Received: 20 June 2017 | Accepted: 24 October 2017

DOI: 10.1002/jmv.24982

RESEARCH ARTICLE

Molecular and epidemiological analysis of pandemic

and post-pandemic influenza A(H1N1)pdm09 virus from
central India

Mahima Sahu1 | Neeru Singh1 | Mohan K. Shukla1 | Varhsa A. Potdar2 |

Ravendra K. Sharma1 | Lalit Kumar Sahare1 | Mahendra J. Ukey1 |
Pradip V. Barde1

1 NationalInstitute for Research in Tribal

Health (NIRTH), ICMR, Jabalpur, Madhya
Prdesh, India
2 National
Institute of Virology, Pune,
Maharashtra, India
Correspondence
Pradip V. Barde, National Institute for
Research in Tribal Health (NIRTH), Jabalpur
482003, Madhya Pradesh, India.
Email: pradip_barde@hotmail.com
Funding information
Indian Council of Medical Research,
Grant number: VIR/43/2011-ECD-1; MP
State Health and Family Welfare Department,
Grant number: IDSP/15/600
Influenza A(H1N1)pdm09 virus pandemic struck India in 2009 and continues to cause
outbreaks in its post-pandemic phase. Diminutive information is available about
influenza A(H1N1)pdm09 from central India. This observational study presents
epidemiological and molecular findings for the period of 6 years. Throat swab samples
referred from districts of Madhya Pradesh were subjected to diagnosis of influenza
A(H1N1)pdm09 following WHO guidelines. Clinical and epidemiological data were
recorded and analyzed. Hemagglutinin (HA) gene sequencing and phylogenetic analysis
were performed. The H275Y mutation responsible for antiviral resistance was tested
using allelic real-time RT-PCR. Out of 7365 tested samples, 2406 (32.7%) were positive
for influenza A(H1N1)pdm09, of which 363 (15.08%) succumbed to infection.
Significant trends were observed in positivity (2 = 50.8; P < 0.001) and mortality
(2 = 24.4; P < 0.001) with increasing age. Mutations having clinical and epidemiological
importance were detected. Phylogenetic analysis of HA gene sequences revealed that
clade 7, 6A, and 6B viruses were in circulation. Oseltamivir resistance was detected in
three fatal cases. Influenza A(H1N1)pdm09 viruses having genetic diversity were
detected from central India and continues to be a concern for public health. This study
highlights the need of year-round monitoring by establishment of strong molecular and
clinical surveillance program.

KEYWORDS
central India, epidemiology, influenza A(H1N1)pdm09, mutations, phylogenetic analysis

1 | INTRODUCTION
Influenza-like illness (ILI), leading to severe acute respiratory infections
1
(SARI), is the fourth leading cause of death across the world and
responsible for over four million deaths annually.2 It is estimated that
18% of global ARI deaths are due to Influenza viruses; thus, influenza is a
major contributor to disease burden due to SARI.3 The influenza
A(H1N1)pdm09 (A[H1N1pdm09]) virus responsible for 2009 pandemic
arose from multiple re-assortment involving genes from avian (PB2, PA),
human (PB1), classical North American swine (HA, NP, NS), and Eurasian
swine (NA, M) viruses.4 The virus was first detected in April 2009 from
the state of Veracruz, Mexico and rapidly spread over the world
affecting more than 200 countries.5 World Health Organization (WHO)
declared pandemic in June 2009 and in August 2010, declared that the
pandemic is over; however, it warned that the local and focal outbreaks
will continue to occur.6 In India, the first case of A(H1N1)pdm09 was

J Med Virol. 2017;19. wileyonlinelibrary.com/journal/jmv 2017 Wiley Periodicals, Inc. | 1

