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Highly Pathogenic Avian Influenza Virus Subtype H5N1 in Mute Swans (Cygnusolor) in Central BosniaAuthor(s): Teufik Goletić, Abdulah Gagić, Emina Rešidbegović, Aida Kustura, Aida Kavazović,Vladimir Savić, Timm Harder, Elke Starick, and Senad PrašovićSource: Avian Diseases, 54(s1):496-501. 2010.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/8705-031609-ResNote.1URL: http://www.bioone.org/doi/full/10.1637/8705-031609-ResNote.1

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Research Note—

Highly Pathogenic Avian Influenza Virus Subtype H5N1 in Mute Swans (Cygnus olor)in Central Bosnia

Teufik Goletic,AD Abdulah Gagic,A Emina Residbegovic,A Aida Kustura,A Aida Kavazovic,A Vladimir Savic,B Timm Harder,C

Elke Starick,C and Senad PrasovicA

ASarajevo Veterinary Faculty, National Reference Laboratory for Avian Influenza and Newcastle Disease, Zmaja od Bosne 90, 71000 Sarajevo,Bosnia and Herzegovina

BCroatian Veterinary Institute, Poultry Centre, Heinzelova 55, 10000 Zagreb, CroatiaCFriedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Sudufer 10, 17493 Greifswald-Insel Riems, Germany

Received 16 March 2009; Accepted and published ahead of print 5 July 2009

SUMMARY. In order to determine the actual prevalence of avian influenza viruses (AIVs) in wild birds in Bosnia andHerzegovina, extensive surveillance was carried out between October 2005 and April 2006. A total of 394 samples representing 41bird species were examined for the presence of influenza A virus using virus isolation in embryonated chicken eggs, PCR, andnucleotide sequencing. AIV subtype H5N1 was detected in two mute swans (Cygnus olor). The isolates were determined to behighly pathogenic avian influenza (HPAI) virus and the hemagglutinin sequence was closely similar to A/Cygnus olor/Astrakhan/Ast05-2-10/2005 (H5N1). This is the first report of HPAI subtype H5N1 in Bosnia and Herzegovina.

RESUMEN. Nota de Investigacion—Presencia del virus de la influenza aviar de alta patogenicidad en cisnes comunes (Cygnusolor) en la parte centra de Bosnia.

Con la finalidad de determinar la prevalencia real de los virus de la influenza aviar en aves silvestres en Bosnia y Herzegovina, serealizo un extensivo muestreo de vigilancia entre Octubre del 2005 a Abril del 2006. Un total de 394 muestras que representaban41 especies aviares se examinaron para la presencia de la influenza aviar A mediante aislamiento viral en embriones de pollo, PCR yanalisis de las secuencias de nucleotidos. El subtipo H5N1 fue detectado en dos muestras de cisnes comunes (Cygnus olor). Sedetermino que los aislamientos eran virus de la influenza aviar de alta patogenicidad (con las siglas en ingles HPAI) y la secuencia denucleotidos de la hemaglutinina resulto ser similar al virus A/Cygnus olor/Astrakhan/Ast05-2-10/2005 (H5N1). Este es el primerreporte del la presencia en Bosnia y Herzegovina del subtipo H5N1 del virus de la influenza aviar de alta patogenicidad.

Key words: highly pathogenic avian influenza, H5N1, mute swan, influenza A

Abbreviations: AI 5 avian influenza; AIV 5 avian influenza virus; BIH 5 Bosnia and Herzegovina; FAO 5 Food and AgricultureOrganization; HA 5 hemagglutinin; HA0 5 hemagglutinin precursor; HPAIV 5 highly pathogenic avian influenza virus;IAV 5 influenza A viruses; LPAIV 5 low pathogenicity avian influenza virus; MEGA 5 molecular evolutionary genetics analysis;NA 5 neuraminidase; OIE 5 World Organization for Animal Health; nt 5 nucleotides; RT-PCR 5 reverse transcription–polymerase chain reaction; WHO 5 World Health Organization

