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Viral evolution and pathogenesis
The use of HPC/GRID Technologies
to make intelligent biological
inferences
Outline
• Viral Bioinformatics Resource Center
• Biodefense/Emerging diseases
• Poxvirus genomics and evolution
• Bioinformatics Research
• Development and use of HPC/GRID
technologies
• Monkeypox pathogenesis
• Real-world case study
The UAB MGBF Contingent
(Microbial Genomics and Bioinformatics Facility)
• Graduate Students
• Chunlin Wang
• Mary Odom
• Programmers
• Jim Moon
• Don Dempsey
• Fellows
• Shankar Changayil
• Curtis Hendrickson
• Elizaveta Karpova
• Technical Writer
• Cathy Galloway
• UAB CIS Collaborators
• Puri Bangalore, CIS
• Barrett Bryant, CIS
• Students
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Najaf Shah
Ritu Arora
Pavithran Sathyanarayana
Catherine Dong
Collaborators
• University of Victoria
• Chris Upton
• David Esteban
• St. Louis University
• Mark Buller
• Medical College of Wisconsin
• Paula Traktman
UAB Grid Development
• Department of Computer and Information
Sciences
• Department of Information Technology,
Academic Computing
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John-Paul Robinson
Pravin Joshi
Silbia Peechakara
Jill Gemmill
Viral Bioinformatics Resource Center
www.biovirus.org
www.poxvirus.org
Bioinformatics Resource Centers for Biodefense
and Emerging or Re-Emerging Infectious Diseases
• Eight centers established by NIH
• Focus on NIH/CDC Category A-C priority
pathogens
• Each Center maintains data related to a
specific set of pathogens
• Each multi-disciplinary team consists of
pathogen domain experts, microbiologists,
bioinformaticians and computer scientists.
BRCs are Designed to Support
Basic and applied research on priority pathogens
including the development of:
• Environmental Detectors
• Diagnostic Reagents
• Animal Models
• Vaccines
• Antimicrobial Compounds
and…
• Basic Bioinformatics Research
• Mining the data for meaningful patterns that can then
provide inferences on biological function that can be
tested in the laboratory
Family
Arenaviridae
Genus
Arenavirus: LCMV-complex
Filoviridae
Flaviviridae
Paramyxoviridae
Disease
Category
Lymphocytic Chorimeningitis Virus
Lymphocytic choriomeningitis
A
Lassa Virus
Lassa Fever
A
Junin Virus
Argentine hemorrhagic fever
A
Machupo Virus
Bolivian hemorrhagic fever
A
Guanarito Virus
Venezuelan hemorrhagic fever
A
Sabia Virus
Sabia-associated hemorrhagic fever
A
Hanta Virus
Hantavirus Pulmonary Syndrome
A
Hantaan virus
Korean hemorrhagic fever
C
Puumala virus
Hemorrhagic Fever with Renal Syndrome
C
Nairovirus
Crimean-Congo Hemorrhagic Fever Virus
Crimean-Congo Hemorrhagic Fever
C
Orthobunyavirus
California encephalitis group (La Crosse)
Encephalitis
B
Phlebovirus
Rift Valley Fever Virus
Rift Valley Fever
A
Ebola-Like Viruses
Ebola Virus
Ebola Hemorrhagic Fever
A
Marburg-Like Viruses
Marburg Virus
Marburg Hemorrhagic Fever
A
Flavivirus
Dengue Virus
Dengue hemorrhagic fever
A
Kyasanar Forest Disease Virus
Kyasanur Forest disease
B
Omsk Hemorrhagic Fever Virus
Omsk hemorrhagic fever
B
Japanese encephalitis virus group
West Nile encephalitis
B
Yellow Fever Virus
Yellow fever
C
Tick-borne Encephalitis virus
Tick-borne encephalitis
C
Arenavirus: Tacaribe complex
Bunyaviridae
Species
Hantavirus
Unclassified
Nipah virus
C
Morbillivirus
Equine morbillivirus
C
Poxviridae
Orthopoxvirus
Variola major
Smallpox
A
Togaviridae
Alphaviruses
Venezuelan equine encephalitis virus
Venezuelan encephalitis
B
Eastern equine encephalitis virus
Eastern equine encephalitis
B
Western equine encephalitis virus
Western equine encephalitis
B
Genomic and Evolutionary
Analysis of Poxviruses
Objectives
• To better understand the role individual genes and
groups of genes (or other genetic elements) play in
poxvirus (especial smallpox ) host range and
virulence
• Try to describe and understand poxvirus diversity
via reconstruction of the families evolutionary
history
• Analyze differences in evolutionary patterns of
conserved core replicative genes vs. divergent host
range/immunomodulatory/virulence factor genes
Orthopoxvirus Phylogeny
132 gene tree possible
Genomic and Evolutionary
Analysis of Poxviruses
We have a problem…
