Complete-Proteome Mapping of Human Influenza A Adaptive Mutations: Implications for Human Transmissibility of Zoonotic Strains

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DOI: 10.1371/journal.pone.0009025 · Source: PubMed
Abstract
There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains, to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates. We constructed a dataset of influenza A protein sequences from 92,343 public database records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses reassort frequently, causing both gains and losses of adaptive mutations. Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins. Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually high number of adaptive mutations. However, these mutations appear unstable, suggesting low pandemic potential of H5N1 in its current form. In addition, adaptation signatures indicate that pandemic H1N1/09 strain possesses multiple human-transmissibility mutations, though not an unusually high number with respect to swine strains that infected humans in the past. Adaptation signatures provide a novel tool for identifying zoonotic strains with the potential to infect humans.

Figures

Complete-Proteome Mapping of Human Influenza A
Adaptive Mutations: Implications for Human
Transmissibility of Zoonotic Strains
Olivo Miotto
1,2
*, A. T. Heiny
3
, Randy Albrecht
4
, Adolfo Garcı
´
a-Sastre
4,5,6
, Tin Wee Tan
3
, J. Thomas
August
7
, Vladimir Brusic
8
1 Centre for Genomics and Global Health, University of Oxford, Oxford, United Kingdom, 2 Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University,
Rajthevee, Bangkok, Thailand, 3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore, 4 Department of
Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America, 5 Division of Infectious Diseases, Department of Medicine, Mount Sinai
School of Medicine, New York, New York, United States of America, 6 Emerging Pathogens Institute , Mount Sinai School of Medicine, New York, New York, United States of
America, 7 Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 8 Cancer
Vaccine Center, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America
Abstract
Background:
There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they
become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires
important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains,
to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates.
Methodology/Principal Findings:
We constructed a dataset of influenza A protein sequences from 92,343 public database
records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify
characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic
sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin
and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence
between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting
their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation
signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three
decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal
protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses
reassort frequently, causing both gains and losses of adaptive mutations.
Conclusions:
Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins.
Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually
high number of adaptive mutations. However, these mutations appear unstable, suggesting low pandemic potential of
H5N1 in its current form. In addition, adaptation signatures indicate that pandemic H1N1/09 strain possesses multiple
human-transmissibility mutations, though not an unusually high number with respect to swine strains that infected humans
in the past. Adaptation signatures provide a novel tool for identifying zoonotic strains with the potential to infect humans.
Citation: Miotto O, Heiny AT, Albrecht R, Garcı
´
a-Sastre A, Tan TW, et al. (2010) Complete-Proteome Mapping of Human Influenza A Adaptive Mutations:
Implications for Human Transmissibility of Zoonotic Strains. PLoS ONE 5(2): e9025. doi:10.1371/journal.pone.0009025
Editor: Art F. Y. Poon, Providence Health Care, Canada
Received October 16, 2009; Accepted December 27, 2009; Published February 3, 2010
Copyright: ß 2010 Miotto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors acknowledge support by the Medical Research Council , United Kingdom; Federal funds from the National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Department of Health and Human Services, United States of America, under Grant No. 5 U19 AI56541 and
Contract Nos. HHSN2662-00400085C and HHSN2662-00700010C. VB acknowledges support in part by the ImmunoGrid project, under EC contract FP6-2004-IST-4,
NO 028069. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: olivo.miotto@ndm.ox.ac.uk
Introduction
Influenza A is a virus belonging to the Orthomyxoviridae family,
which circulates amongst various animal species. Although aquatic
wildfowl are the natural reservoir, influenza A viruses routinely
infect many types of domestic birds and several mammalian
species. In humans, influenza A viruses are the cause of
widespread annual epidemics and of less frequent pandemics,
four of which were recorded within the last century [1]. The threat
of a new pandemic is cause of the greatest concern, because of the
prospect of high death tolls: the Spanish flu in 1918/19 claimed
over 40 million lives, making it possibly the most destructive event
in medical history [2]. The rapid spread and large-scale effect of
pandemics are enabled by the presence of novel surface
glycoproteins, hemagglutinin (HA) or neuraminidase (NA), for
which the human population has no immune memory. Sixteen
PLoS ONE | www.plosone.org 1 February 2010 | Volume 5 | Issue 2 | e9025
serologically distinct HA types, and nine NA types, are known to
circulate in the avian host population; over 100 avian influenza
subtypes have been catalogued to date, resulting from the
combination of different HA and NA types. Only three of these
subtypes, (H1N1, H2N2 and H3N2) are known to have circulated
amongst humans in the last century. Other influenza subtypes of
avian origin have infected humans without acquiring the ability to
spread in the human population [3]. Current concerns focus on
two pandemic threats: from the H5N1 highly pathogenic avian
influenza (HPAI) and from the swine-origin H1N1/09 strain.
H5N1 viruses have been responsible for a considerable number of
human infections and deaths: according to the WHO, 395
individuals were infected by H5N1 between 2003 and 2008,
resulting in 250 deaths (www.who.int/csr/disease/avian_
influenza/). Although no definitive evidence of human-to-human
(H2H) transmission of H5N1 has been reported, there is
widespread concern that these viruses could cause a new
devastating pandemic if they acquire such capabilities. The
H1N1/09 swine strain was first reported to infect humans in
April 2009 [4], and has been classified as pandemic by the WHO
(http://www.who.int/csr/disease/swineflu/). Although this virus
currently appears to cause mild disease [5], its rapid global spread
is causing public alarm as the next epidemic season approaches.
Despite the urgency, it is currently impossible to reliably predict
the emergence of a new pandemic [1], and new tools are needed
for scientists and policymakers to evaluate the pandemic risk posed
by zoonotic viruses.
Limited spread of zoonotic influenza in humans indicates that
immunological naivety of the host population is not a sufficient
condition for initiating a human pandemic, and additional
adaptive mutations in the virus are required. Such mutations
appear not to be limited to the HA and NA proteins, but are also
distributed across its nine internal proteins [6] (for conciseness, we
will refer to all proteins other than HA and NA as ‘‘internal’’,
although a small domain of the M2 protein is externally exposed).
