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 ~ Results
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  Table of Contents  
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 324-337

Genetic sequencing of influenza A (H1N1) pdm09 isolates from South India, collected between 2011 and 2015 to detect mutations affecting virulence and resistance to oseltamivir

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Submission02-Mar-2020
Date of Decision14-Aug-2020
Date of Acceptance31-Aug-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Sujatha Sistla
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_20_83

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 ~ Abstract 

Background: Influenza A viruses evolve continuously and the two surface antigens, hemagglutinin (HA) and neuraminidase (NA) have been the target proteins for research as they are vital components in determining the virulence, immune effectiveness, pathogenicity, transmission and resistance. Methods: Both HA and NA (partial genes) of 45 pandemic influenza A(H1N1)pdm09 isolates were sequenced. Phylogenetic analysis was performed with reference to representative global isolates retrieved from Influenza Virus Resource (IVR), GISAID EpiFluTM and GenBank and evolutionary analyses. Nucleotide and amino acid sequences were aligned using ClustalW/ Clustal Omega/MEGA version 6 with reference to vaccine strain (A/California/07/2009). Results: All the isolates clustered along with the clade 7 virus, irrespective of the year of isolation. The study isolates exhibited 98.5% and 98.8% nucleotide homology to the reference strain A/California/07/2009(H1N1) for HA and NA, respectively. Overall, there was limited genetic diversity observed over a period of 3 years (2012-2015). Two samples collected from expired patients had D239N (D222G or D225G) mutation in HA. This mutation which is associated with dual-binding specificity of the virus has been well-correlated with severe disease outcomes. All the study isolates possessed H274 residue and 7 strains had N295S, the next most common mutation found in oseltamivir-resistant variants. Conclusion: In this study, although H274Y mutation associated with oseltamivir resistance has not been noted, significant mutations have been noted in both HA and NA genes including D239N, N295S, V106I, Q136K, N248D, V267A. In both HA and NA gene analysis, multiple mutations were found more in 2015 strains when compared to 2012 strains. Hence such accumulation of mutations has to be monitored continuously to determine the efficacy of annual flu vaccines and anti-influenza drugs.

Keywords: Genetic sequencing, H1N1, influenza, mutation, oseltamivir, phylogenetic analysis

How to cite this article:
Nandhini P, Sistla S. Genetic sequencing of influenza A (H1N1) pdm09 isolates from South India, collected between 2011 and 2015 to detect mutations affecting virulence and resistance to oseltamivir. Indian J Med Microbiol 2020;38:324-37

How to cite this URL:
Nandhini P, Sistla S. Genetic sequencing of influenza A (H1N1) pdm09 isolates from South India, collected between 2011 and 2015 to detect mutations affecting virulence and resistance to oseltamivir. Indian J Med Microbiol [serial online] 2020 [cited 2021 Feb 27];38:324-37. Available from:

 ~ Introduction Top

Influenza viruses, particularly type A viruses, are recognised for their high mutation rates and ability to generate antigenically distinct variants.[1],[2] Exploring the evolutionary mechanisms occurring in global influenza A viruses to anticipate and prevent future influenza pandemics has been the subject of intense research for several decades. This herculean task has been achieved effectively by closely monitoring the two important surface antigens, haemagglutinin (HA) and neuraminidase (NA).

HA, coded by the fourth segment of influenza genome, is responsible for attachment of virion to host cells and is a vital component of influenza virus to determine the immune effectiveness, pathogenicity and virulence.[3] Unfortunately, this gene presents the highest mutation rates leading to the instability of influenza genome.[4] Evidence suggests that a mutation leading to amino acid substitution at position 229 (D222G) in the receptor-binding site of HA is associated with severe disease and mortality.[5],[6] A latest report from Massachusetts Institute of Technology, USA showed amino acid changes that are known to improve receptor binding and increase virulence (T200A, D225N) in 2014 Indian strains. This commentary raised concerns over under-reporting of such significant mutations in a high population density country like India where person-to-person transmission occurs with ease.[7]