Gene Synteny MPXV vs. VARV
Poxvirus Gene Prediction
• Little consistency from one genome to
another
• Methods employed
• Minimum ORF size
• Similarity with previously described proteins
Consistently predict and annotate the
gene set for all Poxvirus genomes
• Development of a comprehensive gene
prediction tool
• Discovery of new or “missed” genes
• Removal of “pseudo” genes
• As an added bonus:
• Computational annotation of each predicted
gene
Poxvirus Gene Prediction and Annotation
• Chunlin Wang (Graduate Student)
• Poxvirus Genome Annotation System
VBRC Computational Tools
• Similarity searching
• SS-Wrapper
• NCBI BLAST, WU BLAST, FASTA, PC_SCAN, HMMPFAM…
• HPC – Cluster/Grid
• Refinement of genome-scale multiple sequence alignments
• GenAlignRefine
• HPC Cluster
• Poxvirus gene prediction
• Sequence Signals (Promoter prediction, Glimmer)
• Similarity (BLAST and HMMPFAM)
• Comparative analyses (Orthologs and Gene synteny)
Poxvirus promoter detection
• Distinct promoters for each phase of gene
expression
• Two conserved regions (core and initiator)
separated by variable spacing
• Sequence conservation generally within
each genus.
Early promoter alignment
(DNA polymerase)
Late promoter alignment
(RAP94)
VACV Early Promoter Dependencies
Base frequencies
Sequence Logo
Base Dependencies
Poxvirus Promoter Prediction
• Obtain experimentally verified vaccinia virus promoters
from the literature
• Align known promoter sequences to assess sequence
conservation
• Determine statistically significant interactions
(dependencies)
• Build Interpolated Context Models (ICMs) based on
VACV early and late promoter sequences
• Predict the VACV promoters using the ICMs
• Predict Promoter sequences in other Poxviridae species
• Evaluate promoter variation for Orthopoxvirus species
High Performance Computing Tools
• Computationally-intensive Bioinformatics
analyses
• Similarity searching
• Multiple sequence alignment
• Linux Clusters
• Grid Computing
SS-Wrapper
• QS_search—query splitting approach
• Accommodate most database searching
application effortlessly
• All variants of NCBI BLAST, WU BLAST,
FASTA, PC_SCAN, HMMPFAM…
• DS_BLAST—database splitting approach
• A wrapper application tailored for NCBI
BLAST
The performance of QS-search
for BLAST
The performance of QS-search
for HMMPFAM
G-BLAST
• A native Grid Service Interface for BLAST
• G-BLAST provides automatic BLAST
algorithm selection based on # of queries,
length of queries, size of the database used,
and machines available
• BLAST algorithms employed: multithreaded BLAST, database-splitting BLAST
(e.g., mpiBLAST), query-splitting BLAST
GridBLAST User-Friendly Interface
• Access using BlazerID and
password
• Queries and Results easily
uploaded & downloaded
• Web UI can be hosted on
your server
• Web UI can be written in
any development language
GenAlignRefine
• Refinement of multiple whole-genome
sequence alignments
• Supports comparative genomics
• Identification of genotypic differences
• Identify changes related to particular phenotypes
• pathogenic/non-pathogenic strains
• Evolutionary relationships
• Annotation of newly sequenced genomes
“Anchoring-Extension” Strategy
Optimally-aligned
Blocks
“Fuzzy” Regions
• Realign “fuzzy” regions using a genetic algorithm
• Computationally slow
• Parallelize process by sending each region to a
separate node of the cluster/grid
The Poxvirus Genome Annotation System (PGAS) Pipeline
PGAS Gene Layout Panel
Open reading frame (no gene prediction)
Predicted gene
Predicted gene with alternate start codon
Gene fragment
PGAS Genome Comparison Panel
Orthologous Gene Transcriptional Environment
Predicted coding region
Predicted late promoter
Predicted early promoter
T5NT early transcription terminator
ATG start codon
Predicted Gene Map CMLV-M96
Re-prediction of Orthopoxvirus Genes
Gene Count
Virus
Initial
Gene Differences
Revised
New
Removed
CMLV-CMS
265
178
5
92
CMLV-M96
211
177
4
38
CPXV-BR
233
209
3
27
CPXV-GRI
212
211
5
6
ECTV-MOS
175
176
5
4
ECTV-NAV
180
176
22
26
MPXV-ZRE
191
182
6
15
RPXV-UTR
184
180
5
9
VACV-COP
265
176
5
94
VACV-MVA
157
162
10
5
VACV-WR
218
183
4
39
VARV-BSH
189
164
6
31
VARV-GAR
206
164
6
48
VARV-IND
197
164
6
39
Early
Intermediate
Late
E/I
E/L
N.D.
