Characterization of Avian H9N2 Influenza Viruses from UnitedArab Emirates 2000 to 2003

U. B. Aamira, Ulrich Werneryb, N. Ilyushinaa, and R. G. Webstera,*aDivision of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital,332 N. Lauderdale Street, Memphis, Tennessee 38105; uzma.bashir@stjude.org,natalia.ilyushina@stjude.orgbCentral Veterinary Research Laboratory, P.O. Box 597 Dubai, UAE cvrl@cvrl.ae

AbstractOur aim was to establish the phylogenetic relation of H9N2 avian viruses in the Middle East to otherAsian H9N2 lineages by characterization of 7 viruses isolated from United Arab Emirates(2000-2003). All these viruses had an additional basic amino acid at the hemagglutinin-connectingpeptide; 6 contained a mutation associated with increased affinity toward human-like sialic acidsubstrates. The viruses' surface glycoproteins and most internal genes were >90% similar to thoseof A/Quail/Hong Kong/G1/97 (H9N2) lineage. The hemadsorbing site of neuraminidase had up to4 amino acid substitutions, as do human pandemic viruses. M2 sequence analysis revealed aminoacid changes at 2 positions, with increasing resistance to amantadine in cell culture. They replicatedefficiently in inoculated chickens and were successfully transmitted to contacts. They continue tomaintain H5N1-like genes and may augment the spread of H5N1 viruses through regional co-circulation and inapparent infection. These viruses may present as potential pandemic candidatesthemselves.

KeywordsH9N2; United Arab Emirates; Hemadsorbing site; Receptor affinity; Amantadine resistance

IntroductionBirds and migratory waterfowl are natural reservoirs of avian influenza viruses (Kawaoka etal., 1988; Slemons et al., 1974). Currently, 16 hemagglutinin (HA) and 9 neuraminidase (NA)subtypes have been identified in avian species, but H3, H4, and H6 have been more frequentlyisolated in domestic and commercial poultry (Woolcock et al., 2003; Kaleta et al., 2005). Onlya few subtypes such as H5, H7, and H9 cause respiratory and systemic disease. During the lastcentury, strains either partially or entirely derived from avian influenza viruses have caused 4human influenza pandemics (Webster et al., 1992; Cameron et al., 2000; Guan et al., 2000;Shortridge et al., 1998).

*Corresponding author: Robert Webster, Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital,332 N. Lauderdale Street, Memphis, Tennessee 38105-2794. Phone: (901)495-3400. Fax: (901) 523-2622. Email: robert.webster @stjude.org.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

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Published in final edited form as:Virology. 2007 April 25; 361(1): 45–55. doi:10.1016/j.virol.2006.10.037.

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During the last decade, scientists have observed repeated episodes of direct transmission ofH5, H7, and H9 viruses from birds to humans (Wong, 2006; Horimoto & Kawaoka, 2005). Atpresent, they are primarily concerned by avian H5N1 influenza viruses, which have resultedin the deaths of 148 of 252 diagnosed humans cases since 1997 (CDC report.,1997; Chan.,2002; Tran et al., 2004; WHO Update on influenza October 3, 2006). However, theunprecedented spread of H5N1 across many countries underscores the importance ofcharacterizing the pandemic potential of other subtypes such as H9 and H7, because theseviruses have been successfully transmitted to humans in the past and have crossed the speciesbarrier to infect humans (Guo, 2002; Fouchier et al., 2004; Tweed et al., 2004).

Human infection with avian influenza viruses has underscored the continuing threat of a newpandemic virus with global health implications. H9N2 avian influenza viruses have beenenzootic in Asia since the early 1990s, and have been repeatedly isolated from terrestrial poultry(Guo et al., 2000). Since 1997, 2 distinct lineages of H9N2 viruses have become establishedin terrestrial poultry in Asia. They are represented by 2 prototypes: A/Duck/Hong Kong/Y280/97 (A/Dk/HK/Y280/97) and A/Quail/Hong Kong/G1/97 (A/Qa/HK/G1/97) (Guan et al.,1999; Guan et al., 2000; Butt et al., 2005). A/Qa/HK/G1/97 is thought to be the donor of 6“internal” genes to the poultry and human H5N1 viruses isolated in 1997 (Guan et al., 2000).

H9N2 viruses generally cause decreased egg production and mild to moderate infections, butthey can also cause heavy morbidity and mortality when the birds are co-infected with otheragents (Nili et al., 2002, 2003; Brown et al., 2006). Documented cases of human infection withH9N2 avian influenza viruses, first detected in 1999 in Hong Kong and China (Guo, 2002),indicate that these viruses can be directly transmitted from birds to humans. Previous studieshave documented the ability of H9N2 viruses to progressively acquire receptor and bindingcharacteristics that mirror those of the human pandemic influenza viruses (Lin et al., 2000;Matrosovich et al., 2001). The function of the hemagglutinating (HB) site on the neuraminidasesurface is unclear (Laver et al., 1984; Air et al., 1995). It is postulated to have a yet-undefinedrole in host-receptor interactions with the virions, and changes in the HB site are believed tobe important in host adaptation of a virus from avian to mammalian subtype (Webster et al.,1987; Varghese et al., 1997). In addition to altered HA receptor affinity, H9N2 viruses haveshown striking changes in the HB sites similar to pandemic human viruses. Furthermore, since2000, H9 viruses have shown increasing resistance to M2 channel blockers such as amantadinethat are used in the prevention and treatment of human influenza (Ilyushina et al; 2005). Thenearly ubiquitous nature of these viruses, coupled with their role in emergence of human H5N1viruses in 1997 (Guan et al, 1999; Lin et al, 2000), emphasizes the need to characterize andinvestigate H9N2 viruses circulating worldwide.

