# Isolation of Highly Pathogenic Avian Influenza H5N1 Virus from Saker Falcons (Falco cherrug) in the Middle East

Authors: Henju Marjuki; Ulrich Wernery; Hui-Ling Yen; John Franks; Patrick Seiler; David Walker; Scott Krauss; Robert G. Webster
Journal: Advances in Virology
 (2009)

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## Abstract

There is accumulating evidence that birds of prey are susceptible to fatal infection with highly pathogenic avian influenza (HPAI) virus. We studied the antigenic, molecular, phylogenetic, and pathogenic properties of 2 HPAI H5N1 viruses isolated from dead falcons in Saudi Arabia and Kuwait in 2005 and 2007, respectively. Phylogenetic and antigenic analyses grouped both isolates in clade 2.2 (Qinghai-like viruses). However, the viruses appeared to have spread westward via different flyways. It remains unknown how these viruses spread so rapidly from Qinghai after the 2005 outbreak and how they were introduced into falcons in these two countries. The H5N1 outbreaks in the Middle East are believed by some to be mediated by wild migratory birds. However, sporting falcons may be at additional risk from the illegal import of live quail to feed them.


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## Body

## 1. Introduction

Infection of birds
of prey with highly pathogenic avian influenza (HPAI) has been reported only in isolated cases. In 2000, Manvel et al. reported the
discovery of HPAI H7N3 isolated from a peregrine falcon [1]. Another HPAI virus of the H7
subtype was found in a Saker falcon during an outbreak in poultry in Italy in
2000 [2]. Hong
Kong has had a series of cases. In January 2004, Hong Kong
authorities confirmed that a peregrine falcon had died of H5N1 infection [3, 4]. In March 2006, HPAI H5N1
infections were discovered in another peregrine falcon [5]. Most recently, in March 2008,
a wild peregrine falcon found sick was confirmed to be infected with H5N1 virus
[6]. In Belgium, 2 crested hawk eagles (Spizaetus nipalensis) smuggled from Thailand
for a commercial raptor breeding farm were discovered to be infected with HPAI
H5N1 virus and were killed by authorities [7].
Kuwait reported 39 confirmed cases of H5N1 infection in February 2007; 20
infected birds were falcons at a zoo and a farm in Southern Kuwait, and the
remainder were domestic birds caged outdoors [8, 9]. The first confirmed case of lethal
HPAI H5N1 infection in Saudi Arabia was reported in January 2006 in a Saker
falcon [8].  In November 2007, an H5N1 outbreak killed 1500
chickens at a farm in the Al-Kharj region of Saudi Arabia, about 150 kilometers
South of Riyadh [8]. No human cases have been
reported to date in Saudi Arabia or Kuwait.The increasing
number of falcons infected with HPAI H5N1 viruses demonstrates their
susceptibility to these pathogens. Because many falcon species are migratory or
occupy extensive territories, infected falcons may contribute to the spread of
avian influenza viruses within or between countries. The best-studied falcon species
are the peregrine falcon (Falco peregrinus) and the Saker falcon (Falco cherrug),
which feed exclusively on live, medium-sized birds, such as pigeons,
shorebirds, waterfowl,
and songbirds
[10, 11]. The susceptibility of some of
these prey species to HPAI H5N1 infection [12] increases the risk of virus
transmission to falcons. Peregrine falcons, which can fly and dive at high
speed, have long been used in the sport of falconry
in the Middle East. Although there is still no
direct evidence of virus transmission from falcons to humans, the practice of falconry
brings birds of prey into close contact with humans, thereby increasing the
risk of transmission to humans or to poultry.It is not known how
HPAI H5N1 virus spread so rapidly westward after the 2005 outbreak at Qinghai Lake,
Western China, or how falcons in the Middle East contracted HPAI H5N1 virus. To
acquire information that may answer these questions, we characterized two HPAI
H5N1 viruses isolated from dead female Saker falcons in the region: A/Falcon/Saudi
Arabia/D1795/2005 (Fa/SA/05) and
A/Falcon/Kuwait/D286/2007 (Fa/KW/07). The falcon that died in Saudi Arabia
was believed to have been in the country for the past 2 years, and it seems
highly likely that the falcon had been kept in captivity during the entire
period. The falcon had lost its appetite and was passing green feces during its
last 2 days of life. It was confirmed to have died of H5N1 avian influenza virus
[8]. Unfortunately, there is no
information available about the falcon found dead in Kuwait. Organs removed from the two
falcons were sent to the Central
Veterinary Research Laboratory in Dubai,
United Arab Emirates
(UAE), in November 2005 and February 2007, respectively.


