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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Complete-Proteome Mapping of Human Influenza A Adaptive Mutations:
Implications for Human Transmissibility of Zoonotic Strains</title>
<author><name sortKey="Miotto, Olivo" sort="Miotto, Olivo" uniqKey="Miotto O" first="Olivo" last="Miotto">Olivo Miotto</name>
<affiliation><nlm:aff id="aff1"><addr-line>Centre for Genomics and Global Health, University of Oxford, Oxford, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2"><addr-line>Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Rajthevee, Bangkok, Thailand</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Heiny, A T" sort="Heiny, A T" uniqKey="Heiny A" first="A. T." last="Heiny">A. T. Heiny</name>
<affiliation><nlm:aff id="aff3"><addr-line>Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Albrecht, Randy" sort="Albrecht, Randy" uniqKey="Albrecht R" first="Randy" last="Albrecht">Randy Albrecht</name>
<affiliation><nlm:aff id="aff4"><addr-line>Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Garcia Sastre, Adolfo" sort="Garcia Sastre, Adolfo" uniqKey="Garcia Sastre A" first="Adolfo" last="García-Sastre">Adolfo García-Sastre</name>
<affiliation><nlm:aff id="aff4"><addr-line>Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5"><addr-line>Division of Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff6"><addr-line>Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tan, Tin Wee" sort="Tan, Tin Wee" uniqKey="Tan T" first="Tin Wee" last="Tan">Tin Wee Tan</name>
<affiliation><nlm:aff id="aff3"><addr-line>Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="August, J Thomas" sort="August, J Thomas" uniqKey="August J" first="J. Thomas" last="August">J. Thomas August</name>
<affiliation><nlm:aff id="aff7"><addr-line>Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Brusic, Vladimir" sort="Brusic, Vladimir" uniqKey="Brusic V" first="Vladimir" last="Brusic">Vladimir Brusic</name>
<affiliation><nlm:aff id="aff8"><addr-line>Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">20140252</idno>
<idno type="pmc">2815782</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2815782</idno>
<idno type="RBID">PMC:2815782</idno>
<idno type="doi">10.1371/journal.pone.0009025</idno>
<date when="2010">2010</date>
<idno type="wicri:Area/Pmc/Corpus">000A99</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A99</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Complete-Proteome Mapping of Human Influenza A Adaptive Mutations:
Implications for Human Transmissibility of Zoonotic Strains</title>
<author><name sortKey="Miotto, Olivo" sort="Miotto, Olivo" uniqKey="Miotto O" first="Olivo" last="Miotto">Olivo Miotto</name>
<affiliation><nlm:aff id="aff1"><addr-line>Centre for Genomics and Global Health, University of Oxford, Oxford, United Kingdom</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2"><addr-line>Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Rajthevee, Bangkok, Thailand</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Heiny, A T" sort="Heiny, A T" uniqKey="Heiny A" first="A. T." last="Heiny">A. T. Heiny</name>
<affiliation><nlm:aff id="aff3"><addr-line>Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Albrecht, Randy" sort="Albrecht, Randy" uniqKey="Albrecht R" first="Randy" last="Albrecht">Randy Albrecht</name>
<affiliation><nlm:aff id="aff4"><addr-line>Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Garcia Sastre, Adolfo" sort="Garcia Sastre, Adolfo" uniqKey="Garcia Sastre A" first="Adolfo" last="García-Sastre">Adolfo García-Sastre</name>
<affiliation><nlm:aff id="aff4"><addr-line>Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5"><addr-line>Division of Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff6"><addr-line>Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tan, Tin Wee" sort="Tan, Tin Wee" uniqKey="Tan T" first="Tin Wee" last="Tan">Tin Wee Tan</name>
<affiliation><nlm:aff id="aff3"><addr-line>Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="August, J Thomas" sort="August, J Thomas" uniqKey="August J" first="J. Thomas" last="August">J. Thomas August</name>
<affiliation><nlm:aff id="aff7"><addr-line>Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Brusic, Vladimir" sort="Brusic, Vladimir" uniqKey="Brusic V" first="Vladimir" last="Brusic">Vladimir Brusic</name>
<affiliation><nlm:aff id="aff8"><addr-line>Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America</addr-line>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">PLoS ONE</title>
<idno type="eISSN">1932-6203</idno>
<imprint><date when="2010">2010</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p>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.</p>
</sec>
<sec><title>Methodology/Principal Findings</title>
<p>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 <italic>mutual information</italic>
, to identify
<italic>characteristic sites</italic>
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 <italic>adaptation
signatures</italic>
, 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.</p>
</sec>
<sec><title>Conclusions</title>
<p>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.</p>
</sec>
</div>
</front>
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</TEI>
<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id>
<journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id>
<journal-id journal-id-type="publisher-id">plos</journal-id>
<journal-id journal-id-type="pmc">plosone</journal-id>
<journal-title-group><journal-title>PLoS ONE</journal-title>
</journal-title-group>
<issn pub-type="epub">1932-6203</issn>
<publisher><publisher-name>Public Library of Science</publisher-name>
<publisher-loc>San Francisco, USA</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">20140252</article-id>
<article-id pub-id-type="pmc">2815782</article-id>
<article-id pub-id-type="publisher-id">09-PONE-RA-13598R1</article-id>
<article-id pub-id-type="doi">10.1371/journal.pone.0009025</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
<subj-group subj-group-type="Discipline"><subject>Computational Biology</subject>
<subject>Genetics and Genomics</subject>
<subject>Infectious Diseases</subject>
<subject>Biochemistry/Bioinformatics</subject>
<subject>Computational Biology/Comparative Sequence Analysis</subject>
<subject>Computational Biology/Genomics</subject>
<subject>Evolutionary Biology/Bioinformatics</subject>
<subject>Evolutionary Biology/Microbial Evolution and Genomics</subject>
<subject>Genetics and Genomics/Bioinformatics</subject>
<subject>Molecular Biology/Bioinformatics</subject>
<subject>Virology/Emerging Viral Diseases</subject>
<subject>Infectious Diseases/Viral Infections</subject>
</subj-group>
</article-categories>
<title-group><article-title>Complete-Proteome Mapping of Human Influenza A Adaptive Mutations:
Implications for Human Transmissibility of Zoonotic Strains</article-title>
<alt-title alt-title-type="running-head">Human Influenza Adaptation Map</alt-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Miotto</surname>
<given-names>Olivo</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup>
</xref>
<xref ref-type="corresp" rid="cor1"><sup>*</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Heiny</surname>
<given-names>A. T.</given-names>
</name>
<xref ref-type="aff" rid="aff3"><sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Albrecht</surname>
<given-names>Randy</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>4</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>García-Sastre</surname>
<given-names>Adolfo</given-names>
</name>
<xref ref-type="aff" rid="aff4"><sup>4</sup>
</xref>
<xref ref-type="aff" rid="aff5"><sup>5</sup>
</xref>
<xref ref-type="aff" rid="aff6"><sup>6</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Tan</surname>
<given-names>Tin Wee</given-names>
</name>
<xref ref-type="aff" rid="aff3"><sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>August</surname>
<given-names>J. Thomas</given-names>
</name>
<xref ref-type="aff" rid="aff7"><sup>7</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Brusic</surname>
<given-names>Vladimir</given-names>
</name>
<xref ref-type="aff" rid="aff8"><sup>8</sup>
</xref>
</contrib>
</contrib-group>
<aff id="aff1"><label>1</label>
<addr-line>Centre for Genomics and Global Health, University of Oxford, Oxford, United Kingdom</addr-line>
</aff>
<aff id="aff2"><label>2</label>
<addr-line>Mahidol-Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Rajthevee, Bangkok, Thailand</addr-line>
</aff>
<aff id="aff3"><label>3</label>
<addr-line>Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore</addr-line>
</aff>
<aff id="aff4"><label>4</label>
<addr-line>Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</aff>
<aff id="aff5"><label>5</label>