A full reconstruction of this complex landscape of adaptive
mutations is needed for elucidation of biological mechanisms of
viral adaptations to humans. Detailed knowledge of adaptive
mutations may also reveal whether zoonotic strains have the
potential for acquiring H2H transmissibility without needing to
reassort with human strains. A cost effective approach to
identifying mutations of critical importance for host range is to
conduct comparative analyses of large groups of human and avian
protein sequences, to identify candidate mutation sites that can
subsequently be experimentally validated. At such characteristic sites,
a residue that is highly conserved within the human group but
rarely observed in the pool of avian strains (a characteristic variant)is
likely to be associated with an important adaptive mutation, whose
loss would affect the ability of viruses to propagate amongst human
hosts. Studies based on visual inspection of small numbers of
representative isolates found characteristic sites in matrix proteins
[7] and polymerases [8,9]. Large-scale computational methods
have used statistical variability measures such as information entropy
to identify characteristic sites for human transmissibility. An
analysis of 401 full viral proteomes [10] identified characteristic
sites by comparing entropy in the avian and human groups, which
limited its applicability to positions that are highly conserved in
both groups. Finkelstein et al. [11] employed statistical tests that
compared residue frequencies, to construct a catalogue of 32
characteristic mutations in five influenza proteins from the analysis
of more than 23,000 sequences.
This report describes a large-scale complete-proteome analysis
of influenza A sequences: a form of genome-wide association
analysis in which a statistical measure is applied to compare two
alignments of sequences, characterized by phenotype (human-
adapted vs. non-adapted). This method, which uses mutual
information as the statistical measure, was previously applied
successfully to the study of the PB2 polymerase [12]. The
catalogue of characteristic sites identified by our analysis was then
applied to derive adaptation signatures of viral proteomes, which
summarize the residue profiles at all characteristic sites for any
given isolate. By rendering these signatures graphically, we
reconstructed the history of the emergence of adaptive mutations
in human-infecting influenza A viruses. We also used signatures to
analyze the presence of H2H adaptive mutations in avian and
swine viruses, and discussed the implications on the pandemic
potential of zoonotic influenza.
Materials and Methods
Data Collection and Preparation
We compiled a dataset of all available influenza A sequences
(as of September 2006) from the NCBI GenBank and GenPept
databases [13], including entries mirrored from UniProt [14]. A
total of 92,343 records were retrieved from these databases,
using taxonomy-based queries; entries from different databases
that referred to the same sequences were subsequently merged.
If sufficient information was available, sequences were anno-
tated with descriptive metadata properties: isolate name, host
organism, subtype, year of isolation, geographic origin, and
protein name. The resulting dataset was verified by two
independent curators, who discarded duplicates, laboratory
strains, sequences with missing key metadata, and sequences
with quality issues. The final set comprised a total of 40,169
unique sequences, including both full-length sequences and
fragments, covering all influenza A proteins. The data
collection and cleaning process was largely automated by the
Aggregator of Biological Knowledge (ABK) tool, which uses a
rule-based approach to aggregating data from multiple database
sources [15].
For each of the eleven influenza proteins, a master multiple
sequence alignment (MSA) was constructed using the MUSCLE
3.6 [16] software. The MSAs were manually inspected and
corrected. Multiple subset alignments, to be used in comparative
analyses, were extracted from the master alignments based on
their metadata values, using the Antigenic Variability Analyzer
(AVANA, http://avana.sourceforge.net), developed by the au-
thors to support information-theoretical analysis tasks [17,18].
AVANA was also used to conduct all comparative analyses
described in this paper.
Subset Selection
The objective of this study was to identify sites where
characteristic mutations are present in the majority of human
influenza A viruses. Two major lineages of human influenza A are
currently co-circulating: H3N2 and H1N1. In spite of their
common origin (Figure 1), the internal protein constellations of
these two lineages have evolved independently, following the
disappearance of H1N1 in 1957 and its reintroduction in 1977
[19]. Because of genetic similarity and common descent, the
internal proteins of subtypes H2N2 and H1N2 were grouped with
H3N2 in a lineage named HxN2, while H1N1 formed the other
lineage. For each of the nine internal proteins, three subsets were
therefore extracted: A2A (all avian sequences, except for H1N1,
H2N2, H1N2, H3N2 and H5N1 subtypes), H1N1H (all H1N1
human sequences) and HxN2H (all human sequences of subtypes
H2N2, H1N2 and H3N2). Since true adaptive mutations are
expected to be present in both lineages, we analyzed each lineage
Human Influenza Adaptation Map
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separately, discarding sites that are not shared by both human
influenza lineages. Although subtype H1N2 has not been
confirmed to be a stable circulating human subtype, it was
included in the HxN2H group because of the strong sequence
similarity of its internal proteins to that group: we found that
removing this subtype from the analysis does not change our
catalogue of characteristic sites. H5N1 sequences were removed
from both avian and human subsets because of this subtype’s
pronounced ability to jump the species barrier.
In a subsequent refinement of the NS1 analysis, we separated
sequences for this protein into two groups, corresponding to the
two major NS variants known as Alleles A and B, whose
nucleotide sequences exhibit approximately 70% identity
[20,21]. Sequences were classified based on similarity to a
reference Allele B isolate (A/pintail duck/ALB/121/1979
(H7N8)). While 31% of A2A sequences were classified as Allele
B, all H2H sequences were found to belong to Allele A, supporting
the hypothesis that human influenza A evolved from an avian
Allele A lineage [22]. Comparisons restricted to Allele A sequences
are therefore more likely to identify mutations caused by host
adaptation rather than lineage difference. H2H sequences were
compared against the Allele A subset of A2A sequences, and only
characteristic sites that met our selection criteria in this
comparison were selected for our final catalogue.
To analyze adaptation signatures for different virus groups, we
also collected subsets of avian H5N1 (H5N1A) and human H5N1
(H5N1H)sequences,aswellassubsetsofswine(SW)andequine
(EQ) sequences. The number of sequences in each of the
extracted datasets is included in Table 1. Because of the high
degree of genetic divergence in the glycoproteins, we compared
separately each subtype that has circulated amongst humans: H1,
H2 and H3 subtypes of the HA protein, and N1 and N2 subtypes
of NA. Table 2 shows the number of sequences included in each
HA subset, and Table 3 shows the subset sizes for the NA
proteins.
Identification of Characteristic Sites and Variants
The method for identifying characteristic sites has previously
been described in detail [12]. Briefly, an aligned set of adapted
sequences (capable of H2H transmissibility) was compared against
a reference set (not H2H-transmissible) to reveal mutations common
in the adapted set, but rare in the reference set. To measure the
strength of association between mutations and sequence sets, we
used mutual information (MI), an information theoretical statistic that
measures the strength of association between a pair of variables
[23]. MI is defined in terms of information entropy, a measure of
variability. The information entropy H(x) of a discrete variable x is
given by:
H(x)~{
X
e[E
p
e
(x)log
2
p
e
(x)ðÞ ð1Þ
where E ={e
1
, e
2
e
n
} is the set of all possible discrete values of
x,andp
e
(x) is the probability that e ME is the value of x.The
mutual information between two variables A and B is then
defined by:
MI(A,B)~H(A)zH(B){H(A,B) ð2Þ
where H(A)andH(B)areentropiesofAandB,whileH(A,B)is
their joint entropy, computed from equation (1) by replacing E
with the set of all unique pair of values (A,B).
