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1 Institute of Immunology, National Public Health Laboratory, 20A rue Auguste Lumière, L-1950 Luxembourg, Luxembourg
2 Department of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria
3 Institut de Recherche en Sciences de la Santé, 399 Avenue de la liberté, BP 545 Bobo-Dioulasso, Burkina Faso
4 Laboratoire National de l'Elevage, 03 BP 7026 Ouagadougou, Burkina Faso
5 Department of Virology, Erasmus Medical Center, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands
Correspondence
C. P. Muller
Claude.Muller{at}LNS.ETAT.LU
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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These authors contributed equally to this work. 
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AM262524–AM262578 and AM502998–AM503074.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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) reported high mortality, which initially was attributed to Newcastle disease. However, on 7 February 2006, the Food and Agricultural Organization reported that the highly pathogenic avian influenza (HPAI) H5N1 virus was the cause of havoc in these farms. Poultry farming is reportedly one of the most important industries in the country, second only to oil production, providing both high-quality animal protein for human consumption and secure jobs for an impoverished population. Many of the large commercial poultry farms maintain a high biosecurity level, but in other large farms and most small farms biosecurity is inadequate. In addition, backyard rearing of chicken and live-poultry markets are common throughout most of Nigeria. The main hub of the poultry industry is located in the small south-western states of the country (Oyo, Osun, Ogun and Lagos).
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). Thus, south-west Nigerian commercial poultry farms seem to have been free of AIV at least until May 2004. Similar studies are not known from other west African countries. Since the first outbreak in Nigeria, seven other African countries have officially reported HPAI H5N1 to the World Organization for Animal Health (OIE): Niger, Egypt, Cameroon, Burkina Faso, Côte d'Ivoire, the Sudan and Djibouti (www.oie.int).
In Burkina Faso, poultry is mainly reared in backyards. Poultry imports arrive from all over the world to west Africa, and biosecurity measures such as quarantine are not applied rigorously. Moreover, migratory birds of both the east African/west Asian flyway and the Black Sea/Mediterranean flyway (Olsen et al., 2006) seek refuge in a number of important bird areas in west Africa, adding to the risk of introduction of HPAI H5N1 in the region. Therefore, the origin of the outbreaks and the routes of virus transmission remain unclear.
The aim of this study was to investigate and compare the molecular evolution of HPAI H5N1 in Nigeria, Burkina Faso and throughout Africa, utilizing partial and full-length sequences.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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RNA isolation, RT-PCR, sequencing and data analysis.
RNA was isolated and RT-PCR and sequencing were carried out as described previously (Ducatez et al., 2006a). Phylogenetic and molecular evolutionary analyses were conducted using PAUP version 4.0 beta 10 (Swofford, 2003) with the maximum-likelihood method. The model was calculated with MODELTEST (Posada & Crandall, 1998).
Substitution rates were calculated using the BEAST program (http://evolve.zoo.ox.ac.uk/Beast/) (Drummond et al., 2002, 2006). All estimates incorporated the HKR model of DNA substitution as it was the best supported model in MODELTEST (Posada & Crandall, 1998).
Omega (dN/dS) values correspond to nucleotide substitution rate ratios of non-synonymous and synonymous mutations. These were calculated for each gene using PAML version 3.14 (Yang, 1997; http://abacus.gene.ucl.ac.uk/software/paml.html). A test was carried out also with PAML version 3.14 to detect individual codons under positive natural selection (Nielsen & Yang, 1998; Yang et al., 2000). The model (M0–M13) best adapted to the dataset was selected with likelihood ratio tests with P<0.01 (Anisimova et al., 2003; Sainudiin et al., 2005; Yang et al., 2000). The Bayes empirical Bayes method was preferred to the naive empirical Bayes method (Yang et al., 2005). Residues with positive natural selection positions were selected only if 
<0.05.
Virus isolation and characterization.
Virus isolates were obtained by inoculation of embryonated chicken eggs or Madin–Darby canine kidney cells, as described previously (Ducatez et al., 2006a).
