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H5N1 Clade 2.2 Polymorphism Tracing Identifies Influenza Recombination and

Potential Vaccine Targets

Niman HL*, Saad MD

, Boynton BR

, Tjaden, J

,Earhart KC

, Mansour MM

Elsayed NM

, Nayel AE

, Abdelghani AS

, Esmat HM

, Labib EM

, Ayoub EA

Aly MM

, Arafa A-SA

, Monteville, MR

*Recombinomics, Inc., Pittsburgh, Pennsylvania, USA,

U.S. Naval Medical

Research Unit 3 (NAMRU-3), Cairo, EGY,

Central Laboratory for Veterinary

Quality Control, Giza, EGY,

Ministry of Health, Arabic Republic of Egypt, Cairo,

EGY.

Department of Influenza Recombination, Recombinomics, Inc, 648 Field Club
Road, Pittsburgh, PA 15238, USA

Correspondence to Henry L Niman, E-mail:

henry_niman@recombinomics.com

617.877.0987, 412.963.1362 FAX

Text word count: 1656
2 Figures
Running Title: H5N1 Polymorphism Tracing
Submitted to Proceedings of Options for the Control of Influenza VI

June 17-23, 2007 Toronto, Ontario, Canada

Nature Precedings : doi:10.1038/npre.2007.553.2 : Posted 7 Aug 2007

Highly pathogenic Influenza A H5N1 was first identified in Guangdong Province
in 1996, followed by human cases in Hong Kong in 1997

. The number of

confirmed human cases now exceeds 300 and the associated Case Fatality Rate
exceeds 60%

. The genetic diversity of the serotype continues to increase. Four

distinct clades or sub-clades have been linked to human cases

3.4

. The gradual

genetic changes identified in the sub-clades have been attributed to copy errors
by viral encoded polymerases that lack an editing function, thereby resulting in
antigenic drift

. We traced polymorphism acquisition in Clade 2.2 sequences. We

report here the concurrent acquisition of the same polymorphism by multiple,
genetically distinct, Clade 2.2 sub-clades in Egypt, Russia and Ghana. These
changes are not easily explained by the current theory of "random mutation"
through copy error, and are more easily explained by recombination with a
common source. This conclusion is supported by additional polymorphisms
shared by Clade 2.2 isolates in Egypt, Nigeria and Germany including
aggregation of regional polymorphisms from each of these areas into a single
Nigerian human hemagglutinin gene.

The study of influenza evolution in nature has been aided by the emergence of a
new strain (Clade 2.2) first identified at Qinghai Lake in central China in the
spring of 2005. Sequencing of all eight genes

6,7

showed that isolates from

migratory waterfowl were easily distinguishable from previous isolates linked to
poultry and human infections in eastern and southeastern Asia. The new strain
was subsequently found in outbreaks in Russia, Kazakhstan and Mongolia

Prior to these Clade 2.2 outbreaks the highly pathogenic Asian version of H5N1
had never been reported west of China. The H5N1 detection in migratory
waterfowl in summer 2005 in Russian and Mongolian migratory bird sanctuaries
signaled the start of a major geographical expansion of H5N1. In the following 12
months, almost 50 countries west of China reported H5N1 for the first time. All
infections were Clade 2.2. The expanded geographical reach included Europe,
the Middle East and Africa.

This expansion offered a unique opportunity to study the evolution of H5N1 as it
migrated into new regions, including human cases in Turkey, Iraq, Azerbaijan,
Egypt, Djibouti in 2006 and Nigeria in 2007. Sequence analysis indicated all
cases were Clade 2.2. The outbreaks were due to multiple introductions and
isolates had regional specific polymorphisms.

We isolated H5N1 from patients and poultry in Egypt. The first poultry isolates
were collected February 2006. The first human case developed symptoms in
March 2006. Analysis of the H5N1 isolates collected between February and May
2006 defined a series of HA and NA regional markers. These markers were also
found in the human case from Djibouti and poultry isolates from Israel and Gaza.