<addr-line>Division of Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</aff>
<aff id="aff6"><label>6</label>
<addr-line>Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, New York, United States of America</addr-line>
</aff>
<aff id="aff7"><label>7</label>
<addr-line>Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America</addr-line>
</aff>
<aff id="aff8"><label>8</label>
<addr-line>Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America</addr-line>
</aff>
<contrib-group><contrib contrib-type="editor"><name><surname>Poon</surname>
<given-names>Art F. Y.</given-names>
</name>
<role>Editor</role>
<xref ref-type="aff" rid="edit1"></xref>
</contrib>
</contrib-group>
<aff id="edit1">Providence Health Care, Canada</aff>
<author-notes><corresp id="cor1">* E-mail: <email>olivo.miotto@ndm.ox.ac.uk</email>
</corresp>
<fn fn-type="con"><p>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.</p>
</fn>
</author-notes>
<pub-date pub-type="collection"><year>2010</year>
</pub-date>
<pub-date pub-type="epub"><day>3</day>
<month>2</month>
<year>2010</year>
</pub-date>
<volume>5</volume>
<issue>2</issue>
<elocation-id>e9025</elocation-id>
<history><date date-type="received"><day>16</day>
<month>10</month>
<year>2009</year>
</date>
<date date-type="accepted"><day>27</day>
<month>12</month>
<year>2009</year>
</date>
</history>
<permissions><copyright-statement>Miotto et al.</copyright-statement>
<copyright-year>2010</copyright-year>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>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 properly credited.</license-p>
</license>
</permissions>
<abstract><sec><title>Background</title>
<p>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.</p>
</sec>
<sec><title>Methodology/Principal Findings</title>
<p>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 <italic>mutual information</italic>
, to identify
<italic>characteristic sites</italic>
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 <italic>adaptation
signatures</italic>
, 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.</p>
</sec>
<sec><title>Conclusions</title>
<p>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.</p>
</sec>
</abstract>
<counts><page-count count="13"></page-count>
</counts>
</article-meta>
</front>
<body><sec id="s1"><title>Introduction</title>
<p>Influenza A is a virus belonging to the <italic>Orthomyxoviridae</italic>
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 <xref rid="pone.0009025-Taubenberger1" ref-type="bibr">[1]</xref>
. 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 <xref rid="pone.0009025-Potter1" ref-type="bibr">[2]</xref>
. 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 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 <xref rid="pone.0009025-Capua1" ref-type="bibr">[3]</xref>
. 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 (<ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/disease/avian_influenza/">www.who.int/csr/disease/avian_influenza/</ext-link>
). 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 <xref rid="pone.0009025-Fraser1" ref-type="bibr">[4]</xref>
, and has been classified as pandemic by the WHO
(<ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/disease/swineflu/">http://www.who.int/csr/disease/swineflu/</ext-link>
). Although this virus
currently appears to cause mild disease <xref rid="pone.0009025-Peiris1" ref-type="bibr">[5]</xref>
, 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 <xref rid="pone.0009025-Taubenberger1" ref-type="bibr">[1]</xref>
,
and new tools are needed for scientists and policymakers to evaluate the pandemic
risk posed by zoonotic viruses.</p>
<p>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 <xref rid="pone.0009025-Neumann1" ref-type="bibr">[6]</xref>
(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 <italic>characteristic sites</italic>
, a residue
that is highly conserved within the human group but rarely observed in the pool of
avian strains (a <italic>characteristic variant</italic>
) 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
<xref rid="pone.0009025-BucklerWhite1" ref-type="bibr">[7]</xref>
and polymerases <xref rid="pone.0009025-Naffakh1" ref-type="bibr">[8]</xref>
, <xref rid="pone.0009025-Taubenberger2" ref-type="bibr">[9]</xref>
. Large-scale
computational methods have used statistical variability measures such as
<italic>information entropy</italic>
to identify characteristic sites for human
transmissibility. An analysis of 401 full viral proteomes <xref rid="pone.0009025-Chen1" ref-type="bibr">[10]</xref>
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
<italic>et al.</italic>
<xref rid="pone.0009025-Finkelstein1" ref-type="bibr">[11]</xref>
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.</p>
<p>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 <italic>mutual
information</italic>
as the statistical measure, was previously applied
successfully to the study of the PB2 polymerase <xref rid="pone.0009025-Miotto1" ref-type="bibr">[12]</xref>
. The catalogue of
characteristic sites identified by our analysis was then applied to derive
<italic>adaptation signatures</italic>
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.</p>
</sec>
<sec sec-type="materials|methods" id="s2"><title>Materials and Methods</title>
<sec id="s2a"><title>Data Collection and Preparation</title>
<p>We compiled a dataset of all available influenza A sequences (as of September
2006) from the NCBI GenBank and GenPept databases <xref rid="pone.0009025-Wheeler1" ref-type="bibr">[13]</xref>
, including entries
mirrored from UniProt <xref rid="pone.0009025-UniProt1" ref-type="bibr">[14]</xref>
. 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 annotated 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 <xref rid="pone.0009025-Miotto2" ref-type="bibr">[15]</xref>
.</p>
<p>For each of the eleven influenza proteins, a master multiple sequence alignment
(MSA) was constructed using the MUSCLE 3.6 <xref rid="pone.0009025-Edgar1" ref-type="bibr">[16]</xref>
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, <ext-link ext-link-type="uri" xlink:href="http://avana.sourceforge.net">http://avana.sourceforge.net</ext-link>
), developed by the authors to
support information-theoretical analysis tasks <xref rid="pone.0009025-Khan1" ref-type="bibr">[17]</xref>
, <xref rid="pone.0009025-Heiny1" ref-type="bibr">[18]</xref>
.
AVANA was also used to conduct all comparative analyses described in this
paper.</p>
</sec>
<sec id="s2b"><title>Subset Selection</title>
<p>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 (<xref ref-type="fig" rid="pone-0009025-g001">Figure 1</xref>
),
the internal protein constellations of these two lineages have evolved
independently, following the disappearance of H1N1 in 1957 and its
reintroduction in 1977 <xref rid="pone.0009025-Kendal1" ref-type="bibr">[19]</xref>
. 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: <bold>A2A</bold>
(all avian sequences, except for H1N1, H2N2, H1N2, H3N2 and H5N1 subtypes),
<bold>H1N1H</bold>
(all H1N1 human sequences) and <bold>HxN2H</bold>
(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
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.</p>
<fig id="pone-0009025-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g001</object-id>
<label>Figure 1</label>
<caption><title>Human Influenza A reassortment events of the 20<sup>th</sup>
Century.</title>
<p>The reassortment events associated with human pandemics in the
20<sup>th</sup>
century (adapted from Webster <italic>et
al.</italic>
<xref rid="pone.0009025-Webster1" ref-type="bibr">[39]</xref>
). 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
<xref rid="pone.0009025-Scholtissek2" ref-type="bibr">[40]</xref>
. 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 <xref rid="pone.0009025-Kendal1" ref-type="bibr">[19]</xref>
. 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.</p>
</caption>
<graphic xlink:href="pone.0009025.g001"></graphic>
</fig>
<p>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 <xref rid="pone.0009025-Baez1" ref-type="bibr">[20]</xref>
, <xref rid="pone.0009025-Treanor1" ref-type="bibr">[21]</xref>
. 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 <xref rid="pone.0009025-Buonagurio1" ref-type="bibr">[22]</xref>
. 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.</p>
<p>To analyze adaptation signatures for different virus groups, we also collected
subsets of avian H5N1 (<bold>H5N1A</bold>
) and human H5N1 (<bold>H5N1H</bold>
)
sequences, as well as subsets of swine (<bold>SW</bold>
) and equine
(<bold>EQ</bold>
) sequences. The number of sequences in each of the
extracted datasets is included in <xref ref-type="table" rid="pone-0009025-t001">Table 1</xref>
. 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.