Antigenic properties of H5N1 virus strains were compared by haemagglutination inhibition using post-infection ferret or duck antisera generated against the following influenza viruses: A/tern/South Africa/1961 (H5N3) (TE/SA), A/duck/Hong Kong/205/1977 (H5N3) (DK/HK), A/Hong Kong/156/1997 (H5N1) (HK/97), A/mallard/Netherlands/3/1999 (H5N2) (MA/NL), A/mallard/Sweden/49/2002 (H5N9) (MA/SE) and A/turkey/Turkey/1/2005 (H5N1) (TY/TY) (Table 2). Haemagglutination inhibition assays were performed in duplicate. All serum samples were treated overnight with receptor-destroying enzyme at 37 °C and subsequently incubated at 56 °C for 1 h. Twofold serial dilutions of antiserum, starting at 1 : 20, were tested for their ability to inhibit the agglutination of turkey red blood cells using 4 haemagglutinating units of influenza A virus.
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RESULTS |
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Virus isolates from Nigeria were recovered from animals of 15 flocks and six farms in the south-west of Nigeria (Fig. 1
). Increased morbidity was reported in the SO layer farm from mid-January and mortality eventually reached 100 % in the different flocks (mean±SD mortality of 77±33 %). Chickens were 29–79 weeks of age at the time of sample collection and no obvious correlation between flock age and mortality was observed. At necropsy, the chickens showed pinpoint haemorrhages on peritoneal and pericardial fat, congested lungs and greenish faecal pasting of the vent, as well as subcapsular hepatic blood clots. In farms in the south-west, layers of 35–45 weeks of age were found to be infected by the H5N1 virus.
In Burkina Faso, H5-positive strains originated from three adult chickens from two backyards with 100 % mortality in Bobo-Dioulasso and in Sokoroni (in the western part of the country, Fig. 1). H5-positive adult hooded vultures (Falconidae, Accipitridae, Aegyptiinae, Necrosyrtes monachus) collected in Ouagadougou suffered from paralysis, dyspnoea, stiff neck, asthenia and locomotion problems (Ducatez et al., 2007).
Viral sequence analyses
Sequencing of haemagglutinin (HA) and neuraminidase (NA) gene segments revealed in all cases that the virus was of the H5N1 subtype. The HA cleavage site sequence PQGERRRKKRG was identical to the cleavage site of the recent HPAI strains in Europe, Russia, central Asia and Africa, but differed by one to three amino acids from south-east Asian HPAI strains. The HA cleavage site of the west African strains contained only one basic amino acid less than the lineages in south-east China, Vietnam and Cambodia (PQRERRRKKRG) and Thailand (PQREKRRKKRG).
Full-length genomes of four strains from the Nigerian SO farm (A/chicken/Nigeria/SO300/2006, -SO452/2006, -SO493/2006 and -SO494/2006), two strains from the Nigerian BA farm (A/chicken/Nigeria/BA210/2006 and -BA211/2006), A/chicken/Nigeria/IF10/2006, A/chicken/Nigeria/FA7/2006, A/chicken/Nigeria/AB14/2006, A/chicken/Nigeria/OD9/2006, A/chicken/Burkina Faso/13.1/2006 and A/hooded vulture/Burkina Faso/1-2/2006 were compared with full-length sequences available from GenBank. Only sequences from 2005/2006 were closely related and are included in Figs 2 and 3. In addition, partial genomic sequences were obtained from seven birds (seven HA, four M, three NA, five NS, three NP, five PA, four PB1 and two PB2 sequences). These sequences were used in the substitutions analysis, for reassortment evaluation and for substitution rate calculations; but they were not included in the phylogenetic analysis.
