Arch Virol (2008) 153:945–949
DOI 10.1007/s00705-008-0038-5
BRIEF REPORT
Effects of oseltamivir phosphate (TamifluÒ) in human sera
on results of microneutralization and hemagglutinin-inhibition
tests for H5N2 avian influenza virus
Yoshinao Yamazaki Æ M. Doy Æ S. Yamato Æ
Y. Kawada Æ T. Ogata
Received: 6 November 2007 / Accepted: 20 December 2007 / Published online: 29 January 2008
Ó Springer-Verlag 2008
Abstract To determine the influence of oseltamivir
phosphate (TamifluÒ) on the results of microneutralization
and hemagglutinin-inhibition (HI) tests in human sera with
H5N2 influenza virus, ten volunteers were administered
TamifluÒ and blood samples were collected. In the mi-
croneutralization test, no consistent effects were observed.
However, in the HI test, specimens from all volunteers
taken at 4 and 7 h after drug administration showed a
higher titer as compared to 0 and 24 h after administration
when mammalian cells (horse, guinea pig, and human)
were used. These results suggest that the administration of
TamifluÒ may affect the results of HI tests for H5N2 virus.
Influenza viruses belong to the family Orthomyxoviridae,
which comprises influenza virus A, B, and C [10]. Influenza
A viruses commonly infect humans and are also known to
Y. Yamazaki (&)
Department of Genetic Science, Ibaraki prefectural Institute
for Public Health, 993-2, Kasahara-cho, Mito,
Ibaraki 310-0852, Japan
e-mail: petitmadam@power.odn.ne.jp
M. Doy
Division of Health and Welfare, Ibaraki prefectural Government,
978-6, Kasahara-cho, Mito, Ibaraki 310-8555, Japan
S. Yamato
Tsuchiura Public Health Center, 2-7-46, Shimotakatsu,
Tsuchiura, Ibaraki 300-0812, Japan
Y. Kawada
Koga Public Health Center, 6-22, Kitamachi, Koga,
Ibaraki 306-0005, Japan
T. Ogata
Chikusei Public Health Center, 114, Koh, Chikusei,
Ibaraki 308-0021, Japan
infect a variety of animal species, including pigs, horses,
poultry, and other fowls [25]. During previous H5N1 influ-
enza virus infections in the human population, the H5N1
virus was isolated from patients [4, 22]; further, type-specific
antibodies were detected by H5-specific ELISA, microneu-
tralization assay, or western blotting of human sera
specimens [3, 8] and it was found that the H5N1 virus is
virulent in humans [27]. In contrast, humans are not sus-
ceptible to the H5N2 subtype [1]. When an H5N2 influenza
infection occurred among poultry in Italy, no anti-H5 anti-
bodies were detected in human sera and no viral agents were
isolated from humans, suggesting that avian-to-human
transmission of the H5N2 virus had not occurred [5]. In 2005,
in Ibaraki prefecture, which is located in central Japan, an
H5N2 subtype of influenza virus was isolated from com-
mercial chickens, and the prototype strain was named A/
chicken/Ibaraki/1/05 [17]. This outbreak resulted in the
death of 5.68 million commercial chickens; it took approx-
imately 1 year to suppress this outbreak [16, 17].
The outbreak of the H5N2 subtype of influenza virus
was the first case of an outbreak in Japan [19]. Antigenic
analysis revealed that this virus differed from other previ-
ously isolated H5 strains, and that it showed high
nucleotide sequence homology with Latin America strains
[11] (93.7–98.0%) [16]. This outbreak involved 342 indi-
viduals—employees of the chicken farm or prefectural
government staff engaged in epidemic control. The mi-
croneutralization test (MNT) was used to determine
whether these individuals had been infected with the H5N2
virus. The MNT yielded positive results in the case of 70
(20.5%) subjects, indicating that they may have been
infected with the H5N2 virus. However, they were all
asymptomatic, and no virus was isolated. This is the first
report that suggested infection of the human population
with the H5N2 virus [19].
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During this outbreak, many MNT-positive individuals
were prescribed oseltamivir phosphate (OP; TamifluÒ). OP
is an orally active prodrug of oseltamivir carboxylate (OC),
which is particularly effective against the influenza virus
neuraminidase [24]; it is designed to target conserved
residues at the neuraminidase enzyme active site of influ-
enza A and B viruses [23]. Koopmans et al. [9] suggested
that the prophylactic use of oseltamivir was associated with
the development of anti-influenza A virus antibodies. This
may be regarded as a false-positive result caused by the
presence of unknown agents. Clarification of the MNT-
positive results in Ibaraki as true positives or false positives
is required.
