We proposed two new mathematically proper distance metrics based on the lengths of the chords constructed from unit vectors and on proportions of the sample correlation function of unit vectors to replace the pseudo-distance in the dynamical language approach [5] . The results showed improvements with this replacement from a biological perspective. These results confirm their usefulness in phylogenetic analysis. A Single Immunization with Soluble Recombinant Trimeric Hemagglutinin Protects Chickens against Highly Pathogenic Avian Influenza Virus H5N1 BACKGROUND: The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. In addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. Effective vaccination against HPAI H5N1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sH5(3)) produced in mammalian cells. The secreted, purified sH5(3) was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. It was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with H5N1 after a single vaccination. Protected animals did not shed challenge virus as determined by a quantitative RT-PCR on RNA isolated from trachea and cloaca swabs. Also in mice, vaccination with sH5(3) provided complete protection against challenge with HPAI H5N1. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that sH5(3) constitutes an attractive vaccine antigen for protection of chickens and mammals against HPAI H5N1. As these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses. Influenza A viruses are enveloped, negative-strand RNA viruses with a segmented genome. They infect a large variety of animal species, although birds are considered to constitute the reservoir from which all influenza A viruses in other species originate [1, 2] . On the basis of the antigenic properties of their two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are classified into 16 HA (H1-16) and 9 NA (N1-9) subtypes. The highly pathogenic avian influenza (HPAI) virus H5N1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. HPAI H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. It is widely accepted that continued human exposure to influenza viruses circulating in wild and domestic avian species poses a permanent pandemic threat [3, 4] . Effective vaccination against HPAI H5N1 would protect com-mercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird as well as birdto-human transmission. Therefore, the development of efficacious influenza vaccines is of high veterinary and public health importance.