The other vital component, NA, is responsible for releasing the newly produced viral particles and this glycoprotein is the primary target of antiviral drugs. Oseltamivir is a NA inhibitor (NAI) and it is the drug of choice against influenza and is stockpiled for pandemic influenza containment and control. Before the NAIs licensure in 1999, global surveillance of influenza H1N1 isolates did not find naturally occurring resistance[8] and until 2007, only 0.3% of NAI resistance was reported. However, from late 2007 to June 2008, 15% of H1N1 isolates were resistant to oseltamivir without correlation to drug use.[9] In the WHO report on “Influenza A (H1N1) virus resistance to oseltamivir' released in January 2009, the percentage of resistance ranged from 13% (Chile) to 100% (Senegal and South Africa).[10] Reports showed H274Y or N294S amino acid substitutions in NA gene are commonly found in clinically derived influenza A NAI-resistant variants. Currently, nearly ten amino acid substitutions in NA have been linked to a reduction in oseltamivir susceptibility of influenza viruses.[11] Apart from these, many random mutations occur in influenza viruses, but only a few mutations were reported to benefit the viruses by means of enhanced virulence, host receptor binding and drug resistance.[12] Experimental data showed reduced rates of transmissibility of seasonal influenza viruses carrying significant mutations in NA like H274Y,[13] however, the influenza H1N1pdm09 isolates carrying the same mutations were as virulent as its wild-type without loss of fitness, transmissibility or pathogenicity.[14] Such intricate and diverse evolutionary mechanisms leading to drug resistance with unimpaired fitness in influenza viruses underlines the importance of continuous monitoring for effective pandemic planning.

 ~ Methods Top

Study isolates

In 1976, NIV, Pune initiated studies on influenza and in 2000 it became part of the WHO FLUNET program as the nodal centre in India. After outbreaks of avian flu in 2006, a second nodal centre for surveillance of circulating respiratory viruses was established in 2008 by National Centre for Disease Control (NCDC) through funding from the World Bank. Our Institute became a part of this National Influenza Surveillance Programme in 2008 and in July 2009 Regional influenza laboratory was set up. The 12 laboratory network under NCDC was functional during the Influenza A (H1N1) pandemic in 2009 and played a crucial role in contact tracing and successful containment of the pandemic in the country. In 2012, Central Government of India in collaboration with WHO took over this project renaming it the influenza-like illness surveillance by financial and technical support through Integrated Disease Surveillance Programme (IDSP) and NCDC respectively and this surveillance is continued actively till date. This study duration is from November 2011 to March 2015. The study protocol was approved by our Institute Ethics Committee (Human studies). Written informed consent was obtained from all the patients or their guardians as appropriate, before specimen collection.

As a part of surveillance, of total 109 pandemic influenza A (H1N1) pdm09 isolates, a total of 45, collected in different years (one from 2011, 16 from 2012 and 28 from 2015) were selected for genetic sequencing. Due to budgetary constraints, it was decided to sequence representative 50 isolates total, choosing isolates from each year of the duration of study and hence randomisation ( was done to choose the 50 isolates and 45 was successfully sequenced. Patient characteristics are shown in [Table 1]. All the 45 patients were treated with Oseltamivir, 41 recovered and 4 died due to acute respiratory distress syndrome (ARDS).
Table 1: Patient details and GenBank accession numbers of the sequenced influenza A (H1N1) pdm09 isolates (n=45)

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RNA extraction, polymerase chain reaction amplification and sequencing

RNA was extracted from 45 nasopharyngeal samples (which was stored as per guidelines at-80°C and which were positive for pandemic influenza A [H1N1] pdm09) virus by real-time polymerase chain reaction [PCR]), using QIAmp viral RNA kit (Qiagen, Germany) according to manufacturer's specifications. Primers were designed for HA and NA genes [Table 2] and one step RT-PCR was performed to amplify HA and NA gene segments using WHO-CDC protocol. The amplicons were analysed by 1% agarose gel electrophoresis and the PCR products were purified using PCR purification kits (Qiagen, Germany). DNA sequencing was carried out using ABI 3730 Genetic Analyzer (Applied Biosystems, USA) and sequences were aligned using MEGA version 6. Sequences of the influenza A (H1N1) pdm09 isolates have been deposited in GenBank and accession numbers obtained [Table 1].
Table 2: Primers of haemagglutinin and neuraminidase genes

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Phylogenetic analysis

Phylogenetic analysis was performed with reference to representative global isolates retrieved from influenza virus resource, GISAID EpiFlu and GenBank and evolutionary analyses. Nucleotide sequences were aligned using ClustalW and phylogenetic trees were constructed by the neighbour-joining (NJ) method using MEGA ver. 6. We analysed the evolutionary relationships of the HA and NA sequences of 45 isolates collected in Union territory of Puducherry and compared the data with other global isolates collected between 2011 and 2015 by the NJ method, with the bootstra values of 1000 replicates.