The MNT is a sensitive and specific assay suitable for
the identification of virus-specific antibodies [20], but it is
time-consuming and laborious. Alternatively, the HI test is
a simple and convenient method for the detection of anti-
influenza virus antibodies [18]; moreover, it possesses the
advantage of detecting antibodies against avian viruses,
such as those of the H5 or H7 subtype, by using horse
erythrocytes [12, 21].
In this study, we hypothesized that the administration of
TamifluÒ might affect the result of the MNT to some
extent; moreover, the relationship between TamifluÒ
administration and the results of the serological tests were
investigated.
Before initiating this investigation, a research committee
was established in the Ibaraki prefectural government, and
informed consent was obtained from each subject.
The volunteers (ten) comprised six males and four
females. They were aged between 39 and 58 years
(Table 1). They received a dose of 1 capsule of TamifluÒ
(it included 75 mg of OP; F. Hoffmann-La Roche, Basel,
Switzerland). Blood samples were collected prior to
administration of the drug (0 h) and at 4, 7, and 24 h after
administration. Prior to serological testing, the serum
samples were treated with receptor-destroying enzyme
(RDE II; Denka seiken, Tokyo, Japan).
The H5N2 subtype of influenza virus strain, A/chicken/
Ibaraki/1/05 [16], was kindly provided by the Division of
Virology, National Institute of Animal Health, Ibaraki,
Japan. The virus was propagated in 10-day-old hen’s eggs.
MNT was performed according to the method previ-
ously described [26]. The neutralization titer was the
reciprocal of the highest dilution of serum that inhibited a
50% tissue culture infection dose. The HI test was also
performed according to the methods described previously
[26] using horse, guinea pig, human O-type [0.75% in
phosphate buffered saline, PBS(-)], chicken, goose, and
turkey erythrocytes [0.5% in PBS(-)]. The HI titer was the
reciprocal of the highest dilution of serum that inhibited
hemagglutination. A hyper-immunized anti-A/duck/Hong
Kong/342/78 strain (H5N2 subtype) goat serum was used
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Arch Virol (2008) 153:945–949
as a positive control in these tests; it was kindly provided
by the Division of Virology III, National Institute for
Infectious Diseases, Tokyo, Japan. Examinations were
repeated at least twice.
As shown in Table 1, the sera obtained from volunteers
No. 1 and 2 showed a neutralization titer of 380 when
tested at 4 and 7 h after TamifluÒ administration, respec-
tively, and the specimens obtained from volunteers No. 6
and 9 showed a titer of 20. On the other hand, sera from the
remaining volunteers showed a titer of \10.
The serum samples taken at 4 and 7 h after TamifluÒ
administration showed a transient increase in the HI titer
when mammalian erythrocytes were used (Table 1).
However, most specimens showed a titer of \10 when
avian erythrocytes were used.
In the H5N2 virus outbreak in Ibaraki prefecture, Japan,
an MNT was performed to detect anti-H5 antibodies in the
serum samples of employees engaged in epidemic control,
which clarified that some samples were seropositive for the
H5N2 influenza virus [19]. This may be a true-positive
result due to the presence of the H5N2 influenza virus or a
false-positive result due to the presence of unknown agents.
We hypothesized that the administration of TamifluÒ might
have affected the results of MNT because no clinical
symptoms were observed and no viral agents were isolated
from the patients.
In the MNT, volunteers No. 1, 2, and 6, whose MNT
titers were higher than those of the other subjects, were
all medical doctors. It has been epidemiologically sug-
gested that poultry workers and veterinarians are exposed
to some subtypes of influenza viruses [2, 15]. Moreover,
in Great Britain, a significant increase in HI titer was
observed in 23% of health care workers; this was pos-
sibly due to exposure to influenza viruses [6]. These
results suggest that individuals engaged in particular
occupations might be at a higher risk of exposure to
certain pathogens, including influenza viruses, than the
individuals described above. High MNT and HI titers can
be attributed to infection by the H5 subtype of the
influenza virus; however, this increase in the MNT titer
could also be due to other unknown agents in the serum,
suggesting that further studies are required to explain this
phenomenon.
In the HI test, samples taken at 4 and 7 h after TamifluÒ
administration showed higher titers than those taken at 0 h
(Table 1); these high titers could be caused by the presence
of the components in serum samples. It is reported that the
biological halftime of OC in human plasma is 6–10 h, and
that the highest concentration of OC is observed at 3–4 h
after administration [7]. These results suggest that OC
remained in the serum samples taken at 4 or 7 h after
administration; this may influence the attachment of virus
particles to mammalian erythrocytes in the HI test.