<xref ref-type="table" rid="pone-0009025-t002">Table 2</xref>
shows the
number of sequences included in each HA subset, and <xref ref-type="table" rid="pone-0009025-t003">Table 3</xref>
shows the subset sizes for the NA
proteins.</p>
<table-wrap id="pone-0009025-t001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.t001</object-id>
<label>Table 1</label>
<caption><title>Count of influenza A internal protein sequences used in the current
study.</title>
</caption>
<alternatives><graphic id="pone-0009025-t001-1" xlink:href="pone.0009025.t001"></graphic>
<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
</colgroup>
<thead><tr><td align="left" rowspan="1" colspan="1"></td>
<td colspan="4" align="left" rowspan="1"><italic>Sequences used in MI Analysis</italic>
</td>
<td colspan="5" align="left" rowspan="1"><italic>Additional sequences used in signature analysis</italic>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">A2A</td>
<td align="left" rowspan="1" colspan="1">H1N1H</td>
<td align="left" rowspan="1" colspan="1">HxN2H</td>
<td align="left" rowspan="1" colspan="1">Total</td>
<td align="left" rowspan="1" colspan="1">H5N1A</td>
<td align="left" rowspan="1" colspan="1">H5N1H</td>
<td align="left" rowspan="1" colspan="1">SW</td>
<td align="left" rowspan="1" colspan="1">EQ</td>
<td align="left" rowspan="1" colspan="1">Total</td>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1"><bold>M1</bold>
</td>
<td align="left" rowspan="1" colspan="1">1047</td>
<td align="left" rowspan="1" colspan="1">300</td>
<td align="left" rowspan="1" colspan="1">1521</td>
<td align="left" rowspan="1" colspan="1"><bold>2868</bold>
</td>
<td align="left" rowspan="1" colspan="1">458</td>
<td align="left" rowspan="1" colspan="1">105</td>
<td align="left" rowspan="1" colspan="1">190</td>
<td align="left" rowspan="1" colspan="1">22</td>
<td align="left" rowspan="1" colspan="1"><bold>775</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>M2</bold>
</td>
<td align="left" rowspan="1" colspan="1">736</td>
<td align="left" rowspan="1" colspan="1">286</td>
<td align="left" rowspan="1" colspan="1">1517</td>
<td align="left" rowspan="1" colspan="1"><bold>2539</bold>
</td>
<td align="left" rowspan="1" colspan="1">289</td>
<td align="left" rowspan="1" colspan="1">95</td>
<td align="left" rowspan="1" colspan="1">104</td>
<td align="left" rowspan="1" colspan="1">21</td>
<td align="left" rowspan="1" colspan="1"><bold>509</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>NP</bold>
</td>
<td align="left" rowspan="1" colspan="1">884</td>
<td align="left" rowspan="1" colspan="1">316</td>
<td align="left" rowspan="1" colspan="1">1645</td>
<td align="left" rowspan="1" colspan="1"><bold>2845</bold>
</td>
<td align="left" rowspan="1" colspan="1">420</td>
<td align="left" rowspan="1" colspan="1">114</td>
<td align="left" rowspan="1" colspan="1">230</td>
<td align="left" rowspan="1" colspan="1">22</td>
<td align="left" rowspan="1" colspan="1"><bold>786</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>NS1</bold>
</td>
<td align="left" rowspan="1" colspan="1">1123</td>
<td align="left" rowspan="1" colspan="1">303</td>
<td align="left" rowspan="1" colspan="1">1448</td>
<td align="left" rowspan="1" colspan="1"><bold>2874</bold>
</td>
<td align="left" rowspan="1" colspan="1">457</td>
<td align="left" rowspan="1" colspan="1">95</td>
<td align="left" rowspan="1" colspan="1">172</td>
<td align="left" rowspan="1" colspan="1">38</td>
<td align="left" rowspan="1" colspan="1"><bold>762</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>NS2</bold>
</td>
<td align="left" rowspan="1" colspan="1">810</td>
<td align="left" rowspan="1" colspan="1">292</td>
<td align="left" rowspan="1" colspan="1">1419</td>
<td align="left" rowspan="1" colspan="1"><bold>2521</bold>
</td>
<td align="left" rowspan="1" colspan="1">288</td>
<td align="left" rowspan="1" colspan="1">81</td>
<td align="left" rowspan="1" colspan="1">113</td>
<td align="left" rowspan="1" colspan="1">21</td>
<td align="left" rowspan="1" colspan="1"><bold>503</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>PA</bold>
</td>
<td align="left" rowspan="1" colspan="1">701</td>
<td align="left" rowspan="1" colspan="1">279</td>
<td align="left" rowspan="1" colspan="1">1362</td>
<td align="left" rowspan="1" colspan="1"><bold>2342</bold>
</td>
<td align="left" rowspan="1" colspan="1">402</td>
<td align="left" rowspan="1" colspan="1">102</td>
<td align="left" rowspan="1" colspan="1">161</td>
<td align="left" rowspan="1" colspan="1">11</td>
<td align="left" rowspan="1" colspan="1"><bold>676</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>PB1</bold>
</td>
<td align="left" rowspan="1" colspan="1">716</td>
<td align="left" rowspan="1" colspan="1">303</td>
<td align="left" rowspan="1" colspan="1">1385</td>
<td align="left" rowspan="1" colspan="1"><bold>2404</bold>
</td>
<td align="left" rowspan="1" colspan="1">400</td>
<td align="left" rowspan="1" colspan="1">101</td>
<td align="left" rowspan="1" colspan="1">163</td>
<td align="left" rowspan="1" colspan="1">19</td>
<td align="left" rowspan="1" colspan="1"><bold>683</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>PB2</bold>
</td>
<td align="left" rowspan="1" colspan="1">719</td>
<td align="left" rowspan="1" colspan="1">281</td>
<td align="left" rowspan="1" colspan="1">1369</td>
<td align="left" rowspan="1" colspan="1"><bold>2369</bold>
</td>
<td align="left" rowspan="1" colspan="1">404</td>
<td align="left" rowspan="1" colspan="1">97</td>
<td align="left" rowspan="1" colspan="1">161</td>
<td align="left" rowspan="1" colspan="1">17</td>
<td align="left" rowspan="1" colspan="1"><bold>679</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>PB1-F2</bold>
</td>
<td align="left" rowspan="1" colspan="1">352</td>
<td align="left" rowspan="1" colspan="1">262</td>
<td align="left" rowspan="1" colspan="1">1280</td>
<td align="left" rowspan="1" colspan="1"><bold>1894</bold>
</td>
<td align="left" rowspan="1" colspan="1">-</td>
<td align="left" rowspan="1" colspan="1">-</td>
<td align="left" rowspan="1" colspan="1">-</td>
<td align="left" rowspan="1" colspan="1">-</td>
<td align="left" rowspan="1" colspan="1">-</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>Total</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>7088</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>2622</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>12946</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>22656</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>3118</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>790</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>1294</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>171</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>5373</bold>
</td>
</tr>
</tbody>
</table>
</alternatives>
<table-wrap-foot><fn id="nt101"><label></label>
<p>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.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<table-wrap id="pone-0009025-t002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.t002</object-id>
<label>Table 2</label>
<caption><title>Count of influenza A hemagglutinin protein sequences used in the
current study.</title>
</caption>
<alternatives><graphic id="pone-0009025-t002-2" xlink:href="pone.0009025.t002"></graphic>
<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
</colgroup>
<thead><tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">Avian</td>
<td align="left" rowspan="1" colspan="1">Human</td>
<td align="left" rowspan="1" colspan="1">Total</td>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1"><bold>H1</bold>
</td>
