1
High genetic diversity of Newcastle disease virus in poultry in 1
West and Central Africa: co-circulation of genotypes XIV and 2
newly defined genotypes XVII and XVIII 3
Running title: Genetic diversity of NDV in West and Central Africa 4
5
Chantal J. Snoeck1, Ademola A. Owoade2, Emmanuel Couacy-Hymann3, Bello R. Alkali4, 6
Mbah P. Okwen5, Adeniyi T. Adeyanju6, Giscard F. Komoyo7, Emmanuel Nakouné7, Alain 7
Le Faou8, Claude P. Muller1# 8
9
1Institute of Immunology, Centre de Recherche Public de la Santé/Laboratoire National de 10
Santé, 20A rue Auguste Lumière, L-1950 Luxembourg, Luxembourg; 11
2Department of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria 12
3Central Laboratory for Animal Diseases, Bingerville, Côte d’Ivoire 13
4Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Usmanu 14
Danfodiyo University, Sokoto, Nigeria 15
5District Hospital Bali, North West Regional Delegation of Public Health, Bamenda, 16
Cameroon 17
6A.P. Leventis Ornithological Research Institute, University of Jos, Jos, Plateau State, 18
Nigeria and Department of Wildlife and Ecotourism Management, Faculty of Agriculture and 19
Forestry, University of Ibadan, Ibadan, Nigeria. 20
7Institut Pasteur de Bangui, Bangui, Central African Republic 21
8Cibles Thérapeutiques, Formulation et expertise préclinique du Médicament, Faculté de 22
Pharmacie, Université de Lorraine, Nancy, France 23
24
Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00684-13 JCM Accepts, published online ahead of print on 8 May 2013
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#Corresponding author: Claude P. Muller, Institute of Immunology, Centre de Recherche 25
Public de la Santé/Laboratoire National de Santé, 20A rue Auguste Lumière, L-1950 26
Luxembourg. Tel: +352 49 06 04 220. Fax: +352 49 06 86. E-mail address: 27
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Abstract 30
Despite rampant Newcastle disease virus (NDV) outbreaks in Africa since decades, the 31
information about the genetic characteristics of the virulent strains circulating in West and 32
Central Africa is still scarce. In this study, 96 complete NDV fusion gene sequences were 33
obtained from poultry sampled in Cameroon, Central African Republic, Côte d’Ivoire and 34
Nigeria between 2006 and 2011. Based on rational criteria recently proposed for the 35
classification of NDV strains into classes, genotypes and sub-genotypes, we revisited the 36
classification of virulent strains in particular from West and Central Africa, leading to their 37
grouping into genotypes XIV and newly defined genotypes XVII and XVIII, each with two 38
sub-genotypes. Phylogenetic analyses revealed that several (sub-)genotypes are found in 39
almost every country. In Cameroon, most strains were related to vaccine strains, but a single 40
genotype XVII strain was also found. Only three highly similar genotype XVII strains were 41
detected in Central African Republic. Sub-genotypes XVIIa, XVIIIa and XVIIIb co-42
circulated in Côte d’Ivoire, while sub-genotypes XIVa, XIVb, XVIIa, XVIIb and XVIIIb 43
were found in Nigeria. While these genotypes are so far geographically restricted, local and 44
international trade of domestic and exotic birds may lead to their spread beyond West and 45
Central Africa. A high genetic diversity, mutations in important neutralizing epitopes paired 46
with suboptimal vaccination, various levels of clinical response of poultry and wild birds to 47
virulent strains, strains with new cleavage site and other genetic modifications found in these 48
genotypes tend to undermine and complicate NDV management in Africa. 49
50
Keywords 51
Newcastle disease virus, Africa, epidemiology, phylogeny, genotype. 52
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Introduction 54
Newcastle disease, caused by virulent Newcastle disease virus (NDV), is one of the most 55
important diseases in poultry worldwide. This viral pathogen is also a major challenge for the 56
commercial and traditional poultry industry in West Africa (1). 57
NDV is a member of the Avulavirus genus of the Paramyxoviridae family, subfamily 58
Paramyxovirinae (2). Its single-stranded RNA genome is composed of 6 genes 3’-NP-P-M-F-59
HN-L-5’ encoding for 6 major proteins as well as a V protein resulting from mRNA-editing 60
of the P gene (3). The hemagglutinin-neuraminidase (HN) and the fusion (F) proteins are 61
both glycoproteins expressed at the surface of the enveloped virus. They mediate attachment 62
of the viral particle to sialic acid-containing cell receptors, its fusion with the plasma cell 63
membrane and the release of progeny virions from the surface of infected cells (4). Both 64
proteins also induce virus neutralizing antibody responses (5). 65
NDV strains differ in genome length. The smallest NDV genomes are 15 186 nucleotides 66
long, but some genomes are longer due to insertions of either 6 nucleotides in the 5’ non-67
coding region of the NP gene (6), or 12 nucleotides in the P gene (7). NDV strains are 68
genetically highly diverse and their variability continues to unfold (8, 9). Two nomenclature 69
systems have been proposed and are currently used. The first one, separating NDV strains 70
into two classes and several genotypes was based on restriction site mapping, genome length 71
and F gene sequences (7, 10). The second, dividing NDV strains into six lineages, was based 72
on phylogenetic analyses performed on partial F gene sequences (11). 73
Several pathotypes (asymptomatic enteric, lentogenic, mesogenic, viscerotropic or 74
neurotropic velogenic) of NDV are recognized depending on the clinical symptoms observed 75
in chickens (12). Avirulent and virulent strains may also be distinguished on the basis of the 76
cleavage site sequence of their F protein. During replication, the fusion gene is translated into 77
