Different names have been used in publications to describe emerging lineages of the highly pathogeni

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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 [phone] or
3004 Fax [phone] or 4498 E-mail
perduem_at_who.int
Towards a Unified Nomenclature System for the
Highly Pathogenic H5N1 Avian Influenza
Viruses WHO/OIE/FAO H5N1 Evolution Working Group
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Results 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., lt 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 Clade descriptions 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)
Introduction 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
genotypes. 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.
2.3.4
2.3.4
2.3.3
2.3.3
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2.3.2
100
2.3.2
2.3.1
2.3.1
2.4
100
2.2
2.2
100
2.5
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2.5
Methodology Sequence alignments 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 Phylogenetic
trees 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). Smal
l 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.
2.4
2.1.3
Conclusions The results support the hypothesis
that the HPAI H5N1 viruses can be grouped into
several clades designated by a numbering system
that can continue to be expanded as the virus
continues to evolve. Establishes a system and
guidelines to name/number existing clades of
highly pathogenic H5N1 avian influenza, including
criteria for assigning a new clade name/number to
a newly emerging group of variants. Universal
acceptance of this nomenclature system will -
unify the classification of isolates so that
interpretation of sequence/surveillance data from
different laboratories becomes easier -
remove stigmatizing labelling of clades by
geographical reference - provide a system for
future expansion of all-inclusive phylogenetic
trees that can be used to follow the global
patterns of viral evolution - provide a starting
point for a more extensive nomenclature system
that can be used to follow the ever-changing
antigenic variation of H5N1 and the emergence of
novel genotypes from reassortment of the viral
internal genes - may 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 Large and small trees containing
publicly available sequences will be posted on
the WHO GIP website and OFFLU website and
maintained as up-to-date (evergreen)
evolutionary trees of the H5 hemagglutinin to
keep an open forum for following H5N1
evolution - WHO GIP website -
http//www.who.int/csr/disease/influenza/en -
OFFLU website www.offlu.net
2.1.3
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2.1.2
2.1.1
2.1.2
100
100
2.1.1
1
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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) - gt60
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
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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 Collaborators 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
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