2 |
detected in May 2009 from Hyderabad, Andhra Pradesh and the first
death was recorded at Pune, Maharashtra in August 2009.7 Subse-
quently, cases were reported across the country and so far more than
100 thousand laboratory-confirmed cases and over 7700 deaths
have been reported.8,9 The demographic and seasonal patterns of
influenza in tropical and subtropical regions differ every year and this
seasonal fluctuations correlate with factors like connectivity, herd
immunity, social behavior practices, humidity, indoor crowding,
temperature at particular latitude.10,11 In central India, peak influenza
activity is observed during monsoon and post-monsoon season, though
waves were also recorded during winter in recent past.11
The paradigm of shifts and drifts is characteristic of Influenza A virus
and has clinical and epidemiological implications. The A(H1N1)pdm09
12
is divided into eight clades. Viruses belonging to different clades
were detected from India; in the year 2009, clade 1 and in subsequent
years clade 2, 3, 5, 6, 7 viruses were reported circulating.1315 During
recent past, clade 6 viruses became predominant.15,16
It is reported that amino acid changes in different genes at defined
positions such as D222N/G mutation in HA gene which is responsible
for altered receptor binding17,18; the K163Q change that it is
hypothesized to lead escape from neutralizing antibody19 and
A197T that lead to improve human glycan and the intra-host evolution,
2022
leading to the generation of quasispecies may increase severity.
The H275Y amino acid change in NA gene, that is, responsible for
antiviral resistance is also detected in the viruses circulating in India.23
Therefore, it is not only important to monitor and characterize the
circulating virus at molecular level but also to generate the information
regarding epidemiology of disease that will be helpful for planning the
interventions and focusing on resources. However, very limited
information is available about epidemiological and molecular trends
of A(H1N1)pdm09 from central India.
In response to the 2009 influenza pandemic, Indian Council of
Medical Research (ICMR) established a network of virology laborato-
ries for providing reliable and timely diagnosis. The laboratory for the
state of Madhya Pradesh (MP) was established at National Institute for
Research in Tribal Health. The laboratory conducted this study with
the objective to characterize circulating virus at molecular level and
understand epidemiological trends of the disease over a period of
6 years from November 2009 to October 2015.
2 | M ATERIA LS AN D METH ODS
2.1 | Ethical statement
The study had ethical clearance from institute's ethical committee.
2.2 | Study period, samples, and study area
This is an observational laboratory-based study for the period of
6 years from November 2009 to October 2015. The throat swab
samples from patients suffering with ILI were collected at the state-
authorized hospitals in virus transport medium and were referred to
this laboratory for diagnosis. The clinical and demographic information
SAHU ET AL.
about the patients was collected in a predesigned format.24 Samples
were received from almost all parts of MP (2310N 7713E/23.17N
77.21E/23.17; 77.21), which is geographically second largest state
and situated in central part of the country. The state comprises 51
districts of which 21 are designated as tribal districts.25 Population of
the state is 72 597 565 which is about 6% of country's population.26
July to October is rainy season and November to March is the winter
months when temperature drops upto 0C.
2.3 | Inclusion and exclusion criteria
As per the Ministry of Health and Family Welfare, Government of India
guidelines, category-C patients, that is, the hospitalized person having
severe ILI were considered for diagnosis24 and included in the study.
Sample received with minimal or no clinical and demographic details,
insufficient quantity, transported without maintaining the cold chain,
referred after 8 post-illness days were excluded from the study.
2.4 | Diagnosis and reporting
The TaqMan-based real-time RT-PCR assay, recommended by WHO
for detection A(H1N1pdm09), was used.27 Results were conveyed
electronically to the concerned persons preferably within 24 h of the
receipt of sample. Samples were stored at ultra-low temperature
freezer (70C) for further use.
2.5 | Hemagglutinin gene sequencing and analysis
The HA gene sequencing was performed for identifying genetic lineage
and to characterize molecular changes in circulating strains of A(H1N1)
pdm09. Samples (n = 69) having low Ct values (Ct value <30) in
molecular diagnosis were randomly picked taking care that they
scattered over 6-year period, with seasonal representation (Table 1).
Twenty-two complete and 40 partial (targeting amino acid position 89-
396) HA gene fragments were amplified using one-step RT-PCR as
recommended by WHO.28 PCR products were sequenced using Big