Nature Precedings : doi:10.1038/npre.2007.553.2 : Posted 7 Aug 2007

After a lull in reported infections over the summer, H5N1 re-emerged in Egypt in
September 2006. The more recent isolates had the same regional markers seen
the previous season. However, the more recent poultry and human isolates had
acquired a series of new polymorphisms. Non-synonymous polymorphisms were
identified in samples collected from a cluster of three family members from the
Gharbiya governorate (Nile Delta). HA gene polymorphisms were identified in or
near the receptor binding domain, including V223I and M230I, as well as the
oseltamivir resistance polymorphism, N294S (NA gene). The patients first
developed symptoms in December 2006 and all three infections were fatal (a
detailed report on patients and polymorphism tracing will be described
elsewhere).

Additional cases in early 2007 included HA sequences with a 3 BP deletion of the
nucleotides encoding Ser at position 133 (H3 Numbering), and a case with a
novel HA cleavage site, REGRRRKR. The changes were found in multiple
patients in central and southern Egypt. The above non-synonymous changes
were associated with additional synonymous and non-synonymous changes in
the HA and NA sequences. However, these isolates from the 2006/2007 season
maintained the regional markers seen in early 2006 in isolates from Egypt,
Djibouti, Israel and Gaza.

Chicken isolates from Gharbiya samples collected on February 15, 2007 included
one sequence that was closely related to the sequences from the human
Gharbiya cluster. The sequence, A/chicken/1892N3-HK49/2007, had the regional
markers previously seen in the 2006 and 2007 isolates, as well as HA non-
synonymous changes, V223I and M230I. Additionally, an NA synonymous
polymorphism, G743A, was appended onto the genetic background of the human
Gharbiya cluster, as seen in an NA cladogram (data not shown). This
polymorphism was found in two additional chicken isolates, A/chicken/1890N3-
HK45/2007 and A/chicken/1891N3-CLEVB/2007, collected the same day in the
Gharbiya governorate, but these two isolates fell onto a separate branch of the
tree.

The G743A was subsequently found in human isolates from patients who
developed symptoms in April 2007. Included were siblings with HA sequences
that had the 3 BP deletion seen in earlier patients from central Egypt. Like the
chicken sequences above, the G743A polymorphism was appended onto
sequences identified earlier in Egypt. Similarly, distinct sequences from another
patient, A/Egypt/2630-NAMRU3/2007, that acquired G743A, also fell onto a
separate branch

The distinct branches displayed in the NA cladogram were also seen in the HA
cladogram (data not shown). The isolates with G743A are also located at the
tips of the branches, supporting a recent polymorphism acquisition.

Nature Precedings : doi:10.1038/npre.2007.553.2 : Posted 7 Aug 2007

In February 2007, an H5N1 Clade 2.2 outbreak occurred near Moscow,
Russia. Isolates from infected chickens were most closely related to 2006
sequences from Azerbaijan. Figure 1 is an expanded NA cladogram with isolates
from Europe, the Middle East and sub-Saharan Africa.

Figure 1 NA phylogram of Clade 2.2 isolates.
NA phylogram of positions 43-1337. Isolates with G743A marked either with red
arrows or red bars.

Like the acquisitions in Egypt, the isolates with G743A mapped onto the tip of a
branch composed of earlier isolates that did not have the acquisition.

Similarly, in April 2007, an H5N1 clade 2.2 outbreak occurred near Tema,
Ghana. Sequences from three chickens were most closely related to turkey
isolates collected in December 2006 in the Ivory Coast. Like the Egypt and
Moscow isolates above, the recent isolates with G743A mapped to the tip of a
branch containing earlier isolates that did not have the polymorphism.

G743A

Nature Precedings : doi:10.1038/npre.2007.553.2 : Posted 7 Aug 2007

Additional HA polymorphisms are noted in the HA phylogram (data not shown).
Isolates that had the NA polymorphism, G743A, also had a synonymous HA
polymorphism, C689T. This polymorphism was also in human and bird isolates
from the Nile Delta. Another polymorphism, G754A, that encodes M230I, is in
one of the German isolates

, A/eagle owl/Germany/R166/2006, and maps to

another branch with Egyptian human and poultry isolates from the Nile Delta. A
third polymorphism, C1614T, that encodes T517I, is in another German isolate,
A/mute swan/Germany/R797/2006, and another branch with human isolates from
southern Egypt. The isolates also have the novel HA cleavage site initially found
in whooper swan isolates in Mongolia (2005). The polymorphisms found in
German isolates in 2006 were in Russian Clade 2.2 isolates in 2005.

