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Viral evolution and pathogenesis The use of HPC/GRID Technologies to make intelligent biological inferences Slide 2 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 Slide 3 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 Najaf Shah Ritu Arora Pavithran Sathyanarayana Catherine Dong Slide 4 Collaborators University of Victoria Chris Upton David Esteban St. Louis University Mark Buller Medical College of Wisconsin Paula Traktman Slide 5 UAB Grid Development Department of Computer and Information Sciences Department of Information Technology, Academic Computing John-Paul Robinson Pravin Joshi Silbia Peechakara Jill Gemmill Slide 6 Viral Bioinformatics Resource Center www.biovirus.org www.poxvirus.org Slide 7 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. Slide 8 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 Slide 9 FamilyGenusSpeciesDiseaseCategory ArenaviridaeArenavirus: LCMV-complexLymphocytic Chorimeningitis VirusLymphocytic choriomeningitisA Lassa VirusLassa FeverA Arenavirus: Tacaribe complexJunin VirusArgentine hemorrhagic feverA Machupo VirusBolivian hemorrhagic feverA Guanarito VirusVenezuelan hemorrhagic feverA Sabia VirusSabia-associated hemorrhagic feverA BunyaviridaeHantavirusHanta VirusHantavirus Pulmonary SyndromeA Hantaan virusKorean hemorrhagic feverC Puumala virusHemorrhagic Fever with Renal SyndromeC NairovirusCrimean-Congo Hemorrhagic Fever VirusCrimean-Congo Hemorrhagic FeverC OrthobunyavirusCalifornia encephalitis group (La Crosse)EncephalitisB PhlebovirusRift Valley Fever VirusRift Valley FeverA FiloviridaeEbola-Like VirusesEbola VirusEbola Hemorrhagic FeverA Marburg-Like VirusesMarburg VirusMarburg Hemorrhagic FeverA FlaviviridaeFlavivirusDengue VirusDengue hemorrhagic feverA Kyasanar Forest Disease VirusKyasanur Forest diseaseB Omsk Hemorrhagic Fever VirusOmsk hemorrhagic feverB Japanese encephalitis virus groupWest Nile encephalitisB Yellow Fever VirusYellow feverC Tick-borne Encephalitis virusTick-borne encephalitisC ParamyxoviridaeUnclassifiedNipah virusC Morbillivirus Equine morbillivirusC PoxviridaeOrthopoxvirusVariola majorSmallpoxA TogaviridaeAlphavirusesVenezuelan equine encephalitis virusVenezuelan encephalitisB Eastern equine encephalitis virusEastern equine encephalitisB Western equine encephalitis virusWestern equine encephalitisB Slide 10 Genomic and Evolutionary Analysis of Poxviruses Slide 11 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 Slide 12 Slide 13 Orthopoxvirus Phylogeny 132 gene tree possible Slide 14 Slide 15 Genomic and Evolutionary Analysis of Poxviruses We have a problem Slide 16 Gene Synteny MPXV vs. VARV Slide 17 Slide 18 Slide 19 Slide 20 Slide 21 Slide 22 Poxvirus Gene Prediction Little consistency from one genome to another Methods employed Minimum ORF size Similarity with previously described proteins Slide 23 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 Slide 24 Poxvirus Gene Prediction and Annotation Chunlin Wang (Graduate Student) Poxvirus Genome Annotation System Slide 25 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) Slide 26 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. Slide 27 Late promoter alignment (RAP94) Early promoter alignment (DNA polymerase) Slide 28 VACV Early Promoter Dependencies Base frequencies Sequence Logo Base Dependencies Slide 29 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 Slide 30 Slide 31 High Performance Computing Tools Computationally-intensive Bioinformatics analyses Similarity searching Multiple sequence alignment Linux Clusters Grid Computing Slide 32 SS-Wrapper QS_searchquery splitting approach Accommodate most database searching application effortlessly All variants of NCBI BLAST, WU BLAST, FASTA, PC_SCAN, HMMPFAM DS_BLASTdatabase splitting approach A wrapper application tailored for NCBI BLAST Slide 33 The performance of QS-search for BLAST Slide 34 The performance of QS-search for HMMPFAM Slide 35 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: multi- threaded BLAST, database-splitting BLAST (e.g., mpiBLAST), query-splitting BLAST Slide 36 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 Slide 37 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 Slide 38 Anchoring-Extension Strategy Realign fuzzy regions using a genetic algorithm Computationally slow Parallelize process by sending each region to a separate node of the cluster/grid Optimally-aligned Blocks Fuzzy Regions Slide 39 The Poxvirus Genome Annotation System (PGAS) Pipeline Slide 40 PGAS Gene Layout Panel Open reading frame (no gene prediction) Predicted gene