www.fludb.org Sequence Feature Variant Type and Evolutionary Trajectory Analysis using the Influenza Research Database (IRD) 19 July 2011 Richard H. Scheuermann, Ph.D. Department of Pathology U.T. Southwestern Medical Center
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www.fludb.org
Sequence Feature Variant Type and Evolutionary Trajectory Analysis using the
Influenza Research Database (IRD)
19 July 2011
Richard H. Scheuermann, Ph.D.
Department of Pathology
U.T. Southwestern Medical Center
www.fludb.orgOutline
• Brief overview of NIAID-Sponsored Influenza Research Database (IRD)– Comprehensive integrated database– Analysis and visualization tools– U.S. NIH-funded, free access, open to all– Developed by a team of research scientists, bioinformaticians and
professional software developers– www.fludb.org– www.viprbrc.org for other human viral pathogens
• Novel approach to genotype-phenotype association studies – Sequence Feature Variant Type (SFVT) analysis
• Evolutionary Trajectory analysis of the pandemic (H1N1) 2009 strain
www.fludb.orgPublic Health Impact of Influenza
• Seasonal flu epidemics occur yearly during the fall/ winter months and result in 3-5 million cases of severe illness worldwide.
• More than 200,000 people are hospitalized each year with seasonal flu-related complications in the U.S.
• Approximately 36,000 deaths occur due to seasonal flu each year in the U.S.
• Populations at highest risk are children under age 2, adults age 65 and older, and groups with other comorbidities.
• Pandemics– 1918 Spanish flu (H1N1); 20 - 100 million deaths– 1957 Asian flu (H2N2); 1 - 1.5 million deaths– 1968 Hong Kong flu (H3N2); 750,000 - 1 million deaths– 2009 Swine origin (H1N1); > 16,000 deaths as of March 2010
Source: World Health Organization - http://www.who.int/mediacentre/factsheets/fs211/en/index.html
www.fludb.orgInfluenza Virus
Orthomyxoviridae familyNegative-strand RNASegmentedEnveloped
8 RNA segments encode11 proteinsClassified based on serology of HA and NA
www.fludb.orgIRD Overview
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www.fludb.orgSearch Access to Data
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www.fludb.orgData Types
www.fludb.orgCore Query Attributes
www.fludb.orgAdvanced Query Options
www.fludb.orgSegment search results
www.fludb.orgAnalysis and Visualizationwww.fludb.org
www.fludb.orgAnalysis and Visualization Tools
www.fludb.orgWorkbench Access
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www.fludb.orgMy Private Workbench
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www.viprbrc.org
www.fludb.orgIRD Summary
• Funded by U.S. National Institute of Allergy and Infectious Diseases (NIAID)
• Free and open access with no use restrictions
• Developed by a team of research scientists, bioinformaticians and professional software developers
• Comprehensive collection of public data
• Novel derived data, novel analytical tools, unique functions
• Integration – Integration – Integration
• www.fludb.org
• www.viprbrc.org
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NOVEL APPROACH TO GENOTYPE-PHENOTYPE ASSOCIATION STUDIES – SEQUENCE FEATURE VARIANT TYPE (SFVT) ANALYSIS
www.fludb.orgLimitations to Phylogenetics
• Traditional virus phylogenetics focuses on comparative analysis of whole genome/genome segments, and is most useful to understand virus evolution
• However, the genetic determinants of important viral phenotypes, e.g. virulence, host range, replication efficiency, immune response evation, etc., are determined by focused functional regions of viral proteins
• Therefore, specific genotype-phenotype association can be masked by other evolutionary factors that contribute to traditional phylogenetic analysis
www.fludb.orgSFVT approach
VT-1 I F D R L E T L I LVT-2 I F N R L E T L I LVT-3 I F D R L E T I V LVT-4 L F D Q L E T L V SVT-5 I F D R L E N L T LVT-6 I F N R L E A L I LVT-7 I Y D R L E T L I LVT-8 I F D R L E T L V LVT-9 I F D R L E N I V LVT-10 I F E R L E T L I LVT-11 L F D Q M E T L V S
Influenza A_NS1_nuclear-export-signal_137(10)
• Identify regions of protein/gene with known structural or functional properties – Sequence Features (SF)• an alpha-helical region, the binding site for another protein, an enzyme active site, an immune epitope
• Determine the extent of sequence variation for each SF by defining each unique sequence as a Variant Type (VT)
• High-level, comprehensive grouping of all virus strains by VT membership for each SF independently• Genotype-phenotype association statistical analysis, e.g. genetic determinants of host range, virulence,
replication rate
Influenza A_NS1_alpha-helix_171(17)
www.fludb.orgSF definition
• Based on experimentation reported in the literature and 3D protein structures (PDB records)
• Captured by manual curation• Defined by the specific amino acid positions in the
polypeptide chain• Annotated with the know structural or functional
properties
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Influenza A Sequence Features as of 18JUL2011
4128 SFs total
www.fludb.orgNS1 Sequence Features
www.fludb.orgSF8 (nuclear export signal)
www.fludb.orgVT for SF8 (nuclear export signal)
www.fludb.orgVT-1 strains
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DO VARIATIONS IN NS1 SEQUENCE FEATURES INFLUENCE INFLUENZA VIRUS HOST RANGE?
