Typing Sequencing and Phylogeny May 2007 Laboratory Training

Typing Sequencing and Phylogeny May 2007 Laboratory Training for Field Epidemiologists

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

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 Laboratory Training for Field Epidemiologists

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 Laboratory Training for Field Epidemiologists

Evaluating organism similarity …AGCUAAAGCUA… Gene sequence #1 …AACUAAACCUG… Gene sequence #2 …AACUCCACCUA… Gene sequence #3 …GGUUGCACCGG… Gene sequence #4 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… 73% identity 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 Laboratory Training for Field Epidemiologists

Sequence #1 Sequence #2 Sequence #3 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 Laboratory Training for Field Epidemiologists

Molecular Phylogeny of H 5 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 430, 209 -213 (8 July 2004) Genesis of a highly pathogenic and potentially pandemic H 5 N 1 influenza virus in eastern Asia K. S. Li 1, Y. Guan 1, 2, J. Wang 1, 2, G. J. D. Smith 1, 2, K. M. Xu 1, 2, L. Duan 1, 2, A. P. Rahardjo 3, P. Puthavathana 4, C. Buranathai 5, T. D. Nguyen 6, A. T. S. Estoepangestie 3, A. Chaisingh 5, P. Auewarakul 4, H. T. Long 7, N. T. H. Hanh 7, R. J. Webby 8, L. L. M. Poon 2, H. Chen 1, 2, K. F. Shortridge 1, 2, K. Y. Yuen 2, R. G. Webster 2, 8 and J. S. M. Peiris 1, 2 Laboratory Training for Field Epidemiologists

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 Laboratory Training for Field Epidemiologists

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 Laboratory Training for Field Epidemiologists