J. Microbiol. Biotechnol.
(2010), 20(11), 1500–1505
doi: 10.4014/jmb.1006.06031
First published online 29 September 2010
Envelope Proteins Pertain with Evolution and Adaptive Mechanism of the
Novel Influenza A/H1N1 in Humans
Mondal, Shakhinur Islam1, Abdullah Zubaer1, Simrika Thapa1, Chinmoy Saha1, Md. Asraful Alum1,
Md. Salman Reza1, Arzuba Akter2, and Abul Kalam Azad1,3*
1
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh
2
Department of Biochemistry and Molecular Biology, Dhaka University, Dhaka-1000, Bangladesh
3
Department of Life Science and Biotechnology, Shimane University, Shimane 690-8504, Japan
Received: June 22, 2010 / Accepted: August 4, 2010
The novel swine-origin influenza A/H1N1 virus (S-OIV)
first detected in April 2009 has been identified to transmit
from humans to humans directly and is the cause of the
currently emerged pandemic. In this study, nucleotide and
deduced amino acid sequences of the hemagglutinin (HA)
and neuraminidase (NA) of the S-OIV and other influenza
A viruses were analyzed through bioinformatic tools for
phylogenetic analysis, genetic recombination, and point
mutation to investigate the emergence and adaptation of
the S-OIV in humans. The phylogenetic analysis showed
that the HA comes from triple reassortant influenza A/
H1N2 and the NA from Eurasian swine influenza A/H1N1,
indicating that HA and NA descend from different lineages
during the genesis of the S-OIV. Recombination analysis
nullified the possibility of occurrence of recombination in
HA and NA, denoting the role of reassortment in the
outbreak. Several conservative mutations were observed
in the amino acid sequences of the HA and NA, and these
mutated residues were identical in the S-OIV. The results
reported herein suggest the notion that the recent
pandemic is the result of reassortment of different genes
from different lineages of two envelope proteins, HA and
NA, which are responsible for the antigenic activity of the
virus. This study further suggests that the adaptive
capability of the S-OIV in humans is acquired by the
unique mutations generated during emergence.
Keywords: Hemagglutinin, influenza A/H1N1, mutation,
neuraminidase, reassortment, S-OIV
A novel swine-origin influenza A/H1N1 virus (S-OIV)
emerged in Mexico and the United States in March and
*Corresponding author
Phone: +88-0821-717850, Ext. 411; Fax: +88-0821-715257;
E-mail: dakazad-btc@sust.edu
early April 2009 [12]. The virus had spread to 66 countries
with more than 414,000 laboratory confirmed cases
including nearly 5,000 deaths up to 17 October 2009
[World Health Organization. 2009. http://www.who.int/csr/
disease/swineflu/updates/en/index.html]. Since the emergence
of the pandemic-2009 to 13 June 2010, more than 214
countries have reported laboratory confirmed cases of the
S-OIV that caused over 18,172 deaths [http://www.who.int/
csr/don/2010_06_18/en/index.html]. The influenza A/H1N1
pandemic of the 21st century has an evolutionary history of
about 100 years. After the first pandemic emerged in 1918
[12], periodical outbreaks occurred from 1928 to the present
with H1N1 subtypes, and the severe H1N1 epidemic
occurred in 1950-1951 [3, 11, 14, 17].
Influenza A viruses are single-stranded RNA viruses of
negative sense with an eight-segmented genome and belong
to the family Orthomyxoviridae [11]. These eight gene
segments composed in order encode polymerases (PB1,
PA, and PB2), hemagglutinin (HA), nucleoprotein (NP),
neuraminidase (NA), matrix protein (M), and nonstructural
protein (NS) [8]. HA and NA are envelope glycoproteins
and are the key antigens against which humoral immune
responses are directed [11], and hence the study on these
HA and NA is needed for understanding the control and
prevention of H1N1 viruses [17].
Pandemics are believed to arise when a novel avian
influenza HA and/or NA (together with the PB1 gene
segment in the pandemics of 1957 and 1968) are picked up
through reassortment by preexisting human influenza
viruses or by a purely avian virus adapting to efficient
human transmission [11, 14]. Some pathogens can obtain
the ability of cross-species transmission from animal
reservoir species to humans and even human-to-human
transmission via adaptive mutation and reassortment of
various lineages [10], and recombination is a mechanism
driving evolutionary change [4] although it is rare in influenza
1501 Mondal et al.