a precursor protein F0 that must be cleaved by host cell proteases into F1 and F2 subunits for 78
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viral particles to become infectious (13). Most virulent strains exhibit the consensus sequence 79
112R/K-R-Q-R/K-R*F117 at the cleavage site of the F0 precursor, in contrast to 112G/E-K/R-Q-80
G/E-R*L117 in avirulent viruses (12, 14). The additional basic amino acids in the virulent 81
viruses allow the F0 precursor to be cleaved by ubiquitous proteases, such as furin-like 82
enzymes, present in a wide range of cells. Thus virulent viruses have the ability to replicate in 83
a range of tissues and organs causing fatal systemic infections (15). 84
The first records of virulent NDV in West and Central Africa date from the 1950s (Hills et 85
al., cited by (16)). Since then, several viruses from different outbreaks have been investigated 86
by pathogenicity tests (17, 18), but molecular characterization of these viruses has started 87
only recently (19). Based on partial F gene sequences, we have previously classified West 88
African strains (Niger, Nigeria, Burkina Faso) into three new sub-lineages 5f, 5g and 5h (19), 89
while similar full length F gene sequences from Africa were assigned to a new lineage 7 (20), 90
creating some confusion. Here, we report 96 additional full length F gene sequences of NDV 91
detected during surveillance in Nigeria, Cameroon, Central African Republic and Côte 92
d’Ivoire between 2006 and 2011, providing further insights into the genetic diversity of the 93
circulating NDV strains. With this additional genetic information provided, we revisited all 94
full length F gene sequences available on public databases and updated the recent 95
classification. Based on objective classification criteria recently proposed by Diel et al. (9), 96
we classified the strains circulating in West and Central Africa into genotype XIV and newly 97
defined genotypes XVII and XVIII, each with two sub-genotypes. 98
99
Materials and Methods 100
Sample information 101
A total of 3610 domestic birds (mainly chickens but also ducks, geese, guinea fowls and 102
turkeys) were sampled in a variety of locations, including free ranging animals, live-poultry 103
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markets, backyard and commercial farms between 2006 and 2011 in four countries in West 104
and Central Africa (Tables 1 and 2, Fig. 1). Except in Côte d’Ivoire, where only sick birds 105
were sampled, all other samples were collected during active surveillance where birds were 106
sampled regardless of clinical symptoms. The vast majority of collected material consisted of 107
pooled tracheal and cloacal swabs, but cloacal or tracheal swabs, faeces or organs (lung, liver, 108
intestine, trachea, spleen and brain) were also included. All samples were shipped on dry ice 109
to Luxembourg where NDV detection and sequencing were performed. 110
RNA extraction, (RT-)PCRs and sequencing 111
All swabs and fecal samples were discharged in 500 µl of virus transport medium (VTM; 112
(21)). RNA was purified from 140 µl of VTM with QIAamp Viral RNA Mini Kit (Qiagen, 113
Venlo, The Netherlands) or from 50 µl of virus transport medium using the MagMAXTM-96 114
AI/ND Viral RNA Isolation Kit (Life Technologies, Merelbeke, Belgium) with Thermo 115
Electron’s KingFisher (Thermo Fisher, Waltham, MA, USA) following the manufacturers’ 116
instructions. Approximately 30 mg of organs were homogenized with stainless steel beads 117
(Qiagen) and TissueLyser II (Qiagen) in 600 µl of lysis buffer of RNeasy Mini Kit (Qiagen) 118
and RNA was extracted according to the manufacturer’s protocol. Reverse transcription, first 119
round and nested PCRs for NDV detection were performed as described previously (19, 22) 120
on the 3610 samples included in the study. 121
F and HN gene sequences were obtained by generating several overlapping fragments with 122
one-step RT-PCRs followed by (semi-)nested PCRs. All primers used and designed in this 123
study are listed in Table S1 and (RT-)PCR conditions are available in Table S2. All PCR 124
products were visualized on a 1.5% agarose gel stained with SYBR® Safe DNA gel stain 125
(Life Technologies) and purified using JetQuick PCR Purification Spin kit (Genomed, 126
Loehne, Germany) or QIAquick Gel Extraction Kit (Qiagen) when multiple bands were 127
visible. Sequencing was performed in both orientations using the Big Dye Terminator v.3.1 128
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cycle sequencing kit (Life Technologies) and ABI 3130 Avant capillary sequencer (Applied 129
Biosystems). Primers used for generating the PCR fragments were used in the sequencing 130
reaction, and for longer PCR products, additional internal primers were used (Table S1). 131
Contigs were assembled and analyzed using SeqScape v2.5 (Applied Biosystems). 132
Phylogenetic analyses and genotype classification based on complete F sequences 133
All complete fusion gene sequences available on GenBank (February 2013) were aligned 134
using ClustalW (23). Sequences with insertions or deletions resulting in frame shifts were 135
cured from the dataset. Recombinant sequences were identified with the following methods 136
(RDP, Geneconv, MaxChi, Chimaera, Bootscan, Sister Scanning and 3SEQ) as implemented 137
in RDP3 software (24). Sequences were identified as true recombinant when p value was < 138
0.001 for at least two tests. All putative recombinant sequences were removed from the 139
dataset, as well as the recombinants identified based on complete genome analyses by Chong 140
et al. (25) and Diel et al. (9). The final dataset contained 896 previously published sequences 141
and 96 obtained in this study. 142