To identify characteristic sites, we have modified equation (2) to
compute the MI between an observed residue a at position x, and
the label S of the set (alignment) within which residue a is observed:
MI(x)~H
a
(x)zH
S
(x){H
S,a
(x) ð3Þ
H
a
(x) is the entropy in an alignment formed by merging the two
set, while H
S
(x) is derived from the number of sequences in each of
Figure 1. Human Influenza A reassortment events of the 20
th
Century. The reassortment events associated with human pandemics in the
20
th
century (adapted from Webster et al. [39]). A full complement of eight gene segments of zoonotic origin caused the 1918 Spanish flu. In 1957,
the H2N2 Asian flu pandemic replaced the HA, NA and PB1 segments, and in 1968 the H3N2 Hong Kong pandemic replaced the HA and PB1
segments only [40]. In both cases, the new subtype fully replaced the subtypes previously circulating amongst humans. The 1977 Russian epidemic
introduced an H1N1 strain almost identical to that circulating prior to 1957, and may have been caused by the release of 20-year old frozen viruses
[19]. The H1N1 and HxN2 lineages have since co-circulated in the human population; recently, their reassortment has given rise to human strains of
H1N2 subtype.
doi:10.1371/journal.pone.0009025.g001
Human Influenza Adaptation Map
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the two sets (n
1
and n
2
):
H
S
(x)~{
n
1
N
log
2
n
1
N

{
n
2
N
log
2
n
2
N

ð4Þ
where N = n
1
+ n
2
. Finally, H
S,a
(x) is given by:
H
S,a
(x)~{
X
S
X
a[A
p(S,a)log
2
p(S,a) ð5Þ
where p(S,a) is the probability of any given combination of residue
and set label.
When comparing two sets of equal size, the above equations
give MI values in the range 0#MI(x)#1. At alignment sites with
high MI, the observed residues are strongly associated to a given
set; conversely, sites with low MI exhibit similar distributions of
residues in the two sets.
As equation (4) suggests, MI is reduced when the two sets have
unequal sizes. To compensate for this bias and standardize
results, we have applied a statistical correction, based on repeated
resampling. For each pair of sets, we repeatedly compared the
smaller set to a set of equal size, randomly sampled without
replacement from the larger set. The final MI values were the
average over 1000 iterations. This method is effective in
correcting the size bias [12], and confers robustness to the MI
measurement. In addition, by selecting strains randomly across all
phylogenetic groups, it partially corrects for phylogenetic
sampling biases.
Characteristic sites and their characteristic variants (mutations)
were selected based on four empirical criteria, whose rationale is
detailed in [11], and summarized as follows:
N
At a characteristic site, MI$0.4 (found to be the MI threshold
below which avian and human sequences converge to the same
consensus amino acids).
N
A characteristic variant must be 4 times more common in one
set than in the other set (threshold determined from variant
distribution analysis for the PB2 and NS1 proteins)
N
A characteristic variant must occur in at least 2% of the
sequences within the set it represents (a threshold found to be a
good compromise between the minimum representation of
characteristic mutations and the maximum representation of
non-characteristic mutations in PB2)
N
An avian characteristic variant must be uncommon in the
H2H set at a characteristic site. We have manually inspected
all sites where avian variants accounted for more than 2% of
sequences in at least one H2H lineage. All accepted
characteristic sites had less than 5.2% avian variants (average
at all characteristic sites was 0.71%).
Only characteristic sites present in both H2H lineages were
included in the final catalogue.
Reconstruction of Adaptation Signatures
The variants that distinguish H2H sequences and A2A
sequences at characteristic sites form a characteristic variant pattern,
a summary of the significant differences between the two sets of
sequences across the whole proteome. This pattern was used to
Table 1. Count of influenza A internal protein sequences used in the current study.
Sequences used in MI Analysis Additional sequences used in signature analysis
A2A H1N1H HxN2H Total H5N1A H5N1H SW EQ Total
M1 1047 300 1521 2868 458 105 190 22 775
M2 736 286 1517 2539 289 95 104 21 509
NP 884 316 1645 2845 420 114 230 22 786
NS1 1123 303 1448 2874 457 95 172 38 762
NS2 810 292 1419 2521 288 81 113 21 503
PA 701 279 1362 2342 402 102 161 11 676
PB1 716 303 1385 2404 400 101 163 19 683
PB2 719 281 1369 2369 404 97 161 17 679
PB1-F2 352 262 1280 1894 - - ---
Total 7088 2622 12946 22656 3118 790 1294 171 5373
Characteristic site analysis was conducted using the A2A, H1N1H and HxN2H sets. The H5N1A, H5N1H, SW and EQ sets were used for sequence signature analysis.
doi:10.1371/journal.pone.0009025.t001
Table 2. Count of influenza A hemagglutinin protein
sequences used in the current study.
Avian Human Total
H1 48 768 816
H2 80 75 155
H3 115 3105 3220
Total 243 3948 4191
doi:10.1371/journal.pone.0009025.t002
Table 3. Count of influenza A neuraminidase protein
sequences used in the current study.
Avian Human Total
N1 717 360 1077
N2 439 1801 2240
Total 1156 2161 3317
doi:10.1371/journal.pone.0009025.t003
Human Influenza Adaptation Map
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construct the adaptation signatures of several influenza proteomes, by
discarding all residues except those at characteristic sites. Residues
forming the signatures were tagged as A2A-like (i.e. a characteristic
variant of the A2A subset), H2H-like (an H2H characteristic
variant), or as non-characteristic. The resulting signatures thus
provide a succinct summary of H2H adaptive mutations contained
in any influenza proteome. To facilitate the evaluation of multiple
isolates, we developed a software program to graphically display
selected signatures along a timeline, using a contrasting color
scheme to distinguish between A2A-like and H2H-like residues.
Results
Catalogue of Characteristic Sites
Our analysis produced a catalogue of 68 characteristic sites that
met selection criteria (Table 4). Characteristic sites were found in
eight of the nine internal proteins, suggesting that adaptation to
humans requires participation of most products encoded by the
viral genome. The location of characteristic sites found within the
internal proteins is shown in Figures 2, 3 and 4, alongside the
mapping of known functional domains in these proteins. As shown
in Figure 2, the three internal proteins found to contain the highest
number of characteristic sites were PB2 (17 sites), PA (17 sites), and
Table 4. Full catalogue of identified characteristic sites for
H2H transmission of influenza A.
