﻿SHORT REPORT Open Access
Molecular epidemiology of novel swine origin
influenza virus (S-OIV) from Gwalior, India, 2009
Shashi Sharma, Manmohan Parida*
, Jyoti Shukla and PVL Rao
Abstract
Background: The H1N1pandemic virus is a newly emergent human influenza A virus that is closely related to a
number of currently circulating pig viruses in the `classic North American' and `Eurasian' swine influenza virus
lineages and thus referred as S-OIV. Since the first reports of the virus in humans in April 2009, H1N1 virus has
spread to 168 countries and overseas territories. India also witnessed severe H1N1 pandemic virus epidemic with
considerable morbidity and mortality in different parts starting from May 2009.
Findings: The suspected swine flu outbreak from Gwalior India during October- December 2009 was confirmed
through S-OIV HA gene specific RT-LAMP and real time RT-PCR. Positive samples through CDC real time and Lamp
assay were further processed for isolation of the virus. Full HA gene sequencing of the H1N1 isolates of Gwalior,
India revealed 99% homology with California and other circulating novel swine flu viruses. Three major changes
were observed at nucleotide level, while two major amino acid shifts were observed at the position C9W and I30M
corresponding to the ORF with prototype strain. The HA gene sequence phylogeny revealed the circulation of two
genetically distinct lineages belonging to Clade VII and Clade I of S-OIV.
Conclusions: Our findings also supported the earlier report about circulation of mixed genogroups of S-OIV in
India. Therefore continuous monitoring of the genetic makeup of this newly emergent virus is essential to
understand its evolution within the country.
Findings
The Swine Flu is a respiratory viral disease which is
usually found in pigs but can sometimes be transmitted
to humans and cause epidemics or even pandemics (1).
The viral strain involved is type A H1N1. The virus can
be spread amongst humans from direct contact which
can occur through coughing, sneezing or contamination
of hands and surfaces. The severity of symptoms is
highly variable, although with most people suffering
only relatively mild symptoms (2). The pandemic that
began in March 2009 was caused by H1N1 influenza A
virus that had not been recognized previously in pigs or
humans, although six of its eight gene segments were simi-
lar to ones previously detected in triple reassortant swine
influenza viruses in pigs in North America (3). This strain
represents a quadruple reassortment of two swine strains,
one human strain, and one avian strain of influenza. The
largest proportion of genes comes from swine influenza
viruses (30.6 percent from North American swine influ-
enza strains, 17.5 percent from Eurasian swine influenza
strains), followed by North American avian influenza
strains (34.4 percent) and human influenza strains
(17.5 percent). Phylogenetic data even suggest that the
reassortment of swine lineages may have occurred years
before emergence in humans (4). Surprisingly however,
there has been no evidence so far that pigs have played
any role in the epidemiology or in the worldwide spread of
the virus in human populations.
The pandemic H1N1 virus was first detected in India
in May 2009 (5). Since then outbreaks have been
reported from many parts of the country. As of Decem-
ber 6, 2009 the total number of confirmed cases in
India was 19,632 with 621 deaths. Other reports com-
paring the HA gene sequence with those of the earlier
influenza pandemics have shown that human-specific
markers supporting efficient transmissibility of these
viruses in human are present in the H1N1pdm virus (6).
A recent study revealed that the early diversification of
the H1N1pdm virus based on concatenated whole
* Correspondence: paridamm@rediffmail.com
Division of Virology, Defence Research & Development Establishment
(DRDE), Gwalior- 474002, India
Sharma et al. Virology Journal 2011, 8:280
http://www.virologyj.com/content/8/1/280
© 2011 Sharma 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.
genomes resulted into seven lineages, Clade I-VII, that
showed defined spatial patterns (7). A focal outbreak of
swine flu was reported from Military Hospital Gwalior
(Madhya Pradesh), India during October-December
2009. The detail molecular investigation of this Swine
Flu outbreak including the genetic relatedness of this
local isolates with regard to Indian and global isolates is
reported.
Methods
A total of 40 nasopharyngeal swab samples were col-
lected with informed consents from suspected patients
admitted to military hospital, Gwalior. The samples were
collected with HiviralTMnylon flocked swab in HiviralTM
transport medium and were transported immediately to
the laboratory. Initially RNA was extracted from 140 l
of sample using QIAmp viral RNA minikit (Qiagen, Ger-
many) according to the manufacturer's specification.