Avian influenza (AI) is a viral infection of domestic and wild avianspecies with a complex ecology involving reassortment of viral genesegments and transmission among different avian and mammalianspecies carrying the risk of the emergence of pandemic influenza.Populations of aquatic birds, especially those from the Anseriformesand Charadriiformes orders are the natural and the mostheterogeneous reservoir of influenza A viruses (IAVs) in whichIAVs are maintained as biotypes of low pathogenicty (20,24,27,31).A total of 16 hemagglutinin (HA) and nine neuraminidase (NA)subtypes have been detected in the gene pool of avian influenzaviruses (9). Depending on their ability to cause disease in chickens,avian influenza viruses (AIVs) are characterized as having lowpathogenicity (LPAIV) or as being highly pathogenic (HPAIV).Research has shown that the LPAIV strains may, after introductionand circulation in gallinaceous poultry populations, mutate intoHPAIV. This occurs mainly by insertional mutation in the sequenceof the HA gene encoding for the endoproteolytic cleavage siteassociated with visceral dissemination in avian species (1,24,33).From the plethora of different AIVs, only a few strains of H5 and

H7 subtypes have become HPAIVs so far. The currently ongoingoutbreaks caused by HPAIV of the H5N1 subtype are primarily ofconcern for the poultry industry; but as potential zoonotic agents,they present a constant threat to human health as well (14,16).

The first case of HPAIV H5N1 in wild birds in Bosnia andHerzegovina (BIH) was identified in February 2006 (23), after thevirus had been discovered in many parts of Europe in 2005–06,including neighboring countries, including Croatia and Slovenia.The aim of this paper is to describe the AI outbreak caused by AIVsubtype H5N1 among the mute swan (Cygnus olor) population inCentral Bosnia, to characterize the viral strain, and to elucidate itsphylogenetic relationships based on HA and NA gene analysis andcomparison with representative H5N1 strains deposited in Gen-Bank.

MATERIALS AND METHODS

Virus isolation. A total of 394 dead birds representing 41 bird specieswere collected throughout BIH over a 7-mo period from October 2005to April 2006. Pooled organ suspensions (lungs, brain, liver, duodenum,and pancreas) and oropharyngeal and cloacal swabs of these birds wereDCorresponding author. E-mail: teufikg@hotmail.com

AVIAN DISEASES 54:496–501, 2010

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processed separately for virus isolation in specific-pathogen-freeembryonated chicken eggs according to the World Organization forAnimal Health (OIE) recommendations (6,22). HA and NA subtypes ofthe isolated AIV were determined according to standard proceduredescribed elsewhere.

RNA isolation, reverse transcription–PCR (RT-PCR) andsequencing. RNA isolation was done manually from the allantoicfluids of the virus isolate using the QIAamp viral RNA kit (QIAGEN,Hilden, Germany) as described in the kit protocol. One-step RT-PCRassays were performed separately for HA and NA genes using theSuperscript III-based One-Step RT-PCR kit (Invitrogen, Carlsbad, CA)according to the manufacturer’s instructions. The complete NA geneopen reading frame was reverse-transcribed and amplified according toHoffmann et al. (13) with some modifications. Amplification and directsequencing of the HA gene segment was carried out using four primersets generating four overlapping HA gene fragments (primer sequencesand thermocycling profile are available upon request). Each PCRreaction was separately carried out twice, and two independentlygenerated PCR products were sequenced. All RT-PCR products wereeluted from the agarose gels and, after purification with the QIAquickgel extraction kit (QIAGEN), used for direct sequencing. Purifiedcomplementary DNA fragments were cycle-sequenced in both directionsusing the same primers as for the amplification and additional primersN1-F and N1-R for NA gene (8). The Prism Big Dye Terminator v1.1cycle sequencing kit (Applied Biosystems) was used and products wereanalyzed on an automatic ABI PRISM 3130 genetic analyzer (AppliedBiosystems, Foster City, CA). The nucleotide sequences analyzed herecover nucleotides 1–1736 (HA) and 1–1398 (NA).