<td align="left" rowspan="1" colspan="1">48</td>
<td align="left" rowspan="1" colspan="1">768</td>
<td align="left" rowspan="1" colspan="1"><bold>816</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>H2</bold>
</td>
<td align="left" rowspan="1" colspan="1">80</td>
<td align="left" rowspan="1" colspan="1">75</td>
<td align="left" rowspan="1" colspan="1"><bold>155</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>H3</bold>
</td>
<td align="left" rowspan="1" colspan="1">115</td>
<td align="left" rowspan="1" colspan="1">3105</td>
<td align="left" rowspan="1" colspan="1"><bold>3220</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>Total</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>243</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>3948</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>4191</bold>
</td>
</tr>
</tbody>
</table>
</alternatives>
</table-wrap>
<table-wrap id="pone-0009025-t003" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.t003</object-id>
<label>Table 3</label>
<caption><title>Count of influenza A neuraminidase protein sequences used in the
current study.</title>
</caption>
<alternatives><graphic id="pone-0009025-t003-3" xlink:href="pone.0009025.t003"></graphic>
<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
</colgroup>
<thead><tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">Avian</td>
<td align="left" rowspan="1" colspan="1">Human</td>
<td align="left" rowspan="1" colspan="1">Total</td>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1"><bold>N1</bold>
</td>
<td align="left" rowspan="1" colspan="1">717</td>
<td align="left" rowspan="1" colspan="1">360</td>
<td align="left" rowspan="1" colspan="1"><bold>1077</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>N2</bold>
</td>
<td align="left" rowspan="1" colspan="1">439</td>
<td align="left" rowspan="1" colspan="1">1801</td>
<td align="left" rowspan="1" colspan="1"><bold>2240</bold>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>Total</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>1156</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>2161</bold>
</td>
<td align="left" rowspan="1" colspan="1"><bold>3317</bold>
</td>
</tr>
</tbody>
</table>
</alternatives>
</table-wrap>
</sec>
<sec id="s2c"><title>Identification of Characteristic Sites and Variants</title>
<p>The method for identifying characteristic sites has previously been described in
detail <xref rid="pone.0009025-Miotto1" ref-type="bibr">[12]</xref>
. Briefly, an aligned set of
<italic>adapted</italic>
sequences (capable of H2H transmissibility) was
compared against a <italic>reference</italic>
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
<italic>mutual information</italic>
(MI), an information theoretical
statistic that measures the strength of association between a pair of variables
<xref rid="pone.0009025-Shannon1" ref-type="bibr">[23]</xref>
. MI is defined in terms of
<italic>information entropy</italic>
, a measure of variability. The
information entropy <italic>H</italic>
(<italic>x</italic>
) of a discrete
variable <italic>x</italic>
is given by:<disp-formula><graphic xlink:href="pone.0009025.e001.jpg" mimetype="image" position="float"></graphic>
<label>(1)</label>
</disp-formula>
where
<italic>E</italic>
= {<italic>e<sub>1</sub>
</italic>
,
<italic>e<sub>2</sub>
</italic>
…
<italic>e<sub>n</sub>
</italic>
} is the set of all possible discrete values of
<italic>x</italic>
, and <italic>p<sub>e</sub>
</italic>
(<italic>x</italic>
)
is the probability that <italic>e</italic>
∈<italic>E</italic>
is the
value of <italic>x</italic>
. The mutual information between two variables A and
B is then defined by:<disp-formula><graphic xlink:href="pone.0009025.e002.jpg" mimetype="image" position="float"></graphic>
<label>(2)</label>
</disp-formula>
where <italic>H</italic>
(<italic>A</italic>
) and
<italic>H</italic>
(<italic>B</italic>
) are entropies of A and B, while
<italic>H</italic>
(<italic>A</italic>
,<italic>B</italic>
) is their
<italic>joint entropy</italic>
, computed from equation (1) by replacing
<italic>E</italic>
with the set of all unique pair of values
(<italic>A</italic>
,<italic>B</italic>
).</p>
<p>To identify characteristic sites, we have modified equation (2) to compute the MI
between an observed residue <italic>a</italic>
at position <italic>x</italic>
,
and the label <italic>S</italic>
of the set (alignment) within which residue
<italic>a</italic>
is observed:<disp-formula><graphic xlink:href="pone.0009025.e003.jpg" mimetype="image" position="float"></graphic>
<label>(3)</label>
</disp-formula>
<italic>H<sub>a</sub>
</italic>
(<italic>x</italic>
) is the entropy
in an alignment formed by merging the two set, while
<italic>H<sub>S</sub>
</italic>
(<italic>x</italic>
) is derived from the
number of sequences in each of the two sets (<italic>n<sub>1</sub>
</italic>
and
<italic>n<sub>2</sub>
</italic>
):<disp-formula><graphic xlink:href="pone.0009025.e004.jpg" mimetype="image" position="float"></graphic>
<label>(4)</label>
</disp-formula>
where
<italic>N</italic>
= <italic>n<sub>1</sub>
</italic>
+ <italic>n<sub>2</sub>
</italic>
. Finally,
<italic>H<sub>S</sub>
</italic>
<sub>,<italic>a</italic>
</sub>
(<italic>x</italic>
)
is given by:<disp-formula><graphic xlink:href="pone.0009025.e005.jpg" mimetype="image" position="float"></graphic>
<label>(5)</label>
</disp-formula>
where <italic>p</italic>
(<italic>S</italic>
,<italic>a</italic>
)
is the probability of any given combination of residue and set label.</p>
<p>When comparing two sets of equal size, the above equations give MI values in the
range 0≤<italic>MI</italic>
(<italic>x</italic>
)≤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.</p>
<p>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 <xref rid="pone.0009025-Miotto1" ref-type="bibr">[12]</xref>
,
and confers robustness to the MI measurement. In addition, by selecting strains
randomly across all phylogenetic groups, it partially corrects for phylogenetic
sampling biases.</p>
<p>Characteristic sites and their characteristic variants (mutations) were selected
based on four empirical criteria, whose rationale is detailed in <xref rid="pone.0009025-Finkelstein1" ref-type="bibr">[11]</xref>
, and summarized as follows:</p>
<list list-type="bullet"><list-item><p>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).</p>
</list-item>
<list-item><p>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)</p>
</list-item>
<list-item><p>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)</p>
</list-item>
<list-item><p>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%).</p>
</list-item>
</list>
<p>Only characteristic sites present in both H2H lineages were included in the final
catalogue.</p>
</sec>
<sec id="s2d"><title>Reconstruction of Adaptation Signatures</title>
<p>The variants that distinguish H2H sequences and A2A sequences at characteristic
sites form a <italic>characteristic variant pattern</italic>
, a summary of the
significant differences between the two sets of sequences across the whole
proteome. This pattern was used to construct the <italic>adaptation
signatures</italic>
of several influenza proteomes, by discarding all
residues except those at characteristic sites. Residues forming the signatures
were tagged as A2A-like (<italic>i.e.</italic>
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.</p>
</sec>
</sec>
<sec id="s3"><title>Results</title>
<sec id="s3a"><title>Catalogue of Characteristic Sites</title>
<p>Our analysis produced a catalogue of 68 characteristic sites that met selection
criteria (<xref ref-type="table" rid="pone-0009025-t004">Table 4</xref>
).
