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Year : 2012  |  Volume : 30  |  Issue : 3  |  Page : 346-349

Genetic analysis of HA gene of pandemic H1N1 2009 influenza viruses circulating in India

Department of Virology King Institute of Preventive Medicine and Research, Guindy, Chennai-600 032, India

Date of Submission15-Jan-2012
Date of Acceptance04-Apr-2012
Date of Web Publication8-Aug-2012

Correspondence Address:
K Krishnasamy
Department of Virology King Institute of Preventive Medicine and Research, Guindy, Chennai-600 032
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.99500

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 ~ Abstract 

The H1N1 2009 influenza pandemic took the health care workers by surprise in spite of warning about influenza pandemic. Influenza A virus has the ability to overcome immunity from previous infections through the acquisition of genetic changes by shift or drift. Thus, understanding the evolution of the viruses in human is important for the surveillance and the selection of vaccine strains. A total of 23 pandemic A/H1N1 2009 viral HA gene sequences were downloaded from NCBI submitted during March and May 2010 by NIV and were analysed. Along with that the vaccine strain A/California/07/2009 was also downloaded from NCBI. All the sequences were used to analyse the evolution of the haemagglutinin (HA) by phylogenetic analysis. The HA gene could be divided into four groups with shift from 1 to lV revealing that the HA genes of the influenza A viruses evolved in a sequential way, in comparison to vaccine strain A/California/07/2009. Amino acid sequence analysis of the HA genes of the A/H1N1 2009 isolates, revealed mutations at positions 100, 220 and additional mutations in different positions 114, 171, 179, 190, 208, 219, 222, 239, 240, 247, 251, 260 and 285 .The mutations identified showed the adaptation of the new virus to the host that could lead to genetic changes inherent to the virus resulting in a reassortant which could be catastrophic, hence continuous monitoring of strains is mandatory.

Keywords: H1N1 2009, haemagglutinin (HA), influenza virus, multiple-sequence alignment, phylogenetic analysis

How to cite this article:
Gunasekaran P, Krishnasamy K, Arunagiri K, Sambasivam M, Lakshmipathy M, Arunpon, Fathima S G. Genetic analysis of HA gene of pandemic H1N1 2009 influenza viruses circulating in India. Indian J Med Microbiol 2012;30:346-9

How to cite this URL:
Gunasekaran P, Krishnasamy K, Arunagiri K, Sambasivam M, Lakshmipathy M, Arunpon, Fathima S G. Genetic analysis of HA gene of pandemic H1N1 2009 influenza viruses circulating in India. Indian J Med Microbiol [serial online] 2012 [cited 2020 Sep 19];30:346-9. Available from:

 ~ Introduction Top

Influenza A viruses are a genus of the family Orthomyxoviridae. They are RNA viruses with a segmented genome that is comprised of eight negative-sense, single-stranded RNA segments. These eight segments encode 11 proteins. The polymerase complex includes the PB2, PB1 and PA proteins as well as the nucleoprotein (NP). There are two surface glycoproteins: haemagglutinin (HA) and neuraminidase (NA). Matrix protein 1 (M1) is the structural protein of the virus particle, while matrix protein 2 (M2) forms an envelope-spanning proton channel. The genome also encodes two non-structural proteins (NS1 and NS2) as well as an alternate reading frame protein from PB1 known as PB1-F2. [1] According to the viral external HA and NA gene sequences and their serological features, type A influenza viruses have been classified into 16 HA subtypes (H1- H16) and 9 NA subtypes (N1-N9). [2],[3] The combinations of the HA and NA subtypes further formed dozens of subtypes including H1N1, H1N2, H2N2, H3N2 and H5N1.

The influenza A virus surface glycoproteins especially HA is under selective pressure to evade the host immune system. Thus, HA and NA genes of Influenza A mutate at high frequencies [4],[5] resulting in the accumulation of point mutations that may lead to gradual antigenic changes in the surface proteins. The matrix protein gene codes for two viral proteins, M1and M2, contributing to its virulence and growth. [6],[7]

Studies based on sequence analyses of viruses can be utilized as surveillance tools and can contribute to the vaccine selection process when they are combined with classical serological antigenic analysis. [8] In order to elucidate the evolutionary patterns for influenza viruses in India, we have undertaken a genetic analysis of the influenza AH1N1 2009 virus circulating from 2009 to 2010 during the pandemic period, focusing on HA genes which undergo frequent mutation. Continuous monitoring of viral genetic changes throughout the year is necessary for us to develop our ability to precisely define variation among influenza viruses.

 ~ Materials and Methods Top

The HA gene sequences of the pandemic H1N1 2009 vaccine strain A/California/07/2009 and the other Indian pandemic H1N1 2009 nucleotide sequences submitted during March and May 2010 by NIV were downloaded from sequence database NCBI ( and evaluated. All the sequences were assembled by use of the MEGA (version 4.1) program [9] and multiple-sequence alignment was conducted with the ClustalW program for the major coding regions of the HA gene for H1N1 2009 isolates. Phylogenetic tree was constructed by using a neighbour-joining and bootstrap analysis (n _ 1,000) program to determine the best fits for the HA genes. Major branches with bootstrap values of >45% were identified as distinct groups.