A/H1N1 [1]. The influenza A viruses have good potentiality
to rearrange themselves to be more infectious [11]. The
current pandemic S-OIV has divulged a complicated
reassortment of various influenza A viruses [17]. Mutations
are believed to contribute to the adaptation of some
influenza viruses to the host, and others might be fixed
through genomic hitchhiking [3]. In the future, the S-OIV
has the possibility to erupt with higher infectivity and more
stability in human hosts than the present one. Although
many research papers have been published on the S-OIV
[3, 11, 12, 14, 17], extensive studies are still necessary as
the clue is novel. Because the HA and NA are antigenic,
studies should be focused on these proteins. Moreover,
mutation analysis of HA and NA might be informative for
adaptation in the aspect of infection potential and also for
drug discovery or vaccine development. We herein performed
phylogenetic and recombination analyses of HA and NA to
test whether the two events, reassortment and recombination,
have an influence on the evolution of the S-OIV and
mutation analysis was done to investigate the adaptive
mechanism in terms of infection potential.
MATERIALS AND METHODS
Sequences
The nucleotide sequences and the corresponding amino acid sequences
of HA and NA of the novel S-OIV were collected from the NCBI
influenza virus resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/
SwineFlu.html). To investigate the evolutionary relationship, HA
and NA sequences of other influenza A/H1N1 viruses isolated from
North America, Europe, and Asia were included. Because of the
emergence of H1N1 in North American swine, which continued to
circulate in pigs in Asia and Europe [6, 9], the sequences from the
isolates of these areas were taken. Because the S-OIV were
demonstrated to belong to influenza A virus subtype H1N1, HA and
NA sequences of other influenza A/H1N1 sampled from human,
avian, and swine around the world during the period 1918-2009
were also retrieved from the database. In view of the fact that in the

Fig. 1. Phylogenetic tree among influenza A/H1N1 and H1N2 viruses including the S-OIV, based on the HA.
The tree was constructed using the HA sequences of California/09 (ACP41926), A/New [New York/23/2009] (ACR18978), New [New York/19/2009]
(ACP44147), Swine/Ohio (AAL87865), swine/Korea (ACE77937), swine/Minnesota (ABV25641), Sapporo/1/2009 (ACT22037), California/06/2009
(ACP41935), Louisiana/03/2009 (ACR67197), Texas/30/2009 (ACU13096), Sichuan/1/2009 (ACR32986), Taiwan/137/2009 (ACV53909), Toronto/
T9842/2009 (ACV53500), Brevig_Mission/1/18 (AAD17218), New_York/1/18 (AAD17219), South Carolina/1/18 (AAD17229), London/1/1918
(AAO65768), swine/Alberta (ABB86887), swine/Kansas (CAO82678), swine/Minnesota (ABV25639), swine/Wisconsin (ABS49954), swine/Minnesota
(ABR28636), swine/Cotes d’Armor (ABV25642), swine/Shandong (ACK57717), swine/Chachoengsao (BAH02080), pintail duck/Alberta (AAB50963),
mallard/Maryland (ACS68352), mallard duck (ABB19518), and swine/Beijing (ABB19628).
 ORIGIN AND ADAPTIVE MECHANISM OF NOVEL INFLUENZA A/H1N1 1502

triple reassortant, H1N2 has contributed its segment in the novel virus
[5, 17], gene sequences of some swine A/H1N2 were also included.
Phylogenetic Analysis
The nucleotide sequences of HA and NA of the S-OIV were aligned
with that of other influenza A viruses using the ClustalW2 program
(http://www.ebi.ac.uk/tools/clustalw2) [7]. The sequence alignments
were performed under default condition. Genetic distance was estimated
by the p-distance method. Phylogenetic trees were constructed by
the neighbor-joining method. Bootstrap resampling and reconstruction
were performed 1,000 times to confirm the reliability of phylogenetic
trees. The computer software of the Molecular Evolution Genetic
Analysis (MEGA), version 4.0 [13], was utilized in this study for
phylogenetic analysis of selected sequences.
Mutation Analysis
To scrutinize whether the HA and NA gene segments of the S-OIV
are driven by positive selection or not, the codon-based Z-test was
performed in MEGA 4.0. Amino acid sequences of HA and NA of
the S-OIV along with those of the respective neighbor clades were
aligned in ClustalW2 to observe the mutation sites individually.
Only the closest ancestor of HA (A/H1N2) and that of NA (Eurasian
swine A/H1N1) were subjected to alignment one by one in
ClustalW2 to get the consensus sequence for each from the Jalview
[2]. For both HA and NA, the built consensus sequence was further
aligned with the S-OIV, and the mutation profile was constructed in
Mutation Master [15] using the consensus sequence as a reference
sequence.