www.fludb.orgNS1 Sequence Features
www.fludb.orgVT for SF8 (nuclear export signal)
www.fludb.orgVT distribution by host
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Causes of apparent NS1 VT-associated host range restriction
• Virus spread - capability + opportunity– Phenotypic property of the virus – limited capacity– Restricted founder effect – limited opportunity
• Restricted spatial-temporal distribution
• Sampling bias – assumption of random sampling– Oversampling – avian H5N1 in Asia; 2009 H1N1– Undersampling – large and domestic cats
• Linkage to causative variant
www.fludb.orgVT-11 strains
www.fludb.orgVT for SF8 (nuclear export signal)
www.fludb.orgVT lineages
www.fludb.orgVT-4 lineage
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www.fludb.orgVT-4 lineage = B allele/group
www.fludb.orgVT-16 & VT-9 lineages
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EVOLUTIONARY TRAJECTORY ANALYSIS OF THE PANDEMIC (H1N1) 2009 STRAIN
www.fludb.orgPhylogenetic Analysis
• Evolutionary origin– Select a representative pandemic (H1N1) 2009 sequence
from the IRD database– BLAST to identify most similar sequences– Assess phylogenetic relationships
www.fludb.orgPandemic (H1N1) 2009 selection
www.fludb.orgBLAST Result
www.fludb.orgSegment 1 phylogenetic tree
Swine/Ohio/2004
Duck/USA/2000s
Human/USA/2007 (seasonal)
Swine/USA/1990s
Pandemic (H1N1) 2009
www.fludb.orgTemporal component
• Reference strain– A/California/04/2009
• BLAST– Return top 1000 results
• Normalize data• Graph nucleotide differences versus isolation year
differences
www.fludb.orgNP chart
www.fludb.orgNS chart
www.fludb.orgHA chart
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Group 1
Group 3
Group 2
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<= Cali/04/09
NS blue cluster (G1)
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<= Cali/04/09
NS green cluster (G2)
www.fludb.orgPhylogenetic Trees Quantification
• Analysis method– Build tree for Group 1 and Group 2 strains separately– Analyze branch lengths of trees
• Results– Avg. Group 1 Branch Length:0.0034 (S.D. 0.0062)– Avg. Group 2 Branch Length: 0.0075 (S.D. 0.0118)– T-test (2 sample, unequal variance): 3.22 10-05
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Group 1
Group 3
Group 2
www.fludb.orgHA trendline
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Evolutionary Trajectory Slopes vs. Mutation Rate
Segment Group 1 Slope Group 2 Slope Mutation Rate
PB2 6.8 24.9 4.3
PB1 7.6 26.9
PA 5.9 23.2
HA 5.5 28.8 5.7
NP 2.9 18.2 3.6
NA 3.8 23.1 3.2
M 1.3 5.6 1.5
NS 2.0 12.5 1.6
Substitutions/segment/year
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Evolutionary Trajectory (E.T.)
Similar but Distantly Related (SDR)
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Garten, et al. Science 2009
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Garten, et al. Science 2009
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<= Cali/04/09
ET
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<= Cali/04/09
SDR
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North American H1N1 Lineage - HAH1N1 2009
American Swine, 2000’sNorth American H1N1Lineage HA – Group 1
American Swine, 90’s
American Swine, 80’s
American Swine, 70’s
American Swine, 40 - 60’s
www.fludb.orgEvolutionary Trajectory Plots
Evolutionary Trajectory of a strain, with candidates displayed.
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www.fludb.orgSummary
• The Influenza Research Database (IRD) provides a comprehensive resource of data, analysis and visualization tools about influenza virus – www.fludb.org
• SFVT represents a novel tool that can be used to better understand genotype-phenotype relationships for flu
• Use of IRD to illuminate the viral origins of the pandemic (H1N1) 2009 virus
• IRD is continually evolving to capture and integrate addition data and analytical tools to support the needs of the influenza research community
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• U.T. Southwestern– Richard Scheuermann (PI)– Burke Squires– Jyothi Noronha– Victoria Hunt– Shubhada Godbole– Brett Pickett– Yun Zhang
• MSSM– Adolfo Garcia-Sastre– Eric Bortz– Gina Conenello– Peter Palese
• Vecna– Chris Larsen– Al Ramsey
• LANL– Catherine Macken– Mira Dimitrijevic
• U.C. Davis– Nicole Baumgarth
• Northrop Grumman– Ed Klem– Mike Atassi– Kevin Biersack– Jon Dietrich– Wenjie Hua– Wei Jen– Sanjeev Kumar– Xiaomei Li– Zaigang Liu– Jason Lucas– Michelle Lu– Bruce Quesenberry– Barbara Rotchford– Hongbo Su– Bryan Walters– Jianjun Wang– Sam Zaremba– Liwei Zhou
• IRD SWG– Gillian Air, OMRF– Carol Cardona, Univ. Minnesota– Adolfo Garcia-Sastre, Mt Sinai– Elodie Ghedin, Univ. Pittsburgh– Martha Nelson, Fogarty– Daniel Perez, Univ. Maryland– Gavin Smith, Duke Singapore– David Spiro, JCVI– Dave Stallknecht, Univ. Georgia– David Topham, Rochester– Richard Webby, St Jude
• USDA– David Suarez
• Sage Analytica– Robert Taylor– Lone Simonsen
• CEIRS Centers
Acknowledgments
N01AI40041
www.fludb.orgSegment 6 (NA) By Host
0 10 20 30 40 50 60 70 80 90 1000
50
100
150
200
250
300
swineturkeyDuckChickenHuman
Isolation Year Differences
Nuc
leoti
de D
iffer
ence
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