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BIOINFORMATICS OF AVIAN INFLUENZA VIRUS. GROUP 4 Yu Hai Dong Tay Hwee Goon Ling Wen Wan Felicia Loe Loh Shin Shion Clarice Chen Bai Hui Fen Low Soon Wah. INTRODUCTION. Where can one read up more about the Bird Flu?.

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  1. BIOINFORMATICS OF AVIAN INFLUENZA VIRUS GROUP 4 Yu Hai Dong Tay Hwee Goon Ling Wen Wan Felicia Loe Loh Shin Shion Clarice Chen Bai Hui Fen Low Soon Wah


  3. Where can one read up more about the Bird Flu? Avian influenza, or “bird flu” (type A, strain H5N1), is a contagious disease of animals caused by viruses that normally infect only birds and, less commonly, pigs. Avian influenza viruses are highly species-specific, but have, on rare occasions, crossed the species barrier to infect humans. (WHO) www.cdc.gov/flu/avian/ www.who.int/csr/disease/avian_influenza/en/ www.pandemicflu.gov/ www.nature.com/nature/focus/birdflu/ www.ebi.ac.uk/2can/disease/bird_flu/

  4. Mechanisms of infection and pathogenesis • Potential mechanisms of increased virulence • Increased HA cleavage. • H5N1 encodes NS1 to escape anti-viral cytokine responses. Antigenic variation of HA and NA. Antigenic shift is the cause of pandemics. • Bioinformatics and Computational Biology • Modeling of antigenic/ genetic drift (accumulation of mutations) in all segments of the genome, • Prediction of genetic evolution from genetic data to track the emergence of new avian flu strain with high human to human transmission. • Change in receptor binding specificity • Substitution of amino acids in HA and NA. • Wide variety modifications of sialic acids in accordance to the changes of HA and NA.


  6. Extraction of Genomic Data -GenBank

  7. Search Genome forH5N1

  8. Different strains Different segments Different locations

  9. Genomic Sequence Of H5N1

  10. Other Ways to Find Genomic Data • Read paper and find accession number (AF144305) to GenBank from paper • Other sites • EMBL-EBL (European Bioinformatics Institute) • DDBJ (DNA DataBase of Japan)

  11. Diagnosis of Highly Pathogenic Strains of Influenza Virus- Methods Table 1. Molecular diagnosis of influenza Joanna S. Ellis* and Maria C. Zambon

  12. Real Time PCR • Most sensitive and rapid method. http://www.nature.com/nmeth/journal/v2/n4/images/nmeth0405-305-I2.gif

  13. GENBANK/ SEQUENCE NEW MUTANT BLAST/FASTA PRIMER DESIGN PCR KIT Developing a PCR Kit for New Mutant Possibly Human-to-Human Transmissible Virus

  14. PCR Primer Design The critical component for an effective PCR assay are a pair of primers which need to be… • Primers should be 17-28 bases in length • Base composition should be 50-60% (G+C) • Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming • Tms between 55-80oC are preferred; • 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesized preferentially to any other product; • Primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided; • Runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.   adapted from Innis and Gelfand,1991 

  15. Making PCR PrimersOld School way…

  16. Primer Premiere Comprehensive primer design tool • PCR and hybridization primers • Cross species primers • Allele specific primers • Degenerate primers Optimized Primers • Automatic multiple sequence alignment with primer design • Restriction enzyme analysis • Cross homologies • Common Motif

  17. Search Criteria Parameters Bioinformatics way “Primer Premiere”

  18. Primers generated

  19. MySQL Oracle MS SQL Server Etc BUILDING UP OF BIOINFORMATICS DATABASE Factors to consider in choosing a particular software include: - Cost of Initial Purchase - Cost of Maintenance & Support - Size of Database (e.g. amount of data, number of users, exhaustiveness of connectivity) - Compatibility (e.g. supports cross platforms and browsers, flexible data import & export, etc) - Expandability - Transferability - User-Friendliness of the software - Reliability/Stability - Security - Performance (speed, efficiency, etc)

