Virus Research 145 (2009) 329–333

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Phenotypic, genetic, and phylogeographical characterization ofavian influenza virus subtype H5N2 isolated fromnorthern pintail (Anas acuta) in Japan

Alam Jahangir, Sakchai Ruenphet, Dany Shoham, Masashi Okamura,Masayuki Nakamaura, Kazuaki Takehara ∗

Laboratory of Zoonoses, School of Veterinary Medicine, Kitasato University, 35-1 Higashi 23, Towada-shi, Aomori 034-8628, Japan

a r t i c l e i n f o

Article history:Received 7 May 2009Received in revised form 17 July 2009Accepted 19 July 2009Available online 25 July 2009

Keywords:Avian influenza virusNorthern pintail

a b s t r a c t

Intercontinental movements of northern pintail (Anas acuta) ducks wintering in Japan create a high-riskof both incursion and dispersion of avian influenza viruses (AIVs) that circulate in the ducks’ breedinggrounds in Siberia and Alaska. This predisposition is likely amplified by bi-directional conveyance ofAIVs between Japan and China. In this study, H5N2 viruses were characterized by means of HA cleavagesite sequencing and found to be low pathogenic. Through entire genome analysis, as well as in ovo andin vitro pathogenicity tests, one isolate – A/northern pintail/Akita/714/06 H5N2 (Akita/714/06 H5N2) –was characterized. Comparative molecular analysis revealed that genes of this virus have 97.5–99.6% and96.7–100% likeness at nucleotide and at amino acid level, respectively, with genes of different subtypesof viruses isolated from China, Korea, Russia, and Italy. Phylogenetically, Akita/714/06 (H5N2) clustered

Pathogen–host–ecosystem interfaces

Phylogenetic analysis with viruses isolated from Eurasian countries. Partial affinity to a recent Korean porcine strain is notice-y, eced p

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Aquatic birds are known to be the major, usually sub-clinicallynfected, carriers and disseminators of all subtypes of aviannfluenza viruses (AIVs) (Webster et al., 1992). Among them, theorthern pintail (Anas acuta) (thereafter referred to as pintail/s)onstitutes a remarkable migratory waterfowl of considerablepecies biomass and high individual density. Several studieseported that the rate of prevalence of AIVs in pintails ranges fromto 4% (Ip et al., 2008; Jahangir et al., 2008, 2009; Koehler et al.,

008; Parmley et al., 2008; Pearce et al., 2009; Wahlgren et al.,008). However, in one study Runstadler et al. (2007) showed 24.1%revalence rate in pintails sampled in Alaska in 2005 and reportedhe descending order of susceptibility to AIVs within aquatic wildirds to be: mallards > pintails > green winged teal. Infection in wildirds rarely results a disease and whenever noticeable illness doesccur, it is due to the contraction of an HPAI strain from infectedoultry, as is apparently the case of the H5N1 viruses frequently

solated from variedly affected wild birds in Japan (Uchida et al.,

008), China (Ellis et al., 2004), Siberia (Lvov et al., 2006), and else-here. The apparent emergence of HPAI viruses within wild birdsas been observed in several cases, though, while no preceding oroncomitant appearance of the concerned viruses could be pointed

∗ Corresponding author. Tel.: +81 176 23 4371; fax: +81 176 23 8703.E-mail address: takehara@vmas.kitasato-u.ac.jp (K. Takehara).

168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2009.07.015

ological, and phenotypic aspects were discussed, thereby signifying theathogen–host–ecosystem interfaces.

© 2009 Elsevier B.V. All rights reserved.

at elsewhere (Becker, 1966; Becker and Uys, 1967; Gaidet et al.,2008; Kaleta and Hönicke, 2005; Manvell et al., 2000).

In Eastern Asia, roughly, 500,000–1,000,000 pintails congregateduring wintertime, mainly in China, Korea, and Japan (Miyabayashiand Mundkur, 1999). Albeit a much smaller country than China,Japan hosts the largest wintering location of pintails in Asia(Miyabayashi and Mundkur, 1999; Perennou et al., 1994; Wild BirdSociety of Japan, 1992). Intercontinentally, a triangular flyway ofpintails wintering in Japan is evident from band recovery and satel-lite telemetry data – including North America, Siberia, and Japanbeing the three corners of this triangle (Alaska Science Center;Bianki and Dobrynina, 1997; Miller et al., 2005; Rienecker, 1987;Yamashima Institute for Ornithology, 1985, 2004). Bi-directionalmovements of pintails have thus been traced, between Japan andSiberia, as well as between Japan and North America. Since pintailsfrom North America and other areas share common summer loca-tions in Siberia and Alaska, intact virions of AIVs may be exchangedby their hosts (through fecal–oral route), and genetic componentsmay be exchanged (through reassortment or recombination) byco-infecting viruses; consequently, current and new viral strains

would be later on conveyed by pintails to their wintering sites.Hence, it is important to monitor viruses harbored by these pin-tails, in order to detect and comprehend the dynamic of and thechanges occurring within the viruses. In spite of the enormouspotential of pintails, in terms of introducers of AIVs into Japan – and