Protein Position A2A H2H
H1N1
CV
HxN2
CV
CV Cons CV Cons X-pres
M1 115 V 99.70% I 99.39% 0.61% I I
121 T 94.94% A 99.89% 0.11% A A
137 T 99.60% A 99.23% 0.77% A A
M2 11 T 97.28% I 96.89% 3.11% I I
14 G 95.99% E 98.28% 1.72% E E
20 S 97.14% N 97.94% 2.06% N N
28 I 76.36% V 97.72% 2.11% V V
54 R 98.91% LIF 98.94% 0.61% IL LF
55 L 79.18% F 99.33% 0.67% F F
57 Y 99.59% H 97.38% 2.18% H H
78 Q 99.72% KE 99.26% 0.28% EK K
86 V 99.84% A 99.21% 0.45% A A
NP 16 GS 99.16% D 99.49% 0.51% D D
33 V 99.76% I 98.97% 1.03% I I
61 I 98.36% L 99.43% 0.57% L L
100 R 99.65% VI 99.71% 0.06% V VI
136 L 85.41% MI 99.77% 0.11% I MI
214 R 96.64% K 99.32% 0.68% K K
283 L 100.00% P 99.48% 0.47% P P
305 R 99.17% K 99.33% 0.67% K K
313 F 99.31% Y 99.48% 0.52% Y Y
357 Q 98.43% KR 99.90% 0.10% KR K
375 DN 96.93% GEV 99.34% 0.56% V GE
423 A 97.06% STP 98.88% 1.00% T SP
NS1 22 FL 97.07% V 98.21% 0.40% V V
60 AE 97.59% V 99.20% 0.69% V V
81 I 98.66% M 99.08% 0.69% M M
84 VS 96.08% TA 99.20% 0.80% A TA
215 PSA 99.24% T 99.37% 0.63% T T
227 E 98.87% R 99.53% 0.06% R R
NS2 60 S 76.77% NH 98.89% 0.82% H N
70 S 97.46% G 99.88% 0.12% G G
107 L 99.60% F 98.77% 1.17% F F
PA 28 P 100.00% L 99.14% 0.67% L L
55 D 99.69% N 99.63% 0.37% N N
57 R 96.61% Q 98.72% 0.79% Q Q
65 SF 99.08% LP 99.63% 0.37% PL L
66 GS 99.69% DE 98.84% 1.10% ED D
100 V 96.15% A 99.27% 0.37% A A
225 S 98.61% C 99.39% 0.61% C C
268 L 98.84% I 99.14% 0.73% I I
321 NK 97.35% YST 97.30% 0.74% STY Y
337 AT 99.34% S 99.75% 0.25% S S
356 K 98.51% R 99.26% 0.74% R R
382 E 94.34% D 97.37% 2.45% D D
400 PSQ 89.32% L 99.45% 0.31% L L
404 A 99.48% S 99.39% 0.55% S S
Protein Position A2A H2H
H1N1
CV
HxN2
CV
CV Cons CV Cons X-pres
409 S 91.49% N 99.45% 0.49% N N
421 S 98.91% IV 97.79% 0.55% I IV
552 T 99.81% S 99.75% 0.12% S S
PB1 336 V 96.66% I 95.98% 4.02% I I
PB2 9 DE 98.57% NT 99.33% 0.49% N NT
44 A 96.82% S 99.27% 0.61% S S
64 M 97.29% T 99.58% 0.30% T T
81 T 97.93% MV 99.27% 0.30% VM M
105 TA 98.41% VM 99.45% 0.36% VM VM
199 A 99.47% S 99.76% 0.24% S S
271 TI 98.59% A 99.51% 0.37% A A
292 IV 95.54% T 99.15% 0.67% T T
368 R 98.12% K 99.33% 0.67% K K
475 L 99.66% M 99.76% 0.24% M M
567 DE 98.28% N 99.39% 0.55% N N
588 AV 98.45% I 99.63% 0.31% I I
613 VA 98.28% T 96.82% 0.61% TI T
627 E 99.31% K 99.76% 0.12% K K
661 A 86.72% T 99.39% 0.43% T T
674 AS 95.69% T 99.63% 0.18% T T
702 K 89.70% R 99.39% 0.49% R R
The 68 characteristic sites identified by this study are shown in this table,
grouped by protein. Each row represents a site, with the columns detailing the
following: the protein name; th e site position within the protein sequence; the
A2A characteristic variant(s) and their conservation in the A2A subset; the H2H
characteristic variant(s), their conservation in the H2H subset, and the
contamination with avian variants observed in the H2H subset; the
characteristic variant(s) observed in the H1N1 subset alone; and the
characteristic variant(s) observed in the HxN2 subset alone.
doi:10.1371/journal.pone.0009025.t004
Table 4. Cont.
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NP (12 sites). These three proteins, responsible for the transcrip-
tion and replication of viral RNA, bind to each other, to the PB1
polymerase and to viral RNA to form the ribonucleoprotein (RNP)
complex that encases each of the 8 genomic segments packaged
within the virion. However, the PB1 protein was found to contain
only a single characteristic site. PB1 and PB1-F2 are encoded by
an RNA segment that was replaced during the 1957 and 1968
pandemics (Figure 1). As a result, adaptive mutations found in
these two proteins are lineage-specific, with one notable exception:
a single PB1 site has independently produced the same adaptive
mutation (V336I) in both H1N1 and HxN2 lineages (Figure 3). All
remaining internal proteins were found to contain multiple
characteristic sites: M1 (3 sites), M2 (9 sites), NS1 (6 sites) and
NEP/NS2 (3 sites), as shown in Figure 4. The M2 protein
contained the highest density of characteristic sites (almost 1 every
10 residues), including three sites within the M2 extracellular
region (M2e), which has recently been proposed as a universal
vaccine target [24]. The analysis of the HA and NA glycoproteins
revealed a large number of subtype-specific adaptive mutations, as
shown in Figure 5 (details are given in Tables S1, S2, S3, S4, S5 in
the Supplementary Materials S1). However, we were unable to
identify with confidence any adaptive mutation in these proteins as
universal in all human-transmissible strains, even at those positions
where mutations were found to occur in multiple subtypes.
Our catalogue of characteristic sites is considerably more
extensive than those reported in related work. The most
comprehensive previous study [11] used a large-scale dataset
comparable in size to ours to identify 32 of the 68 characteristic
sites found in the present work, indicating that MI may be a more
sensitive measure of association than the statistical tests employed
in that study. Chen et al. [10] identified 52 sites in ten proteins. Of
these, 38 are present in our catalogue; our study discarded 12 sites
shown to be representatives of a single lineage, and we were
unable to identify two characteristic sites in HA reported by [10].