H9N2 viruses circulate widely in the Middle East, and are associated with serious disease inpoultry. In this study, we characterized 7 such viruses isolated from poultry in the United ArabEmirates between 2000 and 2003. The quail isolates caused egg production to decrease by70%, whereas the chicken isolates resulted in rapid mortality due to tracheitis and respiratorycongestion. We delineated the genomes, receptor-binding profile, and HB sites of these fieldisolates and determined their degree of pathogenicity in experimentally inoculated chickens.We also established their phylogenetic relationship to the other Asian H9N2 lineages.

ResultsDisease Signs in the Field

The H9N2 viruses we characterized had been isolated from disease outbreaks on quail andchicken farms in the United Arab Emirates. Outbreaks in quail did not produce any overtdisease signs, but egg production dropped precipitously to 30% accompanied by a 40% dropin food consumption. Outbreaks in chickens occurred annually from 2001 to 2003,

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accompanied by general congestion, severe tracheitis, and acute onset mortality. During the2002 outbreak, all chickens had cyanotic combs and substantial symptomatic respiratorydisease; mortality rose sharply (0.08% to 36%), resulting in considerable economic losses.

Antigenic AnalysisTo determine the antigenic properties of the UAE H9N2 virus isolates, we performedhemagglutination inhibition (HI) assays with polyclonal antisera and monoclonal antibodiesto the two main H9N2 subtypes circulating in Asia, A/Qa/HK/G1/97 and A/Dk/HK/Y280/97(Li et al., 2003). The viruses reacted equally well to polyclonal sera against both subtypes andanti-HK/1073/99. The chicken isolates reacted with 4 to 256-fold higher titers to monoclonalantibodies against A/Dk/HK/Y280/97 than did the quail isolates. These results (Table 1)demonstrate that the chicken isolates are more antigenically similar to A/Dk/HK/Y280/97 thanare the quail viruses. Neuraminidase inhibition assay showed these viruses to be of the N2subtype, reactive in antigenic analysis to α-Singapore/57 and α-ShoreBird/Delaware/1143/97N2 antisera and monoclonal antibodies. Minor antigenic differences were indicated by theresults of monoclonal antibody analysis.

Phylogenetic AnalysisTo determine the genetic diversity of these viruses, all 8 gene segments were partiallysequenced. The partial sequences were then aligned with different sequences from H9 viruseslisted in GenBank. The HA genes of these viruses were related to the Eurasian lineage of H9N2viruses including A/Qa/HK/G1/97 and the human H9N2 isolates A/HK/1073/99 and A/HK/1074/99. In these isolates, hemagglutinin and neuraminidase were most homologous to A/Parakeet/Chiba/1/97, an H9N2 virus of G1 lineage (Table 2). A/Chicken/United ArabEmirates/AG537/99, an earlier H9 isolate from the Middle East, was 99% similar, and itclustered with these newer isolates (Figure 1). The HA sequences of these viruses were verysimilar to those of A/Qa/HK/G1/97; 96% on nucleotide and 94% on amino acid levels. TheHA amino acid sequences showed 95% to 98% concordance with those of A/Parakeet/Chiba/1/97, another isolate from the G1 lineage. The neuraminidase genes of these viruses alsoclustered to the A/Qu/HK/G1/97 lineage, as did isolates from Iran, Saudi Arabia, and Pakistan.However, like other Middle Eastern isolates, these viruses lacked the 2-amino acid deletion inthe stalk region of residues 38 and 39, present in the NA genes of human and quail G1 viruses.

The phylogenetic analysis of the “internal” genes showed that M, NP, PB1, and PA were similarto those of the main G1-like lineage of H9N2 viruses. The matrix genes of these viruses werecloser to those of Middle Eastern and H6N1 isolates such as A/Teal/Hong Kong/W312/97, andthey formed a neighboring clade with A/Qa/HK/G1/97 and A/Dk/HK/Y280/97 lineages. TheirNP, PB1, and PA genes were also similar to those of the Middle Eastern lineage of H9N2viruses, and ultimately to those of the G1 lineage. Only NS and PB2 genes show evidence ofre-assortment with other viral subtypes. The NS genes, 96% to 97% similar to isolates such asA/Duck/Hong Kong/P54/97 (H11N9) and A/duck/Nanchang/1904/92 (H7N4) from theInfluenza Sequence Database, formed a distinct clade with the viruses of G1 and Y280 lineages(88%-91% similarity). The nucleotide sequences of these isolates' PB2 genes were 94% to 95%similar to those of A/Duck/Potsdam/2216-4/84 (H5N6), an isolate from Germany. Thephylogenetic analysis showed no evidence of further reassortment or emergence of newgenotypes within this subgroup of Dubai isolates. All the isolates were clustered in one groupfor all the analyzed genes.

Genetic Characteristics of Surface GlycoproteinsMatrosovich et al. (2001) recently described the human virus-like receptor specificity of avianH9N2 viruses from Asia. To analyze this characteristic of the Dubai isolates, we compared the

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HA amino acid sequences (deduced from the nucleotide sequences) of these viruses with thoseof the representative viruses.