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preaders of AIVs therefrom – only limited sequence data in Gen-ank, is available, indicating specifically that potential. Combiningogether the elements of host (pintail), location (Japan), and H5A subtype, this report presents and discusses, comparatively, theenetic characterization, phylogenetic analysis, ecological featuresnd phenotypic properties of H5N2 viruses isolated from pintailsn Japan (Jahangir et al., 2008), with special reference to the Easternsian–North American interface.

A total of four H5N2 viruses – A/northern pintail/Akita/714/06Akita/714/06 (H5N2)], A/northern pintail/Akita/772/06,/northern pintail/Akita/773/06 and A/northern pin-

ail/Akita/776/06 – were used in this study. All viruses, exceptkita/714/06 (H5N2), were molecularly pathotyped by sequenc-

ng about 300 base pairs surrounding the HA0 cleavage site asescribed earlier (Jahangir et al., 2008). Besides, full length genesf all eight viral gene segments of Akita/714/06 (H5N2) weremplified by RT-PCR (Hoffmann et al., 2001), and PCR productsere cloned into a plasmid. Sequencing was performed both on

he cloned cDNAs and on the PCR products, and the results wereound identical. Phenotypic properties of the isolate Akita/714/06ere also studied in vitro and in ovo. Phylogenetic analyses wereerformed using neighbor joining tree inference analysis with the000 bootstrap replications (Tamura et al., 2007). The nucleotideequences obtained in this study are available from GenBank underccession numbers AB490823–AB490830.

The amino acid sequences of the HA0 cleavage site deduced fromucleotide sequences revealed that all the H5N2 viruses tested pos-ess the same amino acid sequence PQRETR*GLF (*cleavage point)the LPAI motif. The phenotypic traits of the Akita/714/06 (H5N2)

lso comply with this LPAI motif of cleavage site. For example,he mean death time of minimum lethal dose (MDT/MLD) of thekita/714/06 (H5N2) was 126 h. The virus requires trypsin supple-entation for its ongoing replication in the Madin–Darby canine

idney (MDCK) cells and chicken embryo fibroblast (CEF), but doesot when cultured in chicken kidney (CK) cells. The virus produceslaques on CK cells (2.8 × 105 plaque forming unit (PFU) ml−1), butot on CEF or MDCK cells without trypsin supplementation. How-ver, with supplementary trypsin, the number of plaques increasedo 2.5 × 107 from 2.8 × 105 PFU ml−1 on CK cells. On the other hand,he number of plaques found on CEF and MDCK cells with trypsinas 3 × 106 and 2.5 × 108 PFU ml−1, respectively.

The entire genome of Akita/714/06 (H5N2) was found to be3,633 nucleotides long. The viral RNA (vRNA) segments consistf non-translation and translation regions. The non-translationegions (NTR) at the 5 prime end of the vRNA were 19–45ucleotides long, whereas at the 3 prime end length ranged from0 to 58 nucleotides. The HA protein of Akita/714/06 (H5N2) con-ained residues glutamine (Q) and glycine (G) at position 222, and24, respectively reported to be associated with preferential bind-

ng to �2,3-linked sialic acid (Table 1) (Ha et al., 2001; Shinya etl., 2004). Deletion of amino acids from the stalk region of the NArotein was not observed when compared with viruses from wildirds. Moreover, typical catalytic sites 118R, 151D, 152R, 224R, 276E,92R, 371R, and 406Y, as well as framework sites 119E, 156R, 178W,79S, 198D, 222I, 227E, 274H, 277E, 294N, and 425E were found to beompletely conserved in the NA molecule (Colman et al., 1993).kita/714/06 (H5N2) also carried other important residues in pro-

eins not associated with drug resistance and virulence (Table 1).Phylogenetically, Akita/714/06 (H5N2) was clustered with the

