1. Laboratory Training for Field Epidemiologists Typing May 2007 Sequencing and Phylogeny

2. Laboratory Training for Field Epidemiologists Learning objectives At the end of the presentation, participants should: Understand sequencing Interpret phylogenetic trees

3. Laboratory Training for Field EpidemiologistsSequencing 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

4. Laboratory Training for Field Epidemiologists Review of molecular biology …AGCUAAAGCUA… Nucleotides code for amino acids (codons) Genes – DNA and RNA are made of nucleotides …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

5. Laboratory Training for Field Epidemiologists Evaluating organism similarity …AGCUAAAGCUA… Gene sequence #1 …AACUAAACCUG… Gene sequence #2 …AACUCCACCUA… Gene sequence #3 …GGUUGCACCGG… Gene sequence #4

6. Laboratory Training for Field Epidemiologists 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… #2…AACUAAACCUG… #1…AGCUAAAGCUA… #2…AACUAAACCUG… #1…AGCUAAAGCUA… #2…AACUAAACCUG… #1…AGCUAAAGCUA… #2…AACUAAACCUG… 73% identity

7. Laboratory Training for Field Epidemiologists 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

8. Sequence #1 Sequence #2 Sequence #3 Sequence #4 Unrooted phylogenetic tree by unweighted pair group method Sequence #4 Sequence #3 Sequence #2Sequence #1 Distance in characters (nt/aa)

9. Laboratory Training for Field Epidemiologists Comparing genes to evaluate similarity among organisms …AGCUAAAGCUA… Gene sequence #1 …AACUAAACCUG… Gene sequence #2 …AACUCCACCUA… Gene sequence #3 …GGUUGCACCGG… Gene sequence #4 GG LysineGlutamate

10. Molecular Phylogeny of H5 Avian Influenza viruses (HA and M genes) Source: Suarez et. al. 2000: J. Virology 74: 6592-6599

11. Laboratory Training for Field Epidemiologists 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. Peiris1,21 21212121234563547 821212 812

12. Laboratory Training for Field EpidemiologistsConclusions 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

13. Laboratory Training for Field Epidemiologists 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 Typing