Phylogenetic analyses. The nucleotide sequences were assembled,translated, and aligned with the Bioedit software version 7.0.9.0 (12)with an engine based on the ClustalW 1.4 algorithm. Blast homologysearches (http://www.ncbi.nlm.nih.gov/blast) were used to retrieve thetop 50 homologous sequences for the sequenced gene from theGenBank database. Phylogenetic and molecular evolutionary analysesbased on the HA and NA genes alignments were conducted with theMolecular Evolutionary Genetics Analysis (MEGA, version 4.0)software (29) using the neighbor-joining tree inference analysis withthe Tamura–Nei c model, with 2000 bootstrap replications. Thetopology of the constructed trees was confirmed by using minimumevolution on basis of a maximum composite likelihood model, and alsoby maximum parsimony as implemented in MEGA 4.0. Amino acidresidues in HA and NA, if not stated otherwise, are numbered using theHPAIV H5N1 numbering system based on defining the first amino acidof the open reading frame of both genes studied as amino acid 1. In thecase of NA, gaps are included in the numbering (HPAIV H5N1numbering system). Potential N-linked glycosylation sites werepredicted with the NetNGlyc 1.0 Server (10). A threshold value of anaverage potential score .0.5 was set to predict glycosylated sites. Lineagedetermination of studied genes was established by the Influenza A VirusGenotype Tool (17).

RESULTS AND DISCUSSION

We examined 394 birds representing 41 species (Table 1). AIvirus subtype H5 was detected in two mute swans (Cygnus olor)originating from the Central Bosnian district of Jajce. The OIEreference laboratory (Veterinary Laboratories Agency, Weybridge,United Kingdom) confirmed that both birds were H5 positive andidentified isolates as an N1 subtype with the multibasic HA0 aminoacid cleavage sequence PQGERRRKKR/GLF. Additionally, BIHisolates were tested with the restriction enzyme cleavage pattern assayas published by Fereidouni et al. (7), with positive results. TheH5N1-positive birds were found at the shore of Pliva Lake (latitude,44u209N; longitude, 17u119E). Conspicuous symptoms, includingsomnolence, lack of coordination, and ataxia, were noticed in twomute swans in a flock of 15 at Pliva Lake. Those two swans were

humanely killed and immediately sampled at the National ReferenceLaboratory for Avian Influenza and Newcastle Disease at theSarajevo Veterinary Faculty. After confirmation of an HPAIVinfection within 2 days, the rest of the flock was captured and culledbut tested negative for an HPAIV H5N1 infection by PCR. Grosspathology of the two diseased, virus-positive swans included poorconstitution, prostration with severe muscle atrophy, and dehydra-tion. The most consistent and predominant lesions in both birdswere multifocal and partly coalescent hemorrhages with necrosis inthe pancreas. Other findings included multifocal necrosis in theliver, subepicardial petechiation, and multifocal petechial hemor-rhages at the peritoneum. Similarly, the predominant histologiclesions were found in the brain, pancreas, and liver, presenting aslymphoplasmacytic encephalitis in the cerebrum, multifocal necrosisof the acini, and randomly distributed coagulative necrosis,respectively. Sequencing of the entire genome showed that the twoisolates obtained from swans were 100% identical (data forpolymerase acid, polymerase basic 1, polymerase basic 2, nucleo-protein, matrix, and nonstructural genes not shown here). The virusstrain was designated A/Cygnus olor/BIH/1/2006 (H5N1). Accord-

Table 1. Survey of examined bird species.