Dye terminator (Cat No. 4337456, Applied Biosystems, Carlsbad, CA)
on ABI 3130xl DNA analyzer (Applied Biosystems). The nucleotide
sequences were analyzed for quality and converted into amino acid for
identifying changes. All the sequences were assembled, aligned, and
analyzed using ClustalW program within MEGA (version 5.05).29 The
sequences were submitted to NCBI data base GenBank.
2.6 | Phylogenetic analysis
Phylogenetic analysis of A(H1N1pdm09) viruses from the study (n = 22)
was done by comparing with contemporary viruses detected from other
parts of the country and WHO reference viruses (n = 63) representing
different clades. Maximum likelihood (ML) tree using the Tamura-Nei
model was generated using phylogeny tools within MEGA (version
5.05).29 Statistical support was provided by bootstrapping 1000
replicates. The tree was rooted with the vaccine component A/
California/07/2009. H1 numbering for HA gene was used throughout.30
SAHU ET AL.
TABLE 1 Details of 34 amino acid changes in 22 complete HA gene sequences of the samples from central India
Clade-specific changes are highlighted. GenBank accession number for 40 partial HA gene sequences were KT369721-369726, KT426699, KT946852-
946867, KT9364777-9364793. In addition to changes above, five changes were observed in partial HA gene sequences as R113K and I267T (KT936483);
S204P (KT936488); Y230X (KT946852, KT946854-55, KT946858-61, KT946864, KT946866); T310K (KT946864).
2.7 | Detection of Oseltamivir resistance
The samples from fatal cases (n = 213) were tested to identify the
H275Y mutation known to be responsible for antiviral resistance using
allelic qRT-PCR as described by Nakauchi et al.31
2.8 | Data analysis
The clinical, demographic, and other information were entered in
Microsoft Excel spreadsheet for primary analysis. We calculated the
cumulative and weekly incidence, overall and age-specific positivity,
mean age, proportion of suspected and laboratory-confirmed cases,
and season-wise cases. The data were further analyzed using statistical
tests such as Chi square (2) and odds ratio (OR) using SPSS var20.
Data regarding fatality were provided by the MP state health
department.
3 | RE SULTS
A total of 7539 samples were received from 39 districts of MP during
the study period, of which 2406 (32.7%) samples received from 35
districts showed A(H1N1pdm09) activity. First case was detected from
Indore in November 2009 and till October 2010, virus spread to 14
districts, and out of 1524 tested samples, 411 (26.9%) were positive. In
the next year, that is, in 2010-2011, 137 samples from eight districts
were tested, of which only four samples were found positive. In the
year 2011-2012, 142 (17.7%) samples from 13 districts were positive.
Whereas in 2012-2013, out of 624 samples from 12 districts, 114
(18.3%) were found positive. In the following year, only 194 samples
were referred for diagnosis of which 12 (6.45%) were found positive;
however, in 2014-2015, an upsurge occurred and 4090 samples from
| 3
suspected individuals from 38 districts were tested, of which 1723
(42.1%) samples from 35 districts were positive.
Proportion of A(H1N1pdm09)-positive cases 1153 (47.9%) and
deaths (n = 204) was noticeably high in the western MP. About 66% of
the deaths and cases were from major cities like Indore (156/ 641),
Bhopal (96/592), Jabalpur (42/ 402), Ujjain (37/155), and Dewas
(0/179). Six-hundred fifty-three (27.7%) A(H1N1)pdm09 cases were
reported from towns and villages of 16 tribal districts of MP.
Samples of all age group ranging from baby of 14 days to 94 years
were referred and the mean age for female patients was 34.5 (SD
19.3) years, while it was 32.6 (SD 22.2) years in case of males.
Children (<14 years) were least affected and significantly low mortality
was noted when compared with higher age groups (OR = 0.39, 95%
CI = 0.34-0.46) (Table 2). The age distribution curve showed that
positivity (2 = 50.8, P < 0.001) and mortality (2 = 24.4, P < 0.001)
were higher among higher age groups.
The males in higher age groups were more affected as compared to
males in younger age group. More females (34.8%) were positive as
compared with males (30.7%), and this difference was statistically
significant (z = 3.75, P < 0.001). Further, more females (16.4%) suc-
cumbed to infection than males (13.7%) but the differences were not
significant (z = 1.85, P = 0.0645). Highest mortality (20.3%) was noted
in females of age group of 15-29 years (Table 2). Furthermore, year-
wise stratification of the data in gender and age revealed that the trends
were similar over 6 years.
In the initial year of 2009-2010, influenza activity was noted to
peak in winter and monsoon followed by drop in the cases till summer
of 2011-2012. Thereafter, activity increased in monsoon of 2012
followed by minor wave in winter of 2012-2013. Subsequently, in the
year 2013-2014 till November, we observed very low activity of
A(H1N1)pdm09. However, resurgence observed in winter of 2014-
4 | SAHU ET AL.