Amino acid analysis

Amino acid variations in the gene sequences were noted by aligning the amino acid sequences with reference to vaccine strain (A/California/07/2009) using Clustal Omega/MEGA version 6. This was performed using FluSurver ( and each point mutation was analysed to correlate with altered functions.

Statistical analysis

The correlation of site-specific mutations with clinical severity was evaluated using two-tailed Fisher's exact test. Genetic distances between the years, were analysed by t-test.

 ~ Results Top

Phylogenetic analyses of haemagglutinin and neuraminidase gene sequences

A total of 45 isolates were sequenced for genetic analysis of partial HA and NA genes. All the isolates clustered along with the clade 7 virus, irrespective of the year of isolation. The pairwise distance analysis showed that with respect to the reference strain A/California/07/2009(H1N1), the study isolates exhibited 98.5% and 98.8% nucleotide homology for HA and NA, respectively. Overall, there was limited genetic diversity observed over the period of 3 years (2012–2015) [Figure 1] and [Figure 2]. The strains isolated in 2012 were genetically closer to A/California/2009 strain than most of the 2015 strains; however, there was no statistical difference based on the genetic distances of the HA (P = 0.06) and NA (P = 0.06) sequences between the years. With regards to HA sequences, characteristic clustering was observed in 2012 and 2015 strains separately; however, close interrelatedness was observed within the isolates of each year. The NA sequence analysis showed mixed evolutionary pattern among the 2012 and 2015 strains.
Figure 1: The study isolates are marked in pink (2015) and blue (2012): Phylogenetic tree of hemagglutinin gene of influenza A (H1N1) pdm09 isolates

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Figure 2: The study isolates are marked in pink (2015) and blue (2012): Phylogenetic tree of neuraminidase gene of influenza A (H1N1) pdm09 isolates

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Haemagglutinin amino acid analysis

The HA amino acid sequences of the influenza A (H1N1) pdm09 isolates were compared with influenza A/California/07/2009 (vaccine reference strain) [Table 3]. All the influenza A (pdm) 09 isolates from Union territory of Puducherry possessed S220T mutation in the Ca antigenic site of HA1 which is a clade 7 characteristic mutation. Except for one isolate (KX759417), all had S202T mutation in the Sb antigenic motif. The majority of the isolates from Union territory of Puducherry possessed D239 (D225 alternate numbering) in the receptor binding region, except in 5 strains which had D239N; two of these isolates were obtained from fatal cases. However, this mutation was not found to be significantly associated with the fatal cases (P = 0.055). Eleven isolates had S160G mutation at Ca site of HA, many strains had K300E (n = 14) and K180Q (n = 10); 3 isolates had K180I mutation. Alanine to Threonine mutation was found at 214 (n = 8) and 273 positions (n = 9). Two isolates had Valine in place of Isoleucine (I-V) and Glumatic acid instead of Glycine (G-E) at 233 and 279, respectively. A single isolate each with the following mutations were also observed: S138N, D144E, N146D, H155R, A156D, N173d, L178I, V190I, Y223H and M244I.
Table 3: List of mutations in haemagglutinin protein sequences of influenza A (H1N1) pdm09 isolates

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Neuraminidase amino acid analysis

All the study isolates possessed H274 residue (position 275 in N1 numbering), a well-known marker for sensitivity to the NAI, oseltamivir. In other words, none of our isolates exhibited the common mutation associated with oseltamivir resistance. N295S, the next most common mutation found in oseltamivir-resistant variants was found in 7 strains (3–2012, 4–2015) [Table 4]. All but one of these seven patients recovered (KX772189). V106I, Q136K and V267A the other mutations related to drug resistance were found 14, 3 and 1 isolates, respectively. Most of the study isolates had N248D (n = 21), which is known to be present in the dominant variant of 2009 pandemic influenza A (H1N1) viruses. N200S and V241I, two of the frequently reported mutations in the recent strains after 2012 were found in 15 and 19 strains respectively. Influenza isolates collected in 2015 had a few exclusive mutations: N270K, I314M and I321V, many of them co-occurred. All influenza isolates collected from patients who expired during hospitalisation were found to possess three or more mutations in NA gene (One particular isolate, KX772205 obtained from a 31-year-old female had 6 mutations in NA gene: N200S, V241I, N248D, V264I, E287K and I321V).
Table 4: List of mutations in neuraminidase protein sequences of influenza A (H1N1) pdm09 isolates

