Download Epidemiological, Demographic, and Molecular Characteristics of

Jpn. J. Infect. Dis., 63, 239­245, 2010
Original Article
Epidemiological, Demographic, and Molecular Characteristics of
Laboratory­Confirmed Pandemic Influenza A (H1N1)
Virus Infection in Turkey, May 15–November 30, 2009
Mustafa Ertek, Rƒza Durmaz*, Dilek Guldemir, A Basak Altas,
Nurhan Albayrak, and Gulay Korukluoglu
Refik Saydam National Public Health Agency, Ankara, Turkey
(Received March 15, 2010. Accepted May 18, 2010)
SUMMARY: A total of 19,973 clinical specimens obtained from suspected cases of pandemic influenza
A virus infection were analyzed by real­time reverse transcription­polymerase chain reaction. Mutations
in hemagglutinin (HA) gene and alteration at position 275 in neuraminidase (NA) gene of the randomly
selected 29 isolates were detected by sequencing analysis. The virus RNA was detected in 47.3z of the
clinical specimens. The pandemic flu cases increased from the 42nd week and peaked in the 46th week of
2009. This intensity continued to the end of the study period. Pandemic flu mainly affected children in
the 5–14 year age group, without any gender predominance. The analyzed strains had À98.9z homolo­
gy with vaccine strains and with each other. More than 37z of the isolates had mutation at position
D222E/N on HA gene. There was no isolate harbored mutation at the position H275Y of the NA gene,
indicating that the virus isolates currently circulating in Turkey are sensitive to oseltamivir.
the World Health Organization (WHO) Regional Office
for the Americas (5,878 deaths) followed by Europe
(À918 deaths), South­East Asia (766 deaths), Western
Pacific (706 deaths), the Eastern Mediterranean (392
deaths), and Africa (108 deaths) (5). In Turkey, the first
laboratory­confirmed human case was reported on May
15, 2009. This and the first few cases that followed were
imported from outside of the country. Until the 42nd
week of 2009, the number of cases originating from
various provinces of Turkey was at a limited level. After
the beginning of 2009–2010 education period epidemics
were reported in various elementary schools, and by the
end of the 48th week of 2009 the number of laboratory­
confirmed cases reached more than 9,500.
In Turkey, all pandemic influenza A (H1N1) virus in­
fections were reported to the Ministry of Health (MoH).
Nasopharyngeal and/or nasal swabs of the suspected
cases were sent to one of the following laboratories:
Refik Saydam National Public Health Agency
(RSNPHA), National Influenza Center in Ankara, Na­
tional Influenza Reference Laboratory at Istanbul Uni­
versity, Faculty of Medicine in Istanbul; and regional
public health laboratories. At first, clinical specimens
were obtained from all suspected cases. However, since
the number of cases increased tremendously after the
47th week of 2009, clinical specimens were collected
only from patients requiring hospitalization and from
outbreak cases. The clinical samples have been tested by
real­time reverse transcription­polymerase chain reac­
tion (RT­PCR). Data regarding demographic,
epidemiological, and clinical information of the sus­
pected cases are recorded by local healthcare institutes
and the patients' information forms are sent to labora­
tories together with the clinical specimens.
Since the first case was described in California, sever­
al investigations have been performed in many different
countries to analyze clinical, epidemiological, and
INTRODUCTION
Pandemic influenza A (H1N1) virus is a member of
the orthomyxoviruses, and has an envelope and a
negative­sense segmental RNA genome. Because of the
segmental structure of RNA, mutation and reassort­
ment of the gene segment have frequently occurred
among different human and animal strains of the in­
fluenza A virus. Therefore, several pandemic strains of
the influenza A virus such as Spanish influenza H1N1 in
1918–1920, Asian influenza H2N2 in 1957–1958, and
Hong Kong influenza H3N2 in 1968–1969, have oc­
curred at intervals of 10 to 50 years since the beginning
of the 19th century (1). A new pandemic influenza A
(H1N1) virus was described on April 15 and April 17,
2009 in specimens obtained from two epidemiologically
unrelated patients in the United States (2). This virus
contains gene segments from human, swine, and avian
strains (2,3). Two of the polymerase components (PB2
and PA genes) originate from an avian virus, one (PB1)
from the human H3N2, hemagglutinin (HA),
nucleoprotein (NP), and nonstructural (NS) genes
originate from the classical swine virus, and neuramini­
dase (NA) and matrix/membrane proteins (M) genes
originate from Eurasian swine virus (4).
