BIOINFORMATICS OF AVIAN INFLUENZA VIRUS

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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
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INTRODUCTION
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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/avi
an_influenza/en/ www.pandemicflu.gov/ www.nature.c
om/nature/focus/birdflu/ www.ebi.ac.uk/2can/diseas
e/bird_flu/
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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.

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EXTRACTION AND USAGE OF GENOMIC DATA OF
INFLUENZA VIRUS
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Extraction of Genomic Data -GenBank
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(No Transcript)
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Different strains Different segments Different
locations
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Genomic Sequence Of H5N1
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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)

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

• Most sensitive and rapid method.

http//www.nature.com/nmeth/journal/v2/n4/images/n
meth0405-305-I2.gif
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Developing a PCR Kit for New Mutant Possibly
Human-to-Human Transmissible Virus
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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 (GC)
• 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 

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Making PCR Primers Old School way
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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

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Bioinformatics way Primer Premiere
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Primers generated
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BUILDING UP OF BIOINFORMATICS DATABASE
MySQL
Oracle
MS SQL Server
Etc
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)
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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)
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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
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E.g. Find the Mutation Seq. of patients who died
from Bird Flu
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DRUG DEVELOPMENT PROCESS
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Visualizing Pathogenic Proteins

• Obtain protein ID from PDB
• View using tools from the PDB website

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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

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Rational Drug Design
Deterministic approach to develop drugs based on
the molecular structure of the target, Eg
Tamiflu
Step Method Result
1. Biological information and selection of target - Sequence analysis of NA - Modeling of antibody binding NA releases sialic acid (Neu5Ac), which enables the replicated virus to bud from host cell. ? Design transition state analogue to inhibit NA
2. Design of lead compound - X-ray crystallography - Screening using Scanning program Binding sites located pharmacophore constructed. Neu5Ac2en
3. Combinatorial chemistry structure based design - Substituent modification using Builder program Improvement in potency and specificity 4-guanidino-Neu5Ac2en (GG167)
4. Clinical activity - Substituent modification Pharmacokinetics Orallyactive Oseltamivir (GS4071)
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DESIGN OF VACCINES AGAINST BIRD FLU
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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 2. The effect of highly pathogenic H5N1
virus on ducklings in Vietnam (adapted from The
Lancet Infectious Diseases Vol. 4 Aug 2004)
Figure 1. Returning from a market shopping trip
in Vietnam (adapted from The Lancet Infectious
Diseases Vol. 4 Aug 2004)
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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
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• 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
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CONCLUSION
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• 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)