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) and full genome (Fig. 3) of HPAI H5N1 strains showed that, with the exception of the SO farm, strains from all of the farms in the south-west of Nigeria, A/chicken/Nigeria/1047-8/2006, -10/2006, -30/2006, -34/2006, -54/2006 and A/ostrich/Nigeria/1047-25/2006, as well as the strains from Niger, were genetically very close to each other. These were collectively referred to as sublineage A strains. The strains from sublineage A shared five specific mutations in HA (168G, 237A, 1233C, 1431A and 1587C, all synonymous; numbering according to Claas et al., 1998 and Ha et al., 2001) and 45 in the complete genome that were not found in the other sublineages. Two additional sublineages emerging from a common node were found in the SO farm (sublineage B) and in northern Nigeria (sublineage C) (Figs 2 and 3). Sublineage B contained all strains from the SO farm in south-western Nigeria, as well as all Egyptian strains (e.g. A/duck/Egypt/2253-3/2006) and shared six [468A, 633T, 699T, 852G, 990T and 1658T (S
F), the latter being the only non-synonymous mutation] and 26 specific nucleotides in the HA and full genome, respectively. Sublineage C contained A/chicken/Nigeria/641/2006 and all strains from Burkina Faso, Côte d'Ivoire and the Sudan available in GenBank and was characterized by two (405G and 615A) and 13 shared specific nucleotides in the HA and the full genome, respectively.
The phylogenetic analysis also confirmed that the strains from west Africa were more closely related to the central Asian strains than to each other. Sublineages A, B and C were most closely related to strains from Astrakhan, Slovenia and Kurgan, respectively (Figs 2 and 3). For the HA, sequences available from Germany clustered in two of these distinct sublineages: for instance, A/swan/Germany/R65/2006 in sublineage A and A/common bussard/Bavaria/2/2006 in sublineage B. Phylogenetic analyses of all gene segments were carried out individually and phylogenetic trees were similar whichever segment was analysed, excluding any reassortment in the newest strains presented here (Fig. 2 for HA; data not shown for other gene segments).
Substitution rates and selection pressure
The 26 available full-length sequences of African HPAI H5N1 were compared with the most likely common ancestor, A/Bar-headed goose/Qinghai/65/2005, to calculate substitution rates (Table 1). African outbreaks from which sequences were available occurred within 7 months and on average 1 year after the sampling of A/Bar-headed goose/Qinghai/65/2005. The mean substitution rates per gene segment ranged from 5.2x10–4 (for NA) to 3.3x10–3 (for HA) substitutions per site per year. The exact sequence of the strain that initially entered the SO farm and evolved in this farm during the 5 weeks was unknown. A second method was therefore used to calculate substitution rates without the ancestor sequence and values were compared. The virus substitution rate within 5 weeks in the Nigerian SO farm was slightly higher than the virus substitution rate calculated using the likely ancestor of all African strains (A/Bar-headed goose/Qinghai/65/2005) (Table 1).
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values (dN/dS) of 0.097 (for PB1) to 0.435 (for M2) (Table 1).
Antigenic variation
The antigenic variation of the influenza viruses from sublineage B (SO farm) and sublineage A (BA farm) was in agreement with the genetic data for HA (Table 2). In haemagglutination inhibition assays using post-infection ferret and duck antisera, the two virus isolates from the BA farm were quite similar to one another and slightly different from the three virus isolates obtained from the SO farm, in particular when measured with antisera raised against A/turkey/Turkey/1/2005. All five Nigerian virus isolates tested in the haemagglutination inhibition assay were antigenically most similar to influenza virus A/turkey/Turkey/1/2005, a WHO-recommended vaccine strain. The Nigerian strains were antigenically distinct from A/Hong Kong/156/1997 and revealed antigenic differences to strains isolated previously from Vietnam and Indonesia, as measured with ferret antisera to classical H5 strains and the teal antiserum raised against A/turkey/Turkey/1/2005.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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In the new strains reported here, no evidence of reassortment was found, in contrast to A/chicken/Nigeria/1047-62/2006 where HA, NP, NS and PB1 gene segments clustered with sublineage C strains and NA, M, PA and PB2 clustered with sublineage A strains (Salzberg et al., 2007). Human cases reported so far in Africa (Egypt and Djibouti) have all been caused by sublineage B, but it would be dangerous to infer a lower infection potential of sublineages A and C strains for humans.