All these samples were investigated by WHO
approved CDC recommended real time RTPCR assay in
25 l reaction scale using a panel of oligonucleotide pri-
mers and dual labeled hydrolysis probe employing ABI
One-step RT-PCR kit (8). All the 40 samples were also
simultaneously subjected to one step real-time reverse
transcription loop mediated isothermal gene amplifica-
tion (RTLAMP) assay for rapid and real-time detection
of novel H1N1 Swine Flu virus RNA in clinical speci-
mens by targeting the HA gene (9). The gene amplifica-
tion in RTLAMP was accomplished by incubating the
reaction mixture at 63°C for 60 min in routine labora-
tory water bath/dry heating bath. The real-time moni-
toring of RT-LAMP amplification was observed through
spectrophotometric analysis by recording O.D. at 400
nm at every 6 second with the help of loop amp real-
time turbidimeter (LA-200, Teramecs, Japan).
The Real time RT-PCR positive and RT-LAMP posi-
tive Swine flu clinical samples that were collected during
recent 2009 outbreak from Gwalior were subjected to
virus isolation. The nasopharyngeal swabs were filtered
through 0.2 m syringe filters and were passaged in
Madin Darby Canine Kidney (MDCK) cells along with
PBS as negative control (10). All the samples were pro-
cessed under BSL-3+ laboratory. The sequencing of HA
gene was performed with automated ABI Prism 3130
instrument (Applied Biosystems, Foster City CA, USA)
by use of Big Dye3.1 cycle sequencing kits provided by
the same manufacturer. Subsequently, any unincorpo-
rated labeled dNTP's were removed using Dye-X
removal Centrisep column purification kit (Applied Bio-
system). The nucleotide uences were edited and
assembled using DNASTAR Lasergene 5 software pack-
age (Bioinformatics Pioneer DNASTAR, Inc., Wisconsin,
USA), and the sequences are subjected to BLAST to
find closest possible match from available sequences in
GenBank at http://www.ncbi.nlm.nih.gov/BLAST. Multi-
ple sequence alignments were carried out employing
CLUSTALW version 1.83 (11). The phylogenetic analy-
sis was done employing Neighbour-Joining (NJ) and
Maximum-Parsimony (MP) methods using MEGA4
software (12). The tree was constructed using the
Kimura's two-parameter distance model with 1000 boot-
strap replicates. The percent nucleotide identity (PNI)
and percent amino acid identity (PAI) values were cal-
culated as pair wise p-distances.
Results
The first isolate from India (A/India-Hyd/NIV51/2009)
was from a traveler reaching Hyderabad on May 13,
2009 from the USA. Positive cases of H1N1pdm virus
were thereafter detected from major cities (Pune, Delhi,
Mumbai, Chennai and Bangalore) with maximum fatal-
ity reported from Pune and Bangalore (5).
All the clinical specimens having continuous fever,
shivering, cough, with history of traveling history and
closed contact with confirmed patients. All samples
were processed for virus isolation.
The infected culture fluid (ICF) was collected after
observation of virus specific cytopathic changes like
clumping of cells, granulation, syncitia formation, finally
detachment from the flask surface were observed. The
presence of the virus in infected culture fluid was con-
firmed through ELISA using the HA antibody (Sigenics,
USA) as well as by S-OIV HA gene specific RTLAMP and
SYBR Green I real time RTPCR using H1N1 HA specific
primers F3/B3 (Genomic Position 529-547/699-716).
The HA gene target was amplified by WHO recom-
mended primers. All sequences have been submitted in
Gen Bank with the following accession numbers (Acces-
sion No - GU 265731, GU 265730, GU265729, GU
201599, GU201598).
The partial HA gene of sequences of 30 other represen-
tative pdmH1N1 virus of diverse geographical origins
were retrieved from GenBank (Table-1). The phyloge-
netic analysis of the partial HA gene sequences revealed
99% homology at nucleotide level with novel swine flu
virus. The BLAST analysis revealed 99% similarity with
California 2009 prototype strain of novel swine flu virus.
The sequence analysis of these Indian isolates revealed
specific mutation at nucleotide level and amino acid
changes respectively with respect to 22 global H1N1 pan-
demic isolates including few Indian isolates. Among the
isolates, a sequence homology of 99.9% was observed.
