bioinformatics of avian influenza virus

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Oct 2, 2013 (3 years and 9 months ago)

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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

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/

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)

Mechanisms of infection and pathogenesis

Potential mechanisms of
increased virulence




Increased HA cleavage.



H5N1 encodes NS1 to escape
anti
-
viral cytokine responses.

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.

Antigenic variation of HA
and NA. Antigenic shift is
the cause of pandemics.

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.

EXTRACTION AND USAGE
OF GENOMIC DATA

OF INFLUENZA VIRUS

Extraction of Genomic Data
-
GenBank

Search Genome for

H5N1

Different strains


Different segments


Different locations

Genomic

Sequence

Of H5N1

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)


Diagnosis of Highly Pathogenic
Strains of Influenza Virus
-

Methods

Table 1. Molecular diagnosis of influenza Joanna S. Ellis* and Maria C. Zambon

Real Time PCR


Most sensitive and rapid
method.


http://www.nature.com/nmeth/journal/v2/n4/images/nmeth0405
-
305
-
I2.gif

GENBANK/ SEQUENCE NEW MUTANT

BLAST/FASTA

PRIMER DESIGN

PCR KIT

Developing a PCR Kit for New Mutant Possibly
Human
-
to
-
Human Transmissible Virus

PCR Primer Design

The critical component for an effective PCR assay are a pair of primers which

need to be…


1.
Primers should be 17
-
28 bases in length

2.
Base composition should be 50
-
60% (G+C)

3.
Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of
ends and increases efficiency of priming

4.
Tms between 55
-
80oC are preferred;

5.
3'
-
ends of primers should not be complementary (ie. base pair), as otherwise
primer dimers will be synthesized preferentially to any other product;

6.
Primer self
-
complementarity (ability to form 2o structures such as hairpins)
should be avoided;

7.
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




Making PCR Primers



Old School way…


Primer Premiere

Optimized Primers


Automatic multiple sequence alignment with primer
design


Restriction enzyme analysis


Cross homologies


Common Motif


Comprehensive primer design tool


PCR and hybridization primers



Cross species primers


Allele specific primers


Degenerate primers



Bioinformatics way “Primer Premiere”

Search Criteria

Parameters

Primers generated

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)

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)


EXAMPLE:
Database for Avian Influenza Virus Research by a Hospital

R & D Related Data

Statistical Data

Periphery Data


Data for administrative purposes, e.g. :

Staff Records,

Funding / Finances,

Patients’ Personal Records

Patients’ Insurance Records,

Patients’ Medical Records,

Schematic &
R/ship Config

E.g. Find the Mutation Seq. of patients who
died

from
Bird Flu

DRUG DEVELOPMENT
PROCESS

Visualizing Pathogenic Proteins


Obtain protein ID from
PDB


View using tools from the
PDB website

Neuraminidase

(Jmol)


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

Hemagglutinin

Disulphide Bonds

Protein Explorer


Rasmol

Analyzing Pathogenic Proteins

Rational Drug Design

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)



Deterministic approach to develop drugs based on the molecular structure of
the target, Eg: Tamiflu

DESIGN OF VACCINES
AGAINST BIRD FLU

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)

Classical Whole
Cell Heat
-
Killed
Vaccines

Live Attenuated
Vaccines

Subunit Vaccines
via DNA
Recombination
Technologies

Subunit Vaccines
via Non
-
Pathogenic
Carrier

Identification of Vaccine
Candidates via:

Reverse Vaccinology

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

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)


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

A T
-
cell epitope docked on a
MHC Class I molecule

CONCLUSION



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)