Phylogenetic relationships were inferred by comparing the sequences obtained in this study 143
with all complete F gene sequences available on GenBank. The best-fit substitution model 144
was selected based on analyses performed in Topali v2.5 (26) and the model with the lowest 145
Akaike information criterion 1 (AIC1) and Bayesian information criterion (BIC) was 146
selected. Trees were calculated with the Maximum Likelihood method, using the general 147
time-reversible (GTR) substitution model with a gamma (G) and invariant (I) site 148
heterogeneity model as implemented in MEGA v5.03 (27). 992 sequences were included to 149
calculate the tree presented in Fig. S1, Fig. 2 is a detailed view of genotypes XIV, XVII and 150
XVIII as calculated in Fig.S1. 151
Genotypes and sub-genotypes were determined based on (i) tree topology, (ii) bootstrap 152
values, (iii) mean evolutionary distances between (sub-)genotypes and (iv) a minimum of 153
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four sequences from at least two distinct outbreaks per (sub-)genotype. Between group 154
distances, i.e. the mean of all pairwise distances between two groups in the inter-group 155
comparisons, and the mean interpopulation diversity were calculated with Maximum 156
Composite Likelihood model and 500 bootstrap replicates (MEGA v5.03). The mean 157
interpopulation diversity was used to determine the cut-off value between genotypes. For 158
these analyses, class I strains (n=118) were excluded due to the much higher genetic distance 159
of class I (between 0.375 and 0.426 mean genetic distance) to all class II genotypes. 160
Phylogenetic analyses based on complete HN sequences 161
The complete HN sequences generated in this study were compared to all complete HN 162
sequences available on GenBank (February 2013). Phylogenetic analyses were performed 163
similarly to the analyses on complete F sequences. Three to nine representative strains per 164
genotype (as defined on complete F sequences) were then selected to calculate the 165
phylogenetic tree presented in Fig. S2. 166
Statistical analyses 167
Statistical analyses to assess whether there was a correlation between species or sampling 168
location and the outcome of the detection tests were performed using the chi-square test with 169
Yates correction in SigmaPlot software (Systat Software Inc, San Jose, CA, USA). For the 170
variables with a significant correlation according to the chi-square test, the Phi coefficient 171
was calculated in order to assess the direction of the correlation. 172
Nucleotide sequence accession numbers 173
Sequences were submitted to GenBank under accession numbers XXX to XXX. The 174
following strain nomenclature was used: host/country/strain number/year. In the text, the 175
strains obtained in this study are referred to by their strain number. 176
177
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Results 179
Detection of NDV in samples from West and Central Africa 180
Between 2006 and 2011, we collected and screened a total of 3610 samples for the presence 181
of NDV nucleic acids (class II only). In Cameroon and Central African Republic, only 182
chickens were sampled (Table 1). In Côte d’Ivoire and Nigeria, also ducks, guinea fowl and 183
turkeys were included. In Nigeria also samples from geese were analyzed. The species origin 184
of 32 samples was not known (Table 1). In Nigeria, chickens were more often infected by 185
NDV (6.5%, p-value < 0.001), than turkeys (5.1%) and guinea fowls (3.4%; Table 2); ducks 186
had a significantly lower risk of infection, compared to all other bird categories (p-value < 187
0.001) in Nigeria. Chickens sampled in the Nigerian live-poultry markets were statistically 188
more often infected with NDV (11.3%; p-value < 0.001), compared to the other locations. 189
Free-ranging chickens had a lower risk of being infected (0.8%, p-value < 0.001). The overall 190
prevalence of NDV in Nigeria between 2006 and 2011 (129/2342, 5.5%) was higher than the 191
overall prevalence found in Cameroon (16/1096, 1.5%, 2009 and 2011) and in Central 192
African Republic (3/88, 3.4%, 2008), but was comparable to the overall prevalence found in 193
Togo and Benin between 2008 and 2010 (119/2427, 4.9%; (27)). The high overall prevalence 194
found in Côte d’Ivoire (8/53, 15%, 2006-2008) compared to prevalences of 0.3 to 1.4% in 195
2010 (27) and to the other countries could be explained by differences in sampling methods, 196
i.e. targeting exclusively sick animals or not. However, apparent prevalence rates may be 197
somewhat underestimated due to suboptimal local sample storage before shipment and 198
potential PCR inhibitors in fecal material, although this seems to be less of an issue in fecal 199
swabs of poultry (28). 200
Genetic classification based on complete F gene sequences 201
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Sequencing of the fusion gene was attempted for all positive samples (n=157), but only 96 202
full and 27 partial sequences were obtained. These sequences were used for the genetic 203
classification and phylogenetic analyses described below. 204
Two nomenclature systems are currently used to describe the genetic diversity of NDV 205
strains. The first one divides NDV strains into class I and II and several genotypes within 206
each class (7, 10) while the second defines six lineages and multiple sub-lineages (11). 207
However, the latter showed limitations, especially by grouping sub-lineages 3a, 3b, 3c, 3d, 3e 208
and 3g into a single lineage despite being polyphyletic. In addition, the phylogenetic 209
grouping of recent strains from West Africa was not clear. Based on partial F sequences, they 210
were either classified as three new sub-lineages of lineage 5 (19), three clusters in genotype 211
