Continuing progress towards a unified nomenclature system for the highly pathogenic H5N1 avian influenza viruses
Updated March 2009
Background and Objectives
In recent years, a variety of different names have been used to refer to emerging lineages of the currently circulating highly pathogenic avian influenza (HPAI) H5N1 viruses. As a result, discussion, comparison, and analysis of the various lineages proved difficult.
Avian H5N1 viruses continue to spread, continue to infect animals and humans, and continue to evolve and diversify. While most viral genes have been replaced through reassortment yielding many different genotypes, the specific H5 haemagglutinin (HA) gene identified in 1996, remarkably, has remained present in all isolates. Therefore, this H5 HA provides a constant to which the evolving strains may be effectively compared, and it was proposed to develop a standard clade nomenclature system based upon the evolution of this H5 HA. This nomenclature system would enable:
- a unified system to be developed to facilitate the interpretation of sequence/surveillance data from different laboratories;
- the labeling of clades by geographical reference to be replaced by a more representative system;
- the phylogenetic tree to be expanded in the future; and
- a starting point to be established to develop a more extensive system in the near future that takes into consideration antigenic variation and reassortment into multiple genotypes.
The Unified Nomenclature System and Virus Clades
An international core group of eight scientists and their collaborators were convened (as the WHO/OIE/FAO H5N1 Evolution Working Group) to initiate this process with the encouragement and approval of three international agencies: the World Health Organization (WHO), the World Organisation for Animal Health (OIE) and the Food and Agriculture Organization (FAO). Phylogenetic analysis was performed using a variety of approaches on all of the publicly available H5 HA sequences that have evolved from the A/goose/Guangdong/96 H5N1 virus. The initial results showed that the currently circulating H5N1 viruses could be effectively grouped into numerous virus "clades" based on the phylogenetic characterization and sequence homology of the HA gene.
To date, the unified H5N1 nomenclature system has identified 10 distinct initial clades of viruses (numbered 0-9) (WHO/OIE/FAO H5N1 Evolution Working Group, 2008), these are called first order clades. Clades are strictly defined as meeting the following three specific clade definition criteria developed by the WHO/OIE/FAO H5N1 Evolution Working Group:
- sharing of a common (clade-defining) node;
- monophyletic grouping with a bootstrap value of ≥60 at the clade-defining node (after 1000 neighbor-joining bootstrap replicates); and
- average percentage pairwise nucleotide distances between and within clades of >1.5% and <1.5%, respectively.
As the viruses within these 10 clades continue to evolve, new sublineages (potential H5N1 clades) periodically emerge. Once these sublineages meet the same three specific clade definition criteria as the initial clades (listed above), they are designated as separate clades (See Conceptual Diagram of ongoing H5N1 evolution). These new clades are defined as second (or third, etc) order clades and assigned a numerical 'address' which links them to their original clade using a hierarchical decimal numbering system. For example, within the antigenically distinct clade 2.3, third order clades meeting the clade definition were designated as clades 2.3.1 and 2.3.2 and so on. This logical hierarchal numbering system is objectively related to HA phylogeny, and thus removes place designations (e.g., the previously designated 'Fujian-like’ lineage became third order clade 2.3.4, while the 'Qinghai lineage' became second order clade 2.2). The H5N1 Evolution Working Group recommends that emerging sublineages of H5N1 not be classified using the hierarchal decimal numbering system until that sublineage has met all three of the above criteria for designation of a clade. However, the working group fully encourages the designation of newly emerging distinct sublineages of H5N1 virus by independent investigators using alternative, interim, naming conventions (e.g., using letters).
Ongoing circulation of H5N1 viruses in poultry and/or wild birds in Asia, the Middle East, Europe, and Africa leads to ongoing evolution and a continual increase in the cumulative number of isolates available for sequence analysis. Thus, the Evolution Working Group must examine the available data and update the associated phylogenetic trees and clade nomenclature on an ongoing basis. These updated trees (as well as archived trees for reference) will be available on this website (See new versions of full and small trees updated as of October 2008).
Recent divergence of clade 2.2 viruses
Although evolution and divergence continues, all of the viruses so far isolated from Europe, Africa, and the Middle East fall into clade 2.2. Phylogenetic analyses of these viruses are of particular importance due both to their wide geographical spread and their proven ability to cause human disease. Several recent independent reports in the scientific literature (Ducatez et al., 2007; Salzberg et al., 2007; Starick et al., 2007; Kiss et al., 2008) describe the evolution of clade 2.2 viruses and identify emerging sublineages using either novel naming conventions or modifications of the clade definition criteria proposed by the WHO/OIE/FAO H5N1 Evolution Working Group.