## 2. Materials and Methods

### 2.1. Viruses and Cells

The H5N1 isolates
Fa/SA/05 and Fa/KW/07 were obtained from the Central Veterinary Research
Laboratory, Dubai, UAE, and stored in the influenza
virus repository of St. Jude Children’s Research Hospital.
All experiments were conducted in a USDA-approved biosafety level (BSL) 3+ containment facility. Stock viruses were
prepared by inoculating field samples into the allantoic cavities of 10-day-old
embryonated chicken eggs and incubating them at 35°C for 48 hours. Aliquots of stock viruses were stored at −80°C. Madin-Darby
canine kidney (MDCK) cells were obtained
from the American Type Culture Collection (Manassas, Va, USA) and maintained in Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum and 1% antibiotics (penicillin/streptomycin).


### 2.2. Infectivity

The 50% tissue
culture infectious dose (TCID50) was determined in MDCK cells after incubation at 37°C for 3 days, and the 50%
egg infectious dose (EID50) was determined in 10-day-old embryonated chicken eggs after 40 hours of
incubation at 35°C. TCID50 and EID50 values were
calculated by the method of Reed and Muench [13].


### 2.3. Sequencing and Phylogenetic Analysis of Influenza Virus Genes

Viral RNA was
isolated from virus-containing allantoic fluid with the RNeasy Mini kit
(Qiagen, Valencia, Calif, USA), and one-step RT-PCR (Qiagen) was done with a universal primer set [14]. PCR products were purified by
using the QIAquick PCR purification and gel extraction kits (Qiagen). Sequencing was performed by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude using BigDye Terminator (v.3;
Perkin Elmer Applied Biosystems, Foster
City, Calif, USA) chemistry and synthetic oligonucleotides.
Phylogenetic analysis was based on the HA and NA genes. Selected HA and NA
sequences from H5N1 viruses isolated after 1996/1997 were downloaded from the
Influenza Sequence Database at Los Alamos National Laboratory (http://www.flu.lanl.gov/) [15]. Sequences were compared by using
the ClustalW alignment algorithm with the BioEdit Sequence Alignment Editor. To
estimate phylogenetic relationships, nucleotide sequences were analyzed by the neighbor-joining
method with 100 bootstraps by using the phylogeny inference package (PHYLIP) version
3.65.


### 2.4. Pathogenicity in Chickens, Ducks, and Mice

All
animal studies were conducted under applicable laws and guidelines and after
approval from the St. Jude Animal Care and Use Committee. The pathogenicity of
the two falcon H5N1 isolates in chickens was determined in groups of 3 (Fa/SA/05, stock
virus concentration: 108.769 EID50/mL) or 10 (Fa/KW/07,
stock virus concentration: 109.231 EID50/mL) 6-week-old,
specific-pathogen-free white leghorn chickens (Gallus gallus domesticus) by intravenous injection with 0.1 mL of 10-fold
dilutions of virus-containing allantoic fluid. The chickens were
examined daily for 10 days for clinical signs of disease and for mortality. The
experiment was stopped after the birds found dead within 24 hours
postinoculation (p.i.).Pathogenicity in
mallards was determined as described previously [16].
Groups of five 5-week-old Mallard ducks (Anas platyrhynchos) were
inoculated with 106 EID50 of virus in a 1-mL volume (0.3 mL
each was applied to the trachea and the throat, and 0.2 mL each to nares and
eyes). All birds were observed daily for clinical signs and mortality over a
10-day period. Tracheal and cloacal swabs were collected every other day
starting on day 3 p.i., and viral titers (TCID50) were determined.The pathogenicity
of the two falcon H5N1 isolates in mammals was determined in female 6-week-old
BALB/c mice. Groups of mice (n=3) were
inoculated intranasally with 100 plaque-forming units (PFUs) of virus in 50μL of
PBS. Brain, lungs, liver, spleen, and blood were collected from 3 mice in each
group on days 3, 6, and 9 p.i. to evaluate organ tropism. Organs were
homogenized in 1 mL of PBS, and virus was titrated (log10 TCID50/mL)
in MDCK cells.


### 2.5. Hemagglutinin Inhibition (hi) Assay Using H5n1 Ferret Antisera

Ferrets obtained
through the ferret breeding program at St. Jude or from Marshall Farms (North
Rose, NY, USA)
were intranasally inoculated with 106 EID50 of virus in 1 mL of PBS under isoflurane anesthesia. Serum was collected on day 21 p.i. or 10
days after a boost dose of 1 mL of a 1 : 10 dilution of stock virus in PBS by
footpad injection or intranasal inoculation. The antisera were treated with
receptor-destroying enzyme and used for HI assay as previously described by
Palmer et al. [17].