Three major changes were observed at nucleotide level,
while two major amino acid shifts were observed at the
position C9W and I30M corresponding to the ORF with
prototype strain. Phylogenetic analysis based on partial
HA gene classified the geographical diverse pdm H1N1
virus isolates into seven distinct clades (Figure 1).
Sharma et al. Virology Journal 2011, 8:280
http://www.virologyj.com/content/8/1/280
Page 2 of 4
Discussion
Diversity of these Indian isolates based on partial HA
gene phylogeny revealed the co-circulation of Clade VII
and Clade I with predominance of Clade VII as already
reported from Southern India. Multiple lineages of influ-
enza A viruses were found to co-circulate during any
single season and to undergo frequent reassortment.
This, in turn, has had a major impact on antigenic evo-
lution. Our study also further supported the fact that
pandemic (H1N1) 2009 virus has evolved worldwide,
shifting from an initial mixed clade pattern to the pre-
dominance of one Clade (Clade VII) during the course
of the pandemic. The hypothesis that Clade VII virus
enjoyed a marked advantage, in terms of transmissibility,
over other early Clades is intriguing, but has yet to be
demonstrated. The virus constituting this Clade was
therefore responsible for most of the pandemic burden
worldwide. In summary, this is the first report from cen-
tral region of India (Madhya Pradesh) regarding the
emergence, isolation and characterization of novel swine
flu virus.
Author's Information
Ms. Shashi Sharma has been working for last 3 years in
the field of molecular epidemiology of circulating den-
gue virus genotypes.
Acknowledgements
The authors are thankful to Dr. R. Vijayaraghavan, Director, Defence Research
and Development Establishment, Gwalior for providing necessary facilities
and financial grant for this study. The authors are also thankful to
Commandant, MH, Gwalior for providing clinical samples.
C
Clade VII
A/India-Gwl/DRDE-MH04/2009
A/India-Gwl/DRDE-MH02/2009
A/India-Gwl/DRDE-MH01/2009
A/India-Gwl/DRDE-MH06/2009
A/India-Mum/NIV9945/2009
A/Omsk/02/2009
A/India-Delhi/NIV3704/2009
A/India-Pune/NIV10278/2009
A/India-Blore/NIV236/2009
A/India-Pune/NIV9355/2009
A/India-Pune/NIV6447/2009
A/India-Pune/NIV8489/2009
A/India-Delhi/NIV3610/2009
A/India-Mum/NIV5442/2009
A/India-Blore/NIV310/2009
A/Shanghai/1/2009
A/Thailand/CURA9/2009
A/Ohio/07/2009
A/Denmark/523/2009
A/Wisconsin/629-D00008/2009
A/Beijing/3/2009
A/India-Hyd/NIV51/2009
A/Korea/01/2009
A/New York/3177/2009
A/England/195/2009
A/Hamburg/4/2009
A/Kansas/03/2009
A/Osaka/1/2009
A/California/07/2009
A/India-Gwl/DRDE-MH03/2009
A/California/04/2009
A/Shanghai/143T/2009
A/New York/3324/2009
A/India-Pune/NIV10604/2009
A/India-Pune/NIV6196/2009
Clade V
Clade III
Clade II
Clade IV
Clade I
Clade VI
Figure 1 HA Phylogeny of 5 Indian isolates with respect to circulating global isolates. Scale bar indicates number of nucleotide
substitutions per site. Each strain is abbreviated with country of origin and year of isolation.
Sharma et al. Virology Journal 2011, 8:280
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Authors' contributions
All authors read and approved the final manuscript. MMP has designed the
study plan and the experiments were executed by SS and JS. JS has carried
out the sample analysis by RTLAMP while SS has performed the real-time
RTPCR, RTPCR and sequence phylogeny. PVLR has supervised the study plan
and gave critical suggestions for preparing the MS.
Competing interests
The authors declare that they have no competing interests.
Received: 28 December 2010 Accepted: 7 June 2011
Published: 7 June 2011
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doi:10.1186/1743-422X-8-280
Cite this article as: Sharma et al.: Molecular epidemiology of novel
swine origin influenza virus (S-OIV) from Gwalior, India, 2009. Virology
Journal 2011 8:280.
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