HemagglutininSix of seven isolates carry amino acid substitution Q226L (H3 numbering throughout this text)in the receptor-binding site, a mutation that correlates with a shift in affinity of the HA fromthe “avian” type sialic receptors to the human type and from a preference for a 2′-3′ link to apreference for a 2′-6′ link between the sialic acid residues and galactose (Matrosovich et al.,2000). Receptor affinity was confirmed by solid-phase fetuin-binding inhibition assay. Wemeasured the affinity of these H9N2 viruses to high-molecular–weight sialic substrates, bothnatural (fetuin) and synthetic (3′- and 6′-sialyllactose attached to a polyacrylic carrier). Theaffinity to the 3′ substrate was negligible in all the viruses except one (A/Quail/Dubai/303/00)having the L226Q (H3 numbering) change (Table 3). This virus exhibited a higher affinity to3′-sialyllactose-acrylic polymer and an inability to bind sialyl receptors with a 2′-6′ galactoselinkage. In contrast, all H9N2 viruses with leucine at position 226 showed strong affinity top6′SL and fetuin. These viruses had 2 other AA substitutions, H183N (5/7 isolates) and E190T,which are also related to human virus-like receptor specificity. All of these isolates showedthe Arg-Ser-Ser-Arg (R-S-S-R) motif at the connecting peptide of their HA, which is a featureof H9 viruses of land-based poultry (Choi et al., 2004).

Analysis of the potential glycosylation sites in the HA of these viruses revealed 8 sites withthe NXT/S motif (X can be any amino acid except proline); 6 were in the HA1 portion, and 2were in the HA2 part of the molecule. The additional potential site in A/Qa/HK/G1/97 (residue87) was also present in these H9 isolates, but they lacked the Asn 188 potential glycosylationsite seen in A/Qa/HK/G1/97.

NeuraminidaseThe neuraminidase genes of these viruses showed similar glycosylation sites and shared 7amino acids with A/Qa/HK/G1/97 and such Pakistan isolates as A/Ck/Pakistan/2/99 thatdistinguish them from the N2s of other H9N2 viruses (Cameron et al., 2000). Theneuraminidase stalk regions in these viruses lacked the 2-residue deletion (38-39) present inthe NA of A/Qa/HK/G1/97 and the 2 human isolates A/HK/1073/99 and A/HK/1074/99.

HB Site of NeuraminidaseThis HB site is on the NA surface, away from the enzymatic site. Previous studies havedocumented the fact that 6 amino acid residues on 3 loops interact directly with sialic acid; 3serines, on loops carrying amino acids 367-372, Asn-400, Trp-403, and lys-432. These aminoacid positions have been well conserved in the N2 neuraminidase of various aquatic birdviruses. However, as seen in poultry H9N2 isolates from Hong Kong and China, amino acidswere substituted at the sialic acid contact region of the HB site sequences in the UAE viruses.These substitutions are similar to the ones detected previously (Matrosovich et al., 2000) inboth A/Qa/HK/G1/97 and A/Dk/HK/Y280/97 avian lineages, in human H9N2 isolates of 1999,in human pandemic H2N2, and in H3N2 viruses (data not shown).

Antiviral ResistanceThe analysis of M2 protein sequences revealed that 6 viruses carried a V27A substitution atposition 27 and one virus (A/Chicken/Dubai/463/03) possessed S31N at position 31. Thisresistance was tested quantitatively against amantadine, using serially increasingconcentrations of the drug in cell cultures. Correspondingly, all the Dubai isolates showed ahigh level of resistance to amantadine (IC50 ≥ 100μM) in cell culture.

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Pathogenicity in ChickensBecause these viruses had caused moderate disease in quail and severe morbidity and mortalityin chickens in the field, we examined their pathogenicity in chickens. Biological differencesbetween the quail and chicken isolates were clear; quail H9N2 isolates replicated in the infectedchickens, but failed to transmit efficiently. In contrast, the chicken H9N2 isolates replicated inthe inoculated chickens and were transmitted to contact birds. It is notable that none of theH9N2 viruses caused detectable disease signs in chickens. These viruses were comparable toA/Qa/HongKong/G1/97 and A/Dk/HongKong/Y280/97 in pathogenicity and in theirtransmission patterns in infected chickens (Table 4).

All the viruses were shed mainly from the respiratory tract; tracheal titers were ∼106.3 egginfectious doses (EID50)/0.1 ml in tracheal swabs on day 5 post-infection (p.i.). Virus titerswere present in tracheal swabs of the contact birds for the chicken isolates from day 3 post-contact. No virus was detected in most tracheal swabs on day 7 p.i. and only traces (101.5

EID50 /0.1 ml) detectable on day 10 p.i. Fewer virus titers were shed through the cloacal routethan through the trachea (titers of ∼102.7 EID50/0.1 ml in the cloacal swabs [Table 4]) in bothinfected and contact birds. Virus was mostly detected in the cloacal swabs of directly inoculatedbirds up to day 5 p.i. All infected and contact birds had undergone seroconversion by day 14p.i. for chicken isolates, but no serological response was noted in contact birds for quail isolates.

Because of their distinct 2′-6′ sialic acid receptor affinity, we tested the pathogenecity of someof these H9N2 isolates in 6-week-old female BALB-c mice after intranasal infection. Weobserved few signs of weight loss and morbidity in infected animals, although lung titers weresimilar to those observed for A/Qa/HongKong/G1/97 and A/Dk/HongKong/Y280/97(∼104.5on day 3 post-infection, data not shown). On days 3 and 5 p.i., no titers were detectedin samples from the brain, spleen, or blood of infected mice, and the inoculated mice stayedhealthy and gained weight during the observation period.