Arch Virol (2008) 153:945–949 947

Table 1 Results of the microneutralization and hemagglutinin-inhibition tests performed using human specimens
Volunteer Time after Sex Age Viral antigen: A/chicken/Ibaraki/1/2005 (H5N2)
No. medication (h) (Ma/Fb)
Neutralization HI titer with erythrocytes derived from the following animal speciesd
titerc
Horse Guinea pig Human (O-type) Chicken Goose Turkey

1 0 M 58 20 40 20 40 \10 10 \10
4 80 160 160 160 \10 20 10
7 40 160 160 160 \10 20 10
24 40 40 40 80 \10 \10 10
2 0 M 54 10 20 40 160 \10 \10 \10
4 40 80 160 320 \10 \10 \10
7 80 80 160 320 \10 \10 \10
24 40 20 80 80 \10 \10 \10
3 0 M 44 10 \10 \10 40 \10 \10 \10
4 10 80 160 160 \10 \10 \10
7 \10 40 80 160 \10 \10 \10
24 \10 10 20 20 \10 \10 \10
4 0 F 45 10 10 10 20 \10 \10 \10
4 10 80 160 160 \10 \10 \10
7 10 80 80 80 \10 \10 \10
24 10 10 40 20 \10 \10 \10
5 0 F 39 10 10 10 10 \10 \10 \10
4 \10 80 160 160 \10 \10 \10
7 10 40 160 160 \10 \10 \10
24 \10 20 20 40 \10 \10 \10
6 0 M 51 20 10 10 10 \10 20 20
4 20 80 320 320 10 10 10
7 20 80 160 160 10 \10 \10
24 20 10 40 40 \10 \10 \10
7 0 M 55 \10 10 20 20 \10 \10 \10
4 10 80 160 160 \10 \10 \10
7 10 80 160 160 \10 \10 \10
24 10 20 40 40 \10 \10 \10
8 0 M 50 10 10 10 20 \10 \10 \10
4 10 80 320 160 \10 \10 \10
7 10 80 160 160 \10 \10 \10
24 10 10 40 40 \10 \10 \10
9 0 F 57 10 \10 10 10 \10 \10 \10
4 20 80 160 320 \10 \10 \10
7 10 80 160 160 \10 \10 \10
24 10 20 40 40 \10 \10 \10
10 0 F 40 10 10 10 40 \10 \10 \10
4 10 80 320 320 \10 \10 \10
7 10 80 160 160 \10 \10 \10
24 \10 20 40 40 \10 \10 \10
Positive controle 1,600 1,600 3,200 800 400 400 400
a
Male
b
Female
c
Neutralization titer is expressed as the highest dilution of serum that inhibited a 50% tissue culture infection dose of the virus
d
HI titer is expressed as the highest dilution of serum that inhibited hemagglutination
e
Hyper-immunized anti-A/duck/Hong Kong/342/78 strain (H5N2 subtype) goat serum was used as a positive control

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The most important results in this study were the false-
positive results of the HI test with the sera obtained from
TamifluÒ-administered individuals. In the case of all the
volunteers in this experiment, samples taken at 4 and 7 h
showed higher titers than those taken at 0 h after TamifluÒ
administration. However, in the MNT, which is expected to
demonstrate higher specificity and sensitivity than the HI
test [20], samples obtained from volunteers No. 3, 4, 5, and
7–10 showed a low neutralization titer (Table 1). These
results also suggested that the elevation in the HI titer was
caused by the presence of OP or OC in the serum samples.
Moreover, the samples reacted to all types of mammalian
red blood cells used in this study in a similar pattern,
suggesting that this combination may cause the results to
be overestimated or misread.
These results also strongly suggest that HI tests using
mammalian cells and avian viruses should be performed
with great care. In the previous study, it was found that the
recently isolated human influenza viruses lost their ability
to agglutinate chicken erythrocytes [14]; this may have led
to the preferential use of mammalian cells in the HI test. In
order to eliminate agents causing nonspecific reactions,
investigators should examine components of serum sam-
ples, i.e., drugs or any other chemical components, which
are heat-stable and are not influenced by receptor-
destroying enzyme treatment.
For further study, it is recommended that the amount of
OP and OC in serum samples be detected and quantified.
Moreover, it is also important to determine the mechanisms
that influence the results of the HI test or MNT after
TamifluÒ administration. In the previous study, it was
shown that genetic mutations near the receptor-binding site
of influenza virus hemagglutinin molecules resulted in a
decrease in the binding of enzymes and susceptibility to
neuraminidase inhibitors, including oseltamivir [13], sug-
gesting a close relationship between hemagglutinin and
neuraminidase molecules on virus particles. This offers an
important insight into this phenomenon.
Acknowledgments We thank the members of the Division of
Virology III, National Institute for Infectious Diseases, for kindly
providing sera, and the members of the Division of Virology,
National Institute of Animal Health, Tsukuba, Ibaraki, Japan, for
kindly donating the influenza virus strain.
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