3/2006 160 160
A/Brisbane/59/2007 160 160
2007-2008 isolates
A/Yamagata/1/2008 80 160
A/Yamagata/66/2008 160 160
A/Yamagata/67/2008 160 320
A/Yamagata/68/2008 160 160
A/Yamagata/69/2008 160 160
2008-2009 isolates
A/Yamagata/125/2008 20 10
A/Yamagata/126/2008 40 40
A/Yamagata/128/2008 40 40
A/Yamagata/45/2009 20 160
A/Yamagata/80/2009 20 40
Matsuzaki et al. Virology Journal 2010, 7:53
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resistant A(H1N1) viruses circulated in Yamagata in the
2008-2009 season.
Virus growth in MDCK cells
We compared the growth characteristics of oseltamivir-
resistant 2008-2009 isolates with those of oseltamivir-
susceptible and -resistant 2007-2008 isolates. As shown
in Figure 1, the virus titers of A/Yamagata/126/2008
and A/Yamagata/128/2008, which were oseltamivir-
resistant 2008-2009 isolates, increased, as did those of
the oseltamivir-susceptible isolates from the 2007-2008
season. The two 2008-2009 isolates showed rapid
growth and A/Yamagata/126/2008 reached a more than
ten-fold higher final virus titer than did A/Yamagata/68/
2008, an oseltamivir-resistant virus from the 2007-2008
season.
Phylogenetic analysis of the HA and NA genes
Of the 156 A(H1N1) isolates, 13 isolates from the 2007-
2008 season and 22 isolates from the 2008-2009 season
were used for the phylogenetic analysis. All of the 2007-
2008 isolates belonged to two distinct lineages (2B and
2C) in the HA (Figure 2) and NA (Figure 3) gene trees.
Two oseltamivir-resistant 2007-2008 isolates, A/Yama-
gata/66/2008 and A/Yamagata/68/2008, were isolated
three days apart from two respective students from the
same elementary school who had not been treated with
oseltamivir prior to the specimen collection. The
nucleotide sequences of both the HA and NA genes of
these two viruses were identical, and these viruses
showed close genetic similarity to the oseltamivir-sus-
ceptible 2007-2008 isolates belonging to 2B. The
oseltamivir-resistant A(H1N1) viruses that emerged in
Europe in late 2007 are characterized by D354G amino
acid substitutions in the NA protein [2]. However, osel-
tamivir-resistant 2007-2008 isolates from Yamagata did
not have the D354G mutation in the NA gene.
On both the HA and NA phylogenetic trees, all oselta-
mivir-resistant 2008-2009 isolates from Yamagata
belonged to the 2B lineage and were more closely
related to the oseltamivir-resistant viruses isolated in
Europe and North America in the 2007-2008 season
than to the oseltamivir-resistant 2007-2008 isolates from
Yamagata. All 2008-2009 isolates have the D354G muta-
tion in the NA gene and the A189T mutation in the
HA gene. Except for A/Yamagata/45/2008, all 2008-
2009 isolates from Yamagata have an amino acid substi-
tution at residue 185 (G185A, G185V, or G185S) of the
HA protein. This substitution might influence the
decrease in the reactivity with antiserum against A/Bris-
bane/59/2007.
Clinical effectiveness of oseltamivir and zanamivir
We investigated the clinical effectiveness of anti-influ-
enza drugs by comparing the symptoms of children with
oseltamivir-susceptible influenza A(H1N1) virus infec-
tions during the 2007-2008 season to those of children
with oseltamivir-resistant influenza A(H1N1) virus infec-
tions during the 2008-2009 season (Table 4). We found
no significant differences in the age, maximum tempera-
ture, or total febrile period of oseltamivir-treated chil-
dren between cases of the oseltamivir-susceptible and
oseltamivir-resistant influenza A(H1N1) virus infections.
However, for the children treated with oseltamivir, the
1
10
102
103
104
105
106
107
108
0 10 20 30 40 50 60 70 80
Yamagata/1/2008
Yamagata/69/2008
Yamagata/66/2008
Yamagata/67/2008
Yamagata/68/2008
Yamagata/126/2008
Yamagata/128/2008
Viral
load
(pfu/mL)
Time (h)
Figure 1 Growth of oseltamivir-susceptible or -resistant A(H1N1) viruses from the 2007-2008 season and 2008-2009 seasons in
Yamagata. A/Yamagata/66/2008 and A/Yamagata/68/2008 from the 2007-2008 season and A/Yamagata/126/2008 and A/Yamagata/128/2008
from the 2008-2009 season were all oseltamivir-resistant viruses. Viruses were grown in MDCK cells, and the supernatants were harvested at the
indicated time points and titrated by plaque assays. Growth tests were performed in duplicate experiments, and the mean virus titers are shown
in Figure.