## 2.1. Viruses and Cells

The H5N1 isolates
Fa/SA/05 and Fa/KW/07 were obtained from the Central Veterinary Research
Laboratory, Dubai, UAE, and stored in the influenza
virus repository of St. Jude Children’s Research Hospital.
All experiments were conducted in a USDA-approved biosafety level (BSL) 3+ containment facility. Stock viruses were
prepared by inoculating field samples into the allantoic cavities of 10-day-old
embryonated chicken eggs and incubating them at 35°C for 48 hours. Aliquots of stock viruses were stored at −80°C. Madin-Darby
canine kidney (MDCK) cells were obtained
from the American Type Culture Collection (Manassas, Va, USA) and maintained in Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum and 1% antibiotics (penicillin/streptomycin).


## 2.2. Infectivity

The 50% tissue
culture infectious dose (TCID50) was determined in MDCK cells after incubation at 37°C for 3 days, and the 50%
egg infectious dose (EID50) was determined in 10-day-old embryonated chicken eggs after 40 hours of
incubation at 35°C. TCID50 and EID50 values were
calculated by the method of Reed and Muench [13].


## 2.3. Sequencing and Phylogenetic Analysis of Influenza Virus Genes

Viral RNA was
isolated from virus-containing allantoic fluid with the RNeasy Mini kit
(Qiagen, Valencia, Calif, USA), and one-step RT-PCR (Qiagen) was done with a universal primer set [14]. PCR products were purified by
using the QIAquick PCR purification and gel extraction kits (Qiagen). Sequencing was performed by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude using BigDye Terminator (v.3;
Perkin Elmer Applied Biosystems, Foster
City, Calif, USA) chemistry and synthetic oligonucleotides.
Phylogenetic analysis was based on the HA and NA genes. Selected HA and NA
sequences from H5N1 viruses isolated after 1996/1997 were downloaded from the
Influenza Sequence Database at Los Alamos National Laboratory (http://www.flu.lanl.gov/) [15]. Sequences were compared by using
the ClustalW alignment algorithm with the BioEdit Sequence Alignment Editor. To
estimate phylogenetic relationships, nucleotide sequences were analyzed by the neighbor-joining
method with 100 bootstraps by using the phylogeny inference package (PHYLIP) version
3.65.


## 2.4. Pathogenicity in Chickens, Ducks, and Mice

All
animal studies were conducted under applicable laws and guidelines and after
approval from the St. Jude Animal Care and Use Committee. The pathogenicity of
the two falcon H5N1 isolates in chickens was determined in groups of 3 (Fa/SA/05, stock
virus concentration: 108.769 EID50/mL) or 10 (Fa/KW/07,
stock virus concentration: 109.231 EID50/mL) 6-week-old,
specific-pathogen-free white leghorn chickens (Gallus gallus domesticus) by intravenous injection with 0.1 mL of 10-fold
dilutions of virus-containing allantoic fluid. The chickens were
examined daily for 10 days for clinical signs of disease and for mortality. The
experiment was stopped after the birds found dead within 24 hours
postinoculation (p.i.).Pathogenicity in
mallards was determined as described previously [16].
Groups of five 5-week-old Mallard ducks (Anas platyrhynchos) were
inoculated with 106 EID50 of virus in a 1-mL volume (0.3 mL
each was applied to the trachea and the throat, and 0.2 mL each to nares and
eyes). All birds were observed daily for clinical signs and mortality over a
10-day period. Tracheal and cloacal swabs were collected every other day
starting on day 3 p.i., and viral titers (TCID50) were determined.The pathogenicity
of the two falcon H5N1 isolates in mammals was determined in female 6-week-old
BALB/c mice. Groups of mice (n=3) were
inoculated intranasally with 100 plaque-forming units (PFUs) of virus in 50μL of
PBS. Brain, lungs, liver, spleen, and blood were collected from 3 mice in each
group on days 3, 6, and 9 p.i. to evaluate organ tropism. Organs were
homogenized in 1 mL of PBS, and virus was titrated (log10 TCID50/mL)
in MDCK cells.


## 2.5. Hemagglutinin Inhibition (hi) Assay Using H5n1 Ferret Antisera

Ferrets obtained
through the ferret breeding program at St. Jude or from Marshall Farms (North
Rose, NY, USA)
were intranasally inoculated with 106 EID50 of virus in 1 mL of PBS under isoflurane anesthesia. Serum was collected on day 21 p.i. or 10
days after a boost dose of 1 mL of a 1 : 10 dilution of stock virus in PBS by
footpad injection or intranasal inoculation. The antisera were treated with
receptor-destroying enzyme and used for HI assay as previously described by
Palmer et al. [17].


