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UntitledJournal of General Virology (2011), 92, 1172–1183 Reassortant low-pathogenic avian influenza H5N2viruses in African wild birds Chantal J. Snoeck,13 Adeniyi T. Adeyanju,23 Se´bastien De Landtsheer,1Ulf Ottosson,2,3 Shiiwua Manu,2 Ward Hagemeijer,4 Taej Mundkur4and Claude P. Muller1 1Institute of Immunology, CRP-Sante´ and Laboratoire National de Sante´, Luxembourg 2A. P. Leventis Ornithological Research Institute, University of Jos, Jos, Plateau State, Nigeria 3Ottenby Bird Observatory, Degerhamn, Sweden 4Wetlands International, Wageningen, The Netherlands To investigate the presence and persistence of avian influenza virus in African birds, we monitoredavian influenza in wild and domestic birds in two different regions in Nigeria. We found low-pathogenic avian influenza (LPAI) H5N2 viruses in three spur-winged geese (Plectropterusgambensis) in the Hadejia–Nguru wetlands. Phylogenetic analyses revealed that all of the genes,except the non-structural (NS) genes, of the LPAI H5N2 viruses were more closely related togenes recently found in wild and domestic birds in Europe. The NS genes formed a sister group toSouth African and Zambian NS genes. This suggested that the Nigerian LPAI H5N2 viruses foundin wild birds were reassortants exhibiting an NS gene that circulated for at least 7 years in Africanbirds and is part of the African influenza gene pool, and genes that were more recently introducedinto Africa from Eurasia, most probably by intercontinental migratory birds. Interestingly thehaemagglutinin and neuraminidase genes formed a sister branch to highly pathogenic avianinfluenza (HPAI) H5N2 strains found in the same wild bird species in the same wetland only1 year earlier. However, they were not the closest known relatives of each other, suggesting thattheir presence in the wetland resulted from two separate introductions. The presence of LPAIH5N2 in wild birds in the Hadejia–Nguru wetlands, where wild birds and poultry occasionally mix, Received 13 December 2010 provides ample opportunity for infection across species boundaries, with the potential risk of Accepted 18 January 2011 generating HPAI viruses after extensive circulation in poultry.
supports efficient short-range virus transmission of LPAIviruses by the faecal–oral route Wild birds, in particular of the orders Charadriiformes and High densities of mixed bird populations at Anseriformes, are considered natural asymptomatic reser- stopovers and non-breeding sites also promote intra- and voirs of low pathogenic avian influenza (LPAI) virus interspecies virus transmission ).
and the source ofinfluenza viruses in other species including poultry Wetlands in Africa are preferred non-breeding sites for All subtypes (H1–16 and N1–9) of many Eurasian migratory waterbirds. Although Eurasian avian influenza have been found in wild birds species can mix with resident African birds or intra-African Migration after the migrants in many important bird areas ; breeding season along distinct flyways contributes to long- ), only a few avian influenza viruses (AIV) have distance dissemination of influenza viruses been reported from Africa before 2006 ( and the aquatic environment of waterfowl 3These authors contributed equally to this paper.
The first seroprevalence study insub-Saharan Africa, conducted in commercial poultry in The GenBank/EMBL/DDBJ accession numbers for the nucleotide Nigeria between 1999 and 2004, did not detect antibodies to sequences reported in this paper are FR771823–FR771846.
influenza viruses In a first wild-bird Colour versions of Figs 1 and 2, showing phylogenies of the H5 and NS surveillance effort, a wide variety of LPAI viruses were genes, and six supplementary figures showing phylogenies of the NA,PA, PB1, PB2, matrix and NP genes are available with the online version identified in migratory waterbirds of this paper.