Matsuzaki et al. Virology Journal 2010, 7:53
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duration of fever after the start of treatment was signifi-
cantly longer in children with oseltamivir-resistant influ-
enza A(H1N1) virus infections during the 2008-2009
season than in children with oseltamivir-susceptible
influenza A(H1N1) virus infections during the 2007-
2008 season, though no significant differences were
observed among the zanamivir-treated children.
Discussion
Oseltamivir is widely used for patients with influenza A
and B infections in clinical settings in Japan. Therefore,
oseltamivir-resistant viruses induced by oseltamivir
treatment emerge in Japan more readily than in other
countries where oseltamivir is not so widely used. It has
been documented that the infectivity and replicative
ability of neuraminidase inhibitor-resistant viruses are
compromised [14,15]; therefore, until recently it was
thought that oseltamivir-resistant influenza A(H1N1)
viruses were unlikely to circulate among humans.
Antigenic and phylogenetic analyses in this study
revealed that two oseltamivir-resistant influenza A
(H1N1) virus strains isolated in the 2007-2008 season
(A/Yamagata/66/2008 and A/Yamagata/68/2008) were
closely related to other oseltamivir-susceptible A(H1N1)
viruses isolated in Yamagata but were different from the
resistant viruses found in Europe. It appears that
A/Brisbane/59/2007
A/Hawaii/20/2007
A/Yamagata/13/2008
A/Yamagata/67/2008
A/Yamagata/69/2008
A/Yamagata/71/2008
A/Yamagata/63/2008
A/Hawaii/15/2007
A/Yamagata/81/2008
A/SolomonIslands/3/2006
A/NewCaledonia/20/1999
A/Yamagata/50/2008
A/Yamagata/39/2008
A/Yamagata/12/2008
A/Yamagata/74/2008
A/Yamagata/66/2008
A/Yamagata/68/2008
A/Yamagata/1/2008
A/Johannesburg/21/2008
A/IIlinois/10/2007
A/Paris/341/2007
A/Norway/1736/2007
A/Yamagata/137/2008
A/Yamagata/80/2009
A/Yamagata/45/2009
A/England/557/2007
A/NewJersey/15/2007
A/Yamagata/48/2009
A/Yamagata/29/2009
A/Yamagata/61/2009
A/Yamagata/125/2008
A/Yamagata/36/2009
A/Yamagata/128/2008
A/Yamagata/16/2009
A/Yamagata/53/2009
A/Yamagata/10/2009
A/Yamagata/51/2009
A/Yamagata/20/2009
A/Yamagata/77/2009
A/Yamagata/120/2008
A/Yamagata/55/2009
A/Yamagata/57/2009
A/Yamagata/76/2009
A/Yamagata/133/2008
A/Yamagata/26/2009
A/Yamagata/126/2008
70
99
98
99
92
99
80
95
0.005
2B
2C
A189T
G185A
G185V
G185S
Figure 2 Phylogenetic trees for the HA gene of A(H1N1) viruses. The region from nucleotide 33 to 1102 (1070 nucleotides) for the HA gene
were used for the analysis. The numbers below the branches are the bootstrap probabilities (percentages), showing only values greater than
70%. Viruses with a H275Y mutation in the NA gene isolated in Yamagata are shown in red, and those isolated in Europe, the USA, and South
Africa are shown in green. Viruses with H275 strains in Yamagata are shown in blue. Viruses from the 2008-2009 season are indicated in
boldface.