The amino acid sequences of the selected HA genes and the vaccine strain sequences were downloaded from the protein sequence database of NCBI and the multiple-sequence alignment was conducted by ClustalW program to identify the mutations in different positions of H1N1 2009 by comparing the pandemic protein sequences with the corresponding vaccine sequence.

To evaluate the significance of the predictions of a signal peptide, we analysed all the selected HA proteins with multiple software tools including SignalP v3.0 (SignalP-NN and SignalP-HMM), [10],[11] Phobius, [12] SPOCTOPUS [13] and Signal-BLAST. [14]

 ~ Results Top

Twenty-three influenza A/H1N1 2009 virus HA sequences that were collected from NCBI were used in this study [Table 1].
Table 1: Sequence data of influenza isolates

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The phylogenetic tree for the HA genes of the A/H1N1 2009 strains, including 23 isolates from India and one vaccine (A/California/07/2009) reference strain, showed four groupings [Figure 1]. Among the isolates, four isolates from Pune and one isolate from Bangalore were clustered in group I. The two isolates from Bangalore, one isolate from Chennai, one isolate from Delhi and one from Mumbai were clustered in group II. The three isolates from Pune, one isolate from Jalna, two isolates from Mumbai and two from Delhi were clustered in group III. Three isolates collected in Pune, one isolate collected in Dhule and one isolate collected in Mumbai were clustered in group IV.
Figure 1: Phylogenteic analysis of HA gene nucleotide sequences.

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In comparison to vaccine strain A/California/07/2009 [Table 2], the HA genes of the A/H1N1 2009 isolates in groups I, II, III and IV from India had mutations at position 100 in which P100S change had occurred. In position 220, except for five strains (A/Mum/NIV968/09, A/Pune/NIV6196/09, A/Dhule/NIV9433/09, A/Pune/NIV807/09 and A/Pune/NIV10604/09), all the others had mutation at S220T. Along with this, they had additional mutations in different positions 114, 171, 179, 190, 208, 219, 222, 239, 240, 247, 251, 260 and 285. In particular, along with above-mentioned mutations, three amino acid changes occurred at positions V190I, G219W and V260L in the A/Dhule/NIV9433/2009 strain which comes under group IV. The A/Mum/NIV9312/2009 strain which comes under group II had mutations at R222K and D239G. A/Pune/NIV21115/2009 under group III had mutations at Y247H and S285T.
Table 2: Comparison of amino acid sequences of vaccine strain and the pandemic H1N1 isolates collected from India

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A/Che/NIV658/2009 had mutation at L208I and A/Blore/NIV1189/2009 had amino acid change at S179I and K171E change had occurred in A/Delhi/NIV3610/2009. A/Pune/NIV8489/2009 had V251I mutation. D239G had occurred in two strains of A/Pune/NIV9355/2009 and A/Pune/NIV10278/2009. The strains A/Blore/NIV236/2009 and A/Jalna/NIV9436/2009 had amino acid change at Q240R and mutation at D114Q occurred in A/Pune/NIV807/2009 strain.

Analysis of all the selected HA proteins with multiple software tools including SignalP v3.0, Phobius, SPOCTOPUS and Signal-BLAST[15] showed that the N terminal of the HA protein as having a signal peptide which consists of about 17 amino acids and the cleavage site present between positions 17 and 18. HA is cleaved into a signal peptide when mature proteins are produced.

 ~ Discussion Top

Influenza genome has eight RNA segments encoding for viral proteins. It is well known that because of immunological pressure, genetic variability is mostly confined to genomic regions coding for viral surface proteins. [16] The genetic analysis of the HA gene of A H1N1 2009 submitted during various period of the epidemic indicates that genetic change did not arise at the same rate. Furthermore, the HA gene could be divided into four groups with shift from I to lV. This suggests that the HA gene has evolved during the 1 year of the pandemic.

The HA genes of the A/H1N1 2009 isolates in groups I, II, III and IV showed number of amino acid changes from the sequence of the A/California/07/2009 vaccine strain. The HA1 subunit includes the globular head and contains five major antibody binding sites, namely Sa, Sb, Ca1, Ca2 and Cb. [17],[18],[19] A substitution at residue 171 (K171E) occurred in the Ca antigenic site in A/Delhi/NIV3610/2009 isolate, the substitution at residue 208 (L208I) which is located in the Ca site present in the A/Che/NIV658/2009 strain. A substitution at residue 240 (Q240R) also occurred in the Ca antigenic site of two different strains like A/Blore/NIV236/2009 and A/Jalna /NIV9436/2009 isolates. Therefore, we concluded that most of the H1N1 2009 viruses circulated in India during 2009 were antigenically similar to that of strain A/California/07/2009 but had undergone changes more gradually. The mutations were observed in the region of signal peptide of HA in all the 24 strains (inclusive of vaccine strain). This will not be of much relevance as the signal peptide gets cleaved once the mature protein is produced. Therefore, continuous monitoring of viral genetic changes throughout the year is warranted to monitor the variations of influenza viruses. With the arrival of vaccines against H1N1 2009 and being given to the public, monotoring of strains is mandatory. Centres for isolation will have to be set up inclusive of various regions in India to identify a shift or drift.