Predicted gene with alternate start codon Gene fragment Slide 41 PGAS Genome Comparison Panel Slide 42 Orthologous Gene Transcriptional Environment Predicted late promoter Predicted early promoter Predicted coding region ATG start codon T5NT early transcription terminator Slide 43 Predicted Gene Map CMLV-M96 Slide 44 Re-prediction of Orthopoxvirus Genes Gene CountGene Differences VirusInitialRevisedNewRemoved CMLV-CMS265178592 CMLV-M96211177438 CPXV-BR233209327 CPXV-GRI21221156 ECTV-MOS17517654 ECTV-NAV1801762226 MPXV-ZRE191182615 RPXV-UTR18418059 VACV-COP265176594 VACV-MVA157162105 VACV-WR218183439 VARV-BSH189164631 VARV-GAR206164648 VARV-IND197164639 Slide 45 Early Intermediate Late E/I E/L N.D. P. Identical P. Divergent ORF (+) ORF (-) Slide 46 Phylogeny of Poxvirus Gene gain and loss events Slide 47 Orthopoxvirus Evolution Simple Statement: The evolution of all Orthopoxvirus species reflects: Gene loss Protein sequence variation Variation in gene expression Acquisition of new genes does NOT play a role Slide 48 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 Slide 49 Human Monkeypox Bioinformatics, Epidemiology, Evolution, Biology, and Pathogenesis Slide 50 Monkeypox Collaborations CDC Inger Damon Joe Esposito Scott Sammons Anna Likos St. Louis University Nanhai Chen Mark Buller University of Victoria Guiyun Li Chris Upton Ft. Detrick Peter Jarhling UAB Elliot Lefkowitz Chunlin Wang And many others Slide 51 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 Slide 52 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 Slide 53 Characteristics of U.S. Monkeypox Infection No human fatalities No human-human transmission Slide 54 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 Slide 55 CDC 2005; Sammons et. al. Monkeypox Cases in Africa 1970 - 1986 West Africa: 6 cases DRC: 260 cases Origination of rodent shipment to the US Slide 56 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 No fatalities No human-human transmission Genetically distinct strain(s) of virus Equivalent to what was seen for the 2003 US Midwest outbreak Slide 57 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 COP-58 (West African) 1100/3 - 20,0000/3 - ZAI-V79 (Congo) 902/30/3- 50,0003/3 101 Slide 58 MPXV Sequence Comparisons COP-58WRAIR-61SL-V70ZAI-96 COP-58-16 / 78244 / 1245171 / 9859 WRAIR-6110 / 198927 / 0.01%-42 / 1007171 / 9835 SL-V7094 / 198392 / 0.07%94 / 198378 / 0.07%-168 / 9470 ZAI-96892 / 192338 / 0.55%890 / 192219 / 0.55%913 / 192159 / 0.56%- SubstitutionDeletionInsertion #Gaps / Length Gaps #substitutions / #identical / %difference Slide 59 MPXV-ZAI-96 Gene Prediction Slide 60 MPXV-WRAIR-61 Gene Prediction Slide 61 Left-end Orthopoxvirus Genome Alignment Slide 62 Right-end Orthopoxvirus Genome Alignment Slide 63 Presence of OPV virulence ortholog family members in monkeypox and variola viruses Virulence Ortholog Family Predicted Function/motif MPXVVARV SL-V70COP-58WR-61ZAI-96BSH-75 CPXV-BR-045Putative monoglyceride lipase++++- VACV-COP-F1LApoptosis inhibitor+++++ VACV-COP-A38LCD47-like+++++ VACV-COP-A42RProfilin homolog+++++ VACV-COP-A43RMembrane protein+++++ VACV-COP-A44LHydroxysteroid dehydrogenase++++Frag VACV-COP-A45RSuperoxide dismutase-like+++++ VACV-COP-A46RIL-1 signaling inhibitor+++++ VACV-COP-B7RVirulence, ER resident++++- VACV-COP-B8RIFN- BP+++++ CPXV-BR-203Virulence factorFrag + (B10R)- VACV-COP-B12RSer/Thr Kinase++++Frag CPXV-BR-207Serpin-2 (SPI2)+++++ CPXV-BR-209IL-1 BPFrag + (B14R)Frag VACV-COP-B19RIFN-/ receptor+++++ VACV-COP-C12LSerpin-1 (SPI1)+++++ CPXV-BR-219Surface glycoprotein+++++ Slide 64 Presence of OPV virulence ortholog family members in monkeypox and variola viruses Virulence Ortholog Family Predicted Function/motif MPXVVARV SL-V70COP-58WR-61ZAI-96BSH-75 CPXV-BR-003CC-chemokine BP+++++ CPXV-BR-005TNF BP (Crm B)+++++ VACV-COP-K3LEIF-2 homologFrag + VACV-COP-C11RGrowth factor+++++ VACV-COP-C10LIL-1 antagonistFrag + VACV-COP-C4LIL-1 antagonist-like+++++ CPXV-BR-023RING finger/apoptosis+++++ VARV-BSH-D7LIL-18 BP+++++ CPXV-BR-025Chinese Hamster Ovary Host Range++++Frag VACV-COP-C7LHost range, virulence factor+++++ VACV-COP-C3LInhibitor of complement enzymes---+ (D14L)+ VACV-COP-N1LVirulence+++++ VACV-COP-N2L-amanitin sensitivity+++++ VACV-COP-K1LHost range++++Frag VACV-COP-K2LSerpin-3 (SPI3)+++++ VACV-COP-K4LPhospholipase D-like++++- Slide 65 MOPICE structure and function (Monkeypox inhibitor of complement enzymes) MOPICE: cofactor for complement cleavage by serine protease factor I Slide 66 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. Slide 67 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) Slide 68