## 3. Results

### 3.1. Antigenic Characterization

We assessed the
relation of the falcon H5N1 influenza viruses to other H5N1 viruses by hemagglutinin
inhibition (HI) assay with a panel of
reference sera collected from ferrets challenged with various H5N1 isolates
representing clades 1, 2.2, and 2.3. The two falcon H5N1 isolates had slightly
different reactivity patterns (see Table1). Both viruses had relatively high HI
titers (160 for Fa/SA/05 and 320 for Fa/KW/07) against A/chicken/Pakistan (Lahore)/NARC2411.A/06
(clade 2.2) antiserum, suggesting that they
are closely related to the H5N1 virus from that region and to viruses of clade
2.2. The Fa/KW/07 virus was not highly reactive with antisera generated against
Fa/SA/05, suggesting that the two isolates differ antigenically despite their
common origin.Table 1
Antigenic analysis of Fa/SA/05 and Fa/KW/07 hemagglutinin using ferret polyclonal antisera. <: HI antibody titer less than 40. The HI titer for the homologous virus and antiserum is indicated by
bold/underline. Viruses: HK213 (A/Hong Kong/213/03),
VN1203 (A/Vietnam/1203/04), CkPak (A/Chicken/Pakistan(Lahore)/NARC/3320/4/06), FaSA (A/Falcon/Saudi Arabia/D1795/05), FaKW (A/Falcon/Kuwait/D286/06), JweHK (A/Japanese White
Eye/Hong Kong/1038/06), and dkLao25 (A/Duck/Laos/25/06).
HI titer against ferret antiserum toCladeVirusrgHK213rgVN1203CkPakFaSAFaKWJweHKdkLao251rgHK213320̲3201280640160640801rgVN1203<160̲4080<<<2.2CkPak<<320̲160640<<2.2FaSA<<160160̲160<<2.2FaKW<<32040320̲40<2.3JweHK16080804040320̲6402.3dkLao25<<<40<<160̲


### 3.2. Molecular Characterization

There were 4 amino
acid differences in their HA proteins at positions 12, 178, 512, and 553. The amino acid at position 178 is located close to the HA receptor binding sites (H5
HA numbering). The HA proteins of both viruses contained multiple
basic amino acids at the connecting peptide between HA1 and HA2 (PQGERRRKKR at positions 321–330), which is a characteristic
of influenza viruses that are highly pathogenic in chickens [18, 19].
Interestingly, the HA cleavage site of the two falcon H5N1 viruses differed in
a single basic amino acid (R>G) from
that of their ancestor viruses Gs/Gd/1/96 and Ck/HK/220/97 and from that of H5N1
viruses isolated in 2004 in Thailand,
Indonesia, Vietnam, and Eastern China (PQR̲ERRRKKR).We also analyzed
the amino acids of the polymerase genes. A change at position 627 of the PB2 protein
from glutamic acid (E) to lysine (K) was required for the high virulence of the 1997
H5N1 viruses in mice [20, 21]
and for that of H7N7 viruses in humans [22].
Sequence analysis revealed a lysine at that position in both isolates, suggesting
that both are lethal to mice. The PB1 and PA proteins of the two H5N1 isolates
possessed tyrosine at position 436 and threonine at position 515. In contrast, the
T515>A amino acid change in PA and the Y436>H change in PB1 were previously
shown to abrogate the pathogenicity of H5N1 constructs in ducks, and the
Y436>H mutation in PB1 reduced transmission efficiency in ducks. The
T515>A mutation in PA eliminated virus lethality in ducks but not in mice
and ferrets [16].


### 3.3. Potential Sensitivity to Antiviral Treatment

Amantadine-resistant
influenza A variants carry amino acid substitutions at residues 26, 27, 30, 31,
or 34 of the M2 protein [23, 24].
No substitutions at these residues were identified by sequence analysis in either
H5N1 virus (see Table 2), suggesting that both are potentially sensitive to M2 inhibitors.
Mutations in conserved NA framework or catalytic residues (amino
acid residues 119, 274, 292, and 294, N2 numbering) may reduce
virus sensitivity to NA inhibitors [25]. No substitutions were
observed in the conserved NA residues of the studied H5N1 viruses (see Table 2),
suggesting that they may be sensitive to neuraminidase inhibitors.Table 2
Amino acid (aa) residues associated with virus sensitivity and resistance to antiviral drugs.
ProteinM2NAaa position2627303134119274292294SensitiveLVASGEHRNResistantFA/T/S/GV/T/SN/GEV/G/A/DYKSFa/SA/05LVASGEHRNFa/KW/07LVASGEHRN