Interest in AIV in wild birds in Africa further increased in 029728 G 2011 SGM Printed in Great Britain LPAI H5N2 viruses in Nigerian wild birds 2006 when highly pathogenic avian influenza (HPAI) H5N1 the common teal (Anas crecca) from Egypt is not known virus was first identified in Africa in Nigerian poultry farms (After the independent introduction ofthree sublineages of clade 2.2 viruses into poultry, HPAI In this study, we investigated the presence of avian H5N1 viruses have undergone multiple reassortments and influenza in wild and domestic birds in two different reassorted viruses have largely replaced the initial sub- locations in Nigeria in 2008. The Dagona wildlife sanctuary lineages The route of introduction of is a protected area within the Hadejia–Nguru wetlands, H5N1 is unknown and both migratory birds and poultry located in north-eastern Nigeria, and is part of the Chad trade might be suspected Basin national park. Large numbers of migratory birds mix Although efforts to isolate influenza viruses with local wild birds in the Hadejia–Nguru wetlands. The from wild birds in Africa intensified, few met with success Hadejia–Nguru wetlands are also the most important bird area in the region, from which the first HPAI H5N1 probably been infected by scavenging H5N1-infected outbreak in Africa was reported. The Amurum Forest poultry but the infection route of reserve is also a protected area located 15 km north-east of Table 1. List of sampled domestic and wild bird species (order and family) in Dagona wildlife sanctuary and in the Amurum forestreserve and villages around Jos, Nigeria in 2008 For each family, the number of samples from domestic birds is given in parentheses.
Dagona wildlife sanctuary Amurum Forest reserve and villages around Jos No. species per family No. samples per family No. species per family No. samples per family
C. J. Snoeck and others Journal of General Virology 92 LPAI H5N2 viruses in Nigerian wild birds Fig. 1. Dated phylogeny of H5 genes. The horizontal axis represents calendar years and nodes correspond to mean TMRCAs.
Posterior probability values .0.75 are shown. Representative strains from Africa, Europe and Asia are included. Virus strainscharacterized in this study are shown in boldface type. *, HPAI strains. Node I represents the mean TMRCAs of the LPAI H5N2cluster. Node II corresponds to the most recent common ancestor of the Nigerian LPAI and HPAI supported by a high posteriorprobability value.
Jos (Plateau State), very close to the urban community mammals [PB2 627E, 701D, 714S, PB1 678S, PA 615K and ). We did not find any HPAI H5N1 virus, NP 319N (] or drug resistance [M2 26L, but did find LPAI H5N2 strains that contained genes from 27V, 30A, 31S and 34G NA 119E ( the Eurasian gene pool and a non-structural (NS) gene that was most closely related to other African viruses.
294N ] was found.
Interestingly the haemagglutinin (HA) genes were part ofa cluster that also contained HA genes from HPAI H5N2 Phylogenetic analyses of the eight genes reveal a viruses found in the same bird species and in the same separate origin for the NS gene region 1 year earlier.
To explore the evolutionary origin and the time ofintroduction of LPAI H5N2 to Nigeria, tree topologies were assessed and times to the most recent common ancestor(TMRCAs) were estimated using BEAST version 1.5.3. For Avian influenza prevalence is low each gene, the three LPAI H5N2 viruses were geneticallyhighly similar (Kimura distance from 0 to 0.9 %) and always A total of 1024 samples were collected in the Dagona clustered together (100 % posterior probabilities for all wildlife sanctuary from 44 wild bird species, including genes), suggesting that the three strains recently evolved waterfowl, waders and passerines, and from 373 domestic from a common ancestor and resulted from a single poultry such as chickens, ducks and geese introduction event in the Nigerian wetlands. All of the genes Around Jos, cloacal samples were collected from 374 belonged to the Eurasian and not the American lineage, but domestic birds (362 chickens, 5 ducks and 7 turkeys). In none of them were related to HPAI H5N1 strains from addition 154 wild birds corresponding to 52 species, Nigeria, from other parts of Africa or from Eurasia.
mainly passerines, were sampled in the Amurum Forestreserve Only three fresh faecal samples collected Phylogenetic analyses of the HA gene revealed that the from spur-winged geese (Plectropterus gambensis) in a LPAI H5N2 viruses were most closely related to a EuropeanH5N3 isolate (A/duck/Tulcea/RO-AI-LPAI/2009) recently found in a sentinel duck in Romania The NA (12u44.676 N, 10u40.001 E) were influenza A positive.