Bird speciesNumber ofbirds tested

Barn owl (Tyto alba) 2Black-headed gull (Larus ridibundus) 2Buzzard (Buteo buteo) 8Carrion crow (Corvus corone) 5Common coot (Fulica atra) 5Common crane (Grus grus) 10Common pheasant (Phasianus colchicus) 5Common quail (Coturnix coturnix) 2Common raven (Corvus corax) 2Common snipe (Gallinago gallinago) 3Common spoonbill (Platalea leucorodia) 3Eurasian blackbird (Turdus merula) 32Eurasian jay (Garrulus glandarius) 4Eurasian magpie (Pica pica) 9Eurasian nuthatch (Sitta europaea) 2Eurasian pygmy-owl (Glaucidium passerinum) 3Eurasian skylark (Alauda arvensis) 1Eurasian sparrowhawk (Accipiter nisus) 2Eurasian jackdaw (Corvus monedula) 10European goldfinch (Carduelis carduelis) 1European robin (Erithacus rubecula) 6European starling (Sturnus vulgaris) 3European turtle dove (Streptopelia turtur) 3Great spotted woodpecker (Dendrocopos major) 5Grey heron (Ardea cinerea) 16Grey partridge (Perdix perdix) 1Greylag goose (Anser anser) 4Hooded crow (Corvus cornix) 22House sparrow (Passer domesticus) 15Little grebe (Tachybaptus ruficollis) 3Long-eared owl (Asio otus) 1Mallard (Anas platyrhynchos) 53Marsh tit (Parus palustris) 3Mute swan (Cygnus olor) 17Northern goshawk (Accipiter gentilis) 7Northern pintail (Anas acuta) 3Pigmy cormorant (Phalacrocorax pygmaeus) 4Rock dove (Columba livia) 73Song thrush (Turdus philomelos) 37Tawny owl (Strix aluco) 5White stork (Ciconia ciconia) 2

H5N1 HPAI virus in mute swans in BIH 497

ing to the Influenza A Virus Genotype Tool (17) the studied genesof the investigated BIH isolates belong to the following lineages: HA(5J), NA (1J).

Phylogenetic analysis of the HA gene revealed that BIH isolatesbelong to phylogenetic clade 2.2 (32) and that they are closelyrelated to the Qinghai-like viruses circulating in birds throughoutEurope, Russia, Africa, and the Middle East since late 2005. The twoamino acid residues in HA-I99 and N268 (H5 numbering from theinitiating methionine residue used throughout) and NA-R110,assumed to be unique for migratory birds at Qinghai and PoyangLakes (5) were also present in the BIH isolates. The most likelyhypothesis of introduction of HPAIV H5N1 into BIH is by infectedwild birds through an unusual migratory route of the infected birdsdue to harsh weather conditions prevailing in the Black Sea

wintering grounds (30). In February 2006 average temperature indistrict Jajce was 1.1 C (2), which was considerably higher than theunusually low temperatures present at the traditional winteringgrounds of the affected avian species.

Sequence comparison of HA and NA genes segments withsequences in GenBank revealed that the BIH isolates and the onesfrom southern Russia (Astrakhan region) group together phyloge-netically, forming a monophyletic cluster in both genes andindicating that these isolates have evolved from the same origin(Figs. 1, 2). Similar topologies were confirmed using neighbor-joining, maximum parsimony, and maximum likelihood methods asimplemented in MEGA 4.0 software. Besides Astrakhan viruses,BIH virus was highly similar to viruses from Kurgan, Croatia,Germany, Denmark, and Nigeria. Position 403 in HA, used to

Fig. 1. Evolutionary relationship of the HA gene of BIH HPAIV H5N1 compared to the 50 most homologous sequences obtained fromGenBank. The tree comprising nucleotide sequences of the whole coding region (nucleotides [nt] 1–1707) was generated by the neighbor-joiningmethod using the Tamura-Nei c model as implemented in MEGA 4.0. Bootstrap values after 2000 resamplings are indicated in percentages at keynodes. The lengths of the horizontal lines are proportional to the number of nucleotide differences per site. Scale bar indicates number of nucleotidesubstitutions per site. Genetic clades are designated according to the World Health Organization/Food and Agriculture Organization/OIE (WHO/FAO/OIE) H5N1 Evolution Working Group (32). Tree is midpoint-rooted for means of clarity. The BIH virus is highlighted by italic bold letters.