VII (29) or as a new lineage 7 with four sub-lineages (20). With the additional genetic 212
information provided by 96 new complete F gene sequences from West and Central Africa 213
generated in this study, we revisited all full length F gene sequences and updated the recent 214
classification based on objective criteria proposed by Diel and co-workers (9). 215
Each of the 992 complete F gene sequences was assigned to either class I or class II and to 216
genotypes within class II based on (i) tree topology (Fig. S1), (ii) bootstrap values (≥ 60%), 217
(iii) evolutionary distances (Table 3) and (iv) the recent classification proposed (9). The mean 218
interpopulation diversity, which corresponds to the mean evolutionary distance between 219
genotypes, calculated on 873 sequences (excluding class I strains) was 0.097 (± 0.005). A 220
cut-off of 10% evolutionary distance between genotypes was selected, as suggested before 221
(9). Based on these criteria, class II strains were classified into 17 genotypes (I to XIV and 222
XVI-XVIII; Table 3, Fig. S1). Our analyses confirms strain classification into genotypes I to 223
XIII and XVI (8, 9), except for genotype IV strains that did not constitute a monophyletic 224
group. Excluding all putative recombinant strains left genotype XV empty. On the other 225
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hand, only six sequences from West Africa were included at the time by Diel et al. (9) and 226
these were all grouped into genotype XIV. 227
The availability of 18 additional sequences published recently and the 92 generated in our 228
study warranted a more detailed analysis of all West and Central African strains. In our 229
analyses, sequences from West and Central Africa were assigned to three genotypes, XIV and 230
the newly defined genotypes XVII and XVIII. Mean evolutionary distances between 231
genotypes XIV, XVII and XVIII ranged from 0.101 and 0.124, whereas mean evolutionary 232
distances to the other genotypes ranged from 0.1 (between XVIII and XIII) to 0.24 (between 233
XIV and XI; Table 3). One strain from Mali (JF966386 chicken/Mali/ML029_07/2007) was 234
not classified into any genotype (Fig. 2; Fig. S1). Its clustering as an outgroup of genotype 235
XIV and its high genetic distance to genotypes XIV, XVII and XVIII (from 0.1 to 0.118) 236
suggested that it may belong to putative genotype XIX, but more sequences are needed to 237
validate its classification. 238
The high genetic diversity within certain genotypes justified their subdivision into sub-239
genotypes. Diel et al. (9) proposed the following criteria for the definition of sub-genotypes: 240
(i) tree topology, (ii) bootstrap values ≥ 60% and (iii) mean evolutionary distances between 241
sub-genotypes > 0.03 and < 0.1. According to these criteria, the definition of the existing sub-242
genotypes Ia, Ib, Va, Vb, VIa, VIb, VIc, VIe, VIIe and VIIf was confirmed in our study, but 243
bootstrap values for sub-genotypes VIIb and VIId were lower (38% - 59%) than the 60% 244
threshold, suggesting a suboptimal definition of these two sub-genotypes. However, deduced 245
amino acid sequences showed that sub-genotypes VIIb and VIId could be distinguished based 246
on specific residues: most VIIb strains shared the amino acids S24 (124/126), L28 (115/126), 247
K145 (105/126), H279 (108/126), I513 (124/126), and V110 (110/126) with a few 248
exceptions, whereas most sub-genotype VIId strains had G24 (108/124), P28 (77/124) or S28 249
(40/124), N145 and Q279 (124/124), V513 (117/124) and G520 (123/124) residues. New 250
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sub-genotypes were defined within genotypes V (Vc) and VI (VIf and VIg), as well as within 251
genotypes XIV (XIVa and XIVb), XVII (XVIIa and XVIIb) and XVIII (XVIIIa and XVIIIb; 252
Table S3 and Fig. S1). 253
In order to investigate the classification of other strains from Africa that were previously 254
attributed to lineage 5 or genotype VII, phylogenetic analyses were performed on the longest 255
F gene fragment available for each strain with a subset of 208 strains representative of all 256
genotypes I-XIV and XVI-XVIII (data not shown). These analyses established that the strains 257
from Burkina Faso (FM200806-FM200808), previously assigned to sub-lineage 5h (19), 258
clustered with chicken/Mali/ML029_07/2007, strengthening the hypothesis of the existence 259
of a putative additional genotype XIX present in Burkina Faso and Mali (Table S4). 260
Nevertheless further classification based on complete F gene sequences is warranted in order 261
to confirm this grouping. On the other hand, previous sub-lineages 5b or sub-genotype VIIb 262
strains from Burundi (20), Mozambique (30, 31), Zimbabwe and South Africa (31, 32) were 263
grouped into genotype XIII, whereas sub-lineage 5d or sub-genotype VIId strains from South 264
Africa (32, 33) and Sudan (34) still grouped into sub-genotype VIId in our classification. 265
Phylogenetic analyses of the new NDV strains from West and Central Africa 266
Vaccine-like strains (based on 375 nt). Two strains from Cameroon 267
(chicken/Cameroon/CAE11-863/2011 and chicken/Cameroon/CAE11-855/2011) clustered in 268
genotype I, and were identical to the vaccine strain Queensland V4 (JX524203; Kimura 269
distance 0%, 375nt). Strain CAE11-855 had a genetic distance of 0.8% (242 nt) to another 270
isolate from Cameroon (chicken/Cameroon/CS81/2008; FM200839; data not shown). Three 271
strains from 2009 and four from 2011 clustered in genotype II and were very similar to the 272
vaccine strain B1 (JN872150; Kimura distance 0 to 0.5%, 375 nt), suggesting that all 273
genotype I and II strains were related to live vaccine strains (Fig. S2). 274
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Genotype XIV. Fourteen strains from three Nigerian States (Sokoto, Yobe and Lagos States; 275