In order to understand how these new designations are related to the WHO Evolution Working Group H5 HA phylogenetic trees, an updated phylogenetic analysis of the HA of clade 2.2 viruses was performed using approximately 300 publicly available HA sequences. It was determined that one distinct group of viruses within clade 2.2 fits the three specific clade definition criteria described above, and has thus been designated as a new third order clade, named clade 2.2.1. These viruses were isolated primarily from Egypt, Israel, the Gaza Strip, Nigeria, and Europe in 2006 and 2007. One other sublineage of clade 2.2 (comprised of isolates from Europe, Russia, Mongolia, Korea, India, the Middle East, and Nigeria and marked in green in the 2.2 tree, figure 4 link to figure) has evolved extensively in recent years, and although it does not yet meet the clade designation criteria, it is likely to soon assuming further divergence occurs. The large remainder of the clade does not fit the criteria, and thus remains designated as simply clade 2.2.
Although many distinct clade 2.2 sublineages not meeting the clade definition criteria have emerged recently, they are not addressed here. However, the working group fully encourages designation of these sublineages by independent investigators using alternative, interim, naming conventions as suggested above. The emergence of additional third-order clades will be monitored as clade 2.2 viruses continue to evolve. Similarly, monitoring the emergence of new higher order clades among any of the 10 first order clades will continue.
Ducatez MF, Olinger CM, Owoade AA, Tarnagda Z, Tahita MC, Sow A, De Landtsheer S, Ammerlaan W, Ouedraogo JB, Osterhaus AD, Fouchier RA, Muller CP. 2007. Molecular and antigenic evolution and geographical spread of H5N1 highly pathogenic avian influenza viruses in western Africa. J Gen Virol. 88:2297-306.
Kiss I, Gyarmati P, Zohari S, Wilbe Ramsay K, Metreveli G, Weiss E, Brytting M, Stivers M, Lindström S, Lundkvist A, Nemirov K, Thorén P, Berg M, Czifra G, Belák S. 2008. Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006. Virology Journal 5:113.
Salzberg SL, Kingsford C, Cattoli G, Spiro DJ, Janies DA, Aly MM, Brown IH, Couacy-Hymann E, De Mia GM, Dung do H, Guercio A, Joannis T, Maken Ali AS, Osmani A, Padalino I, Saad MD, Savić V, Sengamalay NA, Yingst S, Zaborsky J, Zorman-Rojs O, Ghedin E, Capua I. 2007. Genome analysis linking recent European and African influenza (H5N1) viruses. Emerg Infect Dis. 5:713-8.
Starick E, Beer M, Hoffmann B, Staubach C, Werner O, Globig A, Strebelow G, Grund C, Durban M, Conraths FJ, Mettenleiter T, Harder T. 2008. Phylogenetic analyses of highly pathogenic avian influenza virus isolates from Germany in 2006 and 2007 suggest at least three separate introductions of H5N1 virus. Vet Microbiol. 128:243-52
World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization H5N1 Evolution Working Group. Toward a unified nomenclature system for highly pathogenic avian influenza virus (H5N1). Emerg Infect Dis. 2008 Jul. Available from http://www.cdc.gov/EID/content/14/7/e1.htm
World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization H5N1 Evolution Working Group. 2009. Letter to the Editor: Continuing progress towards a unified nomenclature for the highly pathogenic H5N1 avian influenza viruses: divergence of clade 2.2 viruses. Influenza and Other Respiratory Viruses 3(2)59-62. Available from http://www3.interscience.wiley.com/cgi-bin/fulltext/122211969/HTMLSTART
Technical Resources and Current Trees
Conceptual Diagram of ongoing H5N1 evolution
Full tree (January, 2009)
Small tree (January, 2009)
Clade 2.2 tree (January, 2009)
Letter to Editor regarding clade 2.2 divergence, ISIRV Journal, February 2009
H5N1 HA nucleotide sequence alignment - updated full tree
H5N1 HA nucleotide sequence alignment - updated small tree
WHO/OIE/FAO H5N1 Evolution Working Group, Poster presented at Options meeting, Toronto, 2007
Phylogenetic tree from Evolution Working Group poster (2007)
Small Phylogenetic tree from the Evolution Working Group Poster (2007)
H5N1 HA nucleotide sequence alignment - original big tree
H5N1 HA nucleotide sequence alignment - original small tree
For more information