DiscussionBefore 1992, H9N2 viruses had been isolated only from Asian ducks (Shortridge, 1992).Subsequent surveillance studies identified a widespread presence of this subtype in Asiandomestic poultry as well (Mo et al., 1997; Naeem et al., 1999). The hemagglutinin genes ofH9N2 isolates from South Asia and Middle Eastern countries such as Pakistan, Iran, and SaudiArabia are closely related to the HA genes of the viruses that infected Hong Kong children in1999 (Cameron et al., 2000). The H9N2 viruses from the United Arab Emirates that we studiedcome from this same geographical region, and very few viruses from the region have beencharacterized thus far. Under field conditions, these isolates produced considerable problemsin affected birds such as quail and chickens, such as reduced egg production and high mortalitypreceded by respiratory signs. In this study, we characterized these viruses in detail andevaluated their pathogenicity in chickens.

The UAE isolates are a homogeneous group, cross-reactive to both A/Qa/HK/G1/97 and A/Dk/HK/Y280/97 antisera and monoclonal antibodies. Phylogenetic analysis indicated that thesurface glycoproteins of the UAE isolates were 96% homologous to those of the A/Qa/HK/G1/97 (G1) lineage of H9N2 viruses; in contrast, they were 89% to 90% similar to A/Dk/HK/Y280/97-like lineage. All the “internal” genes, with the exception of the NS and PB2 genes,were highly similar to the genes of isolates from Iran and Pakistan that also belong to the A/Qa/HK/G1/97-like lineage. Pathogenicity and transmission studies in chickens showedeffective transmission to contact birds through the aerosol route, but no clinical disease signswere observed during the study. In 6 of 7 isolates, human-virus-like receptor specificity wasobserved in analysis of the HA sequences, with changes in HB sites of neuraminidase similar

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to those found in human pandemic viruses. All the isolates were highly resistant to amantadine,an M2 ion channel blocker used in the prevention and treatment of human influenza.

The antigenic and phylogenetic analysis of these Middle Eastern isolates shows that theirsurface glycoproteins are related to those of A/Qa/HK/G1/97 lineage, with the highesthomology to A/Parakeet/Chiba/1/97 (97%) and A/Chicken/Pakistan/2/99 (96%) viruses. TheHA and NA genes of these H9N2 viruses have drifted somewhat over the years since the A/Qa/HK/G1/97 lineage was first defined in 1997, most likely as a result of continued circulationin poultry populations in South Asia and the Middle East, but they can be traced back to A/Qa/HK/G1/97-like lineage (Figure 1). The “internal” genes PB1, PA, M, and NP are alsosimilar to those of other Middle and Far Eastern strains, which can be ultimately traced backto the same G1-like lineage (Cameron et al., 2000;Shaw et al., 2002). However, analysis of NSand PB2 genes showed evidence of re-assortment with other avian influenza viruses (Hoffmannet al., 2000;Kou et al., 2005). On the basis of the similarity indices of these genes, it can beargued that the precursors of these viruses underwent reassortment with other subtypes,possibly in the same diverse Hong Kong poultry markets where the main Asian H9N2 lineagesare believed to have emerged (Guan et al., 1999;Guo et al., 2000).

The UAE isolates and South Asian viruses such as A/Ck/Pakistan/2/99 and A/Ck/Iran/IIT/99are highly (94%-98%) homologous. We do not have enough data on viruses from the MiddleEast to conclude whether all Middle Eastern isolates are homologous to those from across thePersian Gulf; however, A/Qa/HK/G1/97-like lineage viruses appear to predominate. Theamount of trade in commercial poultry as well as pet birds between the UAE and Pakistan hasbeen considerable. Therefore, it is prudent to ask whether trade between the 2 regions explainsthe similarity between the viruses. This possibility has been suggested by an earlier study ofH9N2 isolates in exotic pet birds (e.g., parakeet) exported from Pakistan to Japan in 1997 and1998; results showed that A/Qa/HK/G1/97-like viruses were already circulating in South Asiaand possibly the Middle East at approximately the same time that they were isolated in HongKong (Mase et al., 2001). Regional and international pet trade provides a possible route ofdissemination of potentially virulent influenza A viruses, if adequate quarantine andsurveillance systems are not in place. The possibility of such reassortment occurring is furthercomplicated by illegal importation of captive birds to the region (Brown et al., 2006).

Results of previous studies by Shortridge et al., of the 1997 H9N2 viruses from Asia indicategreat variability in host-range restriction and extent of clinical disease produced by H9N2viruses, and they demonstrate that pathogenicity is a polygenic trait, with “internal” geneshaving a definite role in virus transmission and lethality. Genetic analysis of UAE H9N2 virusesrevealed that the amino acid sequences at HA cleavage sites had an RSSR motif identical tothat of human and quail G1 and Dk/HK/Y280/97-like viruses. Although the motif in theseviruses is similar to the RX-RYK-R required for the highly pathogenic H5 and H7 subtypes,the absence of multi-basic amino acids at the cleavage sites in these isolates is associated withthe low pathogenicity of these H9 viruses in poultry. However, the presence of this particularmotif also emphasizes that these viruses have the potential to become pathogenic, should theyacquire any further nucleotide substitutions in the HA-connecting peptide region.