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 <xref ref-type="fig" rid="pone-0009025-g002">Figures 2</xref>
, <xref ref-type="fig" rid="pone-0009025-g003">3</xref>
and
<xref ref-type="fig" rid="pone-0009025-g004">4</xref>
, alongside the mapping
of known functional domains in these proteins. As shown in <xref ref-type="fig" rid="pone-0009025-g002">Figure 2</xref>
, the three internal proteins found
to contain the highest number of characteristic sites were PB2 (17 sites), PA
(17 sites), and NP (12 sites). These three proteins, responsible for the
transcription 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 (<xref ref-type="fig" rid="pone-0009025-g001">Figure 1</xref>
). 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 (<xref ref-type="fig" rid="pone-0009025-g003">Figure 3</xref>
). 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 <xref ref-type="fig" rid="pone-0009025-g004">Figure 4</xref>
. 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 <xref rid="pone.0009025-Tompkins1" ref-type="bibr">[24]</xref>
. The analysis of
the HA and NA glycoproteins revealed a large number of subtype-specific adaptive
mutations, as shown in <xref ref-type="fig" rid="pone-0009025-g005">Figure
5</xref>
(details are given in Tables S1, S2, S3, S4, S5 in the <xref ref-type="supplementary-material" rid="pone.0009025.s001">Supplementary
Materials S1</xref>
). 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.</p>
<fig id="pone-0009025-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g002</object-id>
<label>Figure 2</label>
<caption><title>Characteristic sites identified in components of the RNP assembly of
influenza A (PB2, PA, NP proteins).</title>
<p>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 <xref rid="pone.0009025-Mukaigawa1" ref-type="bibr">[41]</xref>
–<xref rid="pone.0009025-Fechter1" ref-type="bibr">[44]</xref>
, PA <xref rid="pone.0009025-Nieto1" ref-type="bibr">[45]</xref>
–<xref rid="pone.0009025-He1" ref-type="bibr">[48]</xref>
and NP <xref rid="pone.0009025-Ozawa1" ref-type="bibr">[49]</xref>
.</p>
</caption>
<graphic xlink:href="pone.0009025.g002"></graphic>
</fig>
<fig id="pone-0009025-g003" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g003</object-id>
<label>Figure 3</label>
<caption><title>Characteristic sites identified in the PB1 (A) and PB1-F2 (B)
proteins of influenza A.</title>
<p>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 <xref ref-type="fig" rid="pone-0009025-g001">Figure 1</xref>
). 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 <xref rid="pone.0009025-Ohtsu1" ref-type="bibr">[46]</xref>
, <xref rid="pone.0009025-Jones1" ref-type="bibr">[50]</xref>
–<xref rid="pone.0009025-Gonzalez1" ref-type="bibr">[52]</xref>
and
PB1-F2 <xref rid="pone.0009025-Yamada1" ref-type="bibr">[53]</xref>
are also indicated by colored lines
in the upper part of each figure.</p>
</caption>
<graphic xlink:href="pone.0009025.g003"></graphic>
</fig>
<fig id="pone-0009025-g004" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g004</object-id>
<label>Figure 4</label>
<caption><title>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.</title>
<p>Identified characteristic sites are mapped against known functional
domains of M1 <xref rid="pone.0009025-Baudin1" ref-type="bibr">[54]</xref>
, <xref rid="pone.0009025-Hui1" ref-type="bibr">[55]</xref>
, M2 <xref rid="pone.0009025-Lamb1" ref-type="bibr">[56]</xref>
, NS1 <xref rid="pone.0009025-Greenspan1" ref-type="bibr">[57]</xref>
–<xref rid="pone.0009025-Shin1" ref-type="bibr">[64]</xref>
and NEP/NS2
<xref rid="pone.0009025-IwatsukiHorimoto1" ref-type="bibr">[65]</xref>
, <xref rid="pone.0009025-Akarsu1" ref-type="bibr">[66]</xref>
, using the
notation used in <xref ref-type="fig" rid="pone-0009025-g002">Figure
2</xref>
.</p>
</caption>
<graphic xlink:href="pone.0009025.g004"></graphic>
</fig>
<fig id="pone-0009025-g005" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g005</object-id>
<label>Figure 5</label>
<caption><title>Characteristic sites identified in the HA (A) and NA (B)
glycoproteins of influenza A.</title>
<p>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
<xref ref-type="supplementary-material" rid="pone.0009025.s001">Supplementary Materials S1</xref>
). Known domains of these two
proteins are indicated by coloured lines in the upper part of each
figure.</p>
</caption>
<graphic xlink:href="pone.0009025.g005"></graphic>
</fig>
<table-wrap id="pone-0009025-t004" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.t004</object-id>
<label>Table 4</label>
<caption><title>Full catalogue of identified characteristic sites for H2H
transmission of influenza A.</title>
</caption>
<alternatives><graphic id="pone-0009025-t004-4" xlink:href="pone.0009025.t004"></graphic>
<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
</colgroup>
<thead><tr><td align="left" rowspan="1" colspan="1">Protein</td>
<td align="left" rowspan="1" colspan="1">Position</td>
<td colspan="2" align="left" rowspan="1">A2A</td>
<td colspan="3" align="left" rowspan="1">H2H</td>
<td align="left" rowspan="1" colspan="1">H1N1 CV</td>
<td align="left" rowspan="1" colspan="1">HxN2 CV</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">CV</td>
<td align="left" rowspan="1" colspan="1">Cons</td>
<td align="left" rowspan="1" colspan="1">CV</td>
<td align="left" rowspan="1" colspan="1">Cons</td>
<td align="left" rowspan="1" colspan="1">X-pres</td>
<td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1"></td>
</tr>
</thead>
<tbody><tr><td align="left" rowspan="1" colspan="1">M1</td>
<td align="left" rowspan="1" colspan="1">115</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">99.70%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">99.39%</td>
<td align="left" rowspan="1" colspan="1">0.61%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">I</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">121</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">94.94%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">99.89%</td>
<td align="left" rowspan="1" colspan="1">0.11%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">A</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">137</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">99.60%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">99.23%</td>
<td align="left" rowspan="1" colspan="1">0.77%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">A</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">M2</td>
<td align="left" rowspan="1" colspan="1">11</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">97.28%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">96.89%</td>
<td align="left" rowspan="1" colspan="1">3.11%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">I</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">14</td>
<td align="left" rowspan="1" colspan="1">G</td>
<td align="left" rowspan="1" colspan="1">95.99%</td>
<td align="left" rowspan="1" colspan="1">E</td>
<td align="left" rowspan="1" colspan="1">98.28%</td>
<td align="left" rowspan="1" colspan="1">1.72%</td>
<td align="left" rowspan="1" colspan="1">E</td>
<td align="left" rowspan="1" colspan="1">E</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">20</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">97.14%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">97.94%</td>
<td align="left" rowspan="1" colspan="1">2.06%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">N</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">28</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">76.36%</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">97.72%</td>
<td align="left" rowspan="1" colspan="1">2.11%</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">V</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">54</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">98.91%</td>
<td align="left" rowspan="1" colspan="1">LIF</td>
<td align="left" rowspan="1" colspan="1">98.94%</td>
<td align="left" rowspan="1" colspan="1">0.61%</td>
<td align="left" rowspan="1" colspan="1">IL</td>
<td align="left" rowspan="1" colspan="1">LF</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">55</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">79.18%</td>
<td align="left" rowspan="1" colspan="1">F</td>
<td align="left" rowspan="1" colspan="1">99.33%</td>
<td align="left" rowspan="1" colspan="1">0.67%</td>
<td align="left" rowspan="1" colspan="1">F</td>
<td align="left" rowspan="1" colspan="1">F</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">57</td>
<td align="left" rowspan="1" colspan="1">Y</td>
<td align="left" rowspan="1" colspan="1">99.59%</td>
<td align="left" rowspan="1" colspan="1">H</td>
<td align="left" rowspan="1" colspan="1">97.38%</td>
<td align="left" rowspan="1" colspan="1">2.18%</td>
<td align="left" rowspan="1" colspan="1">H</td>
<td align="left" rowspan="1" colspan="1">H</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">78</td>
<td align="left" rowspan="1" colspan="1">Q</td>
<td align="left" rowspan="1" colspan="1">99.72%</td>
<td align="left" rowspan="1" colspan="1">KE</td>
<td align="left" rowspan="1" colspan="1">99.26%</td>
<td align="left" rowspan="1" colspan="1">0.28%</td>
<td align="left" rowspan="1" colspan="1">EK</td>
<td align="left" rowspan="1" colspan="1">K</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">86</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">99.84%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">99.21%</td>
<td align="left" rowspan="1" colspan="1">0.45%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">A</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">NP</td>
<td align="left" rowspan="1" colspan="1">16</td>
<td align="left" rowspan="1" colspan="1">GS</td>
<td align="left" rowspan="1" colspan="1">99.16%</td>
<td align="left" rowspan="1" colspan="1">D</td>
<td align="left" rowspan="1" colspan="1">99.49%</td>