Recombination Analysis
The aligned nucleotide sequences of the HA and NA in FASTA
format obtained from ClustalW2 was applied to the Recombination
Analysis Tool (RAT) for recombination analysis. The Single-
Sequence Viewer of RAT was used to examine the recombination
crossover point (http://cbr.jic.ac.uk/dicks/software/RAT/index.html).
The RAT algorithm is based on the distance method, using pairwise
comparisons between sequences [4].
RESULTS AND DISCUSSION
Evolutionary Relationship
To determine the evolution of the S-OIV, the phylogenetic
analysis was performed among HA and NA sequences of
influenza A/H1N1 and A/H1N2 from human, avian, and
swine (1918 to 2008) including those of the S-OIV. This

Fig. 2. Phylogenetic tree among influenza A/H1N1 and H1N2 viruses including the S-OIV, based on the NA.
The tree was constructed using the NA sequences of Brevig_Mission/1/18 (AAF77036), California/05/2009 (ACP41931), New [New York/06/2009]
(ACQ63214), Mexico2009 (ACQ73395), Ohio2009 (ACQ76385), Indiana2009 (ACQ76379), Texas2009 (ACQ55360), swine/Belgium (ACN67525),
swine/Italy (ABS50325), swine/Hungary (ACN72621), swine/Spain (ABD78107), Taiwan2009 (ACU29991), swine/Thailand (ACM80371), swine/Cotes
d'Armor (CAO82693), duck/Hokkaido (BAG85128), duck/Tsukuba (BAH69237), NJ/11/76 (AAF77043), swine/Tennessee (ABD95715), swine/Ontario
(ABR28672), swine/Kansas (ABR29578), Hanoi (BAF63065), Leiden (BAG72240), Charlottesville (AAL60437), duck/Ohio (AAF77038), duck/Alberta
(AAF77042), pigeon (ABB21775), and muscovy duck/New York (ACA04511).
1503 Mondal et al.

analysis showed that the S-OIV isolates clustered together,
suggesting that these had evolved independently (Fig. 1
and 2). The clade of the HA gene segment was divulged to
cluster closely with the triple reassortant swine A/H1N2
lineage (Fig. 1), which had originated from the reassortment
of three H1N1 lineages, namely North American swine
H1N1, seasonal human lineage, and avian lineage [2]. The
triple reassortant swine influenza A/H1N1 lineages had
circulated in swine around the world and occasionally
resulted in human infections over the past several years
[3]. The clade of the NA gene segment revealed that the
NA segment of the S-OIV evolved directly from Eurasian
swine lineage A/H1N1 (Fig. 2). It has been reported that
influenza viruses have a high potentiality to reassort from
various lineages [16] and the complicated reassortment has
been demonstrated to play a role in the evolution of the
current pandemic S-OIV [17] making it more infectious
[11]. This fact of reassortment behind evolutionary history
was achieved when the two proteins analyzed to reveal
the evolutionary relationship resulted in two distinct
phylogenetic trees that highlight on HA and NA of the S-
OIV being originated directly from the triple reassortant
swine A/H1N2 lineage and Eurasian swine A/H1N1
lineage, correspondingly (Fig. 1 and 2).
The sequences were further analyzed with RAT to
investigate whether there is an influence of recombination
on evolution. RAT analysis showed no prevalence of
recombination for the envelope proteins of the S-OIV (data
not shown), supporting the notion that the current
pandemic had originated through reassortment rather than
recombination. However, recombination has been demonstrated
to be a rare case in influenza A viruses, and even if it

Fig. 3. Mutation profiles of HA in the S-OIV.

