Phylodynamics of H5N1 avian influenza virus in Indonesia TOMMY TSAN-YUK LAM* 1 , CHUNG-CHAU HON*, PHILIPPE LEMEY†, OLIVER G. PYBUS‡, MANG SHI*, HEIN MIN TUN*, JUN LI*, JINGWEI JIANG*, EDWARD C. HOLMES§– and FREDERICK CHI-CHING LEUNG* *School of Biological Sciences, The University of Hong Kong, Hong Kong, China, †Laboratory for Clinical and Epidemiological Virology, REGA Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium, ‡Department of Zoology, University of Oxford, Oxford, UK, §Centre for Infectious Disease Dynamics, Pennsylvania State University, USA, –Fogarty International Center, National Institutes of Health, Bethesda, MD 20892, USA Abstract Understanding how pathogens invade and become established in novel host populations is central to the ecology and evolution of infectious disease. Influenza viruses provide unique opportunities to study these processes in nature because of their rapid evolution, extensive surveillance, large data sets and propensity to jump species boundaries. H5N1 highly pathogenic avian influenza virus (HPAIV) is a major animal pathogen and public health threat. The virus is of particular importance in Indonesia, causing severe outbreaks among poultry and sporadic human infections since 2003. However, little is known about how H5N1 HPAIV emerged and established in Indonesia. To address these questions, we analysed Indonesian H5N1 HPAIV gene sequences isolated during 2003– 2007. We find that the virus originated from a single introduction into East Java between November 2002 and October 2003. This invasion was characterized by an initially rapid burst of viral genetic diversity followed by a steady rate of lineage replacement and the maintenance of genetic diversity. Several antigenic sites in the haemagglutinin gene were subject to positive selection during the early phase, suggesting that host-immune- driven selection played a role in host adaptation and expansion. Phylogeographic analyses show that after the initial invasion of H5N1, genetic variants moved both eastwards and westwards across Java, possibly involving long-distance transportation by humans. The phylodynamics we uncover share similarities with other recently studied viral invasions, thereby shedding light on the ecological and evolutionary processes that determine disease emergence in a new geographical region. Keywords: phylodynamics, influenza, evolution, ecology, phylogeography, agriculture Received 22 September 2011; revision received 2 February 2012; accepted 8 February 2012 Introduction The ecological and evolutionary processes that deter- mine species invasion are inextricably linked. Much of our knowledge of these processes has come from a large body of theoretical work (Shigesada & Kawasaki 1997; Edmonds et al. 2004; Travis et al. 2007; Munk- emuller et al. 2011), and until recently, there were rela- tively few genetic studies in this area. Empirical investigations of animal or plant populations are chal- lenging because the comparatively short duration of study means that few mutations are expected to accu- mulate during the timescale of invasion events. RNA viruses are characterized by extremely high evolution- ary rates (in the range of 10 )4 to 10 )3 nucleotide substi- tutions per site per year; Duffy et al. 2008), such that they exhibit measurable molecular evolution within the time frame of human observation (Holmes 2008) and Correspondence: Frederick Chi-Ching Leung, 5N-12, Kadoorie Biological Science Building, The University of Hong Kong, Pokfulam Road, Hong Kong, China. Fax: (852) 2857 4672; E-mail: fcleung@hkucc.hku.hk 1 Present address: Department of Zoology, University of Oxford, Oxford, UK. Ó 2012 Blackwell Publishing Ltd Molecular Ecology (2012) 21, 3062–3077 doi: 10.1111/j.1365-294X.2012.05577.x