### 3.4. Phylogenetic Analysis

The HA and NA
phylogenetic trees were rooted to the HA and NA genes of A/Chicken/Hong
Kong/220/97 virus. The HA gene of the Fa/SA/05 virus was phylogenetically
closely related to that of viruses isolated in 2006 from chickens in Israel and Gaza (clade 2.2), whereas the HA gene of Fa/KW/07 was
closely related to that of a virus isolated in 2007 from chickens in Russia
(clade 2.2) (see Figure1). On the basis of this
analysis, both were assigned to subclade 2 of clade 2 of H5 HA. The viruses clustered
phylogenetically with 2005–2007 isolates
from Asia, Africa, and the Middle East (see Figure
1).Figure 1
Phylogenetic relationships based on the HA (nucleotides 1–1684) genes of the
Fa/SA/05 and Fa/KW/07 viruses. The nucleotide sequences were analyzed by PHYLIP
3.65 software using the neighbor-joining method with 100 bootstraps. The tree
was rooted to the HA gene of A/Chicken/Hong Kong/220/97. Viruses in bold red
type were characterized in this study. Clades are indicated.The general
topology of the N1 tree differs from that of the HA tree because of differences
in stalk length. There is a 20-amino acid deletion in the stalk region (positions 49 to 68) in the current 2003–2007 and 2001-2002 China
poultry isolates. A 19-amino acid deletion (positions
50 to 68) was observed in the H5N1/97 Hong Kong human and
poultry isolates. Both of the falcon H5N1 viruses had a 20-amino acid deletion
in the stalk region of NA. The NA gene of the Fa/SA/05 virus was
phylogenetically closely related to that of A/chicken/Egypt/1129N3-HK9/2007 (clade 2.2), whereas the NA gene of Fa/KW/07 was
phylogenetically closely related to that of A/chicken/Afghanistan/1207/2006 (clade 2.2) (see Figure2).Figure 2
Phylogenetic relationships based on the NA (nucleotides–1388) genes of Fa/SA/05
and Fa/KW/07 viruses. The nucleotide sequences were analyzed by PHYLIP3.65
software using the neighbor-joining method with 100 bootstraps. The NA
phylogenetic tree was rooted to the NA gene of A/Chicken/Hong Kong/220/97.
Viruses in bold red type were characterized in this study. Clades are indicated.


### 3.5. Organ Tropism in A Mouse Model

To determine the
pathogenicity of the two falcon H5N1 isolates in a mammalian host, we
inoculated BALB/c mice intranasally and measured virus replication in the
brain, lungs, liver, spleen, and blood. Inoculation with 100 PFU of Fa/KW/07 virus
resulted in high virus titers (mean 107.35 TCID50/mL) in
the lungs on day 3 p.i.; titers had declined slightly on days 6 and 9 p.i. (see
Figure 3). Virus titers of Fa/SA/05 virus were approximately 1 log lower than
those of Fa/KW/07 on all days studied. On day 6 p.i., the mean Fa/KW/07 virus titer
in the brain was 105.07 TCID50/mL and that of Fa/SA/05 was
103.02 TCID50/mL (see Figure 3). Virus was detected in
the spleens of mice inoculated with Fa/KW/07 virus only 3 and 6 days p.i. All
mice inoculated with either falcon H5N1 virus died by day 9 p.i.Figure 3
Mean titers of Fa/SA/05 and Fa/KW/07 viruses in mouse organ
homogenates. Mice were inoculated with 100 PFU of each stock virus. Dashed
line indicates detection limit (10 TCID50/mL). BLD: below level of
detection. Error bars are SD obtained from mice (n=3) killed at various times.


### 3.6. Virulence and Pathogenicity in Avian Species

To determine
whether the two falcon H5N1 isolates retained pathogenicity for avian species,
we inoculated 6-week-old, specific-pathogen-free white leghorn chickens by intravenous injection. Both viruses
caused 100% mortality within 24 hours.We then inoculated
2 groups of 5 mallard ducks via natural routes (trachea,
throat, nares, and eyes) with 106 EID50 of Fa/SA/05 and Fa/KW/07 stock virus. All ducks survived during the 10-day
observation period. Clinical signs (cloudy eyes and
ataxia) were observed in 2 of 5 ducks on day 5 after
inoculation with Fa/KW/07. Fa/SA/05 caused only cloudy eyes in 1 of  5 ducks during days 5 to 7 p.i. Tracheal and
cloacal swabs were collected every other day starting on day 3 p.i. Both
viruses were shed from the trachea on day 3 p.i. (see Figure 4), and Fa/KW/07 virus
was detected in cloacal samples on day 3 p.i. No virus was detected in
specimens obtained on days 5, 7, and 10 p.i.Figure 4
Mean (±SE) tracheal
and cloacal virus titers in mallard ducks inoculated with Fa/SA/05 and Fa/KW/07
viruses. Ducks (n=5) were inoculated with 106 EID50 of each stock virus. Virus was tittered from tracheal and cloacal swabs
collected 3 days after inoculation to determine the TCID50. Dashed
line indicates detection limit (10 TCID50/mL). BLD: below level of
detection.