Emergence of H2H Adaptive Mutations
To assess the stability of H2H characteristic mutations, and
reconstruct the timeline of their emergence in human strains, we
produced adaptation signatures for all available virus proteomes
isolated from human hosts. Figure 6 shows the chronological
display of signatures from viruses isolated between 1918 and 1972,
a period spanning the three major 20
th
Century pandemics. A2A
and H2H characteristic residues are shown on contrasting
backgrounds, making it easy to discern visually the evolutionary
pattern of their emergence. The Spanish influenza pandemic
isolate A/BrevigMission/1/1918 (H1N1), at the start of the
timeline, is the oldest characterized proteome. Although this strain
had a primarily avian signature, it contained 23 out of 68 H2H
characteristic mutations (34%), distributed in all proteins except
for PB1 and NS1. This number of H2H mutations is far higher
than that of other avian strains in our dataset, all of which contain
no more than eight H2H mutations. The 1918 H2H mutations
were conserved in later human strains, which gradually accumu-
lated additional adaptive changes throughout the 1930s and
1940s. By 1950, viruses with signatures with no avian character-
istic variants were circulating, such as A/FW/50 (H1N1). Both the
1957 and 1968 pandemics (indicated by red lines) left the internal
protein constellation practically unchanged, except for the
replacement of the PB1 segment, which removed from circulation
the V336I mutation developed in the 1950s by the H1N1 strains.
However, this mutation re-emerged shortly after the 1968
pandemic: by 1972, the HxN2 lineage acquired full H2H
signature. Five years later, a new pandemic introduced a
human-adapted H1N1 strain, whose signature was identical to
that of pre-1957 H1N1 strains (not shown in the figure), but
different from that ofHxN2, which had diverged in the intervening
years. Both lineages are still co-circulating today, and their
signatures have remained distinct and stable throughout the
intervening half-century. A comparison of all H1N1 and HxN2
signatures since 1977 revealed no indication of stable reassort-
ments between the two lineages (data not shown). A2A mutations
could only be found in isolates from reported infections of zoonotic
origin, from swine (see A/Victoria/1968 in Figure 6) or avian
hosts (for example, human H5N1 infections). Apart from major
pandemics, we found no evidence that any zoonotic infection over
the past 90 years has generated stable human-transmissible
lineages.
Assessment of Avian Strains for H2H Adaptive Mutations
We investigated the presence of adaptive mutations in avian
strains by constructing adaptation signatures for all avian
sequences analyzed in this study. The majority of avian signatures
(.63%) contained no H2H mutations at all. Although this high
percentage may be an overestimate (many of these signatures were
incomplete due to partial sequencing of the source genomes), it is
clear that H2H variants are rare in the avian influenza population.
In contrast, we found an unusually high number of H2H mutation
in human-infecting H5N1 strains, which are arranged chronolog-
ically in Figure 7, showing that two distinct signatures character-
ized two major ‘‘waves’’ of H5N1 infections. The 1997/98 Hong
Kong isolates present up to ten H2H mutations spread over five
internal proteins (e.g. A/Hong Kong/532/97 (H5N1)), more than
any other avian strains in our dataset. Later strains, which spread
to South-East Asia, Africa and Europe since 2003, also contain
several H2H variants, but their number (between 3 and 6) is
considerably lower than observed in the first wave. Only a single
mutation was present in the majority of isolates in both waves:
IleRVal at position 28 in the transmembrane region of the M2
protein. In both waves, the signatures of human-infecting isolates
were consistent with those of contemporary avian isolates in the
same geographical region. Our study found several sequences with
a high numbers of adaptive mutations from avian subtypes (see
Figure S1 of the Supplementary Materials S1). Most of these
viruses were isolated in Asia over the past decade, and belong
predominantly to three subtypes (H5N1, H9N2 and H6N1). The
presence of shared H2H mutations suggests that reassortments of
multiple internal proteins have occurred between these three
subtypes.
Analysis of Signatures in Swine and Equine Isolates
We obtained adaptation signatures in our SW subsets
(Figure 8), which clearly show that pigs are infected by a wide
variety of influenza A viruses: in addition to signatures derived
from early ‘‘classical swine’’ influenza (group A in Figure 8), we
identified signatures typical of human (group C) and of avian
viruses (group D). This data supports the hypothesis that swine
hosts may be ‘‘mixing vessels’’ for the reassortments of avian and
human influenza viruses, since they possess cell surface receptors
that are bound by the HA of both avian and human influenza
viruses [25]. Additionally, we found a small number of isolates
with radically different signatures (see Figure S2 of the
Supplementary Materials S1), consistent with the hypothesis that
additional adapted lineages circulate among pigs [26]. The
signatures of the pandemic H1N1/09 strains (group B in Figure 8)
present strong similarities to previously circulating swine strains,
although the signatures of three polymerase proteins are atypical,
supporting the hypothesis of a recombinant virus [27]. However,
similarities with the A/Swine/Albert/14722/2005 signature
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suggest that this recombinant virus has been in circulation for
several years in the swine population. Further evidence of
widespread reassortments is clearly visible in Group A signatures
that acquired internal proteins possessing avian signatures. All
internal proteins appear to be susceptible to such reassortments,
for which we found no discernible pattern. Thirteen H2H
adaptive mutations have been continually present in swine
influenza strains over the last 70 years, suggesting they play an
important adaptive role in swine-to-swine transmission. However,
the absence of these mutations in many signatures suggests they
are not a requirement for swine infection. Eleven of these
conserved mutation were present in the 1918 Spanish influenza
signature (M1 121; M2 14, 20; NP 33, 100, 136; NEP/NS2 60;
PA 55; PB2 199, 475, 627), supporting the hypothesis of a
common origin [28].
A similar analysis of equine influenza signatures, conducted
using the EQ dataset, revealed that they have predominantly avian
signatures (Figure 9). Although the limited available data
prevented us from making statistically significant observations,
we note five H2H mutations (in the NP and PA proteins) that have
appeared over several decades and may be conserved in
circulating adapted strains. Of these mutations, only one (PA
D55N) was also conserved in ‘‘classical swine’’ lineages as well as
human lineages.
Discussion
Characteristic Sites Catalogue
The analysis described in this paper produced the most
complete catalogue of H2H adaptive mutations published to date.