2007, 2009, 2011) obtained in our study clustered in sub-genotype XIVa (previously named 276
5f (19) or 7d (20, 35); Table S4) together with two strains from Niger (2006) and one from 277
Nigeria (2008; Fig. 2A). Thirty-three strains from Nigeria (Sokoto, Yobe and Benue States; 278
2008, 2009, 2011) sequenced in this study clustered in sub-genotype XIVb (previously 279
named 5f (35), 7d (36) or cluster #1 (29); Table S4) together with a strain from Katsina State 280
from 2007 (chicken/Nigeria/VRD07-233/2007) and a strain from Benin (2009). Interestingly, 281
the three strains NIE09-1596, NIE09-1597 and NIE09-1599 were found in a commercial 282
farm in Benue State and represent one of the very few examples of virulent NDV from 283
commercial farms in West and Central Africa (Fig. 2A). 284
Genotype XVII. Thirty-eight sequences obtained in this study clustered within genotype 285
XVI, most of them from Nigeria (Sokoto, Yobe and Plateau States; 2007, 2008, 2009, 2011) 286
but also some from Côte d’Ivoire (2007), Central African Republic (2008) and Cameroon 287
(2009; Fig. 2B). Based the analysis of all complete F gene sequences, genotype XVII was 288
divided into two sub-genotypes XVIIa and XVIIb. Sub-genotype XVIIa (previously sub-289
lineage 5g (19), 7a (36), 7b (20) or cluster #3 (29); Table S4) was geographically the most 290
dispersed as it was found in Nigeria, Côte d’Ivoire, Niger, Cameroon, Burkina Faso in 2008, 291
in Benin in 2009 and in Mali in 2008 (Fig. 2B). Sub-genotype XVIIb (previously named 7b 292
(20); Table S4) was constituted exclusively by Nigerian strains. In the future, genotype XVII 293
may need to be further subdivided due to the high genetic distance between strains from 294
Central African Republic (CAF09-014, CAF09-015, CAF09-016; 0.76 and 0.73 mean genetic 295
distance respectively) and the single virulent strain from Cameroon (CAE08-318; 0.59 and 296
0.53 mean genetic distance respectively) to sub-genotypes XVIIa and XVIIb. The three 297
strains from Central African Republic were highly similar to each other and originated from a 298
single farm. 299
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Genotype XVIII. Two strains from Côte d’Ivoire (2007) clustered in sub-genotype XVIIIa 300
(previously 7a (20); Table S4), together with one strain from Mauritania (2006) and three 301
strains from Mali (2007, 2008 and 2009; Fig. 2A). Sub-genotype XVIIIb (previously 7a (20) 302
or cluster #2 (29); Table S4) was composed of three strains from Côte d’Ivoire (2006 and 303
2007), two strains from Nigeria (2011) obtained in our study as well as one strain from Côte 304
d’Ivoire (2008) and one strain from Togo (2009). 305
An additional set of 22 partial sequences were obtained from Nigeria (n=21) and Côte 306
d’Ivoire (n=1), and were assigned to sub-genotypes XVIIa or XVIIb (Nigeria) and XVIIIa 307
(Côte d’Ivoire) (data not shown, Table S4). Taken together, these phylogenetic analyses 308
revealed that several (sub-)genotypes are found in every country, except in Central African 309
Republic where only genotype XVII was identified. In Cameroon, nine strains were related to 310
vaccine strains, either B1 or Queensland V4, but a single genotype XVII strain was also 311
found. Sub-genotypes XVIIa, XVIIIa and XVIIIb circulated in Côte d’Ivoire, while sub-312
genotypes XIVa, XIVb, XVIIa, XVIIb and XVIIIb were found in Nigeria (Fig. 2 and Fig. 3). 313
In Nigeria, which was most extensively sampled, several sub-genotypes and several clusters 314
within each subgenotype were found in the same States (Fig. 2). For instance, two clusters of 315
sub-genotype XIVa, three clusters of sub-genotype XIVb and four clusters of sub-genotype 316
XVIIa were found in Sokoto State. Sub-genotypes XIVa, XIVb and XVIIa were found in live 317
bird markets in Yobe State (Fig. 2). 318
Phylogenetic analyses based on HN sequences. Phylogenetic analyses performed on 41 319
complete HN sequences obtained in this study and compared to selected representative strains 320
of each genotype showed that the tree topology and the genotype assignment of the strains 321
from West and Central Africa was similar for HN and F genes (Fig. S1 and S2). The three 322
new genotypes XIV, XVII and XVIII were clearly identifiable and distinct from all other 323
genotypes of class II. Deduced amino acid sequences of the HN gene showed that the HN 324
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protein of all strains clustering in genotypes XIV, XVII and XVIII was 571 amino acid long, 325
a feature shared by many virulent strains. Strain clustering within sub-genotypes XIVa-XIVb, 326
XVIIa-XVIIb and XVIIIa-XVIIIb was also similar to that based on F sequences. 327
Nevertheless, phylogenetic analyses based on complete genome sequences of representative 328
strains of each West African sub-genotype would be interesting to further investigate and 329
confirm their evolutionary relationship. 330
Analyses of deduced amino acid sequences of F and HN proteins 331
Cleavage site. Genotype I and II strains encoded for the fusion cleavage site motifs 332
112GKQGR*L117 and 112GRQGR*L117 respectively, both typical of avirulent viruses. Deduced 333
amino acid cleavage site sequences of all genotype XIV, XVII and XVIII strains were 334
indicative of virulent viruses. The majority of strains encoded for the cleavage site sequence 335
112RRQKR*F117, but the motif 112RRRKR*F117 was also observed in sub-genotypes XIVb (28 336
strains/35), XVIIa (6/47) and XVIIIb (1/8) (Table 5). Variability was also observed in 337
position 118 and a conservative I118V substitution was found in all strains of sub-genotype 338
XIVb and in three strains of sub-genotype XVIIIa (Table 5). Virulent strains were 339