2015 was worst then the winter of pandemic period. During 6 years

120.0 (17.2)

368.0 (15.1)
90.0 (15.6)

98.0 (16.7)
45.0 (19.1)
of study period, three major waves, two in monsoon and one in

12.0 (5.2)

P < 0.001
2.0 (1.9)

1.0 (7.7)
winter of 2015, were noted. Interestingly, we had no case of
Total

24.4
A(H1N1)pdm09 in the month of June in any year (Figure 1).
We observed significant differences in clinical symptoms such as
Deaths among positive cases (%)

fever, cough, and sore throat between suspected and confirmed

205.0 (16.4)
72.0 (20.3)
62.0 (16.8)
46.0 (15.7)
15.0 (13.0)
A(H1N1)pdm09-positive cases (P < 0.001). However, these differ-
8.0 (11.1)

1.0 (20.0)

P = 0.919
1.0 (2.4)
Female

ences were non-significant when symptoms such as nasal catarrh and

0.01
difficulty in breathing (P > 0.05) were compared. Fifteen percent
(363/2406) mortality was noted among laboratory-confirmed cases
of A(H1N1)pdm09 across MP. The mortality was significantly high
58.0 (17.6)
52.0 (17.6)
30.0 (25.0)

163 (13.7)
18.0 (7.7)

when pneumonia was associated with A(H1N1)pdm09 infection

P < 0.001
4.0 (2.7)
1.0 (1.5)

0.0 (0.0)

(OR = 2.58, 95%CI = 1.43-4.68, P < 0.001).

Male

45.1
Out of 62 selected samples, 22 were sequenced for complete HA
gene and 40 were done partially. When we compared amino acid
sequence of these 62 sequences acquired with 2009-2015 vaccine
2406.0 (32.7)
211.0 (18.6)
116.0 (29.9)
551.0 (34.0)
692.0 (39.5)
588.0 (37.9)
235.0 (26.4)

component A/California/07/2009 strain, a total of 39 changes were

P < 0.001
13 (36.1)
Age-wise distribution of tested, positive, and death cases of influenza-A(H1N1)pdm09 (November 2009-October 2015)

detected (Table 1).

Total

50.8

Among the 62 sequences from 29 fatal and 33 survival cases, we

detected K163Q mutation in 23 fatal and 23 survival cases, whereas
A197T was found in one fatal and two survival cases. Among 29 fatal
1237.0 (34.8)

cases, 17 had different amino acid (G = 2, N = 1, and X = 14) than D at

346.0 (37.2)
369.0 (41.3)
293.0 (38.5)
115.0 (28.4)
63.0 (16.2)
46.0 (27.8)

5.0 (35.7)

P < 0.001

222 position, whereas among 33 survivors, 32 had D at this position.

Female

The fatality was significantly high (risk ratio = 19.34, 95%CI = 2.74-
22.4

136.53, P = 0.003) in patients having amino acid other than D at 222

position. However, there was no significant statistical difference
among survivors versus non-survivors when other two K163Q,
1169.0 (30.7)
Positivity (%)

148.0 (19.9)

205.0 (29.8)
323.0 (37.6)
295.0 (37.3)
120.0 (24.8)
70.0 (31.4)

A197T mutations were compared.

8.0 (36.4)

P < 0.001

The phylogenetic tree of complete HA gene developed along

Male

25.8

with 63 globally circulating sequences belonging to clades 6A, 6B, 6C,

7, and 8 along with vaccine component showed that the sequences in
year 2009 clustered with clade 7. Sequences of samples from 2010 to
1132

1642
1752
1553

7365
Total

2012 belonged to clades 6 and 7. Subsequently, till 2015, clade 6

361

889
36

viruses which are divided into three sub-groups sharing common

amino acid changes D97N, S185T, and S451N were detected. The
circulating viruses belonged to 6A having characteristic marker
Female

3556
389
151
941
894
762
405

H138R, V249L, 6B with K283E, E499K, K163Q, A256T, and 6C with

14
Sample tested

Chi-square trend computed excluding the missing age cases.

V234I were detected.12 We also detected changes responsible for

sub-grouping of the viruses in clades 6B.1 and 6B.2. Four samples
3809

belonged to clade 6 grouped into 6B.1 with S84N change (Figure 2).
Male
743
210
701
858
791
484
22

Two-hundred thirteen samples of A(H1N1)pdm09 cases who

succumbed to death were tested for the presence of H275Y
mutation that confers antiviral (Oseltamivir) resistance. We detected
Age in years

the presence of this mutation in 3 (1.4%) samples; one case had the
Missing

presence of both wild-type and mutant virus.