Substitution rates
HPAI H5N1 substitution rates in Africa were high, reaching on average 2.6x10–3 substitutions per site per year. This is well within the range of substitution rates of avian influenza viruses in general and H5N1 in particular, as described by Chen & Holmes (2006) using the same calculation method. During the 1994 H5N2 outbreak in Mexico, higher substitution rates (0.88x10–2 to 2.8x10–2 substitutions per site per year) were reported (Garcia et al., 1997) but flocks were partially vaccinated.
To date, estimates of mutation rates from a single farm are rare. Under the assumption of a single introductory event into the SO farm, a direct ancestral relationship between strains isolated subsequently in the farm and a 5 week evolution in the farm (15 January to 20 March 2006), the substitution rate of the virus for each gene segment including 11 HA sequences was calculated. The rate of 4.58x10–2 substitutions per site per year (for HA, Table 1) was higher than the substitution rate calculated within Africa using the same calculation method (1.60x10–2 substitutions per site per year; Table 1). This suggests that HPAI H5N1 mutated faster within a farm with a virtually unlimited supply and high density of susceptible (unvaccinated) animals than on its way from Qinghai to Africa.
Based on the HA substitution rate in the SO farm (sublineage B), it would have taken a minimum of 18 weeks for the south-west Nigerian strains (sublineages A and B) to evolve from a common ancestor and 13–14 weeks for each of the south-western strains to develop from the northern strain (A/chicken/Nigeria/641/2006, sublineage C). As the three outbreaks occurred within a maximum of 8 weeks, the strains probably did not spread from one farm to another. In particular, as H5N1 emerged essentially at the same time in the north (10 January 2006) and in the SO farm (15 January 2006), these two sublineages probably did not develop within Nigeria. Thus, these data and the fact that the closest pre-Nigerian relatives of each of the sublineages were geographically separated further support the independent introduction of each of the three H5N1 sublineages in Nigeria as suggested previously (Ducatez et al., 2006a), even if the B and C sublineages emerged from a common ancestor. The same analysis for the sublineage A strains from the south-west revealed that the 5 months between the outbreaks was in agreement with the genetic distances between strains, as calculated with Kimura parameters. The HA substitution rate in the SO farm was also compatible with a single introduction of a sublineage C strain into Burkina Faso. Unexpectedly, but as already observed by Chen & Holmes (2006), the change of host (chicken to hooded vulture) did not seem to influence the mutation rate of the virus.
Nucleotide and amino acid substitutions and biological consequences
As previously discussed for Burkina Faso strains (Ducatez et al., 2007), west African H5N1 strains retained the amino acid pattern associated with preferential binding to 2,3-linked sialic acid (mostly present in avian species): 91Y, 130GVSS134, 149W, 151I, 179H, 186E, 190LY191 and 220NGQSGR225 (Ha et al., 2001; Shinya et al., 2004). Mutations towards increased binding to human receptors (to 2,6-linked sialic acid) did not seem to have occurred in west African H5N1 strains. In contrast to south-east Asian lineages, PB2 segments of west African strains have a lysine instead of a glutamic acid in position 627, lysine corresponding to a more pathogenic H5N1 phenotype with accelerated viral replication, a reduced host defence and higher mortality in mice (Fouchier et al., 2004; Hatta et al., 2001), as well as increased virulence of H7N7 in human (Chen et al., 2006a). Chen et al. (2006a) studied H5N1 viruses in waterfowl in western China in 2005 and observed lysine residues in position 627 for most but not all of the virus isolates. Among the non-south-east Asian strains, some Russian strains, including A/Cygnus olor/Astrakhan/Ast05-2-4/2005, A/duck/Kurgan/08/2005 and A/chicken/Tula/Russia/Oct-5/2005, had a glutamic acid at PB2 position 627, thus also co-existing with the lysine as found in western China. Strains from Russia and western China seem to represent intermediates between the south-east Asian strains with only glutamic acid and the African strains with only lysine.