genes of the LPAI H5N2 viruses were most closely related They were collected on 31 March 2008 (A/spur-winged to an H2N2 strain from the Netherlands (A/mallard/ goose/Nigeria/2/2008) and on 3 April 2008 (A/spur-winged Netherlands/14/2007) (Supplementary Fig. S1, available in goose/Nigeria/210/2008 and A/spur-winged goose/Nigeria/ JGV Online). The LPAI H5N2 viruses formed a sister clade 226/2008). Thus, in our study, the prevalence of AIV in the to strains recently isolated from wild birds in Europe (HA Dagona wildlife sanctuary was 0.3 % (3/1024). All samples and NA genes) and Asia (NA gene). Interestingly they also collected in Plateau State were influenza A negative.
formed a sister branch with highly pathogenic H5N2strains that had been isolated one year earlier in a similar Molecular analyses show no marker of virulence location (10 km away) from the same wild bird species(HA and NA genes). In the same HA cluster, closely related Genotype-specific PCRs were positive for H5 and N2 for the HPAI and LPAI H5N2 viruses, both from South African three viruses and all of the genes were sequenced using ostriches (A/ostrich/South Africa/AI1160/2006, LPAI; and previously published or newly designed primers. Analyses of the HA gene showed that the predicted amino acid sequence another sister branch to the Nigerian LPAI H5N2 viruses.
of the cleavage site of the three viruses corresponded to a low However, all African isolates did not share a direct pathogenic pathotype (PQRETR*GLF). They had a gluta- common ancestor, suggesting that they did not directly mine at position 226 and a glycine at position 228 evolve from each other. The TMRCAs of the LPAI H5N2 (numbering for the H3 subtype) indicating a higher binding HA and NA genes ranged from February 2007 to March affinity for sialic acid a2,3, which is characteristic of avian 2008 (95 % highest posterior density interval, HPD) and cell-surface receptors ( October 2006 to March 2008 (95 % HPD), respectively.
No stalk deletion in the neuraminidase (NA) gene,nor additional predicted glycosylation sites, both proposed The influenza virus PA polymerase subunit (PA) gene of to be associated with poultry adaptation, were detected the LPAI H5N2 viruses formed a sister clade with PA genes from a goose and wild and captive ducks from France, the genetic marker associated with increased virulence in Netherlands and Sweden (Supplementary Fig. S2, available
C. J. Snoeck and others Journal of General Virology 92 LPAI H5N2 viruses in Nigerian wild birds Fig. 2. Dated phylogeny of NS genes clustering in allele B. The horizontal axis represents calendar years and nodes correspondto mean TMRCAs. Posterior probability values .0.75 are shown. Representative strains from Africa, Europe and Asia areincluded. Virus strains characterized in this study are presented in boldface type. Node I represents the mean TMRCAs of theLPAI H5N2 cluster. Node II corresponds to the most recent common ancestor of the African NS genes, including the NigerianLPAI H5N2.
in JGV Online). The TMRCA of the LPAI H5N2 cluster few exceptions (, was estimated as being between July 2005 and February 2008 (95 % HPD). The polymerase basic protein 1 (PB1) gene showed that the LPAI H5N2 viruses formed a sister has received attention in Africa only after the introduction clade with two wild-bird viruses from the Netherlands (A/ of HPAI H5N1 viruses. In our survey, three of 1024 (0.3 %) samples collected in the Dagona wildlife sanctuary, a part Netherlands/17/2007, H11N8) and a virus from one of the Hadejia–Nguru wetlands, in north-eastern Nigeria domestic goose from the Czech Republic (A/goose/Czech were positive for influenza A. This corresponds to a Republic/1848/2009, H7N9; Supplementary Fig. S3, avail- prevalence of 1 % (3/312) in birds from the orders able in JGV Online). The TMRCA of the LPAI H5N2 Charadriiformes and Anseriformes that are considered cluster was estimated as being between June 2006 and the natural reservoir of AIV In spur- March 2008 (95 % HPD). The PB2 genes of the three winged geese we found an infection rate of 2.4 % (3/123) H5N2 strains were most closely related to A/quail/Italy/ compared with 8.2 % (8/97) in the earlier study 4610/2003 (H7N2) and the TMRCA of the LPAI H5N2 The three viruses found were low-pathogenic cluster ranged from June 2005 to March 2008 (95 % HPD; H5N2 viruses most closely related to each other over their Supplementary Fig. S4, available in JGV Online).