Pandemic influenza A (H1N1) virus infection has
continuously increased worldwide. Up to the 48th week
of 2009, more than 207 countries around the world
reported laboratory­confirmed cases, including more
than 8,768 deaths. The highest mortality was reported in
*Corresponding author: Mailing address: Refik Saydam
National Public Health Agency, Molecular Microbiology
Reseach and Applied Laboratory, Cemal G äursel, Ankara,
Turkey. Fax: {90 312 4582348, Tel: {90 312 4582481,
E­mail: rizadurmaz—ymail.com
239
molecular characteristics of this pandemic influenza A
(H1N1) virus (6–10). Molecular studies are critical for
distinguishing 2009 pandemic influenza A (H1N1) virus
from other circulating influenza viruses. Furthermore,
molecular analysis of this new virus is critical for the
monitoring of modifications in virus genome related to
pathogenesis, and susceptibility to antiviral drugs. All
such efforts provide significant improvements to the ef­
fective management of patients and development of
more protective vaccines to control the infection.
In Turkey, up to now there has been only one pub­
lished study reflecting the characteristics of pandemic
influenza A (H1N1) virus infections; a study of the first
128 cases diagnosed from April 29 to July 17, 2009 (11).
The aim of the current study was to gain an insight to
the epidemiological and demographic characteristics of
the laboratory­confirmed cases diagnosed from May 15,
2009, the date of the first index patient, to the end of the
48th week of 2009, and to search mutations in the recep­
tor­specific region of the HA1 domain and resistance to
NA inhibitor of the 29 viral strains randomly selected
among the isolates of the patients admitted to
RSNPHA, Ankara, Turkey.
performed using the ABI 7500. The 25­ml PCR mixture
contained 5 ml of extracted RNA, 1 ml each of forward
and reverse primers, 1 ml probe, 0.5 ml SuperScript III
RT/Platinium Taq DNA polymerase mix, 12.5 ml of 2~
Master mix, and 4 ml nuclease­free water. Amplification
conditions were as follows; reverse transcription at
509
C for 30 min, Taq inhibitor activation at 959
C for 2
min and 45 cycles at 959C for 15 s, 559
C for 30 s.
(iii) Nucleotide sequence of HA gene: Sequences of
HA gene of 29 strains randomly selected among the
laboratory­confirmed cases were determined using the
forward and/or reverse primers designed by the WHO
for sequencing of this virus. Information of the primers
is available at http://www.who.int/csr/resources/
publications/swineflu/GenomePrimers_20090512.pdf.
The extracted RNA was amplified by in­house RT­PCR
using the one­step RT­PCR system (Invitrogene,
Carlsbad, Calif., USA). Each segment of HA gene was
first amplified using fragment­specific reverse and for­
ward primers of HA gene. Amplification conditions
were as follows: cDNA synthesis at 489C for 45 min and
initial denaturation at 949C for 2 min and 30 cycles at
949
C for 20 s, 509C for 30 s, 729C for 1 min with an ad­
ditional extension step at 729
C for 7 min. After purifi­
cation of amplicons with Agencourt Ampure (Beckman
Coulter, Beverly, Mass., USA), sequence reactions were
setup with M13 primers. Each sequence reaction mix­
ture consisted of 3.5–5 ml of purified amplicon, 5 pmol
primer, and 4 ml of Dye terminator cycle sequencing
quick start kit (Beckman Coulter). The sequence reac­
tion was done as follows: initial denaturation at 949C
for 3 min followed by 30 cycles consisting of denatura­
tion at 969
C for 20 s, annealing at 559C for 20 s, and
elongation at 609C for 4 min. The PCR products were
purified with Dye­Terminator removal kit (Agencourt
Cleanseq; Beckman Coulter) and 20 ml of purified
product was sequenced in the Beckman Coulter 8000
instrument. The sequence results were subjected to
BLAST analysis using the National Center for
Biotechnology Information (NCBI) GenBank. The base
numbering of HA gene in the present study is for H1
gene. Therefore, position 222 in H1 HA corresponds to
position 225 in H3 HA.