PAI viruses isolated from Japan, Korea, China, Sweden, France, andtaly (Fig. 1a–d). Uncommonly, the NS gene was clustered with

he avian allele B viruses. All over, when compared with Northmerican lineages, our isolate considerably clustered with viruseselonging to Eurasian lineages. All genes – except PB1 and HA ofhe isolate Akita/714/06 (H5N2) have highest genetic similarity toifferent subtypes of AIVs isolated from China during 2000–2006,

ch 145 (2009) 329–333

namely 97.5–99.6% at nucleotide level (Table 2). The HA gene hasmaximum nucleotide identity to the virus A/swine/Korea/C12/08H5N2 (99.3%), recently isolated from diseased pig in Korea. On theother hand, the PB1 gene has utmost relatedness to a PB1 gene of anItalian virus isolated from goose in 2003 – A/goose/Italy/296426/03(H1N1) (Table 2). At the same time, the amino acid identity levelfor all gene products of Akita/714/06 (H5N2) ranged from 96.7to 100% with that of different subtypes of viruses isolated fromAsia, dominated by Chinese isolates (Table 2). Although the iso-late Akita/714/06 (H5N2) was found to basically be an ordinaryEurasian LPAI H5N2 strain, the biogeographical nature of its phy-logenetic relationship is significant, and includes, among otherfeatures, noticeable affinity to a recent Korean porcine strain (Fig. 1a and b). Overall, we observed in that respect two interconnectedecosystems: the regional Eastern-Asian Japanese–Chinese axis, aswell as the intercontinental Japanese–Siberian–Alaskan triangle.Pintails may constitute an important AIV reservoir and spreaderacross those two distinct eco-systems. Connectedly, pintails win-tering in Japan are regularly being traced, individually, by means ofthe US Geological Survey program (Alaska Science Center; Koehleret al., 2008). AIVs isolated from pintails in Japan are potentiallyof great significance, then, due to the mentioned intercontinen-tal triangle shaped by their migrations. This movement patterncan facilitate intact virus exchange, gene flow and reassortmentbetween Eurasian and North American lineages of AIVs. Moreover,in Siberia, for instance, birds – waterfowl, foremost – from six conti-nents congregate during breeding seasons (Wahlgren et al., 2008).Eventually, intact plus currently forming viruses may readily beconveyed by pintails to Japan during their fall migration, and lateron to further locations – including North American – therefrom.

The interconnected ecosystem, namely the Japanese–Chineseaxis, is essential, as well.

Fundamentally, the virological significance of theChinese–Japanese interface has been pointed at in connectionwith the formation of the HPAI H5N1 virus in 1997 in China. Threegenes of its ancestral strain A/Goose/Guangdong/1/96 (H5N1)derived from two Japanese LPAI strains (Mukhtar et al., 2007).These findings are consistent with the conclusions arrived at byDuan et al. (2007), particularly in that viral strains of migratorybirds gave rise to the goose strain. They are in conformity, as well,with an earlier study by Okazaki et al. (2000), who observed thatthe likely genetic provenance of A/teal/Hong-Kong/W312/97 –reckoned as the chief ancestor of the human prototype HPAI H5N1A/Hong-Kong/97 – was Siberian lakes. It follows, that in termsof both phylogenetic patterns and migration ecology, highestrelatedness represents the interfaces among Japan, China andSiberia, while Japan apparently serves during wintertime as acardinal congregating area and melting pot, in that concern.

Notably, segment 8 of Akita/714/06 (H5N2) has 98.3%nucleotide homology to a Russian isolate namely –A/duck/Chabarovsk/1610/72 H3N8. It means that ongoingmutation rate during 34 years was only 1.7% – markedly lessthan reported earlier (Chen and Holmes, 2006; Kawaoka et al.,1998). Basically, the two reasons thought to possibly accountfor unexplained genetic conservation in influenza A viruses arecross contamination, or interference with vaccine strains (Duanet al., 2007). The strain A/duck/Chabarovsk/1610/72 or relatedstrains are not held by us, and interference with a vaccine strainseems unlikely, in that case. Abiotic virus preservation has beensuggested as an alternative explanation (Shoham, 1993). This,together with the location of Chabarovsk being close to the regular

migration route of pintails between North Japan and Siberia haveto further be looked into.

Noticeably, there is considerable affinity between the HA andNA genes of the reported strain and those of an H5N2 strainthat was isolated from a diseased swine in South Korea in 2008

A. Jahangir et al. / Virus Research 145 (2009) 329–333 331

Table 1Comparison of amino acid residues in various proteins associated with pathogenicity and other important characteristics.