As the winter months are approaching many experts are predicting a second wave of A/H1N12009 infections. The timing of the second wave may be after 6 months to 2 years. [20] The progressive adaptation of the new virus to the host could be the reason for the increase in severity. It is also evident that as the virus continues to spread there could be antigenic drifts and shifts that could increase the virulence, hence continuous monitoring of the various strains will aid to evolve appropriate vaccination strategies and prevention of outbreaks as well.

 ~ References Top

1.Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev 1992;56:152-79.  Back to cited text no. 1
2.Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, et al. Characterization of a novel type A influenza virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 2005;79:2814-22.  Back to cited text no. 2
3.Liu S, Ji K, Chen J, Tai D, Jiang W, Hou G, et al. Panorama phylogenetic diversity and distribution of type a influenza virus. PLoS ONE 2009; 4:e5022.  Back to cited text no. 3
4.Fitch WM, Bush RM, Bender CA, Cox NJ. Long term trends in the evolution of (H3) HAI human influenza type A. Proc Natl Acad Sci U S A 1997;94:7712-8.  Back to cited text no. 4
5.Fitch WM, Bush RM, Bender CA, Subbarao K, Cox NJ. Predicting the evolution of human influenza A. J Hered 2000;91:183-5.  Back to cited text no. 5
6.Enami K, Qiao Y, Fakuda R, Enami M. An influenza virus temperature sensitive mutant defective in nuclear cytoplasmic transport of the negative-sense viral RNAs. Virology 1993;194:822-7.  Back to cited text no. 6
7.Smeenk CA, Brown EG. The influenza virus variant A/FM/1/47-MA possesses single amino acid replacements in the hemagglutinin, controlling virulence, and in the matrix protein, controlling virulence as well as growth. J Virol 1994;68:530-4.  Back to cited text no. 7
8.Cox NJ, Bai ZS, Kendal AP. Laboratory-based surveillance of influenza A (H1N1) and A (H3N2) viruses in 1980-81: Antigenic and genomic analyses. Bull World Health Organ 1983;61:143-52.  Back to cited text no. 8
9.Kumar S, Tamura K, Nei M. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 2004;5:150-63.  Back to cited text no. 9
10.Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 2004;340:783-95.  Back to cited text no. 10
11.Emanuelsson O, Brunak S, von Heijne G, Nielsen H. Locating proteins in the cell using Target P, Signal P and related tools. Nat Protoc 2007;2:953-71.  Back to cited text no. 11
12.Kall L, Krogh A, Sonnhammer EL. Advantages of combined transmembrane topology and signal peptide prediction-the Phobius web server. Nucleic Acids Res 2007;35:W429-32.  Back to cited text no. 12
13.Viklund H, Bernsel A, Skwark M, Elofsson A. SPOCTOPUS: A combined predictor of signal peptides and membrane protein topology. Bioinformatics 2008;24:2928-9.  Back to cited text no. 13
14.Frank K, Sippl MJ. High-performance signal peptide prediction based on sequence alignment techniques. Bioinformatics 2008;24:2172-6.  Back to cited text no. 14
15.Clifford M, Twigg J, Upton C. Evidence for a novel gene associated with human influenza A viruses. Virol J 2009; Nov 16;6:198.  Back to cited text no. 15
16.Reid AH, Fanning TG, Janezewski TA, Taubenberger JK. Characterisation of the 1918 spanish influenza virus neuraminaidase gene. Proc Natl Acad Sci U S A 2000;97:6785-90.  Back to cited text no. 16
17.Caton A, Brownlee GG, Yewell JW, Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell 1982;31:417-27.  Back to cited text no. 17
18.Yates PJ, Bootman JS, Robertson JS. The antigenic structure of a human influenza A (H1N1) virus isolate grown exclusively in MDCK cells. J Gen Virol 1990;71:1683-8.  Back to cited text no. 18
19.Chutinimitkul S, Thippamom N, Damrongwatanapokin S, Payungporn S, Thanawongnuwech R, Amonsin A, et al. Genetic characterization of H1N1, H1N2 and H3N2 swine Influenza virus in Thailand. Arch Virol 2008;153:1049-56.  Back to cited text no. 19
20.Lee CK. MRCP. Influenza A (H1N1) 2009 pandemic Virus: Learning from the First wave, Preparing for the second. Med J Malaysia 2010;65:1-2.  Back to cited text no. 20


  [Figure 1]

  [Table 1], [Table 2]

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