Research Note— Evolutionary Analysis of HA and NS1 Genes of H5N1 Influenza Viruses in 2004–2005 Epidemics Massimo Ciccozzi, Stefania Montieri, Marzia Facchini, Giovanni Rezza, Isabella Donatelli, and Laura Campitelli A Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita `, Viale Regina Elena, 299, Rome 00161, Italy Received 26 April 2006; Accepted 21 June 2006 SUMMARY. H5N1 ?1 avian influenza viruses circulating in early 2004 in eastern Asia appeared to be under strong purifying selection, except for the hemagglutinin (HA) and nonstructural 1 (NS1) genes, where few amino acid positions were found under positive selection pressure. To evaluate whether the widespread circulation of the H5N1 viruses in the following years was accompanied by a change in the evolution of the HA and NS1, phylogenetic and positive selection analyses were performed on 89 HA1 ?2 and 57 NS1 sequences. Results showed that the number of HA positively selected sites decreased compared to 2004; no selection pressure for NS1 was found. These findings suggest a possible change in the adaptation of the H5N1 virus to birds. RESUMEN. Ana ´lisis de la evolucio ´n de los genes HA y NS1 de los virus H5N1 en las epidemias de los an ˜os 2004–2005. Los virus de influenza aviar H5N1 circulando en Asia oriental durante la primera parte del an ˜o 2004, mostraron estar bajo una fuerte seleccio ´n purificadora (negativa), excepto por los genes de la hemaglutinina (por sus siglas en Ingle ´s HA) y el gen no estructural 1 (por sus siglas en Ingle ´s NS1), en los cuales se hallaron algunas posiciones de aminoa ´cidos bajo presio ´n positiva de seleccio ´n. Con la finalidad de evaluar si la amplia circulacio ´n de los virus H5N1 en los an ˜os consecutivos estuvo acompan ˜ada de cambios evolutivos de los genes HA y NS1, se realizaron ana ´lisis filogene ´ticos y de presio ´n positiva en 89 secuencias de HA y 57 secuencias de NS1. Los resultados mostraron una disminucio ´n en el nu ´mero de posiciones bajo seleccio ´n positiva en el gen HA, en comparacio ´n con los resultados del an ˜o 2004. No se encontro ´ presio ´n de seleccio ´n para el gen NS1. Estos hallazgos sugieren un posible cambio en la adaptacio ´n del virus H5N1 a las aves. Key words: avian influenza, H5N1, virus evolution, bioinformatics Abbreviations: HA ¼ hemagglutinin; LTR ¼ likelihood ratio test; ML ¼ maximum likelihood; NS1 ¼ nonstructural protein 1 Starting in 2004, an epidemic of a highly pathogenic avian influenza A H5N1 virus infection among domestic poultry affected 27 countries worldwide, causing human disease in 185 cases, 104 of whom died (10,15). This was the largest outbreak since an H5N1 influenza epidemic in birds, causing multiple bird-to-human trans- missions, was first identified in 1997 in Hong Kong (2). Most human cases represented rare events of direct transmission from infected domestic birds, whereas only one instance of probable human-to-human transmission has been documented so far (11,13). To become efficiently transmitted from human to human, a zoonotic virus must overcome a series of obstacles (i.e., impairment of viral entry and/or viral replication, inhibitory immune response, etc.) which may lead interspecies passage to a dead end (14). To evaluate the possible occurrence of adaptive changes that could favor interspecies transmission of avian influenza, monitoring muta- tion events among poultry and humans is essential. Reassortment events that involved all but the hemagglutinin (HA) gene and gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the recent Asian epidemics have been fully iden- tified (8), whereas there is no clear-cut evidence about the occurrence and the role of stepwise mutations. To contribute to the compre- hension of viral evolution, in a previous study we evaluated H5N1 sequences from birds and humans infected between the 1997 and 2004 outbreaks. Our results indicated clearly that, despite an overall conservative evolution of the genes, a small but increasing number of specific amino acid positions have been under positive selection pressure since 2001 in the HA gene, whereas the nonstructural 1 (NS1) gene segment appeared to be positively selected in viruses circulating in 2004 (1). To evaluate whether the widespread circulation of the H5N1 viruses in the following years ?3 was accompanied by a change in the evolution of the HA and NS1 genes, phylogenetic and positive selection analyses of these genes were performed. MATERIALS AND METHODS All sequences analyzed in this study were downloaded from the Influenza Sequence Database and the Influenza Virus Resource on the National Center for Biotechnology Information web site. After ex- cluding identical sequences from viruses isolated in the same outbreaks, 88 HA and 57 NS1 sequences were selected ( ?9 Tables 1 and 2) ?10 and analyzed. Phylogenetic and positive selection analyses were performed to compare the two coding regions of H5N1 isolates. Separate maximum likelihood (ML) phylogenies were estimated for each gene region. The best-fitting nucleotide substitution models were chosen with the hierarchical likelihood ratio test (LTR) strategy, as implemented in the Modeltest program (12). The Hasegawa–Kishino–Yano þ À model, selected for both genes, assumes different nucleotide frequencies, a transition–transversion bias, and heterogeneity of substitution rates over sites modeled by a À-distribution (4). ML trees were then inferred with the best-fitting model and ML- estimated substitution parameters with a heuristic search performed A Corresponding author. AVIAN DISEASES 50:000–000, 2006