full genomes. A tenfold higher prevalence (3.9 %) wasfound in waterbirds in the Hadejia–Nguru wetlands in the The matrix gene sequences were more closely related to an previous year This is similar to the H9N2 matrix gene from the Netherlands (A/Bewick's overall prevalence (3.5 %) found in a previous study in swan/Netherlands/5/2007) and the TMRCA ranged from wetlands throughout 12 African countries, including June 2005 to January 2008 (95 % HPD; Supplementary Fig.
several neighbouring countries of Nigeria S5, available in JGV Online). The three nucleoprotein (NP) Many factors including the year, season, location, genes clustered with viral genes isolated from four ducks, species and age of birds influence the prevalence of AIV, as one swan and one turnstone from Italy, the Netherlands, is known from wild birds in Europe and America Hungary, Germany and Sweden; the TMRCA of the cluster formed by these three genes ranged from June 2005 to February 2008 (95 % HPD; Supplementary Fig. S6,available in JGV Online).
Phylogenetic analyses revealed that all genes of our LPAIH5N2 viruses were most closely related to genes circulating The NS gene sequences clustered in allele B and formed a in the Eurasian wild-bird influenza gene pool. It has been sister group to three South African NS sequences (A/ shown that migratory birds can carry LPAI viruses from ostrich/South Africa/AI1091/2006, H5N2; A/duck/South one continent to another ; Africa/1108/2004, H3N8; and A/duck/South Africa/1233A/ introducing new genes and mixing gene pools 2004, H4N8) and to an H3N6 gene from Zambia (A/ pelican/Zambia/01/2006) Values larger than 95 % Three main migratory flyways link Africa to Eurasia HPD were observed for the TMRCA of the Nigerian LPAI (the East Atlantic flyway, the Black Sea–Mediterranean H5N2 cluster (January 2002 to February 2007), which may flyway and the East Africa–West Asia flyway; indicate an older origin for that gene, although we cannot and Nigeria is located where they intersect. Thus, low exclude that our dataset for the NS segment does not pathogenic avian influenza genes may have been intro- contain sufficient phylogenetic signal to provide precise duced from Eurasia to Africa by migratory birds. However, estimates. Also, the exclusion of the distantly related NS spur-winged geese are in principle a sedentary sub-Saharan gene of A/duck/NZL/160/1976 (H1N3) from the phylo- species, normally making only short daily flights, and only genetic analysis had little influence on the TMRCA. The rarely longer flights, depending on the availability of water.
close relationship suggests that all seven viruses shared a The species is widespread throughout sub-Saharan Africa, recent common ancestor that was introduced to Africa but does not leave the continent (TMRCA of the African NS cluster, node II in During the dry season, spur-winged geese November 1997 to May 2003).
are highly gregarious around permanent waterbodiessuch as in the Hadejia–Nguruwetlands where they mix with Afro-tropical birds and Eurasian migratory birds such as garganey (Anas querque- Although avian influenza has been extensively studied for dula), northern pintail (Anas acuta) or ferruginous duck decades in wild birds from the northern hemisphere, with (Aythya nyroca) ( C. J. Snoeck and others The mingling of Eurasian migratory and from the Eurasian lineages have been introduced into African bird species in the Hadejia–Nguru wetlands seems North America and gradually replaced the American H6 to be reflected also in the AIV gene pools.