characterized in apparently healthy animals (45/92), in 13/92 sick animals and no information 340
was available for 34/92 birds. 341
Neutralising epitopes. The fusion and hemagglutinin-neuraminidase proteins contain several 342
neutralising epitopes that are known to be important for their structure and function (37, 38). 343
On the F protein, most of our African NDV strains shared the residues D72, E74, A75, K78, 344
A79, L343 and the stretch 151ILRLKESIAATNEAVHEVTDG171. However, all genotype 345
XIV strains shared a K78R substitution and five of 17 sub-genotype XIVa strains had an 346
A79G substitution. The three strains from Central African Republic (genotype XVII) shared 347
the A75T and A79T substitutions. Variability was also observed in position 170, where all 348
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genotype XVIII strains had a D170S substitution but D170G and D170E were also found in 349
other strains. L343P and L343Q were found twice each (Table S5). 350
Several amino acid substitutions in neutralizing epitopes were also observed on the HN 351
protein: R197K (n=1), R263K (in all but three strains), D287E (n=2), R333K (in all but one 352
strain), E347D (n=4), D349G (n=2), Y350H (n=9), R353Q (n=4), K356R (n=1), R513H 353
(n=2), I514V (in all but one strain), S519A (n=1), S521G (n=1) and D569G (n=4; Table S5), 354
suggesting that some of these strains may partially escape antibody neutralization (39). 355
N-glycosylation sites. The F and HN proteins both contain several potential N-glycosylation 356
sites (N-X-S/T, where X is any amino acid but a proline). Predicted N-glycosylation sites on 357
the F protein were conserved among genotype XIV, XVII and XVIII strains (85NRT88, 358
191NNT193, 366NTS368, 447NIS449 or 447NVS449, 471NNS473 or 471NYS473, 541NNT543), similarly 359
to all other strains published so far. Thus there is no expected difference in viral functions 360
such as protein structure, virus replication or virulence modulated by differential 361
glycosylation (40, 41). On the HN protein, five predicted N-glycosylation sites were 362
conserved among all genotype XIV, XVII and XVIII strains (119NNS121, 341NNT343 or 363
341NDT343 or 341NNS343, 433NKT435, 481NHT483, 508NIS510 or 508NTS510). In addition, 19/20 364
genotype XIV strains and 2/29 genotype XVII strains for which the HN sequences were 365
obtained had an additional potential N-glycosylation site 538NKT540, although this site does 366
not seem to be glycosylated (42). 367
HN-L intergenic region 368
Some strains recently described in Togo and Benin (XVIIIb) showed a six nucleotide 369
insertion in the intergenic region between the HN and polymerase (L) genes (29, 43), 370
although insertions are rare in NDV (6, 7). We therefore sequenced this region for 53/92 371
strains of the three new genotypes. The insertion was only found in strains of sub-genotype 372
XVIIIb (NIE11-1286, NIE10-171, CIV08-042 and CIV08-062; this region could not be 373
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obtained for CIV08-069; Fig. 2), suggesting that the insertion of six nucleotides was probably 374
recently acquired by an ancestor of this sub-genotype. 375
376
Discussion 377
Adding almost 100 new complete F gene sequences to the 44 African sequences on 378
GenBank, it became necessary to revisit their genotype classification. We propose to classify 379
the new virulent NDV strains into three new genotypes XIV, XVII and XVIII, each with at 380
least two sub-genotypes. Interestingly, no strains related to these genotypes were ever 381
reported from Northern (Egypt (44), Sudan (34)), Eastern (Uganda (45), Burundi (20), 382
Ethiopia, Kenya, Tanzania (11), Mozambique (30), Zimbabwe, Madagascar (46)) and 383
Southern Africa (Botswana, South Africa (31, 32)), suggesting that their geographic 384
distribution is still restricted to West and Central Africa. Except for South Africa (31-33), the 385
epidemiology of NDV is still poorly understood in Africa. As surveillance may intensify 386
across West and Central Africa, except perhaps Nigeria, the genetic diversity of NDV will 387
further increase. For instance, strains from Mali and Burkina Faso clustered outside genotype 388
XIV and their high genetic distance to genotypes XIV, XVII and XVIII likely suggests yet 389
another genotype in these countries. The current genotypes XIV, XVII and XVIII may also 390
need further subdivision in the future, especially genotype XVI. 391
The detection of vaccine-like strains is mostly due to the use of live vaccines, mainly used in 392
commercial farms in West and Central Africa. In this study, vaccine-related strains were only 393
detected in Cameroon, and not in Nigeria, contrasting to our previous study carried mainly in 394
Nigerian commercial settings (19). On the other hand, sub-genotype XIVb strains were found 395
in a commercial farm in Nigeria that had reportedly vaccinated with an unspecified vaccine 396
strain against NDV. Also genotype XVII strains were found in a commercial farm in Central 397
African Republic. In this country, farms are smaller and less professional than the large 398
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commercial farms in Nigeria, and introduction of chickens purchased at live bird markets in a 399
breeding flock is not unusual. These examples highlight the risk that virulent strains represent 400
for the commercial poultry sector as an important part of the local economies. 401
At least in Nigeria, birds from live bird markets were significantly more often positive for 402
NDV, compared to commercial or backyard farms, suggesting that supply from various 403
origins by semi-professional intermediate traders with poultry from low biosecurity farms 404
likely contribute to the enzootic circulation of NDV. Trade across State borders is also 405