<td align="left" rowspan="1" colspan="1">0.51%</td>
<td align="left" rowspan="1" colspan="1">D</td>
<td align="left" rowspan="1" colspan="1">D</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">33</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">99.76%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">98.97%</td>
<td align="left" rowspan="1" colspan="1">1.03%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">I</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">61</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">98.36%</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">99.43%</td>
<td align="left" rowspan="1" colspan="1">0.57%</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">L</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">100</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">99.65%</td>
<td align="left" rowspan="1" colspan="1">VI</td>
<td align="left" rowspan="1" colspan="1">99.71%</td>
<td align="left" rowspan="1" colspan="1">0.06%</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">VI</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">136</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">85.41%</td>
<td align="left" rowspan="1" colspan="1">MI</td>
<td align="left" rowspan="1" colspan="1">99.77%</td>
<td align="left" rowspan="1" colspan="1">0.11%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">MI</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">214</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">96.64%</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">99.32%</td>
<td align="left" rowspan="1" colspan="1">0.68%</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">K</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">283</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">100.00%</td>
<td align="left" rowspan="1" colspan="1">P</td>
<td align="left" rowspan="1" colspan="1">99.48%</td>
<td align="left" rowspan="1" colspan="1">0.47%</td>
<td align="left" rowspan="1" colspan="1">P</td>
<td align="left" rowspan="1" colspan="1">P</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">305</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">99.17%</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">99.33%</td>
<td align="left" rowspan="1" colspan="1">0.67%</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">K</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">313</td>
<td align="left" rowspan="1" colspan="1">F</td>
<td align="left" rowspan="1" colspan="1">99.31%</td>
<td align="left" rowspan="1" colspan="1">Y</td>
<td align="left" rowspan="1" colspan="1">99.48%</td>
<td align="left" rowspan="1" colspan="1">0.52%</td>
<td align="left" rowspan="1" colspan="1">Y</td>
<td align="left" rowspan="1" colspan="1">Y</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">357</td>
<td align="left" rowspan="1" colspan="1">Q</td>
<td align="left" rowspan="1" colspan="1">98.43%</td>
<td align="left" rowspan="1" colspan="1">KR</td>
<td align="left" rowspan="1" colspan="1">99.90%</td>
<td align="left" rowspan="1" colspan="1">0.10%</td>
<td align="left" rowspan="1" colspan="1">KR</td>
<td align="left" rowspan="1" colspan="1">K</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">375</td>
<td align="left" rowspan="1" colspan="1">DN</td>
<td align="left" rowspan="1" colspan="1">96.93%</td>
<td align="left" rowspan="1" colspan="1">GEV</td>
<td align="left" rowspan="1" colspan="1">99.34%</td>
<td align="left" rowspan="1" colspan="1">0.56%</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">GE</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">423</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">97.06%</td>
<td align="left" rowspan="1" colspan="1">STP</td>
<td align="left" rowspan="1" colspan="1">98.88%</td>
<td align="left" rowspan="1" colspan="1">1.00%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">SP</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">NS1</td>
<td align="left" rowspan="1" colspan="1">22</td>
<td align="left" rowspan="1" colspan="1">FL</td>
<td align="left" rowspan="1" colspan="1">97.07%</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">98.21%</td>
<td align="left" rowspan="1" colspan="1">0.40%</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">V</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">60</td>
<td align="left" rowspan="1" colspan="1">AE</td>
<td align="left" rowspan="1" colspan="1">97.59%</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">99.20%</td>
<td align="left" rowspan="1" colspan="1">0.69%</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">V</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">81</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">98.66%</td>
<td align="left" rowspan="1" colspan="1">M</td>
<td align="left" rowspan="1" colspan="1">99.08%</td>
<td align="left" rowspan="1" colspan="1">0.69%</td>
<td align="left" rowspan="1" colspan="1">M</td>
<td align="left" rowspan="1" colspan="1">M</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">84</td>
<td align="left" rowspan="1" colspan="1">VS</td>
<td align="left" rowspan="1" colspan="1">96.08%</td>
<td align="left" rowspan="1" colspan="1">TA</td>
<td align="left" rowspan="1" colspan="1">99.20%</td>
<td align="left" rowspan="1" colspan="1">0.80%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">TA</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">215</td>
<td align="left" rowspan="1" colspan="1">PSA</td>
<td align="left" rowspan="1" colspan="1">99.24%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">99.37%</td>
<td align="left" rowspan="1" colspan="1">0.63%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">T</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">227</td>
<td align="left" rowspan="1" colspan="1">E</td>
<td align="left" rowspan="1" colspan="1">98.87%</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">99.53%</td>
<td align="left" rowspan="1" colspan="1">0.06%</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">R</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">NS2</td>
<td align="left" rowspan="1" colspan="1">60</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">76.77%</td>
<td align="left" rowspan="1" colspan="1">NH</td>
<td align="left" rowspan="1" colspan="1">98.89%</td>
<td align="left" rowspan="1" colspan="1">0.82%</td>
<td align="left" rowspan="1" colspan="1">H</td>
<td align="left" rowspan="1" colspan="1">N</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">70</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">97.46%</td>
<td align="left" rowspan="1" colspan="1">G</td>
<td align="left" rowspan="1" colspan="1">99.88%</td>
<td align="left" rowspan="1" colspan="1">0.12%</td>
<td align="left" rowspan="1" colspan="1">G</td>
<td align="left" rowspan="1" colspan="1">G</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">107</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">99.60%</td>
<td align="left" rowspan="1" colspan="1">F</td>
<td align="left" rowspan="1" colspan="1">98.77%</td>
<td align="left" rowspan="1" colspan="1">1.17%</td>
<td align="left" rowspan="1" colspan="1">F</td>
<td align="left" rowspan="1" colspan="1">F</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">PA</td>
<td align="left" rowspan="1" colspan="1">28</td>
<td align="left" rowspan="1" colspan="1">P</td>
<td align="left" rowspan="1" colspan="1">100.00%</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">99.14%</td>
<td align="left" rowspan="1" colspan="1">0.67%</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">L</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">55</td>
<td align="left" rowspan="1" colspan="1">D</td>
<td align="left" rowspan="1" colspan="1">99.69%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">99.63%</td>
<td align="left" rowspan="1" colspan="1">0.37%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">N</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">57</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">96.61%</td>
<td align="left" rowspan="1" colspan="1">Q</td>
<td align="left" rowspan="1" colspan="1">98.72%</td>
<td align="left" rowspan="1" colspan="1">0.79%</td>
<td align="left" rowspan="1" colspan="1">Q</td>
<td align="left" rowspan="1" colspan="1">Q</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">65</td>
<td align="left" rowspan="1" colspan="1">SF</td>
<td align="left" rowspan="1" colspan="1">99.08%</td>
<td align="left" rowspan="1" colspan="1">LP</td>
<td align="left" rowspan="1" colspan="1">99.63%</td>
<td align="left" rowspan="1" colspan="1">0.37%</td>
<td align="left" rowspan="1" colspan="1">PL</td>
<td align="left" rowspan="1" colspan="1">L</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">66</td>
<td align="left" rowspan="1" colspan="1">GS</td>
<td align="left" rowspan="1" colspan="1">99.69%</td>
<td align="left" rowspan="1" colspan="1">DE</td>
<td align="left" rowspan="1" colspan="1">98.84%</td>
<td align="left" rowspan="1" colspan="1">1.10%</td>
<td align="left" rowspan="1" colspan="1">ED</td>
<td align="left" rowspan="1" colspan="1">D</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">100</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">96.15%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">99.27%</td>
<td align="left" rowspan="1" colspan="1">0.37%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">A</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">225</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">98.61%</td>
<td align="left" rowspan="1" colspan="1">C</td>
<td align="left" rowspan="1" colspan="1">99.39%</td>
<td align="left" rowspan="1" colspan="1">0.61%</td>
<td align="left" rowspan="1" colspan="1">C</td>
<td align="left" rowspan="1" colspan="1">C</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">268</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">98.84%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">99.14%</td>