Interestingly, the NS gene sequences of the three LPAI The presence of LPAI H5N2 viruses in African wild birds H5N2 viruses from Nigeria shared a common ancestor represents a certain risk also for poultry. Infected wild with South African and Zambian influenza isolates from birds, such as spur-winged geese, may introduce LPAI 2004 and 2006, suggesting another origin for the NS gene, viruses into free-ranging domestic ducks reared in the in contrast to all of the other genes. Also, the TMRCA of wetlands in north-eastern Nigeria with whom they the Nigerian LPAI H5N2 NS genes suggested that the occasionally mix ). Similar situations cluster emerged in approximately October 2004 (January have already been observed in South Africa where wild 2002–February 2007; 95 % HPD; node I in whereas birds were suspected to have infected domestic birds with the mean TMRCAs for the other genes were found to be whom they shared similar genes Farmed ostriches during late 2006 or 2007 The TMRCA of the were infected with H6N8 in 1998 and African monophyletic cluster, which included the Nigerian, 2007 with H5N2 viruses in 2004 and South African and Zambian strains, suggested that a virus was introduced to Africa between 1997 and 2003. This H9N2 in 2008. H10N7 was also found in domestic ducks ancestral virus evolved and probably reassorted with other ) In addition, HPAI viruses viruses present in the African wild bird population, as has are thought to emerge after extensive circulation of H5 already been suggested (and H7) LPAI subtypes and adaptation in poultry The NS gene was acquired by viruses that later spread throughout Africa. Ring This is of particular concern since recoveries have shown that some intra-African migratory the Nigerian LPAI H5N2 strains belong to a genetic cluster birds from South Africa [e.g. comb duck (Sarkidiornis that seems to have an increased propensity to develop the melanotos)] sometimes migrate as far north as West Africa highly pathogenic phenotype. Indeed, from a common and thus would be able to transmit node (node II, that emerged between June 2002 and AIV over long distances within Africa. The presence of May 2004 (95 % HPD), highly pathogenic H5N2 strains similar genes over a four year period (2004–2008; emerged twice in South Africa in 2004 (not shown in and its probable introduction at least 7 years ago suggests because of its shorter sequence; and in that AIV can persist in the African bird population.
2006 and in Nigeria in 2007 The Eurasian–African AIV interface resembles the situationin the Bering Strait where interregional transmission of Interestingly, the latter Nigerian HPAI H5N2 viruses were influenza viruses occurs between North American and found only 1 year earlier, about 10 km away in the same Eurasian birds. Genes from the Asian lineage have been wetlands and in the same wild bird species as the LPAI found in Alaska ) H5N2 described in this study. For both the HA and NA and genes from the American lineages have been found in genes, the HPAI H5N2 formed a sister branch to the LPAI Japan producing a variety of reassortants.
H5N2 but were not the closest known relatives of each Also, similarly to the persistence of Eurasian genes in other, suggesting that their presence in the wetlands Africa, now constituting the African gene pool, H6 genes resulted from two separate introductions. The question Table 2. Estimated TMRCAs of the Nigerian LPAI H5N2 cluster, nucleotide substitution model and sequence length used in theBayesian analyses General time-reversible (GTR) substitution model with a gamma (C) and invariant (I) site heterogeneity model.
Mean TMRCA (95 % HPD) Nucleotide substitution model Sequence length (nt) March 2007 (Jun 05 – Mar 08) June 2007 (Jun 06 – Mar 08) March 2007 (Jul 05 – Feb 08) October 2007 (Feb 07 – Mar 08) January 2007 (Jun 05 – Feb 08) GTR+I+C with simplifications: CP3.cg5CP3.gt, August 2007 (Oct 06 – Mar 08) December 2006 (Jun 05 – Jan 08) GTR+I+C with simplifications: CP3.cg5CP3.at, October 2004 (Jan 02 – Feb 07) GTR+C with simplifications: CP3.cg5CP3.gt Journal of General Virology 92 LPAI H5N2 viruses in Nigerian wild birds Fig. 3. Genetic relationship of African AIV viruses, other than HPAI H5N1 viruses, sequenced since 2004, by year, in wild anddomestic birds. Genes have the same colour code if they share a direct common ancestor. Unsequenced genes are indicatedas dotted bars and genes that have no African sister gene are shown as white bars. Phylogenetic relationships were firstassessed by comparing the African strains with all avian influenza strains downloaded on to the NCBI Influenza Virus Resourcedatabase, but based on shorter fragments, depending on the shortest sequences available for the African strains.