  20. 1. Setup Database Server (e.g. Microsoft SQL Server) 2. Create Database (e.g. BioInformatics) 3.Create Tables Various ways of data entry (e.g. Import from Batch or Excel Files)

  21. EXAMPLE: Database for Avian Influenza Virus Research by a Hospital Periphery Data Data for administrative purposes, e.g. : Staff Records, Funding / Finances, Patients’ Personal Records Patients’ Insurance Records, Patients’ Medical Records, R & D Related Data Schematic & R/ship Config Statistical Data

  22. E.g. Find the Mutation Seq. of patients who died from Bird Flu


  24. Neuraminidase (Jmol) Visualizing Pathogenic Proteins • Obtain protein ID from PDB • View using tools from the PDB website

  25. Disulphide Bonds Rasmol Protein Explorer Analyzing Pathogenic Proteins Hemagglutinin • Visualize using RasMol or Protein Explorer • Structural analysis • Identify basic structures: Alpha helix and beta sheets • Atom spatial distance measurement • Observing specific amino acid residues • Identifying disulphide bridges

  26. Rational Drug Design Deterministic approach to develop drugs based on the molecular structure of the target, Eg: Tamiflu


  28. Bioinformatics in Vaccine Development • Several milestones in the history of Immunization • From early attempts of “variolation” to modern genetically engineered vaccines • Vaccination has prevented illness and death for more than 200 years • Despite the success, infectious diseases still the leading cause of death worldwide • Two major innovations in vaccine design: • Modern molecular biology techniques • Genomic technology • Genomic information used to screen the inclusive set of proteins coded by pathogens, in search of potential vaccine candidates – Reverse Vaccinology Figure 1. Returning from a market shopping trip in Vietnam (adapted from The Lancet Infectious Diseases Vol. 4 Aug 2004) Figure 2. The effect of highly pathogenic H5N1 virus on ducklings in Vietnam (adapted from The Lancet Infectious Diseases Vol. 4 Aug 2004)

  29. Classical Whole Cell Heat-Killed Vaccines Live Attenuated Vaccines • Reverse Vaccinology: • Completed pathogen genome sequence opened up a completely new approach to vaccine discovery • Entire set of potential antigens can be identified by the analysis in silico of the genome sequence • Potential antigens cloned, purified and subjected to immunological screening • Whole procedure leads to the identification of a restricted number of vaccine candidates, thereby lowering cost Subunit Vaccines via DNA Recombination Technologies Subunit Vaccines via Non-Pathogenic Carrier Identification of Vaccine Candidates via: Reverse Vaccinology

  30. Epitope Analysis: • Using statistical mechanics to quantify the immune response that results from antigenic drift in the epitopes of the hemagglutinin and neuraminidase proteins • Able to explain the ineffectiveness of past influenza vaccines • Able to predict the effectiveness of future annual influenza vaccines • Quantitative epitope analysis could be incorporated as part of the regular protocol for construction of the annual influenza vaccine Hemagglutinin protein for an H3N2 strain (pdb 1HGF). Highlightedare the A (red), B (orange), C (brown), D (green), and E (blue) epitopes. The rest of the protein is shown in ribbon format. (Adapted from Vaccine 2006) A T-cell epitope docked on a MHC Class I molecule


  32. Integration of molecular biology, genomics and bioinformatics • Bioinformatics tools selects protein subsets from microbial genome sequence • Epitope mapping allows selection of putative epitopes from selected protein subsets • Confirmation of immunogenicity of selected set of epitopes or proteins using in vitro or in vivo tools • Confirmed epitopes formulated in delivery vehicle for further evaluations in challenge models • Integrated approach to developing vaccines may radically accelerate the vaccine pipeline in years to come (Adapted from Expert Rev. Vaccines 3(1), 2004)

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