<td align="left" rowspan="1" colspan="1">0.73%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">I</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">321</td>
<td align="left" rowspan="1" colspan="1">NK</td>
<td align="left" rowspan="1" colspan="1">97.35%</td>
<td align="left" rowspan="1" colspan="1">YST</td>
<td align="left" rowspan="1" colspan="1">97.30%</td>
<td align="left" rowspan="1" colspan="1">0.74%</td>
<td align="left" rowspan="1" colspan="1">STY</td>
<td align="left" rowspan="1" colspan="1">Y</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">337</td>
<td align="left" rowspan="1" colspan="1">AT</td>
<td align="left" rowspan="1" colspan="1">99.34%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">99.75%</td>
<td align="left" rowspan="1" colspan="1">0.25%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">S</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">356</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">98.51%</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">99.26%</td>
<td align="left" rowspan="1" colspan="1">0.74%</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">R</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">382</td>
<td align="left" rowspan="1" colspan="1">E</td>
<td align="left" rowspan="1" colspan="1">94.34%</td>
<td align="left" rowspan="1" colspan="1">D</td>
<td align="left" rowspan="1" colspan="1">97.37%</td>
<td align="left" rowspan="1" colspan="1">2.45%</td>
<td align="left" rowspan="1" colspan="1">D</td>
<td align="left" rowspan="1" colspan="1">D</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">400</td>
<td align="left" rowspan="1" colspan="1">PSQ</td>
<td align="left" rowspan="1" colspan="1">89.32%</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">99.45%</td>
<td align="left" rowspan="1" colspan="1">0.31%</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">L</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">404</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">99.48%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">99.39%</td>
<td align="left" rowspan="1" colspan="1">0.55%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">S</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">409</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">91.49%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">99.45%</td>
<td align="left" rowspan="1" colspan="1">0.49%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">N</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">421</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">98.91%</td>
<td align="left" rowspan="1" colspan="1">IV</td>
<td align="left" rowspan="1" colspan="1">97.79%</td>
<td align="left" rowspan="1" colspan="1">0.55%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">IV</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">552</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">99.81%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">99.75%</td>
<td align="left" rowspan="1" colspan="1">0.12%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">S</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">PB1</td>
<td align="left" rowspan="1" colspan="1">336</td>
<td align="left" rowspan="1" colspan="1">V</td>
<td align="left" rowspan="1" colspan="1">96.66%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">95.98%</td>
<td align="left" rowspan="1" colspan="1">4.02%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">I</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1">PB2</td>
<td align="left" rowspan="1" colspan="1">9</td>
<td align="left" rowspan="1" colspan="1">DE</td>
<td align="left" rowspan="1" colspan="1">98.57%</td>
<td align="left" rowspan="1" colspan="1">NT</td>
<td align="left" rowspan="1" colspan="1">99.33%</td>
<td align="left" rowspan="1" colspan="1">0.49%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">NT</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">44</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">96.82%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">99.27%</td>
<td align="left" rowspan="1" colspan="1">0.61%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">S</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">64</td>
<td align="left" rowspan="1" colspan="1">M</td>
<td align="left" rowspan="1" colspan="1">97.29%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">99.58%</td>
<td align="left" rowspan="1" colspan="1">0.30%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">T</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">81</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">97.93%</td>
<td align="left" rowspan="1" colspan="1">MV</td>
<td align="left" rowspan="1" colspan="1">99.27%</td>
<td align="left" rowspan="1" colspan="1">0.30%</td>
<td align="left" rowspan="1" colspan="1">VM</td>
<td align="left" rowspan="1" colspan="1">M</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">105</td>
<td align="left" rowspan="1" colspan="1">TA</td>
<td align="left" rowspan="1" colspan="1">98.41%</td>
<td align="left" rowspan="1" colspan="1">VM</td>
<td align="left" rowspan="1" colspan="1">99.45%</td>
<td align="left" rowspan="1" colspan="1">0.36%</td>
<td align="left" rowspan="1" colspan="1">VM</td>
<td align="left" rowspan="1" colspan="1">VM</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">199</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">99.47%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">99.76%</td>
<td align="left" rowspan="1" colspan="1">0.24%</td>
<td align="left" rowspan="1" colspan="1">S</td>
<td align="left" rowspan="1" colspan="1">S</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">271</td>
<td align="left" rowspan="1" colspan="1">TI</td>
<td align="left" rowspan="1" colspan="1">98.59%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">99.51%</td>
<td align="left" rowspan="1" colspan="1">0.37%</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">A</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">292</td>
<td align="left" rowspan="1" colspan="1">IV</td>
<td align="left" rowspan="1" colspan="1">95.54%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">99.15%</td>
<td align="left" rowspan="1" colspan="1">0.67%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">T</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">368</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">98.12%</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">99.33%</td>
<td align="left" rowspan="1" colspan="1">0.67%</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">K</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">475</td>
<td align="left" rowspan="1" colspan="1">L</td>
<td align="left" rowspan="1" colspan="1">99.66%</td>
<td align="left" rowspan="1" colspan="1">M</td>
<td align="left" rowspan="1" colspan="1">99.76%</td>
<td align="left" rowspan="1" colspan="1">0.24%</td>
<td align="left" rowspan="1" colspan="1">M</td>
<td align="left" rowspan="1" colspan="1">M</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">567</td>
<td align="left" rowspan="1" colspan="1">DE</td>
<td align="left" rowspan="1" colspan="1">98.28%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">99.39%</td>
<td align="left" rowspan="1" colspan="1">0.55%</td>
<td align="left" rowspan="1" colspan="1">N</td>
<td align="left" rowspan="1" colspan="1">N</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">588</td>
<td align="left" rowspan="1" colspan="1">AV</td>
<td align="left" rowspan="1" colspan="1">98.45%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">99.63%</td>
<td align="left" rowspan="1" colspan="1">0.31%</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">I</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">613</td>
<td align="left" rowspan="1" colspan="1">VA</td>
<td align="left" rowspan="1" colspan="1">98.28%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">96.82%</td>
<td align="left" rowspan="1" colspan="1">0.61%</td>
<td align="left" rowspan="1" colspan="1">TI</td>
<td align="left" rowspan="1" colspan="1">T</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">627</td>
<td align="left" rowspan="1" colspan="1">E</td>
<td align="left" rowspan="1" colspan="1">99.31%</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">99.76%</td>
<td align="left" rowspan="1" colspan="1">0.12%</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">K</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">661</td>
<td align="left" rowspan="1" colspan="1">A</td>
<td align="left" rowspan="1" colspan="1">86.72%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">99.39%</td>
<td align="left" rowspan="1" colspan="1">0.43%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">T</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">674</td>
<td align="left" rowspan="1" colspan="1">AS</td>
<td align="left" rowspan="1" colspan="1">95.69%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">99.63%</td>
<td align="left" rowspan="1" colspan="1">0.18%</td>
<td align="left" rowspan="1" colspan="1">T</td>
<td align="left" rowspan="1" colspan="1">T</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">702</td>
<td align="left" rowspan="1" colspan="1">K</td>
<td align="left" rowspan="1" colspan="1">89.70%</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">99.39%</td>
<td align="left" rowspan="1" colspan="1">0.49%</td>
<td align="left" rowspan="1" colspan="1">R</td>
<td align="left" rowspan="1" colspan="1">R</td>
</tr>
</tbody>
</table>
</alternatives>
<table-wrap-foot><fn id="nt102"><label></label>
<p>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; the 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.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>Our catalogue of characteristic sites is considerably more extensive than those
reported in related work. The most comprehensive previous study <xref rid="pone.0009025-Finkelstein1" ref-type="bibr">[11]</xref>