The 2-amino acid stalk deletion present in the lineage viruses, A/Qa/HK/G1/97 and the humanH9N2 isolates A/HK/1073/99 and A/HK/1074/99, is absent in the NA of the UAE isolates. Itsabsence may reflect establishment of the UAE lineage in the Middle East independent of theG1 lineage, either before or at approximately the same time the A/Qa/HK/G1/97-like groupbecame prevalent in Hong Kong. It appears that both lineages shared a common ancestor. Itappears unlikely that these viruses descended directly from the G1 lineage, because thelikelihood of their both having acquired 2 amino acid residues is lower than the possibility of

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concurrent circulation of similar viruses (Mase et al., 2001, Cameron et al., 2002) in the MiddleEast and South Asia in approximately 1997.

Amino acids in the receptor binding site of HA are associated with different receptor-bindingspecificities. Avian viruses preferentially bind to sialic acid of receptors by α-2, 3 glycosidicbond (SA α-2, 3 Gal), whereas human viruses preferentially bind to α-2, 6 Gal receptors(Paulson et al., 1985). Six of the seven H9N2 isolates from the United Arab Emirates had theamino acid changes that conferred human-virus-like receptor specificity on them. No human-virus-like substitutions were noted in positions 190 and 225, which are believed to be importantto the receptor-binding properties of H1 viruses. The viruses we studied carried substitutionsat positions 226 and 228 that parallel the receptor-binding profile of H3 human isolates (Vineset al., 1998; Stevens et al., 2006). Data from the receptor-binding assays clearly establish thata single change to leucine at position 226 can alter the receptor-binding properties of avianisolates (Table 4). The respiratory and intestinal epithelial cells of chicken as well as quailcarry α-2, 3 and α-2, 6 receptor linkages (Gambrayan et al., 2003; Perez et al., 2003; Wan etal., 2006). Carrying both types of receptor linkages reflects these viruses' ability to infectdifferent poultry species such as quail, chicken, pheasant, and guinea fowl. The presence ofα-2, 6 linkages on chicken cells probably explains the susceptibility of chickens to an increasinggroup of H9 viruses. Results of work conducted before 2001(Matrosovich et al.) do not indicatethe existence of any lineages of poultry viruses that carried human-virus-like receptorspecificity. Therefore, it is not clear whether these viruses could have emerged in chickensfirst. However, this lineage of H9N2 viruses probably emerged as a result of initial adaptationto a mammalian or avian host and later reintroduction to other species of poultry. These virusesalso successfully infected mammalian hosts, albeit without significant morbidity ordissemination, suggesting that further adaptive changes may be needed for more widespread,markedly disseminated disease.

The variations in both HA and NA, along with receptor specificity of different phylogeneticlineages of H9N2 viruses from Asia, reflect the diverse ways in which viruses adapt to variousspecies.

The sialic–acid-binding pocket on the hemadsorbing (HB) site of NA is highly conserved inequine and aquatic bird influenza viruses. However avian viruses co-circulating in mammalssuch as pigs and humans accumulate various substitutions in the HB site, thus decreasing theirhemadsorbing capacity. Previous studies have shown the HB site in the NA of Asian H9N2viruses to be under positive selection pressure for mutations, which results in compatiblecombinations of HA and NA (Matrosovich et al., 2001). The mutations in the HB site werepreviously seen in viruses that bind to α-2, 6-linked receptors. The NA of these new UAEisolates carried substitutions in the HB site similar to those of other avian H9N2 viruses fromAsia and human pandemic H2N2 and H3N2 viruses. Together, human-virus-like receptorspecificity and similar substitutions in the HB region of the NA in these recent isolates suggestthe possible role of poultry species such as chickens in the zoonotic transmission of influenzaviruses from aquatic birds to humans (Gambarian et al., 2002).

Previous data showed that H9N2 influenza viruses are transmitted mainly by the tracheal routeand to a lesser extent by feces (Shortridge et al., 1998). The data we collected on the H9N2isolates from the United Arab Emirates conformed to already established data regarding anaerosol mode of transmission, with evidence of transfer to contact birds within 48 hours. Thismode of spread has been advantageous to H9N2 viruses in interspecies transmission, resultingin perhaps the widest host range and genetic diversity in Asia (Guan et al., 2000; Li et al.,2005).

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Previous studies of H9N2 isolates from South Asia and the Middle East have shown that theseviruses produce significant disease problems in poultry, especially when poultry are co-infected with other respiratory pathogens such as infectious bronchitis virus (Nili et al.,2003, 2002; Brown et al., 2006). The fact that these isolates, moderate to highly pathogenic inthe field, failed to produce disease after experimental infection in chickens points to the roleof secondary pathogens (other viruses and bacteria) under field conditions (Bano et al., 2003;Kishida et al., 2004).

The resistance of these H9N2 isolates to antiviral drugs such as amantadine is consistent withan increasing trend in both avian and human viruses (Ilyushina et al., 2005; Hayden, 2006).Whether this trait represents a natural advantage to viruses, or is a consequence of amantadineuse is an unanswered question. However, this factor presents a special concern with regard topandemic preparedness, because the newer H5N1 viruses are resistant to M2 blockers. It istherefore important to continuously acquire data on emerging resistant viruses so thatappropriate treatment strategies can be planned accordingly.

