Typing
Sequencing and Phylogeny
May 2007
E P I D E M I C
Laboratory Training for Field Epidemiologists
A L E R T
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R E S P O N S E
Learning objectives
At the end of the presentation, participants should:
• Understand sequencing
• Interpret phylogenetic trees
E P I D E M I C
Laboratory Training for Field Epidemiologists
A L E R T
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Sequencing
Process to determine the exact order of nucleotides in DNA
Parts of the genome are compared with other sequenced samples
• From a databank
• By direct comparison
E P I D E M I C
Laboratory Training for Field Epidemiologists
A L E R T
A N D
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Review of molecular biology
Genes – DNA and RNA are made of nucleotides
…AGCUAAAGCUA… Nucleotides code for amino acids (codons)
…ala-lys-ala… Amino acids make proteins
Proteins 'R' us; enzymes, cell membranes, etc.
Genetic variation (mutation) breeds trial and error – evolution
RNA based organisms mutate (evolve) much faster than DNA based
ones e.g. influenza, HIV
E P I D E M I C
Laboratory Training for Field Epidemiologists
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Evaluating organism similarity
…AGCUAAAGCUA… Gene sequence #1
…AACUAAACCUG… Gene sequence #2
…AACUCCACCUA… Gene sequence #3
…GGUUGCACCGG… Gene sequence #4
E P I D E M I C
Laboratory Training for Field Epidemiologists
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Algorithms & programs to compare genes
Several available: http://research.amnh.org/~siddall/methods/
Different approaches and assumptions but all startcwith computer
alignment e.g. Phylip, PAUP
Parsimony is "a principle that states that the simplest explanation that
explains the greatest number of observations is preferred to more
complex explanations".
#1…AGCUAAAGCUA…
#1…AGCUAAAGCUA…
#1…AGCUAAAGCUA…
#1…AGCUAAAGCUA…
#2…AACUAAACCUG…
#2…AACUAAACCUG…
#2…AACUAAACCUG…
#2…AACUAAACCUG…
73% identity
E P I D E M I C
Laboratory Training for Field Epidemiologists
A L E R T
A N D
R E S P O N S E
Comparing genes to evaluate
similarity among organisms
…AGCUAAAGCUA… Gene sequence #1
…AACUAAACCUG… Gene sequence #2
…AACUCCACCUA… Gene sequence #3
…GGUUGCACCGG… Gene sequence #4
Trees can be built with nucleic acid or protein sequences
E P I D E M I C
Laboratory Training for Field Epidemiologists
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A N D
R E S P O N S E
Sequence #1
Sequence #1
Sequence #2
Sequence #2
Sequence #3
Sequence #3
Sequence #4
Sequence #4
Distance in characters (nt/aa)
Unrooted phylogenetic tree by
unweighted pair group method
Comparing genes to evaluate
similarity among organisms
…AGCUAAAGCUA… Gene sequence #1
Glutamate
Lysine
…AACUAAACCUG…
G G
Gene sequence #2
…AACUCCACCUA… Gene sequence #3
…GGUUGCACCGG… Gene sequence #4
E P I D E M I C
Laboratory Training for Field Epidemiologists
A L E R T
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Molecular Phylogeny of H5 Avian Influenza viruses (HA and M genes)
Source: Suarez et. al. 2000: J. Virology 74: 6592-6599
Evolution of the 'Z genotype'
Letters to Nature
Nature 430, 209-213 (8 July 2004)
Genesis of a highly pathogenic and
potentially pandemic H5N1 influenza virus in
eastern Asia
K. S. Li1, Y. Guan1,2, J. Wang1,2, G. J. D.
Smith1,2, K. M. Xu1,2, L. Duan1,2, A. P.
Rahardjo3, P. Puthavathana4, C.
Buranathai5, T. D. Nguyen6, A. T. S.
Estoepangestie3, A. Chaisingh5, P.
Auewarakul4, H. T. Long7, N. T. H. Hanh7, R.
J. Webby8, L. L. M. Poon2, H. Chen1,2, K. F.
Shortridge1,2, K. Y. Yuen2, R. G. Webster2,8
and J. S. M. Peiris1,2
E P I D E M I C
Laboratory Training for Field Epidemiologists
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Conclusions
A phylogenetic tree is a tool to be used under defined
circumstances – it is not absolute
Phylogenetic relationships offer useful information
Phylogenetic relationships often match antigenic relationships,
particularly with influenza viruses
Phylogenetic relationships can not predict evolutionary pathways ,
specific mutations or likelihood of animal viruses becoming human
viruses
E P I D E M I C
Laboratory Training for Field Epidemiologists
A L E R T
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Typing
Developed by the Department of Epidemic and Pandemic Alert
and Response of the World Health Organization with assistance
from:
European Program for Intervention
Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
E P I D E M I C
Laboratory Training for Field Epidemiologists
A L E R T
A N D
R E S P O N S E