(iv) Nucleotide sequence for NA mutation: Since a
mutation which results in a histidine­to­tyrosine substi­
tution at position 275 (H275Y) in the NA gene, is known
to be associated with oseltamivir resistance (12,13), a set
of primers was used to determine the alterations at this
position of the NA gene (14). The viral RNA was ex­
tracted from the original clinical specimens or Madin­
Darby canine kidney (MDCK) cell isolates. Amplifica­
tion and sequencing conditions of NA gene were similar
to those used for HA gene, except the used primers. Se­
quence data was analyzed to find out whether there was
a mutation at codon 275 or not. The ``CAC'' base se­
quence at position 275 was evaluated as susceptible to
oseltamivir. If the strain was resistant to oseltamivir,
this base sequence would be ``TAC'' or ``TAT'' (12).
Statistical analysis: The distributions of pandemic in­
fluenza A (H1N1) virus positivity among different age
groups and genders were compared by x2 test. Homolo­
gy between the sequence results of the studied isolates
and A/California/07/2009 (H1N1) strain and homolo­
gy of each isolate with the other studied isolates were
MATERIALS AND METHODS
Patients: In this study, a total of 19,973 clinical speci­
mens obtained from the cases suspected with pandemic
influenza A virus infection were analyzed from the 18th
week to the end of the 48th week of 2009. Nasal or
nasopharyngeal samples were taken from suspected
cases. All clinical samples were inserted into viral trans­
port medium (Virocult; Medical Wire & Equipment,
Wiltshire, UK) and transported to the laboratories in a
triple biological safety system by courier. Patients' in­
formation including age, gender, work place, school,
city of residence, and clinical findings were recorded in
a form prepared by the MoH and sent to the laboratory
with the clinical specimens.
Laboratory diagnosis: (i) RNA extraction: The clini­
cal specimens arriving in transport medium were gently
mixed by vortex and 200 or 400 ml of the samples were
transferred into a 1.5­ml microcentrifuje tube in a bio­
logical safety cabinet. Following the kits instructions,
RNA extraction was performed using either a Total
Nucleic Acid Isolation Kit (Roche, Mannheim, Germa­
ny) or an EZ1 Virus Mini Kit (Qiagen GmbH, Hilden,
Germany).
(ii) PCR amplification: Search for viral RNA was
conducted using either an in­house real­time RT­PCR
protocol provided by the Centers for Disease Control
and Prevention (CDC) or a commercial real­time RT­
PCR kit (Qiagen). The commercial kit provided the op­
portunity to search an 80­base pair region of pandemic
influenza A virus (H1N1) using the Rotor­Gene 6000
instruments. Using in­house real­time RT­PCR method,
the clinical samples were first analyzed for influenza A
M gene, pandemic influenza A (H1), and for RNaseP as
an external internal control in the same run. If any sam­
ple yielded a positive result for influenza A virus but
was negative for pandemic influenza A (H1), those sam­
ples were tested for seasonal influenza A (H1) and in­
fluenza A (H3), using subtype­specific primers for the
HA gene segments. In­house real­time RT­PCR was
240
evaluated using MEGA 4.1 software (Beta 3) Alignment
Explorer. The available HA gene sequence results of the
studied isolates were compared with the same length of
the corresponding HA gene sequences of the A/Califor­
nia/07/2009 (H1N1) virus strain and we also compared
the sequence results of the studied isolates with each
other. Homology was calculated as a percentage in ac­
cordance with the number of identical nucleotides be­
tween the compared sequences.
RESULTS
Fig. 1. Weekly distribution of the pandemic influenza­positive
cases. Since laboratory test was performed only on the patients
having hospitalization indication in the 48th week, the number
of cases in this week seems lower than the previous weeks.