It is interesting to compare the fate of the A/Qa/HK/G1/97-like lineage of H9N2 viruses inregions such as Southeast Asia and the Middle East with its fate in Hong Kong, where it wasfirst identified in 1997. Within 5 years of the withdrawal of quail from Hong Kong's live poultrymarkets, this lineage was reported to have almost disappeared from the region (Choi et al.,2004). However, individual internal gene segments continue to circulate all over Asia. Thatthis lineage is still quite widespread is indicated by the similarity (as much as 96%) betweenthe surface glycoproteins and most “internal” genes of the UAE isolates we analyzed and thoseof the G1 lineage. This lineage also continues to evolve in other parts of Asia, whereinterspecies mixing is still prevalent. Evolutionary trends of this and other lineages are difficultto trace due to insufficient surveillance data from the Middle East. This data gap underlinesthe need for further studies. The continued presence of this viral subtype in the Middle and FarEast indicates that it has become endemic to these regions (Alexander, 2003). At this time, itcan be argued that the lineage in this region has remained essentially G1-like. Its stability maybe due to insufficient reassortment in domestic poultry viruses or lack of selective pressure, asituation that contrasts what has been observed in Hong Kong markets since 1999 (Choi et al.,2004).

Prior infection with H9N2 can confer cross-protective cell-mediated immunity in chickens tosubsequent infection with highly pathogenic H5N1 viruses (Seo et al., 2001). Becauseserological methods are unable to show any evidence of antibody-mediated immunity to H5N1,the absence or reduction in disease signs due to previous H9N2 exposure may aid the insidiousintroduction and spread of H5N1 viruses. And, because H9N2 viruses are widespread,relatively inapparent infection could be pivotal in the silent spread of H5N1 viruses across thecontinents.

Our results suggest that the H9N2 viruses from the Middle East carry A/Qa/HK/G1/97-likegene segments previously implicated in human disease, with clear-cut receptor binding profilesthat favor human infection and resistance to first-line antivirals. What emerged in 1997 as alimited H5N1 outbreak and threatens to become a global pandemic today, started from a similar“soup” of H9N2 and H6N1 subtype viruses. With the threat of H5N1 viruses threatening toflare into a global pandemic, it is prudent to understand the interplay between the two subtypesand how the widespread prevalence of H9N2 viruses might be masking the spread of H5N1viruses. The H9N2 viruses continue to evolve in various hosts, and every favorable changefuels their pandemic potential. The need for continuous surveillance of domestic as well aswild bird species in these regions cannot be overemphasized. Continuous surveillance wouldimprove our understanding of the role of various avian hosts in ecology of influenza virusesand thus the underlying phenomena in emergence of pandemic strains.

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Materials and MethodsGeographical Location

The term “Middle East” traditionally refers to the countries of Southwest Asia and NortheastAfrica west of Afghanistan, Pakistan, and India. Thus defined it includes Cyprus, the Asianpart of Turkey, Syria, Lebanon, Israel, the West Bank and Gaza, Jordan, Iraq, Iran, the countriesof the Arabian peninsula (Saudi Arabia, Yemen, Oman, United Arab Emirates, Qatar, Bahrain,Kuwait), Egypt, and Libya. Located in the south of the Persian Gulf, the United Emiratescomprise 7 emirates, including Dubai and Abu-Dhabi.

VirusesWe characterized and analyzed 7 avian H9N2 influenza viruses, of which 3 were quail isolatesand 4 were chicken isolates. The viruses had caused problems in the field such as a 70%decrease in egg production; the chicken isolates produced severe disease signs such astracheitis, respiratory congestion and rapid mortality. The virus subtypes were confirmed byusing a panel of reference antisera recommended by WHO (WHO Animal Influenza Network,2002).

Antigenic AnalysisWe analyzed the cross-reactivity of these viruses by hemagglutination inhibition (HI) assay;we used polyclonal sera and monoclonal antibodies to 4 H9N2 viruses representing the Asianlineages in poultry and humans: A/Quail/Hong Kong/G1/97 (A/Qa/HK/G1/97), A/Chicken/Hong Kong/G9/97 (A/Ck/HK/G9/97), A/Duck/Hong Kong/Y280/97 (A/Dk/HK/Y280/97),and A/Hong Kong/1073/99 (A/HK/1073/99). Methods were as described by Palmer et al.,1975. To analyze the neuraminidase part of these viruses, we used a panel of polyclonal seraand monoclonal antibodies to N2.

Genetic and Sequence AnalysisWe extracted viral RNA from infected allantoic fluid of embryonated chicken eggs usingRNEasy Mini Kit (Qiagen, Valencia, CA). After reverse transcription, cDNA was amplifiedby polymerase chain reaction (PCR) analysis, and the PCR products were sequenced by usingsynthetic oligonucleotides produced by the Hartwell Center for Bioinformatics andBiotechnology (St. Jude Children's Research Hospital, Memphis, Tennessee). Template DNAwas sequenced by using rhodamine dye-terminator cycle sequencing (Ready Reaction Kitsused with AmpliTaq DNA Polymerase FS [Perkin-Elmer/Applied Biosystems, Inc.], FosterCity, California). Samples were separated by electrophoresis and analyzed on PE/AB1 model377 DNA sequencers.

Phylogenetic AnalysisAll sequence data were compiled and edited by using the Wisconsin Sequence AnalysisSoftware Package version 10.0 (GCG, Madison, WI). Nucleotide and deduced amino acidsequences were aligned by using the BioEdit program with ClustalW (Thompson et al., 1994).Phylogenetic trees were generated by using the Phylip program, version 3.65. The nucleotideregions used in phylogenetic analysis were as follows: for the PB2 gene, nucleotides 1 to 1262;for the PB1 gene, nucleotides 66 to 1368; for the PA gene, nucleotides 8 to1290; for the HAgene, nucleotides 1 to 1640; for the NP gene, nucleotides 1 to 962; for the NA gene, nucleotides20 to 1389; for the M gene, nucleotides 1 to 889; and for the NS gene, nucleotides 1 to 842.The nucleotide sequences obtained from this study are available from GenBank underaccession numbers EF063503 to EF063558.