Figure 3. Characteristic sites identified in the PB1 (A) and PB1-F2 (B) proteins of influenza A. RNA segment 2, which encodes both the
PB1 and PB1-F2 proteins, has been replaced at the onset of the 1957 and 1968 pandemics (see Figure 1). As a result, the H1N1 and HxN2 lineages do
not share recent common origin for this segment. Characteristic mutations are therefore shown separately for the two lineages, in the lower part of
each diagram, using blue (H1N1) and green (HxN2) circles. Known functional sites for PB1 [46,50–52] and PB1-F2 [53] are also indicated by colored
lines in the upper part of each figure.
doi:10.1371/journal.pone.0009025.g003
Figure 2. Characteristic sites identified in components of the RNP assembly of influenza A (PB2, PA, NP proteins). Circular markers,
indicating the position of characteristic sites, are placed along the sequence length of the PB2 (A), PA (B) and NP (C) proteins of influenza A. Avian (A2A)
variants are indicated above each marker, while human (H2H) variants are located below. If multiple characteristic variants are present, they are shown in
decreasing order of frequency. In the upper part of each figure, colored lines show reported functional domains of PB2 [41–44], PA [45–48] and NP [49].
doi:10.1371/journal.pone.0009025.g002
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This catalogue describes a complex landscape of adaptations,
involving a greater number of proteins than reported by previous
studies. The presence of characteristic sites in eight of the nine
internal influenza proteins indicates that host adaptation is highly
complex and systemic in nature, requiring the participation of
products from the whole genomic ensemble. Gradual emergence
of H2H mutations in the three decades after the Spanish influenza
pandemic suggests that many of these adaptive mutations are
individually not essential. However, their high level of conserva-
tion over the following decades strongly implies their important
role in adapting to human hosts. A possible explanation is that the
1918 H1N1 proteome contained a non-optimal set of important
components for human transmission, which has been refined over
time to improve equilibrium between virus and host. This model
does not imply that any of the 1918 mutations are individually
sufficient, or necessary, for human-to-human transmission. There
may be multiple combinations of H2H mutations capable of
enabling sufficiently efficient infection and transmission in humans
to allow the gradual refinement of the adaptive mutation
repertoire. Our catalogue of characteristic sites, derived from the
analysis of 90 years of refinements in human lineages, can
therefore be a valuable tool for assessing the potential of zoonotic
viruses to infect and circulate amongst humans.
Our results indicate that concurrent mutations in the internal
protein constellation are required for efficient host range
adaptation, although the role of most internal proteins is still
poorly understood. Internal proteins participate in various cellular
processes, such as nuclear transport, replication and virion
assembly, each of which may require adaptation to the host
organism. The location of characteristic sites within putative
nuclear localization signals (NLS) of various components supports
this model. However, it is unlikely that all characteristic sites
identified in our catalogue play independent roles. The presence of
multiple H2H characteristic sites in both M1 and NEP/NS2,
within their reciprocal binding regions, raises the question of
whether such mutations have co-evolved as a result of preferred
structural interactions. This may also be the case for RNP complex
proteins, which frequently contain characteristic sites in putative
Figure 5. Characteristic sites identified in the HA (A) and NA (B) glycoproteins of influenza A. The characteristic mutations identified for
each of the subtypes present in humans are shown: H1 (blue circles), H2 (green circles), H3 (orange circles) for HA; and N1 (blue circles), N2 (green
circles) for NA (details of these sites are given in Tables S1, S2, S3, S4, S5 of the Supplementary Materials S1). Known domains of these two proteins
are indicated by coloured lines in the upper part of each figure.
doi:10.1371/journal.pone.0009025.g005
Figure 4. Characteristic sites identified in the matrix proteins M1 (A) and M2 (B) and non-structural proteins NS1 (C) and NEP/NS2
(D) of influenza A. Identified characteristic sites are mapped against known functional domains of M1 [54,55], M2 [56], NS1 [57–64] and NEP/NS2
[65,66], using the notation used in Figure 2.
doi:10.1371/journal.pone.0009025.g004
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protein-binding domains. Using our previously published PB2
data [12], a recent reconstruction of the atomic structure of two
PB2 domains [29] has shown that all seven characteristic sites in
these domains were located on the protein surface, suggesting their
interaction with other viral proteins, or with host factors.
Unfortunately, we are currently unable to map these interactions
more accurately, either because of insufficient structural informa-
tion, or because binding regions are identified on only one of the
binding proteins. In addition, the available number of influenza A
sequences prior to 1950 is insufficient for the statistical
identification of co-evolving residues. Even so, our data clearly
indicates that internal protein constellations form stable lineages in
humans, and natural reassortments of internal proteins do not
tend to occur between these lineages. Such lack of reassortments is
remarkable given the genetic similarity and overlapping geo-
graphical spread of the two lineages, and suggests a very strong
interdependency between the elements of the constellation.
The reassortment of the PB1 segment in multiple pandemic
events may indicate that stable PB1 mutations are not required for
human host adaptation. The paucity of adaptive mutation sites in
PB1, when compared to PB2 and PA, suggests it may play a core
enzymatic role in the trimeric polymerase complex, while the
remaining two subunits are responsible for interactions with host
factors. Recent research has proposed a critical role of the PB1
gene in the high virulence of the 1918 pandemic [30], and it is
possible that flexibility in replacing this segment is of benefit to the
virus at the onset of pandemics. The repeated emergence of the
PB1 V336I mutation suggests that it plays an important adaptive
role that should be further investigated.
The unusually high density of characteristic sites in the M2
protein may be explained by its physical arrangement in the virion
assembly: M2 is a transmembrane protein, thought to interact
both with the internal proteins and with the host immune system.
The extracellular region (M2e) of this protein was observed to be
conserved in humans, and thus proposed as a vaccine candidate
[31]. Recently, further studies have claimed that M2e-based
vaccines may confer immune protection against zoonotic strains
[24]. Our results suggest that the M2 and in particular its M2e
domain are prone to developing adaptive mutations. Its conser-
vation in the two human lineages is a poor indicator of its
conservation in avian viruses. In view of our incomplete
knowledge of avian influenza diversity, claims of universal
protection against avian strains should be regarded with caution,
especially because of the ease with which reassortments occur in
these viruses.
One characteristic mutation observed in the NS1 protein (the
introduction of a threonine residue at position 215) affects a Src
homology 3 (SH3) motif that is present in many avian isolates.
This motif is believed to recruit Crk and CrkL adaptive proteins,
and thus modulate signaling pathways that affect the replication
Figure 6. Timeline of adaptation to H2H transmission for the influenza A proteome. Adaptation signatures from human isolates between
1918 and 1972 are arranged in chronological order. Subtype, year and country of isolation, and isolate name are shown in the first column. The
remaining columns show residues at all characteristic sites, in the order given in Table 4. A2A characteristic mutations are shown on a dark blue
background, H2H mutations on a yellow background, while all other variants are on white. Blank cells represent unknown residues in incompletely
sequenced proteomes. Consensus signatures for A2A and H2H proteomes are shown in the first and last row, respectively. Red horizontal lines
indicate the start of the 1957 and 1968 pandemics, which introduced the H2N2 and H3N2 subtypes respectively.
doi:10.1371/journal.pone.0009025.g006
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ability of the virus [32]. The proline residue required by the SH3
motif was observed in 81% of A2A and 93% of H5N1A isolates.
Notably, it was present in the 1918 Spanish influenza isolates and
in all but one human-infecting H5N1 viruses. Two other NS1
characteristic sites (positions 81 and 84) are located in a 5-amino
acid region (80–84) whose deletion is associated with the second
wave of human-infecting H5N1 viruses (see Figure 7).