## 3.1. Antigenic Characterization

We assessed the
relation of the falcon H5N1 influenza viruses to other H5N1 viruses by hemagglutinin
inhibition (HI) assay with a panel of
reference sera collected from ferrets challenged with various H5N1 isolates
representing clades 1, 2.2, and 2.3. The two falcon H5N1 isolates had slightly
different reactivity patterns (see Table1). Both viruses had relatively high HI
titers (160 for Fa/SA/05 and 320 for Fa/KW/07) against A/chicken/Pakistan (Lahore)/NARC2411.A/06
(clade 2.2) antiserum, suggesting that they
are closely related to the H5N1 virus from that region and to viruses of clade
2.2. The Fa/KW/07 virus was not highly reactive with antisera generated against
Fa/SA/05, suggesting that the two isolates differ antigenically despite their
common origin.Table 1
Antigenic analysis of Fa/SA/05 and Fa/KW/07 hemagglutinin using ferret polyclonal antisera. <: HI antibody titer less than 40. The HI titer for the homologous virus and antiserum is indicated by
bold/underline. Viruses: HK213 (A/Hong Kong/213/03),
VN1203 (A/Vietnam/1203/04), CkPak (A/Chicken/Pakistan(Lahore)/NARC/3320/4/06), FaSA (A/Falcon/Saudi Arabia/D1795/05), FaKW (A/Falcon/Kuwait/D286/06), JweHK (A/Japanese White
Eye/Hong Kong/1038/06), and dkLao25 (A/Duck/Laos/25/06).
HI titer against ferret antiserum toCladeVirusrgHK213rgVN1203CkPakFaSAFaKWJweHKdkLao251rgHK213320̲3201280640160640801rgVN1203<160̲4080<<<2.2CkPak<<320̲160640<<2.2FaSA<<160160̲160<<2.2FaKW<<32040320̲40<2.3JweHK16080804040320̲6402.3dkLao25<<<40<<160̲


## 3.2. Molecular Characterization

There were 4 amino
acid differences in their HA proteins at positions 12, 178, 512, and 553. The amino acid at position 178 is located close to the HA receptor binding sites (H5
HA numbering). The HA proteins of both viruses contained multiple
basic amino acids at the connecting peptide between HA1 and HA2 (PQGERRRKKR at positions 321–330), which is a characteristic
of influenza viruses that are highly pathogenic in chickens [18, 19].
Interestingly, the HA cleavage site of the two falcon H5N1 viruses differed in
a single basic amino acid (R>G) from
that of their ancestor viruses Gs/Gd/1/96 and Ck/HK/220/97 and from that of H5N1
viruses isolated in 2004 in Thailand,
Indonesia, Vietnam, and Eastern China (PQR̲ERRRKKR).We also analyzed
the amino acids of the polymerase genes. A change at position 627 of the PB2 protein
from glutamic acid (E) to lysine (K) was required for the high virulence of the 1997
H5N1 viruses in mice [20, 21]
and for that of H7N7 viruses in humans [22].
Sequence analysis revealed a lysine at that position in both isolates, suggesting
that both are lethal to mice. The PB1 and PA proteins of the two H5N1 isolates
possessed tyrosine at position 436 and threonine at position 515. In contrast, the
T515>A amino acid change in PA and the Y436>H change in PB1 were previously
shown to abrogate the pathogenicity of H5N1 constructs in ducks, and the
Y436>H mutation in PB1 reduced transmission efficiency in ducks. The
T515>A mutation in PA eliminated virus lethality in ducks but not in mice
and ferrets [16].


## 3.3. Potential Sensitivity to Antiviral Treatment

Amantadine-resistant
influenza A variants carry amino acid substitutions at residues 26, 27, 30, 31,
or 34 of the M2 protein [23, 24].
No substitutions at these residues were identified by sequence analysis in either
H5N1 virus (see Table 2), suggesting that both are potentially sensitive to M2 inhibitors.
Mutations in conserved NA framework or catalytic residues (amino
acid residues 119, 274, 292, and 294, N2 numbering) may reduce
virus sensitivity to NA inhibitors [25]. No substitutions were
observed in the conserved NA residues of the studied H5N1 viruses (see Table 2),
suggesting that they may be sensitive to neuraminidase inhibitors.Table 2
Amino acid (aa) residues associated with virus sensitivity and resistance to antiviral drugs.
ProteinM2NAaa position2627303134119274292294SensitiveLVASGEHRNResistantFA/T/S/GV/T/SN/GEV/G/A/DYKSFa/SA/05LVASGEHRNFa/KW/07LVASGEHRN