P. Identical
P. Divergent
ORF (+)
ORF (-)
Phylogeny of Poxvirus Gene gain and loss events
Orthopoxvirus Evolution
Simple Statement:
• The evolution of all Orthopoxvirus species
reflects:
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Gene loss
Protein sequence variation
Variation in gene expression
Acquisition of new genes does NOT play a role
Future work
• Apply the tools and techniques developed
for poxviruses to the study of other viral
pathogens
• Identification of significant RNA-virus
sequence co-dependencies
• Identification of amino acid co-dependencies
• RNA virus evolution
Human Monkeypox
Bioinformatics, Epidemiology,
Evolution, Biology, and Pathogenesis
Monkeypox Collaborations
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CDC
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St. Louis University
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Peter Jarhling
UAB
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Guiyun Li
Chris Upton
Ft. Detrick
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Nanhai Chen
Mark Buller
University of Victoria
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Inger Damon
Joe Esposito
Scott Sammons
Anna Likos
Elliot Lefkowitz
Chunlin Wang
And many others…
Monkeypox
• Smallpox-like disease
• Approximately 10% case fatality rate
• Rare human-human transmission
• No more than 2 generations of transmission
from an index case
• Increasing incidence
• Human encroachment on animal reservoir
habitats
U.S. Midwest Monkeypox
Outbreak
• April – June, 2003
• Imported from West Africa
• Shipment of infected rodents from Ghana
• Rodents housed with native prairie dogs
• Infected prairie dogs transmitted virus to humans
• Transmission due to respiratory and direct
mucocutaneous exposure
• 72 confirmed or suspected human cases
Characteristics of U.S.
Monkeypox Infection
• No human fatalities
• No human-human transmission
Possible Explanations for Reduced
Virulence in the U.S. Monkeypox
Outbreak
• Higher natural resistance of the U.S.
population
• Healthier patient population
• Better supportive care
• Viral strain differences with variable
pathogenicity
Monkeypox Cases in Africa 1970 - 1986
West Africa: 6 cases
Origination of rodent shipment
to the US
DRC: 260 cases
CDC – 2005; Sammons et. al.