A total of 9,459 laboratory­confirmed pandemic in­
fluenza A (H1N1) virus infections were defined among
the 19,973 clinical specimens analyzed in Turkey up un­
til the end of the 48th week of 2009. The overall rate of
laboratory­confirmed cases was 47.36z. Up to the 42nd
week of 2009, pandemic influenza cases were reported
rarely and from a limited number of provinces; mainly
in the two biggest cities, Ankara and Istanbul. The first
reports of local outbreaks from primary schools were in
the 42nd week of 2009. In the 43rd week, the number of
cases increased tremendously and reached the highest
level during the 46th week of the year (Fig. 1). By the
48th week of 2009, all 81 provinces in Turkey had con­
firmed pandemic influenza cases, with 33.3z of cases
collected from Ankara and Istanbul.
Demographic data was available for the 9,588 sus­
pected cases analyzed during the study period. More
than half of these cases (51.9z) belonged to two age
groups, 5–14 years old (n  2,380, 24.8z) and 25–44
years old (n  2,600, 27.1z). The 15–24 year age group
included 19.4z (n  1,861) of the suspected cases. The
sampling rates for the other age groups were as follows:
10.9z (n  1,041) in 1–4 years old, 10.1z (n  966) in
45–64 years old, 4.1z (n  397) in Ã1 year old; and
3.6z (n  343) in À64 years old. Of the 9,588 suspect­
ed cases, 4,121 were real­time RT­PCR positive. The
mean age of the 4,121 PCR­positive cases was 20 years
and 48.5z of them were male. The highest PCR positiv­
ity rate belonged to the 5–14 year age group (30.5z),
followed by the 15–24 year age group (21.9z), the
25–44 year age group (20.7z), the 1–4 year age group
(10.6z), the 45–64 year age group (7.5z), the 0–11
month age group (3.4z), and the Æ65 year age group
(2.0z) (Fig. 2). The positivity rates of the 5–14, 15–24,
and 25–44 year age groups were significantly higher
than the rates of the other age groups (P  0.001).
HA genes of the 29 pandemic influenza A (H1N1)
viruses were sequenced. Of these 29 strains, 14 belonged
to patients diagnosed in summer, 2009; the remaining 15
were selected from among the positive cases in autumn,
2009. These strains were randomly selected from differ­
ent parts of Turkey. Twenty of the isolates were ob­
tained from males and nine were from females. Eight of
these pandemic virus isolates belonged to the deceased
patients. Based on the number of identical nucleotides
between the compared sequences, there was 98.96–
100z homology between California strains and the test­
ed isolates (Table 1). Moreover, genetic homology be­
tween each of the tested isolates ranged from 99.01 to
100z.
Mutation analysis of the receptor binding domain of
HA1 gene was available for 25 strains. Nine of the iso­
Fig. 2. The sampling rate and pandemic influenza A (H1N1) vi­
rus positivity among the different age groups. The highest sam­
pling rate belonged to the 25–44 year age group, while the
highest positivity rate was obtained in the 5–14 year age group.
lates (37.5z) had mutations at position D222E/N and
P83S. Eight of the nine mutant isolates had D222E al­
teration, and one had D222N alteration. In addition to
these mutations, these isolates also had mutations at po­
sition P83S, S203T, and I302V (Fig. 3). Two of these
mutant isolates belonged to the deceased patients, two
isolates were obtained from females, and six were from
males (Table 1).
Among the 29 pandemic influenza A (H1N1) virus
isolates, there were no isolates with amino acid substitu­
tion at position 275 in the NA gene. All isolates had a
base sequence of ``CAC'' at the 275th codon. The ana­
lyzed virus strains belonged to patients who had not
been treated previously with oseltamivir. Demographic
and epidemiological characteristics of the patients are
shown in Table 1.
DISCUSSION
This study was conducted to analyze the epidemiolog­
ic and demographic characteristics of pandemic A in­
fluenza (H1N1) virus infections in Turkey between May
15 and November 30, 2009, and to determine the muta­
tions in the HA gene and resistance to oseltamivir
among the selected isolates. During the study period,
more than 47z of the study population had confirmed
pandemic influenza A (H1N1) virus infection which
241
Table 1. Epidemiologic, demographic, and sequencing data of the 29 pandemic influenza A (H1N1) virus strains
Patient
no.