reflected in the similarity of strains found in different Nigerian States. Equally, highly similar 406
strains, especially of the sub-genotypes XVIIa, XVIIIa and XVIIIb, in different countries 407
across West and Central Africa paired with the diversity of strains in each country suggest 408
intensive poultry trade between countries. Selling of live birds is very common in West 409
Africa and birds may sometimes be transported hundreds kilometres between their breeding, 410
rearing, slaughtering and consumption place (1). Seasonal high demand and movement also 411
coincides with increased incidence of NDV in Nigeria (47), and may be compounded by 412
environmental factors (48). 413
In Nigeria, strains exhibiting a virulent cleavage site motif were detected in 43 apparently 414
healthy chickens. Similar observations were made in Mali (49), Benin and Togo (29), leading 415
some authors to question the virulence of those strains (49). Pathotyping showed, however, 416
that so far all strains tested had ICPI values between 1.51 and 1.87, characteristic of 417
velogenic strains (20, 29) (Table 4). In our study, most of the asymptomatic infected chickens 418
(41/43) were sampled in live bird markets where both vaccinated and unvaccinated animals 419
can be found and vaccination would likely reduce the expression of clinical signs but not 420
necessarily suppress viral shedding (29, 50, 51). Viral RNA was also detected in the other 421
poultry species, including ducks, turkeys and guinea fowls. Normally these species are more 422
resistant to the disease and they may be infected without symptoms (52-55). Although they 423
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also shed less virus than chickens, they may spread the virus at live bird markets, in backyard 424
farms or as free-roaming birds. Also exotic birds may be more resistant to Newcastle disease 425
than chickens facilitating their role in spreading typical African NDV genotypes. For 426
instance, two genotype XVIII strains were found in wild birds (finch, JN942101; Green 427
Wood Hoopoe, JN872157) at a quarantine station in the USA. Although the exact origin of 428
the birds could not be confirmed (N. Hines, personal communication), the NDV strains which 429
they carried have otherwise only been found in West and Central Africa indicative of another 430
case of export of virulent NDV by exotic species (56). 431
NDV pathogenicity is largely determined by the amino acid sequence of the F protein 432
cleavage site. Interestingly, an unusual 112RRRKR*F117 cleavage site was observed in sub-433
genotypes XIVb, XVIIa and XVIIIb, while the vast majority of virulent and avirulent strains 434
share a glutamine in position 114 instead of an arginine. Only a few other recent (40/45, ≥ 435
2000) genotypes VI (33/119), VII (4/274), XIII (4/19) strains and the four genotype XI (4/4) 436
strains from Madagascar also have this Q114R or a Q114K substitution. Reverse genetic 437
experiments recently showed that a Q114R substitution reduced viral replication in vitro and 438
in vivo, and attenuated pathogenicity in one day-old chicks (57). The additional I118V 439
substitution – otherwise found in genotype V (69/73), XI (4/4) and class I (58/118) strains 440
and a few other exceptions – further reduced pathogenicity (57). This could, however, only 441
partially explain the apparently reduced virulence observed in the field since both Q114 and 442
R114 or I118 and V118 were both found in healthy and sick animals at least in our study. 443
These reverse genetic experiments performed in a Beaudette C backbone (genotype II) may 444
not be directly applicable to genotype XIV, XVII and XVIII strains due to the high number of 445
amino acid differences of their F and HN proteins (between 10 and 14%). This may also 446
influence the structure of both proteins, their interactions and conformational changes of F 447
protein during fusion of the virus with plasma cell membrane (58). Therefore, additional 448
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experiments should be performed in order to assess the role of Q114R and I118V 449
substitutions in genotype XIV, XVII and XVIII strains. 450
Outbreaks of NDV in vaccinated flocks have been increasingly reported from Nigeria (35), 451
suggesting a suboptimal protection by vaccination. In general, all vaccine strains (genotypes 452
I, II and III) are thought to protect against all virulent strains, except for some variant viruses 453
that overcame vaccine protection (59). Challenge experiments with genotype XVIIa or 454
XVIIIb strains after vaccination with the commonly used LaSota vaccine also confirmed that 455
vaccination conferred efficient protection against clinical disease and death induced by these 456
West African strains, although shedding was not inhibited for all animals (29). Other reasons 457
such as poor vaccine quality, suboptimal vaccination (60), or co-infections (61) may result in 458
reduced vaccine efficacy. Nevertheless, multiple amino acid substitutions in neutralizing 459
epitopes of F and HN proteins in a number of genotypes XIV, XVII and XVIII strains (Table 460
S5), as well as on other viral proteins that have not been investigated in our study, could 461
contribute to partial vaccine resistance and warrant further vaccine challenge experiments. 462
Vaccines better matching this antigenic diversity of current West and Central African NDV 463
strains are probably required. 464
In conclusion, we showed that the genetic diversity of virulent NDV strains enzootically 465
circulating in West and Central Africa continues to grow, requiring a continuously updated 466
classification based on rational criteria for (sub-)genotypes. While genotypes XIV, XVII and 467