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NA genes of the study isolates had other single mutations (not listed in table) like the following: G96R, A98P, L91I, S101T, F115S, P126H, D142Y, F174S, G197S, A204T, G236S and I263V. In both HA and NA gene analysis, multiple mutations were found more in 2015 strains when compared to 2012 strains [Figure 3] and [Figure 4].
Figure 3: Number of mutations in haemagglutinin gene of influenza isolates (n = 45)

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Figure 4: Number of mutations in neuraminidase gene of influenza isolates (n = 45)

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 ~ Discussion Top

Influenza A viruses are known to undergo genetic reassortment (genetic shift) of multiple gene segments from different lineages, and when such an assorted virus adapts to efficient human transmission, it attains pandemic potential. The first recorded pandemic influenza was in 1918. Since then many outbreaks had occurred including the recent one in 2009, and the real problem is the lack of predictability of influenza. Apart from the genetic shift which occurs once in every few decades, minor changes occur more frequently named genetic drift. These are small variations in the influenza genes (mutations) that occur over time during replication, due to the error-prone RNA polymerase enzyme of the virus. Most of the mutations are insignificant, but when these accumulate, they can result in variants that are antigenically different. This is the reason behind the recommendation of annual flu vaccine, which is prepared for each year based on the circulating variant strains.

Literature has shown that a few mutations in HA such as D222G, A156D and N173D increase the virulence of influenza strains causing severe pneumonia. A controversial study by Fouchier and his team has described that just a few substitutions in HA (4) and PB2 (1) can alter the host specificity among the highly pathogenic avian H5N1 viruses to infect mammalian cells via an airborne transmission.[15] Hence, it is imperative to conduct dynamic molecular surveillance worldwide to monitor the transmission and pathogenicity of influenza viruses, to inform policy makers for pandemic preparedness. In this study, we sequenced the two essential genes, HA and NA to genetically characterise the influenza A (H1N1) pdm09 strains isolated from Union territory of Puducherry between November 2011 and March 2015. All the 45 strains belonged to 2012 (16 strains) and 2015 (28 strains) except for one in 2011; 4 isolates (2012-2, 2015-2) were collected from patients who died due to influenza-induced ARDS.

D239N (D222G or D225G), located in the antigenic site Ca2 of HA, has been associated with dual-binding specificity of the virus for both 2, 3 (avian-like, found in lower respiratory tract of humans) and 2, 6-linked sialic acid receptors on host cells.[16] This mutation has been well-correlated with severe disease outcomes. In this study, 2 expired patients had isolates harbouring this mutation (2012-3, 2015-2). In 2009, a report from the WHO proposed that this mutation might not pose a major public problem, but many subsequent reports from many countries including India identified this D239N mutant in patients who died of severe influenza.[18] This mutation is known to emerge de novo and is more resistant to antiviral drugs and clinically is considered as a genetic signature for severe influenza infection and it is recommended that patients are given a prolonged treatment.[18] One of the 2012 isolates possessed the N173D (or S159N) mutation, which has also been seen to play a role in host specificity shift, but only 7 countries have reported this mutation so far (0.12% prevalence).[19]

All the 45 strains had S220T mutation in HA when compared with the reference strain and this has been reported in 88% of the isolates from >90 countries [Table 5]. This is considered as one of the characteristic mutation of clade 7 viruses. All the strains, except one (44/45) had S202T mutation in the Sb antigenic motif of HA gene. Since 2010, a new genetic subgroup characterised by this mutation has been found dispersed geographically and currently, S202T prevalence is 35% as per the reports from 53 countries.
Table 5: Details of haemagglutinin amino acid mutations observed in this study

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K180Q/K180I (or R166G) mutation in HA has been associated with antigenic drift/escape in both human and avian influenza strains.[21] In this study, we observed 10 strains with K180Q and all 10 were isolated in 2015; 3 strains had K180I mutation. More often Lysine has been replaced by Glutamine (K to Q) than Isoleucine (K to I) and the prevalence rate of these mutations lie around 18% and 0.6% respectively. D144E and A156D are the two other mutations related to antigenic drift and these were found in two separate strains isolated in 2012. A156D has also been shown to influence the host specificity shift; however, only a few countries have reported these two mutations (0.2%–0.3%).[20] K300E is the other common mutation in HA gene of our study isolates (n = 14); this particular mutation has been documented from 36 countries (22%) but has not been associated with any adverse outcome. Similarly, no significance of S160G mutation has been documented although it has been reported from 42 countries (6.5%) and we found this mutation in 11 of our isolates.