The NA G743A polymorphism can be traced through public H5N1 sequences

The polymorphism was identified in the first reported sequences linked to the
spread of H5N1 in Asia in 2003/2004 in South Korea and Japan. The
polymorphism was subsequently identified in Clade 1 isolates in southeast Asia,
as well as Clade 2.1 isolates in Indonesia and Clade 2.3 isolates in China. The
first reported Clade 2.2 isolates were in wild birds in Germany collected in
February 2006. The isolates in Germany formed distinct HA and NA branches
due to a series of regional markers.

The first human isolate from Nigeria had several markers that were regionally
distributed in the 2006 Clade 2.2 isolates. These shared HA polymorphisms are
labeled in the HA cladogram (Figure 2).

Nature Precedings : doi:10.1038/npre.2007.553.2 : Posted 7 Aug 2007

Figure 2 HA Phylogram of H5N1 isolates showing polymorphisms shared with
Nigerian isolate.

HA phylogram of positions 93-1688. A/Nigeria/6e/2007 shared polymorphisms
annotated with colored arrows and bars.

One of the Nigerian human polymorphisms, T937C is an Egyptian regional
marker. A/Nigeria/6e/2007 also has T610C matching a small subset of the
Egyptian isolates. G881A is in another small Egyptian subset, and C980T is in
isolates from Egypt and Nigeria. Similarly, the Nigerian isolate has two regional
markers from Germany, G295A and C1480T, and another marker, A778G, found
in one of the German bird isolates, A/great crested grebe/Germany/R1226/2006.
The pattern continues in this same Nigerian isolate with the inclusion of sub-
Sahara African regional markers, A433G and G643A, as well as another marker
from a subset of the Sudan isolates, A1006G. Finally, G1658A from A/whooper
swan/Mongolia/7/2006 appears in A/Nigeria/6e/2007. Hence the demonstration
shows that the Nigerian isolate has recently aggregated H5N1 Clade 2.2
sequence information, including twelve Clade 2.2 SNPs, from a minimum of three
geographically distinct areas. The cobbling together of this series of 2006
regional markers from multiple, geographically distinct locations into a single
sequence is most easily explained by recombination.

C610T

T937C

C289T

G295A

C1480T

A1006G

A433G

G643A

G1708A

Nature Precedings : doi:10.1038/npre.2007.553.2 : Posted 7 Aug 2007

Similarly, the concurrent acquisition of the same polymorphism by multiple Clade
2.2 sub-clades challenges the current theory of influenza evolution that invokes
random mutations as a mechanism for the generation of antigenic drift. The
isolates with the newly acquired polymorphisms map to the tips of the branches
of the phylogenetic trees, indicating recent acquisition. All referenced isolates on
the tips of the branches were collected over a short time frame between February
and April 2007 and in three geographically distinct regions. These data do not
support a common progenitor sequence because the most closely related
sequences to each of the respective recent isolates do not have this
change. Similarly, concurrent mutation / selection by eleven isolates that map to
six branches in three countries and were collected over a short time frame is also
unlikely.

An alternative explanation for this concurrent acquisition of G743A in multiple
Clade 2.2 sub-clades and for the Nigerian isolate aggregating previously
dispersed regional markers is homologous recombination between closely
related sequences. Polymorphism tracing demonstrates that most of the newly
acquired polymorphisms can be traced to the same serotype identified recently at
locations that are linked together by migratory bird flyways, raising the possibility
that the distribution and acquisition of the polymorphism is linked to
recombination between H5N1 sequences transmitted and transported by
migratory birds.

The individual polymorphisms recombine to generate sequences that create
antigenic drift. Mapping of these pathways and associations can be used to
develop novel vaccine targets representing rapidly evolving genomes.

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Nature Precedings : doi:10.1038/npre.2007.553.2 : Posted 7 Aug 2007