882
1304
1057
1230
1082
1247
1585
1808
1850
1595
1862
1712
1845
1329
1712
1637
1667
2081
2078
2074
2070
5341
5440
5441
5492
11402
8051
8245
9738
1):
2):
Gender/age
M/21
M/24
M/12
M/28
M/28
F/52
M/15
M/25
M/20
M/27
M/7
M/21
F/22
F/14
F/14
M/11
M/11
F/0
M/20
F/42
F/12
F/65
M/14
F/2
M/3
M/27
M/4 mo
M/3 mo
F/27
Sampling Localization Outcome
date
NCBI
Available
Homology with
Mutation at
Mutation at
Accession sequence length A/California/07/2009 H275Y in position 222 in
no.
(base pair)
(z)1)
NA gene
HA1 domain2)
06.01.09
07.15.09
06.22.09
07.08.09
06.24.09
07.10.09
08.09.09
08.31.09
09.04.09
08.09.09
09.05.09
08.19.09
09.04.09
07.17.09
09.12.09
08.13.09
08.14.09
10.14.09
10.13.09
10.13.09
10.13.09
10.31.09
11.01.09
11.01.09
11.02.09
11.24.09
11.11.09
11.12.09
11.18.09
GU369649
GU369646
GU369647
GU369648
GU369650
GU369651
GU369652
GU369653
GU369654
GU369655
GU369656
GU369657
GU369658
GU369659
GU369660
GU369661
GU369662
GU369663
GU369664
GU369665
GU369666
GU369674
GU369667
GU369668
GU369669
GU369670
GU369671
GU369672
GU369673
Ankara
Ordu
Denizli
Ankara
Ankara
Ankara
Mersin
Ankara
Ankara
Istanbul
Ankara
Ankara
Ankara
Ankara
Ankara
Hatay
Adana
Ankara
Ankara
Ankara
Ankara
Ankara
Konya
Konya
Batman
Ankara
Ankara
Urfa
Kayseri
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Recovered
Died
Died
Died
Died
Died
Died
Died
Died
364
1701
1754
1701
1770
386
1783
1356
1782
1782
1770
1748
732
1647
1781
1600
1776
1769
1782
1787
1782
1746
881
922
408
792
1782
1774
1782
100.00
99.35
99.64
99.52
99.58
98.96
99.58
99.33
99.52
99.52
99.52
99.58
99.45
99.43
99.52
99.43
99.41
99.52
99.41
99.41
99.41
99.52
99.54
99.42
99.26
99.11
99.35
99.52
99.41
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Not available
No
No
D222E
No
Not available
D222E
No
No
No
D222E
No
D222E
D222E
D222E
No
D222E
No
No
No
No
No
Not available
No
Not available
D222N
No
D222E
No
The homology was calculated using available sequence length and the corresponding region of vaccine strain A/California/07/2009.
D222E, alteration of aspartic acid (D) to glutamic acid (E) at position 222; D222N, alteration of aspartic acid (D) to asparagine (N) at posi­
tion 222; H275Y, alteration of histidine (H) to tyrosine (Y) substitution at position 275; NA, neuraminidase gene; HA, hemagglutinin gene.
obtained in males and females (46.5z versus 48.5z, re­
spectively, P  0.096). However, it has been reported
that this virus mostly affects young adults and persons
aged º24 years (3,7), with the mean age of patients
ranging from 20 to 25 years old (6,8). Similar to this
data, the mean age of our laboratory­confirmed cases
was 20 years old and 66.4z of the positive cases were
Ã24 years old. Additionally, a siginificant portion (one­
fifth) of our cases was in the young adult group,
whereas people aged Æ65 years had the lowest rate. The
lower attack rate of pandemic influenza A (H1N1) virus
infections in older people can be explained by the
presence of a cross­reactive antibody (3,7). Although we
do not have any laboratory­test results regarding the
cross­reactive antibody in our study population, the
lower positive rate in the older age group supports the
view of previous cross immunity within this age group in
Turkey.