Antiviral drugs are the only option available for treatment of influenza and prevention during the emergence of outbreaks. The major strain in circulation, the influenza A (H1N1) pdm09, is resistant to M2 inhibitors, amantadine and rimantadine (S31N in M2 gene). Oseltamivir, the NAI, is the drug of choice to treat pandemic influenza infection, but their increased use has produced selective drug pressure, leading to the global emergence of resistant variants. NA gene of influenza type A viruses have catalytic (R118, D151, R152, R224, E276, R292, R371 and Y406) and framework residues (E119, R156, W178, S179, D/N198, I222, E227, H274, E277, N294 and E425) and mutations in this area related to NAI resistance. Although many mutations associated with NAI resistance have been identified, the amino acid changes that bestow multidrug resistance without reducing viral fitness remain poorly understood.

None of the influenza isolates had Tyrosine in place of Histidine at the 275 position of the NA amino acid sequence suggesting sensitivity to NAI, Oseltamivir. The single nucleotide polymorphism, cytosine to thymine, at 823 position results in H275Y mutation and WHO preliminary report released in June 2008, confirms that virus with this mutation confers high-level resistance to oseltamivir.[10] First reported case with oseltamivir-resistant influenza A(H1N1) harbouring H275Y mutation, was made in December 2013 by Potdar et al., however, the patient recovered after oseltamivir treatment.[22] The recent studies from India have confirmed the absence of this mutation in circulating influenza A (H1N1) pdm09 strains.[23],[24]

Seven strains had N295S (or N294S), the second most common mutation associated with oseltamivir resistance and six patients recovered after oseltamivir treatment while one female patient aged 56 years died despite receiving treatment. However, it is difficult to confirm whether drug resistance or ARDS led to mortality of the patient. Asparagine at 295th position is involved in drug binding activity and replacement by Serine, has been related to strong, multi-drug resistance.[25] More than 70 countries have reported N248D (or A246/S247/G248) mutation and this mutation is associated with multi-drug resistance [Table 6]. In this study, more than 45% (n = 21) of the isolates possessed this mutation and it was observed in three of the expired cases. Valine to Isoleucine change at the 106 position (V106I) has been reported in 71% of the global isolates and this has also been shown to cause multidrug resistance.[26] N248D and V106I are correlated with enhanced viral stability.[28]
Table 6: Details of neuraminidase amino acid mutations observed in this study

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Two potential mutations that are known to cause strong drug resistance, Q136K and V267A (or I266V) have been found in 3 and 1 of the isolates, respectively. Very few countries have reported these two mutations. V241I, which was observed in 19 of the study isolates, is known to enhance surface expression and activity of NA, and it is one of the permissive mutations along with N369K, playing a major role in compensating the destabilising effect of H275Y mutation.[29] The G236S mutation has not been reported elsewhere except in this study and role of Glycine in this position has to be explored further. A few other rare mutations were also found in our study isolates with no previous functional or clinical significance (E278G, S182T, E165D, F174S and F115S).

 ~ Conclusion Top

Multiple mutations were found to a greater extent in isolates, in this study, from 2015 than those from 2012 suggesting accumulation of mutations from the original strain of influenza A (H1N1) pdm09. Previous studies have shown that mutations in HA and NA accumulate over time and lead to functional changes conferring increased replication in human cells (human adaptation). However, globally, as well as with our strains, no changes in the surface antigens have occurred in comparison with the vaccine strain, prompting WHO to continue with the same vaccine strains as the previous years. However, the changes in these genes have to be monitored continuously as they determine the efficacy of current flu vaccines and anti-influenza drugs.


IDSP and NCDC, New Delhi, India for financial and technical support.

Financial support and sponsorship

Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, Integrated Disease surveillance programme and National centre for Disease control, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.

 ~ References Top

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Wagner R, Matrosovich M, Klenk HD. Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev Med Virol 2002;12:159-66.  Back to cited text no. 4
Puzelli S, Facchini M, De Marco MA, Palmieri A, Spagnolo D, Boros S, et al. Molecular surveillance of pandemic influenza A(H1N1) viruses circulating in Italy from May 2009 to February 2010: Association between haemagglutinin mutations and clinical outcome. Euro Surveill 2010;15:pii=19696.  Back to cited text no. 5
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Lackenby A, Thompson CI, Democratis J. The potential impact of neuraminidase inhibitor resistant influenza. Curr Opin Infect Dis 2008;21:626-38.  Back to cited text no. 9
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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