Contact information: Michael L. Perdue, Ph.D. WHO Global Influenza Programme Dept. of Epidemic and Pandemic Alert and Response 1211 Geneva 27, Switzerland Tel: +41 22 791 4935 or 3004; Fax: +41 22 791 4878 or 4498; E-mail: [email protected]
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Contact information: Michael L. Perdue, Ph.D. WHO Global Influenza Programme
Dept. of Epidemic and Pandemic Alert and Response 1211 Geneva 27, Switzerland
Tel: +41 22 791 4935 or 3004;
Fax: +41 22 791 4878 or 4498; E-mail: [email protected]
Towards a Unified Nomenclature System for the Highly Pathogenic H5N1 Avian Influenza Viruses
WHO/OIE/FAO H5N1 Evolution Working Group
9 unique clades defined from analysis of publicly available H5N1 sequences.
Clade 2 is highly divergent with 5 subclades within the clade (2.1-2.5).
- subclades 2.1 and 2.3 further delineated into sub-subclades due to distinct
evolution of more than one lineage
Clade topology nearly identical regardless of phylogenetic analysis/algorithm used (i.e., NJ, ML, MP algorithms produce nearly identical trees)
Some clade topology lost if partial HA sequences are used (i.e., < 800 ntds.)
- partial HA sequences appropriate for checking clade designation
- ideal alignments should have nearly compete HA sequence
Isolates in the small tree match the clade designations of the large tree
- overall tree topology from big tree to small tree varies because fewer isolates
were used in the small tree, which distorts the hierarchical structure of the tree
- despite this, all clade designations remain the same
0 = early progenitors; predominately 1996-2002 from Hong Kong (HK) and China (mostly avian, few human)
3 = 2000-2001 from HK, China, Vietnam (all avian)
4 = 2002/2003 lineage from HK and China; 2005/2006 from Guiyang Prov. (all avian)
5 = 2000-2003 from China and Vietnam; 2004 lineage from Guangxi Province (all avian)
6 = 2002/2004 from China (all avian)
7 = 2002/2004 from China; 2005/2006 from Yunnan, Hebei, Shanxi Provinces (all avian)
8 = 2001-2004 from HK and China (all avian)
9 = 2003-2005 from China (all avian)
1 = 2002/2003 progenitors from HK; 2003-2006 from Vietnam, Cambodia, Thai, Laos, Malaysia (mixed A/H)
2.1 = 2003-2007 from Indonesia (mixed avian/human)
2.2 = 2005 progenitors from Qinghai Lake outbreak and Mongolia; 2005-2007 isolates from
Eastern and Western Europe, the Middle East, and Africa (mixed avian/human)
2.3 = 2003-2006 from China, HK, Vietnam, Thailand, Laos, and Malaysia (mixed avian/human)
2.4 = 2002-2005 from China (predominately Yunnan and Guangxi Provinces) (all avian)
2.5 = 2003/2004 from Korea, Japan, China; 2006 lineage from Shantou Prov. (all avian)
Different names have been used in publications to describe emerging lineages of the highly pathogenic avian influenza A (H5N1) viruses. This has made discussion and comparison of the various lineages difficult. The H5N1 viruses have now appeared in at least 53 countries on three continents and continue to infect humans as well as to evolve and diversify at an alarming rate. Remarkably, the hemagglutinin protein has not been replaced in the various isolates since 1996, while other genes have undergone reassortment yielding many different genotypes. As such, following the evolution of the HA provides an initial constant by which the strains may be effectively compared. It was proposed to develop a clade nomenclature system based upon the evolution of the HA for several reasons:
1. To unify the system so that interpretation of sequence/surveillance data from different labs becomes easier.
2. To remove stigmatizing labelling of clades by geographical reference.
3. To provide for easy future expansion of the phylogenetic tree.
4. To provide a starting point for a more extensive system to follow based upon antigenic variation and reassortment into multiple
An international core group of 8 scientists and their collaborators were convened to initiate this process with the encouragement and approval of three international agencies: World Health Organization (WHO), World Animal Health Organization (OIE) and the Food and Agriculture Organization (FAO). Phylogenetic analysis was performed by a variety of approaches on all of the publicly available H5 HA sequences that have evolved in the A/Goose/Guangdong/96-like H5N1 lineage. The initial results support the idea that the currently circulating HPAI H5N1 viruses could be effectively grouped into numerous clades easily designated by a hierarchical numbering system. For example, the 'Fujian-like lineage' within the antigenically diverse Clade 2 of H5N1 would be designated Clade 2.3.4, with other distinct branches called 2.3.1 and 2.3.2 etc.., while the 'Qinghai lineage' would be designated Clade 2.2. If such a system could become universally accepted, it might serve as a model for other influenza lineages and genes, including seasonal influenza, which currently uses geographical names, and other avian influenza viruses that infect humans. The progress of the international working group and collaborators will be provided in this late breaker presentation.
Nucleotide sequences of the highly pathogenic H5N1 hemagglutinin (HA) (only nearly complete sequences) were collected from publicly available databases: GenBank (NCBI) and Influenza Sequence Database of Los Alamos National Laboratories (LANL)
Large alignment consisted of 884 HA sequences each approximately 1,659 nucleotides
- identical sequences/redundant isolates were removed from the alignment to remove bias
Small alignment consisted of 109 HA sequences each approximately 1,659 nucleotides
- isolates chosen include vaccine strains, reference strains, many human isolates,
pathogenesis study strains, geographically diverse isolates
Neighbor-joining trees were generated using MEGA (Version 3.1) and PAUP (Version 4.0) using the Kimura 2-parameter.
Large tree rooted to highly pathogenic historical Eurasian H5 isolates (turkey/England/91 and chicken/Scotland/59).
Small tree rooted at the clade 0 node (gs/Guangdong/1/96 lineage).
1000 bootstrap replicates were performed to support tree topology.
Maximum likelihood and maximum parsimony trees also generated to confirm clade topology.
Clade designation criteria
1) Maintain previously designated clade numbers when possible (i.e., Clade 2.2 remains 2.2 and Clade 1 remains 1)
2) New clade designations based on phylogenetic tree topology derived from the large tree
- H5N1 progenitors (closest to gs/Guangdong/1/96) designated as Clade 0
- Subsequent clades numbered starting from Clade 3 (i.e., Clades 3-9)
- Clades designated by the presence of a distinct common node shared by at least 4 isolates
- Subclades/sub-subclades designated as a single clade evolves into more than
one distinct lineage (based on sharing of a common node)
3) Average percentage pairwise distances between and within clades (using the Kimura 2-parameter):
- Distinct clades should have ≥ 1.5% average distances between other clades
- Distinct clades should have ≤ 1.5% average distances within the clade (may be
slightly higher in clades that have highly evolved outliers (i.e., ck/Shanxi/2/06
in Clade 7)
4) Bootstrap support for clade defining node (based on 1000 replicates)
- >60 at clade defining node
5) Antigenic properties as measured by the hemagglutination inhibition assay should be used as a correlate of
clade designation when the data is available
WHO/OIE/FAO H5N1 Evolution Working Group participants
Mike Purdue, WHO, GIP, Geneva, Switzerland*
Ian Brown, VLA-Weybridge, UK
Hualan Chen, Harbin Veterinary Research Institute, CAAS, China
Ruben Donis, Influenza Division, CDC, Atlanta, GA USA
Ron A.M. Fouchier, Erasmus University, Netherlands
Yoshi Kawaoka, U of Wisconsin, USA & Inst of Medical Sciences Tokyo, Japan
John Mackenzie, John Curtin School of Medicine, Australia
Gavin Smith, The University of Hong Kong, HK SAR, China
Yuelong Shu, China CDC, China
Ilaria Capua, IZSVE, Padova, Italy
Nancy Cox, Influenza Division, CDC, , Atlanta, GA USA
Todd Davis, Influenza Division, CDC, Atlanta, GA USA
Rebecca Garten, Influenza Division, CDC, Atlanta, GA USA
Yi Guan, The University of Hong Kong, HK SAR, China
Elizabeth Mumford, WHO, GIP, Geneva, Switzerland
Collin A. Russell, Department of Zoology, University of Cambridge, UK
Catherine Smith, Influenza Division, CDC, Atlanta, USA
Derek Smith, Department of Zoology, University of Cambridge, UK
Dhanasekaran Vijaykrishna, The University of Hong Kong, HK SAR, China