Variability of Monkeypox Infections in
Different Regions of Africa
• Prevalence equivalent as determined by antibody titers of
the population
• Central African (Congo basin) Disease
• >90% of reported cases
• All reported fatalities
• 11.5% Case fatality rate
• Human-human transmission
• West African
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No fatalities
No human-human transmission
Genetically distinct strain(s) of virus
Equivalent to what was seen for the 2003 US Midwest outbreak
Aerosol Infection of Cynomolgus Monkeys with
West and Congo Basin Isolates Monkeypox virus
(Ft. Detrick)
MPXV isolate
Aerosol dose
(PFU/monkey)
Morbidity
(Day 7)
Mortality
Mean day
of death
110
0/3
0/3
-
20,000
0/3
0/3
-
90
2/3
0/3
-
50,000
3/3
3/3
10±1
COP-58
(West African)
ZAI-V79
(Congo)
MPXV Sequence Comparisons
Substitution
COP-58
Deletion
Insertion
#substitutions / #identical / %difference
#Gaps / Length Gaps
COP-58
WRAIR-61
SL-V70
ZAI-96
-
16 / 782
44 / 1245
171 / 9859
-
42 / 1007
171 / 9835
-
168 / 9470
WRAIR-61
10 / 198927 / 0.01%
SL-V70
94 / 198392 / 0.07%
94 / 198378 / 0.07%
ZAI-96
892 / 192338 / 0.55%
890 / 192219 / 0.55%
913 / 192159 / 0.56%
-
MPXV-ZAI-96 Gene Prediction
MPXV-WRAIR-61 Gene Prediction
Left-end Orthopoxvirus Genome Alignment
Right-end Orthopoxvirus Genome Alignment
Presence of OPV virulence ortholog family members in
monkeypox and variola viruses
MPXV
Virulence
Ortholog Family
Predicted Function/motif
CPXV-BR-045
VARV
SL-V70
COP-58
WR-61
ZAI-96
BSH-75
Putative monoglyceride lipase
+
+
+
+
-
VACV-COP-F1L
Apoptosis inhibitor
+
+
+
+
+
VACV-COP-A38L
CD47-like
+
+
+
+
+
VACV-COP-A42R
Profilin homolog
+
+
+
+
+
VACV-COP-A43R
Membrane protein
+
+
+
+
+
VACV-COP-A44L
Hydroxysteroid dehydrogenase
+
+
+
+
Frag
VACV-COP-A45R
Superoxide dismutase-like
+
+
+
+
+
VACV-COP-A46R
IL-1 signaling inhibitor
+
+
+
+
+
VACV-COP-B7R
Virulence, ER resident
+
+
+
+
-
VACV-COP-B8R
IFN-γ BP
+
+
+
+
+
CPXV-BR-203
Virulence factor
Frag
Frag
Frag
+ (B10R)
-
VACV-COP-B12R
Ser/Thr Kinase
+
+
+
+
Frag
CPXV-BR-207
Serpin-2 (SPI2)
+
+
+
+
+
CPXV-BR-209
IL-1β BP
Frag
Frag
Frag
+ (B14R)
Frag
VACV-COP-B19R
IFN-α/β receptor
+
+
+
+
+
VACV-COP-C12L
Serpin-1 (SPI1)
+
+
+
+
+
CPXV-BR-219
Surface glycoprotein
+
+
+
+
+
Presence of OPV virulence ortholog family members in
monkeypox and variola viruses
MPXV
Virulence
Ortholog Family
Predicted Function/motif
CPXV-BR-003
VARV
SL-V70
COP-58
WR-61
ZAI-96
BSH-75
CC-chemokine BP
+
+
+
+
+
CPXV-BR-005
TNF BP (Crm B)
+
+
+
+
+
VACV-COP-K3L
EIF-2α homolog
Frag
Frag
Frag
Frag
+
VACV-COP-C11R
Growth factor
+
+
+
+
+
VACV-COP-C10L
IL-1β antagonist
Frag
Frag
Frag
Frag
+
VACV-COP-C4L
IL-1β antagonist-like
+
+
+
+
+
CPXV-BR-023
RING finger/apoptosis
+
+
+
+
+
VARV-BSH-D7L
IL-18 BP
+
+
+
+
+
CPXV-BR-025
Chinese Hamster Ovary Host Range
+
+
+
+
Frag
VACV-COP-C7L
Host range, virulence factor
+
+
+
+
+
VACV-COP-C3L
Inhibitor of complement enzymes
-
-
-
+ (D14L)
+
VACV-COP-N1L
Virulence
+
+
+
+
+
VACV-COP-N2L
α-amanitin sensitivity
+
+
+
+
+
VACV-COP-K1L
Host range
+
+
+
+
Frag
VACV-COP-K2L
Serpin-3 (SPI3)
+
+
+
+
+
VACV-COP-K4L
Phospholipase D-like
+
+
+
+
-
MOPICE structure and function
(Monkeypox inhibitor of complement enzymes)
MOPICE: cofactor for complement
cleavage by serine protease factor I
Conclusions
• Genomic sequence differences may be
responsible for differences in virulence
between Monkeypox strains isolated from
geographically-distinct regions
• Strains with reduced pathogenicity lack the
MOPICE gene that codes for a protein with
complement inhibitory activity.
Future work
• Targeted mutagenesis of the MOPICE gene
• Effect on pathogenesis
• Further analysis of newly-sequenced
Monkeypox isolates
• Analysis of the B10R and B14R genes
• (Among others)