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Animal TestsTwo 6-week-old white leghorn chickens were inoculated with 1 ml of virus-infected allontonicfluid (containing 106 50% egg infectious doses of each virus) via intranasal, intraocular, andintratracheal routes. The contact birds were introduced to the inoculated birds' cage one daypost-inoculation (p.i.). All the birds were observed daily for weight loss, other disease signs,or mortality. Tracheal and cloacal swabs were collected from all birds on days 3, 5, 7, and 10p.i., and virus was titrated in 10-day-old embryonated chicken eggs. Sera samples werecollected from all birds before inoculation and on day 13 p.i., and HI assays (Palmer et al.,1975) were used to analyze seroreactivity to corresponding viruses.

On the basis of their genetic similarity to the Hong Kong lineage of H9N2 viruses and theirreceptor binding profile, we evaluated the replication and pathogenicity of one isolate fromeach group (quail and chicken). We inoculated 6-week-old BALB/c mice via the intranasalroute with 106 EID50 of each virus. Results were then compared to those of representativeviruses from established Hong Kong lineages, A/Qa/HongKong/G1/97 and A/Dk/HongKong/Y280/97 (Li et al., 2005). For every virus, we observed 10 mice for 3 weeks for signs ofmorbidity/weight loss. Organs from 3 mice per virus were collected on days 3 and 5 p.i. forvirus titration in embryonated chicken eggs.

Receptor Specificity AssayBinding of the peroxidase-labeled chicken egg-white ovomucoid and pig α2-macroglobulinwas assayed as described by Matrosovich et al. (1999). The binding to oligosacchrides 3′SLand 6′SLN was determined by the solid-phase fetuin-binding inhibition assay, and results wereexpressed as binding affinity constants (Gambaryan et al., 1992).

Amantadine Sensitivity TestsAmplified M protein sequences were analyzed for amino acid substitutions at the 5 positionsimplicated in amantadine resistance (positions 26, 27, 30, 31 and 34) within the trans-membrane domain of the M2 protein (Hays et al., 1985; Pinto et al., 1992). Amantadinesensitivity was determined by plaque reduction assay on Madin-Darby canine kidney cells(MDCK, ATCC, Manassas, VA), as described previously (Hayden et al., 1980). Six-well plateswere inoculated with virus diluted in minimal essential medium (MEM) to give 80 to 100plaques per well. Cells were incubated for 1 hour at 37°C and then overlaid with MEMcontaining 0.9% agar, 4% bovine serum albumin, 1 μg/ml trypsin treated with L-1-(topsylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) (Worthington Diagnostics,Freehold, NJ) and amantadine (1-aminoadamantane hydrochloride, Sigma-Aldrich, Inc., St.Louis, MO) at increasing concentrations (0.01 to 100 μM). After 3 days of incubation at 37°C, plaques were visualized by staining with 0.1% crystal violet containing 10% formaldehyde.The percentage inhibition of plaque formation relative to untreated controls was calculated foreach drug concentration. The drug concentration that resulted in a 50% reduction of the plaquenumber (IC50) was determined. An amantadine-sensitive H9N2 virus (A/Quail/Hong Kong/YU415/00) was used to compare the susceptibility of amantadine-resistant strains.

AcknowledgmentsDue to the similarity of the surface glycoprotiens of these isolates to those of A/Qa/HK/G1/97 lineage of H9N2 viruses,the pathogenicity studies were carried out in BioSafety Level 3 facilities per USDA regulations. All animal experimentswere approved by the Animal Care and Use Committee of St. Jude Children's Research Hospital and performed inUSDA-inspected and AAALAC-accredited facilities.

This study was supported by U.S. Public Health Service grant A95357 and by the American Lebanese SyrianAssociated Charities (ALSAC).

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We thank Scott Krauss for his excellent support during the study. We are also grateful to Kelly Jones, John Franks,and Jennifer Humberd for their technical help, Margaret Carbaugh for editorial assistance and Carol Walsh formanuscript assistance.

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Figure 1.Phylogenetic Analysis of HA, NA , M and PB2 genes of U.A.E H9N2 viruses to the establishedEurasian lineages. The U.A.E isolates are in bold and underlined.

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Table 2Similarity Indices of the UAE H9N2 Genes at the Nucleotide Levels

Gene Nucleotide Similarity % Lineage

PB2 A/Dk/Potsdam/2216-4/84 H5N6 94-95 Unknown

A/Pheasant/Ireland/PV18/97 H9N2 89-91

PB1 A/Ck/Iran/IIT/99 H9N2 94 G1

PA A/Ck/Iran/IIT/99 H9N2 96 G1

A/Dk/Guangxi/35/01 H5N1 95-96

HA A/Parakeet/Chiba/1/97 H9N2 95-97 G1

A/Quail/HK/G1/97 H9N2 94-95

NP A/ParakeetChiba/1/97 H9N2 98 G1

A/Quail/HK/G1/97 H9N2 97

A/HK/481/97 H5N1 97

NA A/Parakeet/Chiba/1/97 H9N2 95-97 G1

A/Quail/HK/1/97 H9N2 95-96

M A/Chicken/Pakistan/2/99 H9N2 97 G1

A/Quail/HK/1/97 H9N2 97-98

A/HK/156/97 H5N1 97

NS A/Dk/Nanchang/1904/92 H7N4 95-97.3 Unknown

A/Mallard/Italy/43/01 H7N3 95-97.1

A/Duck/Hong Kong/P50/97 H11N9 94-97.3

Virology. Author manuscript; available in PMC 2009 August 31.