Matsuzaki et al. Virology Journal 2010, 7:53
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A/Yamagata/63/2008
A/Yamagata/69/2008
A/Yamagata/81/2008
A/Yamagata/71/2008
A/Hawaii/15/2007
A/SolomonIslands/3/2006
A/NewCaledonia/20/1999
A/Yamagata/13/2008
A/Hawaii/20/2007
A/Brisbane/59/2007
A/Yamagata/67/2008
A/Yokohama/22/2008
A/Yokohama/35/2008
A/Yamagata/12/2008
A/Yamagata/39/2008
A/Yamagata/50/2008
A/Yamagata/68/2008
A/Yamagata/66/2008
A/Yamagata/74/2008
A/Yamagata/1/2008
76
97
99
99
99
83
86
76
A/Paris/341/2007
A/Illinois/10/2007
A/Yamagata/137/2008
A/Yamagata/80/2009
A/Johannesburg/21/2008
A/Norway/1736/2007
A/NewJersey/15/2007
A/Yamagata/45/2009
A/England/557/2007
A/Yamagata/48/2009
A/Yamagata/10/2009
A/Yamagata/120/2008
A/Yamagata/16/2009
A/Yamagata/128/2008
A/Yamagata/26/2009
A/Yamagata/53/2009
A/Yamagata/133/2008
A/Yamagata/125/2008
A/Yamagata/126/2008
A/Yamagata/29/2009
A/Yamagata/51/2009
A/Yamagata/20/2009
A/Yamagata/55/2009
A/Yamagata/57/2009
A/Yamagata/76/2009
A/Yamagata/61/2009
A/Yamagata/77/2009
A/Yamagata/36/2009
83
97
0.005
D354G 2B
2C
Figure 3 Phylogenetic trees for the NA gene of A(H1N1) viruses. The region from nucleotide 21 to 1430 (1410 nucleotides) for the NA gene
were used for the analysis. The numbers below the branches are the bootstrap probabilities (percentages), showing only values greater than
70%. Viruses with a H275Y mutation in the NA gene isolated in Yamagata are shown in red, and those isolated in Europe, the USA, South Africa,
and Japan (except Yamagata) are shown in green. Viruses with H275 strains in Yamagata are shown in blue. Viruses from the 2008-2009 season
are indicated in boldface.
Table 4 Comparison of the effectiveness of oseltamivir and zanamivir against the oseltamivir-susceptible and
oseltamivir-resistant influenza A(H1N1) infections
Oseltamivir-treated cases Zanamivir-treated cases
2007-2008
Oseltamivir-
susceptible
(n = 8)
2008-2009
Oseltamivir-
resistant
(n = 25)
P-
value
2007-2008
Oseltamivir-
susceptible
(n = 4)
2008-2009
Oseltamivir-
resistant
(n = 6)
P-
value
Age, years; median (range) 6 (1-9) 5 (2-9) .8155 8 (7-11) 11 (9-12) .0506
Maximum body temperature, °C;
median (range)
39.3 (38.9-39.8) 39.1 (38.2-40.6) .3874 39.2 (38.6-39.6) 39.0 (38.2-39.6) .5906
Total febrile period, days; median
(range)
3.5 (2-5) 4 (2-7) .0503 3 (2-4) 3 (3-4) .4642
Duration of fever after the start of
therapy, days; median (range)
1.5 (1-2) 2 (1-6) .0356 1 (1-2) 1 (1-3) .6926
Matsuzaki et al. Virology Journal 2010, 7:53
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oseltamivir-resistant viruses found during the 2007-2008
season in Japan emerged independently among persons
treated with oseltamivir and were isolated in various
communities, as previously described [11,12]. Two osel-
tamivir-resistant viruses in the present study have HA
and NA genes sequences that are 100% identical
between the two isolates; these viruses were isolated on
February 29 and March 3 from two different children
from the same elementary school who had not been
treated with oseltamivir. In this school, an outbreak of
influenza occurred on February 29, 2008, and 28 (22%)
of 129 students showed influenza-like symptoms
between February 29 and March 17, 2008. Although
only two samples were collected from which oseltami-
vir-resistant viruses were isolated, there is a possibility
that the transmission of an oseltamivir-resistant virus
had occurred in this school. It was also reported that an
outbreak of an oseltamivir-resistant A(H1N1) virus
occurred in an elementary school in Yokohama City in
Japan during the same season (A/Yokohama/22/2008
and A/Yokohama/35/2008 in the NA gene tree of Figure
3) [12]. Thus, it is likely that oseltamivir-resistant
viruses posses the ability to be transmitted among
humans. However, because the level of oseltamivir resis-
tance remained at 2.5% in Yamagata (2.6% in Japan), it
is apparent that the oseltamivir-resistant viruses did not
spread among humans as easily as the oseltamivir-sus-
ceptible viruses.
The oseltamivir-resistant viruses isolated during the
2008-2009 season in Yamagata were antigenically and
phylogenetically different from those isolated during the
2007-2008 season in Yamagata but were phylogenetically
similar to viruses isolated in Europe and North America
during the 2007-2008 season. Thus, it seems that oselta-
mivir-resistant viruses were imported into Japan, where
they spread across the country during the 2008-2009
season. Some reports have suggested that the emergence
of oseltamivir-resistant viruses in the 2007-2008 season
in Europe was not related to the use of oseltamivir
[16,17], and that natural genetic variations may have
resulted in the change in sensitivity to oseltamivir [18].