Several studies have been performed to evaluate the
mutations which may result in resistant and/or more
virulent strains during the spread of this new virus
among human populations (16–18). Researchers have
mainly focused on monitoring the mutations in HA
gene which is very important for viral attachment and
immunization. Sequencing studies showed that muta­
tions have been predominately observed in HA1 domain
spread throughout all of the provinces of Turkey. The
number of cases began to increase in the 42nd week of
2009, reaching its peak in the 46th week, with this in­
crease continuing for the duration of the study period.
Children aged 5–14 seemed to have an increased risk for
contracting pandemic flu. Gender was not considered to
be a significant risk factor for pandemic influenza A
(H1N1) virus infection. Sequencing of the HA gene
showed that Turkey had a homogenous pandemic in­
fluenza A (H1N1) virus population with more than
98.9z similarity between the vaccine strain and the test­
ed strains, with all tested isolates susceptible to osel­
tamivir. Interestingly, more than 37z of the pandemic
influenza A virus isolates in Turkey had a mutation at
position D222E/N in the HA1 domain.
Since pandemic influenza A virus is easily transmitted
from infected people to healthy people, its positivity
rate rapidly increases all over the world (5–7,11). In
Turkey, only a few cases were reported in only a limited
number of provinces during the summer. By the start of
the education period, outbreaks were occurring in
elementary schools and the rate of the laboratory­con­
firmed cases increased tremendously to 47z with con­
firmed cases observed all over the country. In agreement
with previous reports (7,15), there were no statistically
significant differences between the PCR positivity rates
242
Fig. 3. Amino acid sequences of the HA1 domain of the HA gene of the 9 clinical isolates having alteration at posi­
tion 222 and A/California/07/2009 (H1N1) strain. Alterations between the clinical isolates and the reference
strain are indicated as the gray background.
quency of D222E substitution did not differ significant­
ly between mild and severe cases, while D222N was ob­
served in very few cases (20). In our study, we were able
to evaluate the mutation in HA1 domain in 24 of the 29
isolates sequenced. We found a mutation at position 222
in the nine isolates (37.5z) with eight of these isolates
having an alteration from aspartic acid (D) to glutamic
acid (E), and one having an aspartic acid­asparagines
(N) alteration. In our study there was no isolate with a
D222G substitution, however D222E substitution seems
to be predominant in Turkish isolates. Although it was
indicated that the clinical significance of the D222E and
D222N substitution remains uncertain (19), two of the
mutant viruses (D222N, D222E) in our study were iso­
lated from deceased patients. Interestingly, we found
mutation of D222N in only one fatal case. Since we only
had one case, it is very difficult to speculate about the
importance of this mutation on the fatality; however
this result supports previous data. Kilander et al. (20)
found D222N alteration in only a few cases in Norway
(4/266 cases), however three of the isolates having this
of the HA gene and mostly clustered in five main anti­
genic regions (16,17). Amino acid changes at codons
138, 163, 187, 189, 190, 194, 225, 226, or 228 (H3 num­
bering) in the HA receptor­linked domain are responsi­
ble for receptor binding capacity to either a2­6­sialyl (N­
acetyllactosamine) residues (a2­6SA; human­linked
receptor) or a2­3­sialyl galactose moiety (a2­3SA; avian
like receptor) (17). Recently, some of these mutations
especially D222G/N/E in HA1 regions (corresponding
to position 225 in H3 HA) have been reported among
the pandemic influenza A (H1N1) virus strains from
different countries, such as Norway, China, Japan,
Ukraine, the United States, and the Netherlands (18).