NIH

-PA Author Manuscript

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-PA Author Manuscript

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-PA Author Manuscript

Aamir et al. Page 18Ta

ble

3A

ffin

ity o

f UA

E H

9N2

Viru

ses f

or S

ialy

l Sub

stra

tes

Am

ino

acid

subs

titut

ions

in H

A p

rote

in *

Affi

nity

for

subs

trat

e **

Vir

uses

183

226

228

fetu

inp6′S

Lp3′S

L

A/Q

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± 0

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0.38

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9>1

00

A/Q

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LG

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± 0

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0.47

±0.0

4>1

00

A/Q

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>100

1.01

±0.2

9

A/C

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en/D

ubai

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DL

G0.

45 ±

0.0

60.

92±0

.08

>100

A/C

hick

en/D

ubai

/339

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NL

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45 ±

0.0

61.

50±0

.13

>100

A/C

hick

en/D

ubai

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NL

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50 ±

0.0

91.

30±0

.24

>100

A/C

hick

en/D

ubai

/463

/03

NL

G0.

59 ±

0.0

50.

51±0

.05

>100

* Am

ino

acid

num

berin

g is

bas

ed o

n H

3 H

A (N

obus

awa

et a

l., 1

991)

.

**Κ

d =

mea

n ±

SE×

t α,n

-1 (μ

M si

alic

aci

d), w

here

t α is

Stu

dent

's co

effic

ient

with

pro

babi

lity α

= 0.

95, f

rom

4 in

depe

nden

t exp

erim

ents

.

Virology. Author manuscript; available in PMC 2009 August 31.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

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-PA Author Manuscript

Aamir et al. Page 19Ta

ble

4Tr

ache

al a

nd C

loac

al T

iters

of U

AE

H9N

2 V

iruse

s in

Chi

cken

s

Vir

uses

Vir

us T

iter

[logE

ID 5

0/m

l(SD

) ]

Sick

-dea

d bi

rds/

tota

l num

ber

Tra

chea

Clo

aca

Infe

cted

Cont

act

Infe

cted

Cont

act

35

73

57

35

73

57

A/Q

a/H

K/G

1/97

4.4(

0.8)

2.8(

0.6)

<1<1

<1<1

<12.

5(0.

7)4.

5*<1

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0/4

A/D

k/H

K/Y

280/

976.

5(0.

1)3.

8(0.

5)3

45.

83.

51.

6<1

<1<1

<1<1

0/4

A/Q

a/D

ubai

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2)6.

3 (0

.3)

5.7

<1<1

5.5*

<1<1

<1<1

<1<1

0/4

A/Q

a/D

ubai

/302

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4.9(

0.4)

5.5(

0.7)

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<1<1

<12.

7*<1

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<10/

4

A/Q

a/D

ubai

/303

/00

4.0(

0.4)

4.8(

0.8)

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<13.

75*

5.5*

2.5*

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4

A/C

k/D

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3.4(

0.2)

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0.8)

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74.

54.

0<1

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<1<1

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4

A/C

k/D

ubai

/463

/03

4.1(

0.5)

4.6(

0.6)

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94.

64.

1<1

<1<1

<12*

<10/

4

* One

/two

bird

s had

a d

etec

tabl

e tit

er.

Virology. Author manuscript; available in PMC 2009 August 31.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Aamir et al. Page 20Ta

ble

5A

min

o A

cid

Subs

titut

ions

in th

e H

emad

sorb

ing

Site

s of N

2 N

As C

ompa

red

to E

stab

lishe

d H

9N2

HB

Site

*

366-

373

399-

304

431-

433

Vir

uses

HA

iSkd

SrSg

dNnn

Ws

pQe

Stal

k de

letio

n

A/Q

uail/

HK

/G1/

97H

9.K

......

.Sdi

R.

...38

-39

A/H

K/1

073/

99H

9.K

......

.Sd.

.....

38-3

9

A/ D

k/H

K/Y

280/

97H

9.K

e....

..S

d...

...63

-65

A/Q

uail/

Dub

ai/3

01/0

0H

9.K

..L.A

..S

dn..

pQe

-**

A/Q

uail/

Dub

ai/3

02/0

0H

9.K

..L.A

..S

dn..

pQe

-

A/Q

uail/

Dub

ai/3

03/0

0H

9.K

..L.A

..S

dn..

pQe

-

A/C

hick

en/D

ubai

/338

/01

H9

.K..L

.A.

.Sdn

..pQ

e-

A/C

hick

en/D

ubai

/339

/01

H9

.K..L

.A.

.Sdn

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A/C

hick

en/D

ubai

/383

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H9

.K..L

.A.

.Sdn

..pQ

e-

A/C

hick

en/D

ubai

/463

/03

H9

.K..L

.A.

.Sdn

..pQ

e-

* Subs

titut

ions

are

with

refe

renc

e to

con

sens

us se

quen

ce (t

op li

ne).

**N

o st

alk

dele

tion

seen

Virology. Author manuscript; available in PMC 2009 August 31.