Representative strains were selected for each gene based on these preliminary analyses and trees were calculated using MEGA4by using the neighbour-joining method and by using the Kimura two parameters model and 1000bootstrap replicates. *, Values corresponding to the probability values from The figure suggests, based on the availabledata (from South Africa, Nigeria and Zambia), that some avian influenza viruses may be maintained in the African wild-birdpopulation where reassortment events can occur, and that these viruses can be transmitted from wild to domestic birds. Only A/DK/SA/811/04 (LPAI H5N1) does not share any gene with a domestic bird strain. OS, Ostrich; EG, Egyptian goose; SWG,spur-winged goose; WFWD, white-faced whistling duck; PD, pekin duck; PE, pelican; SA, South Africa.
C. J. Snoeck and others also remains as to where the Nigerian HPAI H5N2 virus were predicted for HA and NA by using the NetNGlyc 1.0 server acquired its HPAI phenotype. The wetlands in north- (http://www.cbs.dtu.dk/services/NetNGlyc/). Kimura distances were eastern Nigeria provide ample opportunity for cross- calculated with MEGA4 (using the Kimura twoparameters model. For each gene, phylogenetic relationships were species infection and perhaps even the generation of inferred by comparing the LPAI H5N2 strains with all avian influenza HPAI viruses, which normally only occur after circulation strains downloaded to the NCBI Influenza Virus Resource database and adaptation in poultry.
(up until 15 December 2009; www.ncbi.nlm.nih.gov./genomes/FLU/)after removing short sequences and sequences with insertions or In conclusion, we report the presence of LPAI H5N2 deletions resulting in frame shifts. Datasets were aligned using viruses in wild birds in an African wetland, which were CLUSTAL W Coding regions were used for reassortants with genes from the Eurasian and African gene phylogenetic analyses and only the first ORFs were used for the pools, as strong evidence of the introduction of low- matrix and NS genes. Trees were calculated using MEGA4 ( pathogenic avian influenza into Africa by Eurasian with the neighbour-joining method by using the Kimura migratory birds. Furthermore, the circulation of LPAI two parameters model and 1000 bootstrap replicates. Representativestrains were selected for each gene based on these preliminary and HPAI H5N2 strains in wild birds in African wetlands that emerged from a cluster that had an increasedpropensity to develop the highly pathogenic phenotype Tree topologies, substitution rates and TMRCAs were estimated by aBayesian Markov-chain Monte Carlo (MCMC) method represents a high risk for poultry, especially in areas with implemented in BEAST version 1.5.3 low biosecurity that provide opportunities for cross-species Depending on the available details on isolation dates, the exact isolation dates, the mid-month dates (15th), the mid-interval dates or the mid-year dates were used as calibration points.
For each dataset, different substitution models with two codon partitions to allow independent estimates for the third codonposition, two uncorrelated relaxed-clock models (log-normal and Wild bird surveillance. Wild birds were captured with mist nets in exponential distributions; and two the Dagona wildlife sanctuary in north-eastern Nigeria between 28 coalescent models (constant population size and Bayesian skyline; March and 22 April 2008. Oropharyngeal and cloacal swabs as well as were compared visually in TRACER version fresh faecal samples were collected after ensuring the species of origin.