498 T. Goletic et al.

differentiate between 2.2.1 and 2.2.2 subclades (27), was shown tobe D in BIH isolates, assigning BIH isolates to the 2.2.2 subclade(Fig. 1). The previously mentioned multibasic HA0 amino acidstretch, PQGERRRKKR/GLF, marker of strains highly pathogenicto poultry (21), was present at the cleavage site of BIH strain. Thiscleavage sequence is present in the majority of the European HPAIVH5N1 strains from the same time period and for the Qinghai virusesthat infected wild birds in 2005. Regarding pathogenicity, recentresearch has also shown that single amino acid residues at positionsD113, I124, E142, L154, K228, and S233 of HA of highlypathogenic viruses play an important role in the pathogenicity ofinfluenza viruses (11,14). In case of the BIH strain all these positions

but Q154 are occupied by residues typical for highly pathogenicviruses. Analysis of the deduced HA amino acid sequences of BIHHPAIV H5N1 isolates showed that host-specific residues in the HAprotein, according to the lists of Shaw et al. (26) and Chen et al. (4),were all avian-like including receptor specificity for the avian a-(2,3)linkage indicated by Q238 and G240 at the receptor binding pocket(11,19,28). Six potential glycosylation sites, four in HA1 and two inHA2, were identified and mapped with NetNglyc server (threshold:0.5) at the following positions: 27, 39, 181, 302, 500, and 559. Aunique mutation, L562V, was observed in BIH HA which was notpresent in the 50 most homologous sequences obtained from theGenBank database.

Fig. 2. Evolutionary relationship of NA gene of BIH HPAIV H5N1 compared to the 50 most homologous sequences obtained from GenBank.The tree comprising the whole protein coding region (nt 36–1385) was generated by neighbor-joining analysis with the Tamura-Nei c model, usingMEGA 4.0. Numbers below key nodes indicate bootstrap values of 2000 replicates by percentage. The lengths of the horizontal lines areproportional to the number of nucleotide differences per site. Scale bar indicates number of nucleotide substitutions per site. Genetic clades aredesignated according to WHO/FAO/OIE H5N1 Evolution Working Group (32). Tree is midpoint-rooted for means of clarity. The BIH virus ishighlighted by italic bold letters.

H5N1 HPAI virus in mute swans in BIH 499

The NA protein of BIH isolates was characterized by a 20–aminoacid deletion at position 49 to 68 in the stalk region of NA. Thisdeletion has been observed in many recent highly pathogenic H5N1isolates (3,15,30) and is suggested to be an adaptation to efficientreplication in chickens (18). Three N-linked glycosylated sequonswere predicted for N1 using the NetNglyc server (threshold: 0.5) atpositions 88, 146, and 235 (HPAIV H5N1 numbering system). Fiveamino acid residues—E119, V149, D151, R156, and H275—responsible for binding the drug Oseltamivir at the active site of NA(25) were conserved in BIH virus, predicting full susceptibility tothis drug. Two unique mutations, I50 and A342, observed in BIHHA sequences were not present in the 50 most homologoussequences obtained from the GenBank database.

In conclusion, HPAIV H5N1 infection in wild birds was observedfor the first time in BIH in February 2006. The identified BIH 2.2.2subclade virus was highly similar to viruses from southern Russia(Astrakhan region) isolated in the second half of 2005. Based on thisit is highly likely that the introduction of HPAIV H5N1 into BIHoccurred due to wild birds’ unusual migration routes during harshwinter conditions during 2005–06 in southern Russia and Ukraine.

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H5N1 HPAI virus in mute swans in BIH 501