It is likely that influenza A(H1N1) viruses having an epi-
demiological advantage over previous viruses emerged
with natural resistance to oseltamivir and spread
throughout the world. Two representative viruses iso-
lated during the 2008-2009 season in Yamagata (A/
Yamagata/126/2008 and A/Yamagata/128/2008) showed
the same level of growth in MDCK cells as the oseltami-
vir-susceptible 2007-2008 isolates. The maintenance of
replicative ability and the acquirement of antigenic dif-
ferences are vital for viruses to continue transmission
among humans. Hereafter, A(H1N1) viruses might con-
tinue to circulate with the H275Y mutation in the NA
gene preserved. The pandemic H1N1 viruses emerged in
2009 were oseltamivir-susceptible without H275Y muta-
tion in the NA gene. Cocirculation of oseltamivir-resis-
tant seasonal A(H1N1) viruses and the novel H1N1
pandemic viruses may give rise to the potential risk of
genetic reassortment acquiring the H275Y resistance
mutation in the NA gene.
There have been few reports concerning the clinical
effectiveness of oseltamivir against oseltamivir-resistant
influenza viruses. We found that the duration of fever
after the start of oseltamivir treatment was significantly
longer in children with oseltamivir-resistant influenza A
(H1N1) virus infections than in children with oseltami-
vir-susceptible A(H1N1) virus infections. This result
suggests that oseltamivir-resistant A(H1N1) viruses cir-
culating during the 2008-2009 season were resistant to
oseltamivir not only in vitro but also in vivo. Zanamivir,
however, reduced the duration of fever in oseltamivir-
resistant cases. In Japan, zanamivir is used as well as
oseltamivir, especially for the treatment of patients older
than five years. Thus, in cases of oseltamivir-resistant
influenza virus infection, zanamivir would be a more
appropriate treatment than oseltamivir.
Conclusions
Oseltamivir resistance was observed in 2.5% and 100%
of A(H1N1) isolates from Yamagata from the 2007-2008
and 2008-2009 seasons, respectively. Oseltamivir-resis-
tant isolates from Yamagata in the 2007-2008 season
might have acquired resistance through the use of osel-
tamivir. In contrast, the 2008-2009 oseltamivir-resistant
isolates from Yamagata were similar to viruses isolated
in Europe and North America; therefore, they might
have been introduced into Japan and circulated through-
out the country. It is certain that influenza surveillance
to monitor oseltamivir resistance will benefit clinicians
in determining an effective antiviral treatment strategy.
Timely monitoring and reporting of antiviral drug-resis-
tance is important at the global, national, and the local
community levels.
Methods
Specimen collection and virus isolation
Nasopharyngeal swab specimens from patients with
acute respiratory infection were collected at pediatric
clinics collaborating with the local health authorities in
Yamagata Prefecture for the surveillance of viral diseases
in Japan. Specimens were transported to the Yamagata
Prefectural Institute of Public Health and were grown in
a virus culture as previously described [19]. A total of
79 A(H1N1) viruses isolated from 690 specimens
between December 2007 and March 2008 and a total of
77 A(H1N1) viruses isolated from 831 specimens
between December 2008 and March 2009 were used in
this study. Of the 156 isolates, 84 (53.8%) were isolated
Matsuzaki et al. Virology Journal 2010, 7:53
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Page 6 of 8
from children under 6 years of age, 55 (35.3%) were
from children aged between 6 and 10 years, 15 (9.6%)
were from children aged between 11 and 15 years, and
2 (1.3%) were from patients >15 years.
NA inhibition assay
A chemiluminescent NA inhibition assay was performed
using a commercially available kit, NA-Star (Applied
Biosystems, Foster City, CA), according to the manufac-
turer's protocol. The NA inhibitors (oseltamivir and
zanamivir) susceptibility of influenza virus isolates was
expressed as the concentration of NA inhibitor needed
to inhibit the NA enzyme activity by 50% (IC50). Oselta-
mivir carboxylate, the active form of the prodrug oselta-
mivir phosphate, was provided by F. Hoffmann-La
Roche Ltd, Switzerland, and zanamivir was provided by
GlaxoSmithKline Research and Development Ltd., Uni-
ted Kingdom.
Hemagglutination inhibition test
The hemagglutination inhibition (HI) test was per-
formed in microtiter plates with 1% guinea pig erythro-
cytes and post-infection ferret antisera against A/
Solomon Islands/3/2006 [2007-2008 A(H1N1) vaccine
strain] and A/Brisbane/59/2007 [2008-2009 A(H1N1)
vaccine strain] (Denka Seiken Co., Ltd., Tokyo, Japan),
as described previously [20]. Sera were treated with
receptor-destroying enzyme before use. For the vaccine
antigen, purified HA protein (Denka Seiken Co., Ltd)
was diluted in a hemagglutination titer of eight, while
MDCK culture fluid was also diluted in hemagglutina-
tion titer of eight and used as the isolate antigen.