A. Amino acids substituted in HA of the S-OIV lineage compared with their neighboring clade (triple reassortant swine A/H1N2 lineage as shown in Fig. 1)
are boxed, and the position of the amino acid residues is indicated below the box. The alignment was performed by the ClustalW2 computer program. Amino
acid residues not shown are identical in all sequences. B. Analysis with Mutation Master shows the conservative nature of mutations in the HA. Stars (*) in
the horizontal line indicate the unique mutation sites. The bottom horizontal line represents the amino acid position. The second line indicates the total
number of mutant amino acids at each position. The vertical axis shows the BLOSUM value, and the one-letter amino acid in the box represents the most
common substitution. The black line at the “0” position indicates the BLOSUM score of a certain mutation in a highly conserved protein to be “0”.
 ORIGIN AND ADAPTIVE MECHANISM OF NOVEL INFLUENZA A/H1N1 1504

occurs during the course of emergence, it has a minor role
in the evolution of the virus [1].
Adaptive Mechanism
Positive selection drives viral evolution during cross-
species transmission, and negative selection assures the
viruses to be adapted to the new host [3]. The codon-based
Z-test that compares the amount of synonymous and non-
synonymous mutations among the species was used for
positive and negative selections. In the test, the null
hypothesis (dN=dS) was rejected in favor of purifying
selection (dN<dS) at the 5% level of significance. It
assumes that the null hypothesis is H0 :dN=dS [13]. The
rejection of H0 in favor of positive selection was insignificant,
indicating that the HA and NA are not evolved during
cross-species transmission. On the other hand, the null
hypothesis being rejected in favor of purifying selection
(dN>dS) established the prevention of fixation of harmful
mutation in both HA and NA gene segments, and thus
these two genes play a significant role in the adaptation of
the S-OIV to the new human host [12].
The amino acid sequences of the HA and NA in the S-
OIV and in their neighboring clade were compared to
analyze the mutations. The ClustalW2 alignments showed
that 17 and 16 unique amino acid residues in HA and NA,
respectively, are substituted (Fig. 3A and 4A). These newly
evolved amino acid residues in HA and NA might be
important in the adaptation of the S-OIV [3]. The conservative
or nonconservative nature of the unique mutations was
studied using Mutation Master, which signifies the fact
by calculating the BLOSUM values. The positive and
negative BLOSUM values point out the conservative and
nonconservative substitutions, respectively [15]. Fig. 3B
and 4B show the point mutation profile obtained from
Mutation Master. Among the 17 substituted amino acids in
HA, 13 residues had positive (3 of 13 have zero) and only
4 had negative BLOSUM values (Fig. 3B). These data
indicate that most of the substitution mutations in HA of
the S-OIV are conservative mutations. Despite the 7 amino
acids that had faintly negative BLOSUM values, 10
substituted amino acid residues in NA of the S-OIV had
zero to positive BLOSUM values (Fig. 4B). These data

Fig. 4. Mutation profiles of NA in the S-OIV.

A. Amino acids substituted in NA of the S-OIV lineage compared with their neighboring clade (Eurasian swine A/H1N1 lineage as shown in Fig. 2) are
boxed, and the position of the amino acid residues is indicated below the box. The identical amino acids in all sequences are not shown. B. Analysis with
Mutation Master shows the conservative nature of mutations in NA and is illustrated as in Fig. 3B.
1505 Mondal et al.
also support the same view that the maximum of the
substitution mutations are conservative. Being conservative
in nature, these unique sites in the HA and NA of the S-
OIV lineage remain exclusively identical and may play a
key role in the adaptation of these viruses to human hosts.
Based on the results reported in this study, it can be
concluded that the S-OIV is the outcome of a reassortment
of the HA and NA gene segments from different lineages,
and conservative substitution mutations in the unique sites
of the HA and NA prevail the S-OIV for adaptation in the
human host and gaining the infectivity with the start of the
new era.
Acknowledgments
We are thankful to Professor Haseena Khan (Department
of Biochemistry and Molecular Biology, Dhaka University,
Bangladesh) for her valuable discussions related to this
research. We also give indebted thanks to Professor
Yasmeen Haque (Dean, School of Life Sciences, Shahjalal
University of Science and Technology, Bangladesh) for the
support in developing a bioinformatics laboratory.
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