1.5.3 and statistically using a Bayes Domestic poultry in the villages around the wetlands were also factor test (the ratio of the marginal likelihoods of two models) sampled. All samples were collected in triplicate with cotton swabs, as implemented in TRACER stored in virus transport medium [PBS pH 7.0 with 2000 U version 1.5.3, in order to identify the model that fitted the data best.
streptomycin ml-1, Evidence against the null model, which is the model with the lowest B ml-1, 250 mg gentamicin ml-1, 60 mg ofloxacin ml-1, 200 mg marginal likelihood, was assessed by the method proposed by sulfamethoxazole ml-1 and 2.5 mg amphotericin B ml-1] and placed When there was no evidence against the null model or directly into liquid nitrogen in the field. In addition, between 4 when this evidence was weak, the simplest model was kept to avoid December 2007 and 5 March 2008, swabs were collected from wild unnecessary overparameterization. Analyses revealed that the GTR birds in the Amurum forest reserve (Plateau State) and from backyard substitution model, assuming an uncorrelated exponential relaxed poultry in five villages around Jos.
clock and a constant population size, was the model that best fittedthe data for all genes. For matrix, NP and NS genes, the GTR model RNA Extraction, RT-PCR and sequencing. RNA was extracted was further simplified to avoid overparameterization Two from 50 ml of virus transport medium by using a MagMAX-96 AI/ND to three runs of 50–1006106 generations of the MCMC method were Viral RNA Isolation kit (Ambion) and a KingFisher 96 (Thermo performed and sampled to produce 10 000 trees each. Convergence of Fisher). Influenza A-positive specimens were detected by using a real- the runs was confirmed in TRACER version 1.5.3. The results of time RT-PCR assay targeting the matrix gene and previously multiple runs were combined using LogCombiner version 1.5.3 published gene-specific primers and probe with a burn-in of 10–25 %, RT-PCRs were carried out using the following summarized into the maximum clade credibility tree using cycling conditions: reverse transcription for 30 min at 50 uC, TreeAnnotator version 1.5.3 and denaturation at 95 uC for 15 min followed by 40 cycles of visualized in FigTree version 1.3.1 amplification at 95 uC for 10 s, 60 uC for 20 s. Amplifications wereperformed with a Qiagen OneStep RT-PCR kit using 2 ml of RNA in afinal volume of 25 ml. Matrix-positive samples were tested for H5 H7 (http://www.defra.gov.uk/vla/science/docs/sci_ai_vi536.pdf) and N1 genotypes. The The authors wish to thank A. Sausy and E. Charpentier for technical eight genes were then amplified by using several PCRs targeting help, D. Kihlberg, P. O ¨ sterman, A. Eriksson, Dr Ullarama and the overlapping fragments (primer sequences and details available upon A. P. Leventis Ornithological Research Institute (APLORI) for their request). PCR products were purified using a JetQuick PCR expertise in sample collection and the Nigerian Authority for Purification Spin kit (Genomed). Sequencing was performed as authorization and support. They also acknowledge the Wild Bird previously described using PCR primers as Global Avian Influenza Network for Surveillance (GAINS) project of sequencing primers.
the Global Health Program, Wildlife Conservation Society, funded byUSAID and Wetlands International, for financial support. They Molecular and phylogenetic analyses. Sequence assembly and gratefully acknowledge the Ministry of Cooperation of Luxembourg, analyses were performed using SeqScape version 2.5 (Applied the Ministry of Health, the Ministry of Research and the Centre de Biosystems) and BioEdit The nucleotide sequences are Recherche Public-Sante´ for their generous financial and moral available in the GenBank/EMBL/DDBJ databases under the accession support. C. J. S. was supported by an AFR fellowship from the numbers FR771823–FR771846. Potential N-linked glycosylation sites Fonds National de la Recherche, Luxembourg. This study was also Journal of General Virology 92 LPAI H5N2 viruses in Nigerian wild birds supported by the contribution No. 242 from Ottenby Bird Drummond, A. J., Ho, S. Y., Phillips, M. J. & Rambaut, A. (2006).
Observatory and contribution No. 43 from APLORI.
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