15-29
30-44
45-59

Total
6-14

60
0-5

4 | DISC US SION
Chi-square trenda

Our study presents a comprehensive picture of demographic spatial

TABLE 2

and molecular analysis of A(H1N1)pdm09 circulating in central India

for the period of 6 years, that is, from 2009 to 2015. The study
No.
1
2
3
4
5
6
7

reports higher positivity (32.7%) in comparison to few other studies

a
SAHU ET AL.
FIGURE 1 Graph showing year-wise and season-wise samples tested and detected positive for influenza A(H1N1)pdm09 from
November 2009 to October 2015 in central India. The year and season are shown on X axis, the number of cases on Y axis. The percentage
of positivity is marked on the top of bar
from India because of two reasons: one, we tested only the cases in
category-C; second, most of the studies are done prior to the upsurge
of 2015, wherein we observed almost 45% positivity. A study from
north MP also documented 38% positivity during the upsurge of
15
2015. The first case in MP was detected in the month of
November 2009, almost 6 months after the index case was diagnosed
in Andhra Pradesh, India.32 Indore is industrial capital and tourist hub
of the state; the first case had no history of traveling outside country or
contact with A(H1N1)pdm09-confirmed case, indicating that the virus
was circulating causing mild infection in the city.
Although cases were detected from all over MP, western MP was
the worst affected. The cities such as Bhopal, Indore, Dewas, and
Jabalpur which are well connected to each other and other metros of
the country were worst hit during these years. Furthermore, 27.7% of
cases were from tribal districts of MP because of local transmission,
indicating that the virus has reached and established in the rural
and tribal areas of central India, posing new challenge for health
system.
The positivity, mortality increased with age, adults were worst
affected. These observations are similar to observation reported from
33,34 7,8,35
other parts of the world and India. Generally during pandemic,
the incidence is reported higher,36 which declined during post-
pandemic.9 Interestingly, in contrast to the other studies,37,38 children
were found least affected due to A(H1N1)pdm09 in our study
throughout the period. This could be because of health-seeking
behavior and early treatment, leading to the avoidance of hospitaliza-
tion. Second, childhood hospitalization due to ILI could be because of
other viral infections such as RSV, rhinovirus, or bacterial infection.39,40
The higher positivity and mortality in females could be attributed
to delayed treatment-seeking behavior, which is seen in general in
| 5
India.41 Unfortunately, we do not have data regarding pregnancy, a
known risk factor, for severity in A(H1N1)pdm09 infection,42 but it is
worth to mention that we noted highest mortality in females in most
reproductive age group.
Year wise, maximum cases were detected in 2015, followed by
2009-2010 and 2011-2012. This was in agreement with the national
trend.9,15 We observed three major waves of A(H1N1)pdm09 with the
positivity of 30% or more. The influenza activity peaks generally with
monsoon and post-monsoon season in India11,43; however; we
observed a major peak in winter of 2015, sporadic cases almost in
each month of the year, indicating the importance of surveillance of the
ILI cases round the year.
Among the symptoms, nasal catarrh and shortness of breath had
no association with positivity, indicating that theses nonspecific
symptoms had little role in clinical diagnosis; on the other hand, typical
influenza symptoms of fever cough and sore through were strongly
associated with positive cases. We observed significant high number
of death among A(H1N1)pdm09 positives having pneumonia than
those who had no pneumonia. Pneumonia is reported to be major
cause of hospitalization and death in A(H1N1)pdm09 cases.44
Different states have reported varying mortality due to A(H1N1)
pdm09 in pandemic and post-pandemic period which ranged between
3.6% and 23.3% with the average of 6.9%.9 Whereas this study reports
15.1% case fatality, which is one of the highest among states having
more than 400 cases in the country. There could be several reasons for
it, the most important being delayed treatment. Second, we have
samples only from the hospitalized severe ILI cases. The large-scale
publicity campaigns persuade vaccination for those who can afford and
encouraging early antiviral treatment following national guidelines in
case of ILI will help in reducing morbidity and mortality.
6 | SAHU ET AL.

FIGURE 2 Phylogenetic tree of A(H1N1)pdm09 HA gene for 22 complete nucleotide sequences from central India. Multiple sequences
alignment and phylogenetic tree were constructed using Claustal W and maximum likelihood (ML) tree using the Tamura-Nei model running
within MEGA 5.05 software. Sequences from this study are indicated by red dot whereas Indian strains are marked with blue triangles.
Reference strains representing known genotype are indicated by their respective clade at the right