None of the west African H5N1 strains showed a signatory amino acid pattern associated with resistance to oseltamivir or to amantadine. 274H was indeed present in all Nigerian and Burkina Faso NA sequences, whilst 274Y has been observed in oseltamivir-resistant patients in Vietnam (de Jong et al., 2005). Moreover, all west African strains had a 27V/I, 30A, 31S and 34G pattern in M2 and none of the amantadine-resistance markers (27V, 30A, 31S and 34D) (Scholtissek et al., 1998).
Unlike the suggestion by Chen et al. (2006b), 99I and 268N in HA as well as 111R in NA are not always associated with wild birds, as shown by new sequences from domestic poultry in Africa, Europe and Russia. All Nigerian and Burkina Faso H5N1 strains from chickens had these three amino acids, whilst A/hooded vulture/Burkina Faso/1/2006 had 268Y instead of the predicted 268N.
In all of the available NS gene sequences from Africa, Europe and Russia, a 563GC substitution was observed, which may correspond to a change in RNA conformation as described by Gultyaev et al. (2007) for the recent H5N1 strains. Thus, the NS gene of the African HPAI H5N1 2006 viruses would be likely to show a hairpin rather than a pseudoknot conformation towards the 3' extremity, which may play a role in mRNA splicing regulation (Gultyaev et al., 2007).
Selection pressure
In Africa, Europe and Russia, most H5N1 genes are under purifying selection with 0.10<<0.43 (Table 1), suggesting that a non-synonymous mutation has only 10–43 % as much chance as a synonymous mutation of being fixed in the population. Only the PB1-F2 gene seems to be under positive natural selection (=8.476, data not shown), but, as discussed by Holmes et al. (2006), this is probably due to the overlap of the PB1 and PB1-F2 ORFs (a shift of 1 nt compared with PB1 ORF) and therefore represents an artefact. In south-east Asian 2002–2005 genotype Z H5N1 strains, M2 was also under positive natural selection, which was not the case in the 26 African strains analysed here or in the available strains from western Asia, Russia and Europe (data not shown). No positively selected codons were detected here in HA, unlike observations in south-eastern strains (Smith et al., 2006).
Our data indicate that the geographical origin of west African HPAI H5N1 was not south-east Asia but the central Asian/European region. The three sublineages A, B and C already existed prior to the arrival of HPAI H5N1 in Africa. They have continued to circulate and to evolve both in Africa and in Europe, e.g. in Germany. Although the relative roles of bird migration and the poultry trade in the spread of recent H5N1 remain a matter for debate, the present study contributes to a better understanding of the evolution and geographical spread of HPAI H5N1 in Africa.
H5N1 virus has been reported from 14 of the 31 Federal Nigerian States and has caused at least five outbreaks in poultry and one outbreak in wild birds in Burkina Faso. It is difficult to see how the virus can be contained without depopulation combined with surveillance and large-scale vaccination. Despite the high mortality caused by H5N1, surveillance is complicated by uncharacteristic organ lesions at necropsy and multiple viral and bacterial co-infections (Ducatez et al., 2006b; Igbokwe et al., 1996; Owoade et al., 2006, 2004a, b), many of which are associated with significant levels of mortality. Reservations of the developed countries towards vaccination against H5N1 delay crucial decisions and prevent rapid action in Africa. Socio-economic and political hurdles further compromise control measures, whilst the virus continues to spread and threatens to become endemic.
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ACKNOWLEDGEMENTS |
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Received 21 February 2007;
accepted 21 April 2007.
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and African, non-Nigerian strains by 
. The tree was calculated using the maximum-likelihood method implemented in PAUP 4.0. The substitution model was obtained using MODELTEST. A/duck/Vietnam/01/2005 was used as the outgroup and is indicated by an arrow.