Growth of H1N1 isolates in culture
MDCK cells were infected with an A (H1N1) isolate at
an MOI of 0.001 and incubated for 72 hours at 37°C in
the presence of 2 g/ml trypsin. At 12 h, 24 h, 48 h,
and 72 h post-infection, the supernatants were harvested
and virus titers were determined by a plaque assay on
MDCK cells. The growth test was performed in dupli-
cate experiments, and the mean virus titer was
calculated.
Sequence analyses
The extraction of viral RNA and cDNA synthesis using
random primers were performed as described previously
[21,22]. The cDNA was then used as the template for the
amplification of the HA and NA genes by PCR. The HA
gene was amplified using primers 5'-AGCAAAAG-
CAGGGGAAAATAA-3' and 5'-AACCATCTACCATTC-
CAGTC-3', and the NA gene was amplified using primers
5'-AGCAAAAGCAGGAGTTTAAAATGA-3' and 5'-
GTAGAAACAAGGAGTTTTTTCAAC-3'. The PCR pro-
ducts were purified with a QIAquick PCR purification kit
(QIAGEN, Hilden, Germany) and then sequenced using a
Big Dye Terminator V1.1 cycle sequencing kit (Applied
Biosystems) on an Applied Biosystems 3130 automatic
sequencer. The nucleotide sequences determined in this
study were assigned the accession numbers as follows at
GenBank: AB521053-AB521112 and AB539701-
AB539710. Published A(H1N1) virus sequences were
obtained from GenBank (Accession numbers AB465320,
AB465322, CY030230, CY030233, CY033622, CY033624,
EU124136, EU124137, EU516057, EU516085, EU516083,
EU516116, EU516118, EU516200, EU516257, EU516148,
EU551811, EU551832, EU914903, EU914910, FJ403550,
FJ445031, FJ445089, FJ654304). The sequence data were
analyzed with the CLUSTAL W version 1.83 software, and
a phylogenetic tree was constructed via the neighbor-join-
ing method using the same software.
Clinical information
We used the clinical information of children who visited
the Sanjoh Clinic and from whom A(H1N1) influenza
virus was isolated. Data were obtained retrospectively
from their medical records. To compare the data between
oseltamivir-resistant and oseltamivir-susceptible A(H1N1)
virus infections, we used the Mann-Whitney U test. A P
value of < 0.05 was regarded as statistically significant.
Abbreviations
HA: hemagglutinin; HI: hemagglutination inhibition; MDCK: Madin Darby
canine kidney; MOI: multiplicity of infection; NA: neuraminidase; PCR:
polymerase chain reaction.
Acknowledgements
This work was supported by a grant-in-aid for Scientific Research from the
Japanese Ministry of Education, Culture, Sports, Science and Technology and
by a research grant from the Yamagata Prefectural Society of Child Health.
Author details
1
Course of Clinical Nursing, Yamagata University Faculty of Medicine,
Yamagata 990-9585, Japan. 2
Yamagata Prefectural Institute of Public Health,
Yamagata 990-0031, Japan. 3
Sanjoh Clinic, Yamagata 996-0084, Japan.
4
Department of Infectious Diseases, Yamagata University Faculty of Medicine,
Yamagata 990-9585, Japan. 5
Influenza Virus Research Center, National
Institute of Infectious Diseases, Tokyo 208-0011, Japan. 6
Yamanobe Pediatric
Clinic, Yamagata 990-0301, Japan. 7
Katsushima Pediatric Clinic, Yamagata
990-2461, Japan.
Authors' contributions
YM was responsible for the research design, antigenic and sequence
analysis, and writing of this manuscript. KM, YA, AS, and CA performed the
cell culture experiments, RT-PCR, and sequencing of influenza A isolates. KS
(Sanjoh), TI, and YK collected specimens from patients and clinical
information. KS (Sugawara) and ET performed the growth test of viruses in
culture. MU, MO, TO, and MT performed the NA inhibition assay and
sequencing of viruses. KM, MO and MT participated in the study design and
helped to draft the manuscript. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 29 December 2009 Accepted: 5 March 2010
Published: 5 March 2010
Matsuzaki et al. Virology Journal 2010, 7:53
http://www.virologyj.com/content/7/1/53
Page 7 of 8
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doi:10.1186/1743-422X-7-53
Cite this article as: Matsuzaki et al.: A two-year survey of the
oseltamivir-resistant influenza A(H1N1) virus in Yamagata, Japan and
the clinical effectiveness of oseltamivir and zanamivir. Virology Journal
2010 7:53.
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