The surface proteins of utmost viruses are recognized by the pressure of the host immune response and unusual in that it evolves
immune system, and influenza HA is no exception with crucial role in remarkably and possesses highest variation among eight genes, thus
binding and infecting host cells. HA gene of influenza is under constant targeted by maximum studies for molecular characterization.45,46 On
SAHU ET AL.
the other hand, several studies recommend full genome sequencing
especially to detect the mutations in PB1, PA, and M genes that have
importance in severity and fatality owing to quasispecies genera-
tion.2022,47
In this study, 62 samples were targeted for HA gene sequencing
and observed for high polymorphism over a period of time in
circulating A(H1N1)pdm09 virus. We detected three changes that
are reported clinically and epidemiologically important. Of these three
mutations we focused, two mutations (K163Q and T197A) showed no
significant role as far as mortality was concerned. However, the
mutation at D222 leading either to N or G and the infection by mixed
population of D/G/N/X at 222 amino acid position in HA gene was
found to be significantly associated with mortality. This mutation and
infection by quasispecies is reported to be associated with increased
viremia, severe pneumonia, and cellular tropism of A(H1N1)pdm09
virus leading to diseases severity and death.17,4850 However, since we
do not have several details of patients such as other underlying
diseases, immune status, time laps between onset of illness and
treatment, etc.; thus, it is difficult to directly correlate only the D222N/
G/X mutation to severity and death. Hospital-based studies with
detailed information supported by molecular studies need to be done
which will elucidate the phenomena.
Viruses belonging to clades 6 and 7 were found circulating during
study period in central India. Although, in the year 2009 clade 1,13
clade 2, clade 3, clade 5, and clade 614 virus circulation was reported
from India. We detected clade 7 virus in the samples collected from
central India in 2009, probably because our first sequence was from
December 2009 and by then clade 7 was established as a predominant
clade.14 Subsequently this clade was replaced by the viruses belonging
to clade 6, which continues to be dominant to date. It is worth to
mention that we detected clade-7 virus in 2012 as well, indicating that
the virus belonging to clades 6 and 7 were co-circulating. However,
post-2013, we only detected virus belonging to clade 6, sub-group 6A,
6B further 6B.1 and 6B.2 and 6C similar to a study from northern India
that reported detection of virus belonging to clade 6B during 2015
upsurge.15 There was no history of travel associated cases, indicating
that the viruses were evolving at local level, underlining the importance
of vigilant molecular surveillance.
Oseltamivir is a drug of choice in A(H1N1)pdm09 infection,
H275Y mutation in NA gene confers the resistance. Recent study from
India had reported detection of Oseltamivir resistance.51 Our study
showed that majority of A(H1N1)pdm09-positive cases remained
sensitive for oseltamivir and only three cases had H275Y mutation
leading to reduced susceptibility which was only 1.4% of tested cases.
Further, one sample showed the presence of both resistance and
susceptible A(H1N1)pdm09, indicating that the virus is undergoing
evaluation. Our results also indicate the importance of monitoring the
viral strains as the resistant virus is detected in population and may flair
up in coming seasons.
This study is from the leading laboratory providing diagnosis for
A(H1N1)pdm09 presenting comprehensive picture of the A(H1N1)
pdm09 situation in central India. We have analyzed data from more
than 7000 patients, over a period of 6 years, giving sufficient power
| 7
to data for understanding meaningful demographic, seasonal, and
virological patterns of A(H1N1)pdm09 infections in the region. On
the other hand, it has some limitations such as not testing samples for
other etiologies, typing for other influenza viruses, and not
sequencing other genes of A(H1N1)pdm09 such as PB1, PA, and
M.47 We had limited clinical data especially about underlying
conditions of the patients and had no information about patients in
categories A and B.
In conclusion, the study presents overall epidemiological and
molecular characteristics of A(H1N1)pdm09 in central India, highlight-
ing the importance of molecular surveillance owing to ever changing
virus genotype and emerging antiviral resistance.
ACKNOWLEDGMENT
The authors are thankful to Influenza Group, National Institute of
Virology, Pune for their support by providing reagents for diagnosis
and technical help in sequence analysis and MP state health and family
welfare department for providing mortality data. This work was
supported by Indian Council of Medical Research (grant number VIR/
43/2011-ECD-1) and MP state health and family welfare department
(IDSP/15/600).
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
AUTHORS CONTRIBUTION
PVB and NS conceived the idea, designed the study. LS, MJU, and MS
did diagnosis and managed the data. MS and MKS did sequencing, and
MS and VAP did sequence analysis. RKS did statistical analysis. PVB,
VAP, NS, MS, and MKS prepared the MS. All authors read and agreed
upon the manuscript.
ORCID
Pradip V. Barde http://orcid.org/0000-0002-4767-6599
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