Strain HA1 NA M2 NS PB2

aa 222a aa 224 Cleavage site (aa 321–329) aa 274 aa 26 aa 31 aa 92 aa 627

Akita/714/06 Q G PQRETRGLF H L S D EGa/Sanjiang/160/06 Q G PQRETRGLF H L S D ESw/Kor/C12/08b Q G PQRETRGLF H L S D ESw/Kor/C13/08b Q G PQRETRGLF H L S D E

a For each protein amino acid (aa) positions are shown.b Lee et al. (2009).

Fig. 1. Phylogenetic analysis of Akita/714/06 H5N2. An unrooted neighbor-joining trees of nucleotide sequences of HA – 1695 bp (a), NA – 1410 bp (b), PB2 – 2280 bp (c) andNS – 838 bp (d) genes were generated, followed by 1000 replications of bootstrap re-sampling. The number at each branch point indicates percentage probability that theresultant topology is correct. Akita/714/06 (H5N2) was marked by black circle (�). For NS gene representatives of alleles A (�) and B (�) are marked in the tree. Abbreviation:SPG, spar-winged goose.

332 A. Jahangir et al. / Virus Research 145 (2009) 329–333

Table 2Nucleotide and amino acid sequence comparisons of Akita/714/06 (H5N2) to its nearest genetic associations.

Gene Virus with maximum % similarity with Akita/714/06 (H5N2)a

Nucleotide Amino acid

PB2 A/duck/Nanchang/2-0527/00 H4N6 (98.2%) A/duck/Nanchang/2-0527/00H4N6 (99.9%)PB1 A/goose/Italy/296426/03 H1N1 (97.9%) A/migratory duck/HongKong/MP206/04H5N2 (100%)PB1-F2 A/duck/Italy/775/04 H5N3 (96.7%)PA A/mallard/Yan chen/05 H4N6 (99.3%) A/chicken/Guiyang/4059/05 H5N1 (99.4%)HA A/swine/Korea/C12/08 H5N2 (99.3%) A/swine/Korea/C12/08 H5N2 (99.3%)NP A/chicken/Nanchang/7-010/00 H3N6 (97.5%) A/mallard/Astrakhan/244/82 H14N6 (100%)NA A/garganey/SanJiang/160/06 H5N2 (99.6%) A/garganey/SanJiang/160/06 H5N2 (99.6%)M1 A/mallard/Zhalong/88/04 H4N6 (98.9%) A/duck/Korea/S8/03 H3N2 (99.6%)

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Lee et al., 2009). The latter’s HA gene has the highest (99.3%)imilarity to Akita/714/06 (H5N2), and it is regarded to deriverom a Chinese wild duck strain – A/garganey/SanJiang/160/065N2 (SanJiang/160/06 H5N2), a virus showing >99.5% similarityf its NA and NS proteins, respectively, to Akita/714/06 (H5N2).igh identity (99.8%) was found between the swine-originatedorean strain NS gene and that of the Japanese waterfowltrain Dk/Hokkaido/120/01, a virus of the subtype H6N2 isolated

years before the Korean one. It follows, that Korea consti-utes an additional significant phylogeographical link within theapanese–Chinese ecosystem. Our isolate and the Korean porcinesolate showed unremarkable similarity (69.9–91.8%) to the cur-ent pandemic avian–porcine–human H1N1 swine influenza virusnternal protein genes. However, the biogeographical interface webserved between our isolate and the Korean porcine virus mighte contributive to the formation of genotypes bearing pandemicotential.

The Chinese and Korean viruses mostly related to our isolateere commonly obtained during 1999–2008. Most of the related

solates derived from mallards (Anas platyrhynchos) and garangeyucks (Anas querquedula) (Table 2), once again two migratory hostpecies, the same genus of the host from which our isolate derived,he pintail. The related SanJiang/160/06 H5N2 strain is also derivedrom mallard. Expectably, the species barrier between host specieselonging to the same genus can more readily be crossed by AIVs, inhat case while pintails and mallards congregate in China, Japan, orlsewhere. Furthermore, virus transmissibility between mallardsnd domestic ducks does not involve species barrier. All over, theeported virus appears to be most closely related to strains affili-ted with the four principal host classes shaping the ecology andenetic dynamics of AIVs. Meaning, migratory ducks (in that casewo host species affiliated with same genus); domestic waterfowltwo genera – duck and goose – the former being represented by

species identical to one of the migratory ducks); gallinaceousoultry (chicken); and a distinct AIV mammalian host (pig). Thisomplex properly illustrates the pivotal role of ducks, both wildnd domestic, in the genetic dynamics of AIVs.

cknowledgements

This study was supported in part by a grant from Ministry ofgriculture, Forestry, and Fisheries (MAFF), Japan.

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