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.
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 [
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 (
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
[
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).
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 [
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 [
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 (
Pathogenicity in
mallards was determined as described previously [
The pathogenicity
of the two falcon H5N1 isolates in mammals was determined in female 6-week-old
BALB/c mice. Groups of mice (
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. [
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 Table
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 to | ||||||||
---|---|---|---|---|---|---|---|---|
Clade | Virus | rgHK213 | rgVN1203 | CkPak | FaSA | FaKW | JweHK | dkLao25 |
1 | rgHK213 | 320 | 1280 | 640 | 160 | 640 | 80 | |
1 | rgVN1203 | < | 40 | 80 | < | < | < | |
2.2 | CkPak | < | < | 160 | 640 | < | < | |
2.2 | FaSA | < | < | 160 | 160 | < | < | |
2.2 | FaKW | < | < | 320 | 40 | 40 | < | |
2.3 | JweHK | 160 | 80 | 80 | 40 | 40 | 640 | |
2.3 | dkLao25 | < | < | < | 40 | < | < |
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 [
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 [
Amantadine-resistant
influenza A variants carry amino acid substitutions at residues 26, 27, 30, 31,
or 34 of the M2 protein [
Amino acid (aa) residues associated with virus sensitivity and resistance to antiviral drugs.
Protein | M2 | NA | |||||||
---|---|---|---|---|---|---|---|---|---|
aa position | |||||||||
Sensitive | L | V | A | S | G | E | H | R | N |
Resistant | F | A/T/S/G | V/T/S | N/G | E | V/G/A/D | Y | K | S |
Fa/SA/05 | L | V | A | S | G | E | H | R | N |
Fa/KW/07 | L | V | A | S | G | E | H | R | N |
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 Figure
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 Figure
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.
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
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 (
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
Mean (±SE) tracheal
and cloacal virus titers in mallard ducks inoculated with Fa/SA/05 and Fa/KW/07
viruses. Ducks (
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 [
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 [
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 [
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 Table
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 Figure
The Saudi Arabian
and Kuwaiti falcon H5N1 viruses were replicated in multiple mouse organs,
particularly brain and lungs, without prior adaptation (see Figure
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. [
This work was supported in part by Grants A195357, A157570, and CA21765 and Contract no. HHSN266200700005C from the U.S. Department of Health and Human Services and by the American Lebanese Syrian Associated Charities (ALSAC). The authors gratefully acknowledge editorial assistance of Sharon Naron, the administrative assistance of James Knowles, the technical support of Natasha Lyushina and Jacco Boon, and the contributions of the Hartwell Center for Bioinformatics & Biotechnology and the Animal Resources Center at St. Jude Children’s Research Hospital.