In spite of their demonstrated flexibility in the composition of
their protein constellations, swine viruses have established at least
one lineage that has retained several H2H mutations for over
three-quarters of a century. Such continuity is in marked contrast
with the frequency of reassortment events, which would lead one
to expect a number of lineages to have emerged. This suggests that
conserved adaptive mutations in ‘‘classic swine’’ are important for
swine-to-swine transmission, and essential to the establishment of
stable lineages, but not prerequisites for infecting this host. This
has important implications for human influenza, since nearly all of
these mutations are also conserved in human strains, and have
been present since the 1918 Spanish pandemic. The eleven
adaptive variants shared by ‘‘classical swine’’ and human
signatures may constitute a basic ‘‘adaptive suite’’ for within-
species transmission, essential for founding stable lineages.
However, this set accounts for only about half of the 1918 H2H
mutations, suggesting that major additional changes were required
before influenza viruses could spread efficiently among humans.
The pandemic H1N1/09 virus appears to possess H2H
mutations similar to those of other swine viruses. Normal ‘‘classic’’
swine viruses possess several such mutations, and it is possible that
low-pathogenicity human infections by these strains occur
frequently. The avian signature of the PA and PB2 proteins of
H1N1/09 suggests suboptimal replication in human hosts, but this
might be compensated by the human PB1 recombinant protein.
Although the rapid pandemic spread of H1N1/09 may appear
inconsistent with suboptimal host adaptation, it is possible that the
advantages conferred by the antigenic novelty of its HA and NA
proteins were sufficient for the virus to overcome replication
disadvantages. As more people develop immunity and its antigenic
novelty decreases, H1N1/09 may disappear, or establish itself as a
stable human lineage. Either way, the outcome will be extremely
informative in assessing the H2H mutations present in this virus’
signature.
Assessment of Avian Influenza Viruses
In our analysis of avian influenza, signatures from H5N1
isolates stood out as the richest in H2H mutations. This result was
by no means expected, and it strongly supports the utility of our
characteristic site catalogue as an assessment tool. A comparison of
1997 Hong Kong H5N1 signatures against those of contemporary
H9N2 and H6N1 isolates from the same geographical region
reveals a dynamic interplay between these three subtypes, in which
viral segments appear to have been transferred through reassort-
ments (Figure S2 of the Supplementary Materials S1). This
observation supports previous studies, which have proposed that
the 1997 Hong Kong H5N1 epidemic followed the reassortment
Figure 7. Adaptation signatures of human-isolated H5N1 influenza A proteomes. This figure shows the adaptation signatures of H5N1
sequences that infected humans in the period 1997–2008. For display clarity and conciseness, only a selection of representative signatures is
presented. The same coloring scheme was used as in Figure 6. Dashes (in the NS1 protein signature) indicate amino acid deletions. A red horizontal
line separates the early wave of infections in Hong Kong (1997–8) from more recent South-East Asian infections (since 2003).
doi:10.1371/journal.pone.0009025.g007
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of H5N1 and H9N2 viruses [33], and that H6N1 viruses were also
involved [34]. Such highly dynamic composition of the avian
influenza proteome puts into question the validity of labeling
influenza isolates exclusively by their HA and NA subtypes. H5N1
isolates of 1981, 1997 and 2004 clearly present distinct internal
protein constellations, and grouping them into a homogeneous set
reveals little about their ability to adapt to humans. In addition, an
excessive focus on the HA/NA subtype deviates attention from the
analysis of co-circulating strains with a potential for reassortment,
impairing effective surveillance of the potential for human
infectivity and transmissibility. Glycoproteins must be considered
as important components of a larger systemic ensemble of
adaptations, some of which can only be modeled by new
approaches that transcend current subtype definitions.
Remarkably, the two H5N1 waves only share one conserved
H2H mutation (M2 I28V), while all other mutations involved in
the 1997 waves have been replaced by avian variants. Thus, it
appears that H5N1 viruses are not only acquiring, but also losing
H2H mutations through reassortments. The lack of stability of
adaptive variants is evidenced by the instability of the crucial PB2
E627K mutation, implicated in replication in humans [35] and
high virulence of human H5N1 infections [36]. Overall, there is
no evidence of a trend of gradual accumulation of H2H mutations
in H5N1 viruses. This may indicate that H5N1, in its current
form, poses a relatively low pandemic risk. On the other hand, the
abundant evidence of reassortments among H5N1 raises the
concern that these avian viruses may reassort with a human
lineage, combining a human-adapted internal protein constella-
tion with an immunologically novel set of glycoproteins. Such
reassortants have been produced under laboratory conditions,
using human H3N2 viruses, but have failed to propagate amongst
mammalian models [37]. Even if reassortants acquired the ability
to circulate efficiently among humans, it is impossible to predict
how such adaptation would affect the extreme pathogenicity that
Figure 8. Adaptation signatures of selected swine influenza A proteomes. H2H signatures for a number of representative swine proteomes
are shown. Subtype, year and country of isolation, and isolate name are shown in the first column, while the remaining columns show the signature
residues, using the same coloring scheme as in Figure 6. The isolates are shown in four groups, according to signature similarity: (A) isolates with
signatures similar to that of ‘‘classical swine’’ influenza, such as A/Swine/Iowa/15/30; (B) isolates from the H1N1/09 pandemic; (C) isolates with
signatures that are very similar to those of contemporarily circulating human isolates; (D) isolates with predominantly ‘‘avian’’ signatures, con taining
very few or no H2H adaptive mutations. In most of groups, reassortment events are evidenced by discontinuities in the signature timeline.
doi:10.1371/journal.pone.0009025.g008
Figure 9. Adaptation signatures of selected equine influenza A proteomes. The signatures of selected equine isolates, spanning a period of
nearly 50 years are shown. For conciseness, a number of similar signatures were removed from this set. Subtype, year and country of isolation, and
isolate name are shown in the first column, while the remaining columns show the signature residues, using the same colouring scheme as in
Figure 6.
doi:10.1371/journal.pone.0009025.g009
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has characterized human H5N1 infections: like transmissibility,
pathogenicity appears to be systemically determined, and is likely
to be affected by the replacement of internal proteins. Since there
is no evidence of a link between virulence and host adaptation, our
results cannot help make such a prediction.