## 3.4. Phylogenetic Analysis

The HA and NA
phylogenetic trees were rooted to the HA and NA genes of A/Chicken/Hong
Kong/220/97 virus. The HA gene of the Fa/SA/05 virus was phylogenetically
closely related to that of viruses isolated in 2006 from chickens in Israel and Gaza (clade 2.2), whereas the HA gene of Fa/KW/07 was
closely related to that of a virus isolated in 2007 from chickens in Russia
(clade 2.2) (see Figure1). On the basis of this
analysis, both were assigned to subclade 2 of clade 2 of H5 HA. The viruses clustered
phylogenetically with 2005–2007 isolates
from Asia, Africa, and the Middle East (see Figure
1).Figure 1
Phylogenetic relationships based on the HA (nucleotides 1–1684) genes of the
Fa/SA/05 and Fa/KW/07 viruses. The nucleotide sequences were analyzed by PHYLIP
3.65 software using the neighbor-joining method with 100 bootstraps. The tree
was rooted to the HA gene of A/Chicken/Hong Kong/220/97. Viruses in bold red
type were characterized in this study. Clades are indicated.The general
topology of the N1 tree differs from that of the HA tree because of differences
in stalk length. There is a 20-amino acid deletion in the stalk region (positions 49 to 68) in the current 2003–2007 and 2001-2002 China
poultry isolates. A 19-amino acid deletion (positions
50 to 68) was observed in the H5N1/97 Hong Kong human and
poultry isolates. Both of the falcon H5N1 viruses had a 20-amino acid deletion
in the stalk region of NA. The NA gene of the Fa/SA/05 virus was
phylogenetically closely related to that of A/chicken/Egypt/1129N3-HK9/2007 (clade 2.2), whereas the NA gene of Fa/KW/07 was
phylogenetically closely related to that of A/chicken/Afghanistan/1207/2006 (clade 2.2) (see Figure2).Figure 2
Phylogenetic relationships based on the NA (nucleotides–1388) genes of Fa/SA/05
and Fa/KW/07 viruses. The nucleotide sequences were analyzed by PHYLIP3.65
software using the neighbor-joining method with 100 bootstraps. The NA
phylogenetic tree was rooted to the NA gene of A/Chicken/Hong Kong/220/97.
Viruses in bold red type were characterized in this study. Clades are indicated.


## 3.5. Organ Tropism in A Mouse Model

To determine the
pathogenicity of the two falcon H5N1 isolates in a mammalian host, we
inoculated BALB/c mice intranasally and measured virus replication in the
brain, lungs, liver, spleen, and blood. Inoculation with 100 PFU of Fa/KW/07 virus
resulted in high virus titers (mean 107.35 TCID50/mL) in
the lungs on day 3 p.i.; titers had declined slightly on days 6 and 9 p.i. (see
Figure 3). Virus titers of Fa/SA/05 virus were approximately 1 log lower than
those of Fa/KW/07 on all days studied. On day 6 p.i., the mean Fa/KW/07 virus titer
in the brain was 105.07 TCID50/mL and that of Fa/SA/05 was
103.02 TCID50/mL (see Figure 3). Virus was detected in
the spleens of mice inoculated with Fa/KW/07 virus only 3 and 6 days p.i. All
mice inoculated with either falcon H5N1 virus died by day 9 p.i.Figure 3
Mean titers of Fa/SA/05 and Fa/KW/07 viruses in mouse organ
homogenates. Mice were inoculated with 100 PFU of each stock virus. Dashed
line indicates detection limit (10 TCID50/mL). BLD: below level of
detection. Error bars are SD obtained from mice (n=3) killed at various times.


## 3.6. Virulence and Pathogenicity in Avian Species

To determine
whether the two falcon H5N1 isolates retained pathogenicity for avian species,
we inoculated 6-week-old, specific-pathogen-free white leghorn chickens by intravenous injection. Both viruses
caused 100% mortality within 24 hours.We then inoculated
2 groups of 5 mallard ducks via natural routes (trachea,
throat, nares, and eyes) with 106 EID50 of Fa/SA/05 and Fa/KW/07 stock virus. All ducks survived during the 10-day
observation period. Clinical signs (cloudy eyes and
ataxia) were observed in 2 of 5 ducks on day 5 after
inoculation with Fa/KW/07. Fa/SA/05 caused only cloudy eyes in 1 of  5 ducks during days 5 to 7 p.i. Tracheal and
cloacal swabs were collected every other day starting on day 3 p.i. Both
viruses were shed from the trachea on day 3 p.i. (see Figure 4), and Fa/KW/07 virus
was detected in cloacal samples on day 3 p.i. No virus was detected in
specimens obtained on days 5, 7, and 10 p.i.Figure 4
Mean (±SE) tracheal
and cloacal virus titers in mallard ducks inoculated with Fa/SA/05 and Fa/KW/07
viruses. Ducks (n=5) were inoculated with 106 EID50 of each stock virus. Virus was tittered from tracheal and cloacal swabs
collected 3 days after inoculation to determine the TCID50. Dashed
line indicates detection limit (10 TCID50/mL). BLD: below level of
detection.