Although currently available data showed that the
D222G substitution does not appear to pose a major
public health issue, this substitution has been frequently
observed in the HA gene of pandemic influenza A
(H1N1) viruses isolated from severe and fatal cases in
several countries (19). While the prevalence of D222G
substitution is º1.8z, it increased to 7.1z among the
isolates of fatal cases (19). On the other hand, the fre­
243
mutation were isolated from severe and fatal cases. In
Ukraine, two of the five sequenced viruses had D222N
substitution and both of these viruses were isolated
from fatal cases (http://www.recombinomics.com/
News/12090901/D225N_Ukraine.html). In Brazil, two
isolates of fatal cases also had D222N substitution
(http: //www. recombinomics. com / News / 11090902 /
Ukraine_1918.html). Although the number of strains
analyzed in our study was limited, since our sequenced
isolates were collected from patients of different ages,
gender, geographic origin, and survival, we can con­
clude that these results may represent the current situa­
tion of Turkish isolates. However, further analyses on
additional strains from different patients including fatal
cases must be continued to highlight the real frequency
of the mutation at 222 and the relationship between mu­
tations and virulence.
Comparison of the sequence data of the patients' iso­
lates to the vaccine virus A/California/07/2009 (H1N1)
provides valuable information for understanding the ef­
ficacy of the vaccines. In the WHO report Pandemic
(H1N1) 2009­update 78, it was reported that all pan­
demic influenza viruses analyzed worldwide were anti­
genically and genetically closely related to the vaccine
strain (18). In concurrence with this data, the sequenc­
ing results of the 29 pandemic A (H1N1) virus isolates in
Turkey were closely related to the sequence of the vac­
cine strain. The homology rate was similar among the
isolates collected from different provinces, gender,
ages, and patient outcome. This data showed that the
current pandemic A virus vaccines cover all pandemic
virus strains that were circulating in Turkey.
Based on present data, it has been accepted that pan­
demic influenza A (H1N1) strains are mainly susceptible
to NA inhibitor but resistant to adamantine (21).
However, once oseltamivir resistance develops, this
resistant virus becomes predominant (22). For this rea­
son, tracking oseltamivir resistance among pandemic in­
fluenza A virus isolates is of great importance. Up to
December 11, 2009, a total of 102 oseltamivir­resistant
isolates were reported from 31 countries, and worldwide
more than 10,000 isolates tested for oseltamivir
resistance were found to be sensitive (18). A study which
conducted analysis of more than 1,000 pandemic in­
fluenza A isolates found that only 3 strains from Den­
mark, China, and Japan were resistant to oseltamivir
but susceptible to zanamavir (23). It was reported that
the patients infected with these resistant variants did not
have severe illness and all recovered. A case report study
described a 16­year­old girl who was infected with an
oseltamivir­resistant pandemic influenza virus in Hong
Kong (21). In the Netherlands, oseltamivir resistance
was found in 11 strains, 5 of which were isolated from
fatal cases (http://www.recombinomics.com/News/
12110901/H274Y_Fatal_Netherlands_Spike.html). On
the other hand, similar to our study, in New Zealand all
pandemic influenza A (H1N1) virus strains tested were
found to be as sensitive to oseltamivir (24). Additional­
ly, the 13 isolates of the patients diagnosed in California
were tested for resistance to antiviral drugs and all
viruses were susceptible to oseltamivir and zanamivir
(13). Similar to the previous studies, our data confirmed
that the NA inhibitors are important antiviral drugs for
controlling influenza A (H1N1) virus epidemics and/or
pandemics in Turkey (25). However, since some coun­
tries reported mutations at position H275Y and there is
a possibility of transmission of the resistant isolates to
other patients, oseltamivir resistance should be closely
followed, especially in patients at risk of developing
resistance. Patients having prolonged postexposure
prophylaxis or treatment with subtherapeutic dosages
are at risk of developing resistance to oseltamivir
(26,27).
In conclusion, pandemic influenza A (H1N1) virus
mainly affected children aged 5–14 years old in Turkey,
without any gender predominance. Molecular studies
indicated that the current vaccine prepared from
A/California/07/2009 (H1N1)­like virus covered all
pandemic A (H1N1) virus strains in Turkey, and all
pandemic influenza A (H1N1) viruses currently circulat­
ing in Turkey were susceptible to oseltamivir. Observa­
tion of mutations in the receptor binding domain ena­
bles us to closely track alterations in new isolates in Tur­
key.
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