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 <italic>et al.</italic>
<xref rid="pone.0009025-Chen1" ref-type="bibr">[10]</xref>
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 <xref rid="pone.0009025-Chen1" ref-type="bibr">[10]</xref>
.</p>
</sec>
<sec id="s3b"><title>Emergence of H2H Adaptive Mutations</title>
<p>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. <xref ref-type="fig" rid="pone-0009025-g006">Figure 6</xref>
shows the
chronological display of signatures from viruses isolated between 1918 and 1972,
a period spanning the three major 20<sup>th</sup>
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 accumulated
additional adaptive changes throughout the 1930s and 1940s. By 1950, viruses
with signatures with no avian characteristic 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 reassortments 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 <xref ref-type="fig" rid="pone-0009025-g006">Figure 6</xref>
) 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.</p>
<fig id="pone-0009025-g006" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g006</object-id>
<label>Figure 6</label>
<caption><title>Timeline of adaptation to H2H transmission for the influenza A
proteome.</title>
<p>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 <xref ref-type="table" rid="pone-0009025-t004">Table 4</xref>
. 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.</p>
</caption>
<graphic xlink:href="pone.0009025.g006"></graphic>
</fig>
</sec>
<sec id="s3c"><title>Assessment of Avian Strains for H2H Adaptive Mutations</title>
<p>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 chronologically in <xref ref-type="fig" rid="pone-0009025-g007">Figure 7</xref>
, showing that two
distinct signatures characterized two major “waves” of H5N1
infections. The 1997/98 Hong Kong isolates present up to ten H2H mutations
spread over five internal proteins (<italic>e.g.</italic>
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: Ile→Val 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 <xref ref-type="supplementary-material" rid="pone.0009025.s001">Supplementary
Materials S1</xref>
). 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.</p>
<fig id="pone-0009025-g007" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g007</object-id>
<label>Figure 7</label>
<caption><title>Adaptation signatures of human-isolated H5N1 influenza A
proteomes.</title>
<p>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 <xref ref-type="fig" rid="pone-0009025-g006">Figure 6</xref>
. 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).</p>
</caption>
<graphic xlink:href="pone.0009025.g007"></graphic>
</fig>
</sec>
<sec id="s3d"><title>Analysis of Signatures in Swine and Equine Isolates</title>
<p>We obtained adaptation signatures in our SW subsets (<xref ref-type="fig" rid="pone-0009025-g008">Figure 8</xref>
), 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
<xref ref-type="fig" rid="pone-0009025-g008">Figure 8</xref>
), 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 <xref rid="pone.0009025-Scholtissek1" ref-type="bibr">[25]</xref>
. Additionally,
we found a small number of isolates with radically different signatures (see
Figure S2 of the <xref ref-type="supplementary-material" rid="pone.0009025.s001">Supplementary Materials S1</xref>
), consistent with the hypothesis that
additional adapted lineages circulate among pigs <xref rid="pone.0009025-Webby1" ref-type="bibr">[26]</xref>
. The signatures of
the pandemic H1N1/09 strains (group B in <xref ref-type="fig" rid="pone-0009025-g008">Figure 8</xref>
) present strong similarities to
previously circulating swine strains, although the signatures of three
polymerase proteins are atypical, supporting the hypothesis of a recombinant
virus <xref rid="pone.0009025-Novel1" ref-type="bibr">[27]</xref>
. However, similarities with the
A/Swine/Albert/14722/2005 signature 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 <xref rid="pone.0009025-Gorman1" ref-type="bibr">[28]</xref>
.</p>
<fig id="pone-0009025-g008" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g008</object-id>
<label>Figure 8</label>
<caption><title>Adaptation signatures of selected swine influenza A
proteomes.</title>
<p>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 <xref ref-type="fig" rid="pone-0009025-g006">Figure 6</xref>
. 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, containing very
few or no H2H adaptive mutations. In most of groups, reassortment events
are evidenced by discontinuities in the signature timeline.</p>
</caption>
<graphic xlink:href="pone.0009025.g008"></graphic>
</fig>
<p>A similar analysis of equine influenza signatures, conducted using the EQ
dataset, revealed that they have predominantly avian signatures (<xref ref-type="fig" rid="pone-0009025-g009">Figure 9</xref>
). 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.</p>
<fig id="pone-0009025-g009" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0009025.g009</object-id>
<label>Figure 9</label>
<caption><title>Adaptation signatures of selected equine influenza A
proteomes.</title>
<p>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 <xref ref-type="fig" rid="pone-0009025-g006">Figure 6</xref>
.</p>
</caption>
<graphic xlink:href="pone.0009025.g009"></graphic>
</fig>
</sec>
</sec>
<sec id="s4"><title>Discussion</title>
<sec id="s4a"><title>Characteristic Sites Catalogue</title>
<p>The analysis described in this paper produced the most complete catalogue of H2H
adaptive mutations published to date. 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 conservation 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.</p>
<p>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 protein-binding domains.
Using our previously published PB2 data <xref rid="pone.0009025-Miotto1" ref-type="bibr">[12]</xref>
, a recent
reconstruction of the atomic structure of two PB2 domains <xref rid="pone.0009025-Tarendeau1" ref-type="bibr">[29]</xref>
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 information, 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 geographical spread of the two lineages, and
suggests a very strong interdependency between the elements of the
constellation.</p>
<p>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 <xref rid="pone.0009025-Pappas1" ref-type="bibr">[30]</xref>
, 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.</p>
<p>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
<xref rid="pone.0009025-Neirynck1" ref-type="bibr">[31]</xref>
. Recently, further studies have claimed that
M2e-based vaccines may confer immune protection against zoonotic strains <xref rid="pone.0009025-Tompkins1" ref-type="bibr">[24]</xref>
.
Our results suggest that the M2 and in particular its M2e domain are prone to
developing adaptive mutations. Its conservation 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.</p>
<p>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 ability of the virus <xref rid="pone.0009025-Heikkinen1" ref-type="bibr">[32]</xref>
. 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 <xref ref-type="fig" rid="pone-0009025-g007">Figure 7</xref>
).</p>
<p>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.</p>
<p>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.</p>
</sec>
<sec id="s4b"><title>Assessment of Avian Influenza Viruses</title>
<p>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 reassortments (Figure S2 of the <xref ref-type="supplementary-material" rid="pone.0009025.s001">Supplementary
Materials S1</xref>
). This observation supports previous studies, which have
proposed that the 1997 Hong Kong H5N1 epidemic followed the reassortment of H5N1
and H9N2 viruses <xref rid="pone.0009025-Guan1" ref-type="bibr">[33]</xref>
, and that H6N1 viruses were also involved
<xref rid="pone.0009025-Hoffmann1" ref-type="bibr">[34]</xref>
. 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.</p>
<p>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 <xref rid="pone.0009025-Subbarao1" ref-type="bibr">[35]</xref>
and high
virulence of human H5N1 infections <xref rid="pone.0009025-Hatta1" ref-type="bibr">[36]</xref>
. 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 constellation 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 <xref rid="pone.0009025-Maines1" ref-type="bibr">[37]</xref>
. 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 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.</p>
</sec>
<sec id="s4c"><title>Conclusions</title>
<p>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. Characteristic 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 the 1977 H1N1 pandemic strain, H1N1/09 may have
enjoyed rapid spread because of partial serological novelty, but may ultimately
be limited by cross-reactive immune memory in a large portion of the
population.</p>
<p>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
verification by researchers interested in the epidemiology and evolutionary
behavior of influenza viruses, including characterization 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 adaptations should be studied.</p>
</sec>
</sec>
<sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title>
<supplementary-material content-type="local-data" id="pone.0009025.s001"><label>Supplementary Materials S1</label>
<caption><p>(0.27 MB DOC)</p>
</caption>
<media xlink:href="pone.0009025.s001.doc"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
</sec>
</body>
<back><ack><p>The authors thank Asif M. Khan for helpful discussions and suggestions.</p>
</ack>
<fn-group><fn fn-type="COI-statement"><p><bold>Competing Interests: </bold>
The authors have declared that no competing interests exist.</p>
</fn>
<fn fn-type="financial-disclosure"><p><bold>Funding: </bold>
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.</p>
</fn>
</fn-group>
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