## 4. Discussion

Accumulating data
demonstrate that falcons are extremely susceptible to HPAI H5N1 infection. We
investigated the molecular, phylogenetic, antigenic, and pathogenic properties
of 2 H5N1 viruses isolated from dead falcons in the Middle
East. Phylogenetic and antigenic analyses indicated that both viruses
originated from Qinghai-like H5N1 viruses that caused a massive die-off among
wild birds in China
in 2005. However, it is still not clear how these viruses were introduced into the
region, and how the falcons in the affected countries contracted the HPAI H5N1
virus.There are several
possibilities that may explain the introduction of these viruses. Since no
cases of H5N1 infection were reported in the Middle East before 2005, the first
possibility is that the virus was introduced by wild birds migrating westward
after the 2005 outbreak in China.
The virus rapidly spread westward through various regions, including the Middle
East [26], and subsequently spread to
domestic poultry and probably to other avian species, including falcons.A second
possibility is that the virus was introduced by infected falcons illegally
imported into the Middle East from China,
Mongolia, or Russia.
Thousands of wild-caught falcons are traded on the black market
withoutveterinary inspection, licensing from the Convention on
International Trade in Endangered Species of Wild Fauna and Flora, or
border controls [27]. Illegal trading of falcons
can contribute to the global spread and transmission of HPAI viruses, and
therefore export/import activity should be strictly regulated. As an example,
falcons brought into the UAE must be quarantined for 21 days; blood and nasal
mucus are tested during the first and second weeks for H5N1 virus [28]. These extraordinary security
measures have so far prevented any cases of HPAI H5N1 infection in falcons in
the UAE.A third
possibility is that the falcons were infected via diseased pigeons and/or
quail, which are their normal food source. To feed hunting falcons, tens of
thousands of live quail areflown from the Arabian Gulf into royal
falconry camps across Central Asia without veterinary inspection [27]. Many falcons in North
America, Germany, and the Persian Gulf area have died after feeding on birds (especially pigeons and quail) and other prey infected
with avian influenza viruses [29]. Therefore, falcons in
captivity should be fed only birds that have been inspected by a veterinarian.The phylogenetic
tree of H5 HA revealed that although the two falcon H5N1 viruses originated
from the same ancestor viruses (clade 2.2, Qinghai-like),
they are likely to have spread westward via different flyways. Because the HA
of Fa/SA/05 shares strong sequence similarity with the HAs of 2006 chicken
isolates from Gaza and Israel, this virus appears to have spread from Qinghai
Lake toward the African continent via a “southern” flyway. In contrast, the HA
of Fa/KW/07 is closely related to that of a 2007 chicken isolate from Russia,
suggesting that the Kuwaiti isolate was carried toward Europe via a “northern”
flyway. The two viruses showed slightly different reactivity patterns with
antisera from ferrets inoculated with various H5N1 viruses. The fact that the Fa/KW/07
virus was poorly reactive with antisera is generated against the Fa/SA/05 virus
(see Table1), suggesting that it may have undergone minor antigenic drift in
its HA gene. In addition, the fourfold difference between the homologous and
heterologous titer also suggests only minor antigenic differences. Therefore, based
on this observation, an indication of different origin of isolates should be predicated
on the HA phylogenetic analysis rather than on the slight antigenic differences
of the two falcon isolates. Furthermore, sequence analysis revealed a 20-amino
acid deletion in the NA stalks of both viruses; this feature is a characteristic
of chicken-adapted influenza viruses.Interestingly, although
both falcon H5N1 isolates were uniformly lethal to chickens, their replication was
limited in mallard ducks. Only about one third of inoculated ducks showed
clinical signs of illness, and virus was shed only on day 3 p.i. (see Figure4).
This low pathogenicity may allow ducks to passively carry and shed these and
other HPAI H5N1 viruses.The Saudi Arabian
and Kuwaiti falcon H5N1 viruses were replicated in multiple mouse organs,
particularly brain and lungs, without prior adaptation (see Figure3). H5N1 influenza
virus lethality and spread to the brain in mice usually require multiple
passages for adaptation [30].
The high lethality of these isolates in mice is probably related to the K627 residue
of the PB2 protein [20, 21].
Interestingly, the PB2s of all H5N1 viruses isolated from humans and those of
all avian H5N1 isolates from the recent outbreaks in Qinghai Lake (Western China),
Russia, and Nigeria, possess lysine (K) at
position 627 [21, 31],
whereas the PB2s of their progenitor avian viruses contain glutamic acid (E) at this position.All subtypes of
influenza A virus have the potential to become pandemic strains, but the
currently circulating HPAI H5N1 viruses are of greatest concern. Our study
provides the first molecular and biological insights into the HPAI H5N1 viruses
isolated from dead falcons in the Middle East.
This information will be useful in determining which measures should be taken
to provide effective surveillance and protection of these birds in the affected
countries and around the globe. During the preparation of this manuscript,
Lierz et al. [31] reported that falcons
survived HPAI H5N1 infection after vaccination with inactivated H5N2 virus. The
vaccinated birds did shed virus but at reduced titers. This approach may
provide a partial solution to the protection of falcons in captivity.



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*Source: 294520-2008-11-24.xml*