Conclusions
In this study, we performed analysis based on mutual
information to a dataset of over 40,000 influenza protein
sequences, to identify adaptive mutations associated with
human-to-human transmissibility. The 68 characteristic sites
found by our analysis constitute the most comprehensive
catalogue published to date, and a useful tool for assessing the
potential for human transmission of influenza viruses. Charac-
teristic sites were found in eight internal proteins, suggesting
that adaptation to the human population is complex and may
require the orchestration of concurrent mutations in multiple
proteins. A remarkable product of this complex adaptive system
is the stability of internal protein constellations in the two
lineages that circulate amongst humans. These lineages
developed most of their repertoire of adaptive mutations
gradually over the three decades following the 1918 Spanish
influenza pandemic, and have conserved these mutations to the
present day. Their internal protein constellations appear so
interdependent that the two lineages never reassort with each
other or with zoonotic viruses. By contrast, we observed that
avian strains are subject to frequent reassortments, which may
have helped the accumulation of human adaptive mutations in
avian H5N1 prior to the 1997 Hong Kong wave. However, such
accumulation has not been stable, and H5N1 adaptations to
human hosts are often lost through reassortments. By this
measure, the current genetic forms of H5N1 viruses appear to
present a limited pandemic risk, except in the event of a
reassortment with a human lineage, which is likely to affect the
viruses’ pathogenicity. H1N1/09 viruses appear similar to
existing swine lineages, which multiple H2H mutations that
may account for its capability to transmit among humans. Like
the1977H1N1pandemicstrain,H1N1/09mayhaveenjoyed
rapidspreadbecauseofpartial serological novelty, but may
ultimately be limited by cross-reactive immune memory in a
large portion of the population.
The catalogue produced by this study is consistent with the
results of previous studies, and our mutual information
method appears to be the most powerful and sensitive amongst
those proposed thus far. Whilst our catalogue of adaptive sites
might contain some false positives, it is clear that this set of
positions serves as a useful starting point for verification
studies and further research. These studies will include both
advanced computational analyses and experimental verifica-
tion by researchers interested in the epidemiology and
evolutionary behavior of influenza viruses, including charac-
terization of molecular mechanisms. Our results show that our
understanding of the mechanisms involved in influenza
adaptation to humans is incomplete; we propose that a
systemic perspective that considers constellations of adapta-
tions should be studied.
Supporting Information
Supplementary Materials S1
Found at: doi:10.1371/journal.pone.0009025.s001 (0.27 MB
DOC)
Acknowledgments
The authors thank Asif M. Khan for helpful discussions and suggestions.
Author Contributions
Conceived and designed the experiments: OM TWT JTA VB. Performed
the experiments: OM ATH. Analyzed the data: OM RAA AGS TWT JTA
VB. Contributed reagents/materials/analysis tools: OM ATH. Wrote the
paper: OM JTA VB.
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    • "Avian proteomics An interesting field of application of proteomics is also the study of the pathogenesis of infectious disease affecting the avian species. The importance of this topic ranges from the economical aspect, to reduce the impact of avian diseases on production by characterizing the pathogenesis and by identifying new biomarkers of vaccines, to the need to study some avian diseases as a zoonosis, for example, avian flu, where human host adaptation signatures have been identified (Miotto et al., 2010) and responses to the virus characterised in mice (Zhao et al., 2012) and chicken (Sun et al., 2014). Proteomics has been already utilized to study the pathogenesis of herpes viruses (Kunec, 2013), with a special focus on Marek disease (Thanthrige-Don et al., 2010; Hu et al., 2012), which is of particular interest as a model for human tumours (Buza and Burgess, 2007). "
    [Show abstract] [Hide abstract] ABSTRACT: Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002 - Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.
    Full-text · Article · Oct 2014
    • "Alteration of viral surface proteins to recognize a range of host receptors is the strategy by which influenza increases its host range [2], [14]. Interestingly, despite its complexity, recent studies shows that a few amino acid substitutions (4–5) has the potential of altering A/Indonesia/5/2005 avian A/H5N1 and A/Vietnam/1203/2004 A/H5N1 to be transmissible between ferrets via respiratory droplets [2], [17]–[19]. "
    [Show abstract] [Hide abstract] ABSTRACT: The evolution of the influenza A virus to increase its host range is a major concern worldwide. Molecular mechanisms of increasing host range are largely unknown. Influenza surface proteins play determining roles in reorganization of host-sialic acid receptors and host range. In an attempt to uncover the physic-chemical attributes which govern HA subtyping, we performed a large scale functional analysis of over 7000 sequences of 16 different HA subtypes. Large number (896) of physic-chemical protein characteristics were calculated for each HA sequence. Then, 10 different attribute weighting algorithms were used to find the key characteristics distinguishing HA subtypes. Furthermore, to discover machine leaning models which can predict HA subtypes, various Decision Tree, Support Vector Machine, Naïve Bayes, and Neural Network models were trained on calculated protein characteristics dataset as well as 10 trimmed datasets generated by attribute weighting algorithms. The prediction accuracies of the machine learning methods were evaluated by 10-fold cross validation. The results highlighted the frequency of Gln (selected by 80% of attribute weighting algorithms), percentage/frequency of Tyr, percentage of Cys, and frequencies of Try and Glu (selected by 70% of attribute weighting algorithms) as the key features that are associated with HA subtyping. Random Forest tree induction algorithm and RBF kernel function of SVM (scaled by grid search) showed high accuracy of 98% in clustering and predicting HA subtypes based on protein attributes. Decision tree models were successful in monitoring the short mutation/reassortment paths by which influenza virus can gain the key protein structure of another HA subtype and increase its host range in a short period of time with less energy consumption. Extracting and mining a large number of amino acid attributes of HA subtypes of influenza A virus through supervised algorithms represent a new avenue for understanding and predicting possible future structure of influenza pandemics.
    Full-text · Article · May 2014
    • "doi:10.1371/journal.pone.0084638.g007 genomic signatures without considering their phylogenetic relationships [10,12,16] . Other studies considered the phylogenetic structures, but applied theoretical modeling of site substitution rates [13]. "
    [Show abstract] [Hide abstract] ABSTRACT: An increase in the availability of data on the influenza A viruses (IAV) has enabled the identification of the potential determinants of IAV host specificity using computational approaches. In this study, we proposed an alternative approach, based on the adjusted Rand index (ARI), for the evaluation of genomic signatures of IAVs and their ability to distinguish hosts they infected. Our experiments showed that the host-specific signatures identified using the ARI were more characteristic of their hosts than those identified using previous measures. Our results provided updates on the host-specific genomic signatures in the internal proteins of the IAV based on the sequence data as of February 2013 in the National Center for Biotechnology Information (NCBI). Unlike other approaches for signature recognition, our approach considered not only the ability of signatures to distinguish hosts (according to the ARI), but also the chronological relationships among proteins. We identified novel signatures that could be mapped to known functional domains, and introduced a chronological analysis to investigate the changes in host-specific genomic signatures over time. Our chronological analytical approach provided results on the adaptive variability of signatures, which correlated with previous studies' findings, and indicated prospective adaptation trends that warrant further investigation.
    Full-text · Article · Jan 2014
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