XVIII strains are so far geographically restricted, local and international trade of domestic 468
and exotic birds may lead their spread beyond West and Central Africa. A high genetic 469
diversity, mutations in important neutralizing epitopes paired with suboptimal vaccination, 470
various levels of clinical response of poultry and wild birds to virulent strains, strains with 471
new fusion protein cleavage sites and other genetic modifications tend to undermine and 472
complicate NDV management in Africa. 473
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474
Acknowledgments 475
The authors wish to thank A. Sausy, E. Charpentier, R. Brunnhöfer and F. Leenen for their 476
excellent technical help and J. Garba, H. Iyiola and I. Nwankwo, D. Sambo and D. Semaka 477
for sample collection in Nigeria, T.E. Fon of Science For Life Foundation for help in sample 478
collection in Cameroon. They also acknowledge the Ministry of Agriculture of Central 479
African Republic and the Agence Nationale pour le Développement de l’Elevage, particularly 480
Dr E. Balete and Dr M.-N. Mbaikoua, for technical support. They gratefully acknowledge the 481
Ministry of Cooperation of Luxembourg, the Ministry of Health, the Ministry of Research 482
and the Centre de Recherche Public de la Santé for their generous financial and moral 483
support. C.J.S. was supported by an AFR fellowship TR_PHD BFR08-095 from the Fonds 484
National de la Recherche, Luxembourg. This study was also supported by the contribution 485
No. XXX from A.P. Leventis Ornithological Research Institute, Nigeria. 486
487
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Protective efficacy of commercial inactivated Newcastle disease virus vaccines in 695
chickens against a recent Korean epizootic strain. J Vet Sci. 9:295-300. 696
697
698
699
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Figure legend 700
Fig. 1. Collection sites in Côte d’Ivoire, Nigeria, Cameroon and Central African Republic. 701
Provinces or States visited are indicated by black points. In Côte d’Ivoire, numbers 702
correspond to the following provinces: 1. Savanes; 2. Worodougou; 3. Sud Bandama; 4. 703
Zanzan; 5. Moyen Comoé; 6. Agnéby; 7. Bas Sassandra; 8. Sud Bandama; 9. Lagunes. 704
Fig. 2. Detailed view of genotypes XIV and XVIII (A) and genotype XVII (B) as calculated 705
in Fig. S1, which shows the phylogeny of 992 complete F gene sequences analyzed with the 706
Maximum Likelihood method and the GTR+G+I nucleotide substitution model. Sequences 707
obtained in this study are shown in bold. For Nigeria, the States are indicated as follows: full 708
circle, Sokoto State; grey circle, Yobe State; open circle, Plateau State; black square, Benue 709
State; grey square, Lagos State; open square, Oyo State. Accession numbers of previously 710
published sequences available on GenBank are indicated. Only bootstrap values ≥ 60% are 711
shown. The scale corresponds to number of base substitutions per site. GWH: Green Wood 712
Hoopoe. 713
Fig. 3. Summary of (sub-)genotypes XIV, XVII and XVIII distribution in West and Central 714
Africa (including previously published results from (19, 20, 29, 49)). (Sub-)genotypes are 715
represented as follows: full circle, XIVa; grey circle, XIVb; full square XVII; grey square, 716
XVIIa; open square, XVIIb; full triangle, XVIIIa; grey triangle, XVIIIb; open circle, putative 717
XIX. 718
719
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Tables 720
Table 1: Distribution of collected and positive samples according to country, year of 721
sampling and species 722
Country Year No. of positive sample/No. of sample tested by species (%)
chicken duck goose guinea fowl
turkey not
specified
Cameroon 2009 4/644 2011 12/452
Central African Republic
2008 3/88
Côte d’Ivoire 2006 1/26 1/7 0/1 2007 6/13 0/1 0/1 0/1 2008 0/1 0/2
Nigeria 2006 0/49 0/15 0/6 0/1 0/6 2007 1/185 0/17 0/1 0/6 1/24 2008 5/558 0/177 0/9 0/15 1/28 0/1 2009 69/576 0/72 3/51 2/42 2011 46/484 0/5 1/44 1/1
Total
147/ 3076
(4.8%)
1/296 (0.3%)
0/9 (0%)
4/118 (3.4%)
4/79 (5.1%)
1/32 (3.1%)
723
724
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Table 2: NDV prevalence in bird species by type of birds in Nigeria 725
Sampling location
No. of positive animals/No. of animals tested (%)
chicken duck goose guinea fowl
turkey total
market 99/873
(11.3 %) 0/74
4/90
(4.4 %) 3/19
(15.8 %) 106/1056
(10%)
backyard farm 11/204 (5.4%)
0/17
0/12 0/28 11/261 (4.2 %)
commercial farm 6/115
(5.2 %) 0/3
6/118 (5.1%)
free ranging 5/638
(0.8 %) 0/151 0/7
1/9
(11.1 %) 6/805 (0.7%)
unknown 0/22 0/44 0/2 0/15 0/19 0/102
Total 121/1852 (6.5 %)
0/286 (0%)
0/9 (0%)
4/117 (3.4 %)
4/78 (5.1 %)
129/2342 (5.5%)
726
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Table 3: Estimates of evolutionary distances (in number of base substitutions per site) over sequence pairs between genotypes of class IIa 727
728 I II III IV V VI VII VIII IX X XI XII XIII XIV XVI XVII XVIII
I (n=81)b
[0.008] [0.007] [0.007] [0.01] [0.01] [0.01] [0.008] [0.007] [0.007] [0.01] [0.011] [0.01] [0.011] [0.009] [0.01] [0.011]
II (n=112)
0.116
[0.01] [0.009] [0.011] [0.011] [0.012] [0.01] [0.009] [0.009] [0.012] [0.013] [0.011] [0.013] [0.011] [0.013] [0.012]
III (n=9)
0.104 0.13
[0.007] [0.01] [0.01] [0.011] [0.009] [0.007] [0.01] [0.01] [0.011] [0.01] [0.012] [0.009] [0.011] [0.011]
IV (n=6)
0.094 0.12 0.079
[0.009] [0.008] [0.01] [0.007] [0.007] [0.009] [0.008] [0.011] [0.009] [0.011] [0.008] [0.01] [0.011]
V (n=73)
0.17 0.184 0.163 0.137
[0.007] [0.008] [0.008] [0.011] [0.011] [0.011] [0.009] [0.008] [0.01] [0.008] [0.011] [0.011]
VI (n=119)
0.161 0.179 0.159 0.125 0.146
[0.008] [0.007] [0.01] [0.011] [0.011] [0.008] [0.007] [0.009] [0.008] [0.01] [0.009]
VII (n=274)
0.16 0.185 0.155 0.128 0.145 0.121
[0.008] [0.011] [0.011] [0.012] [0.008] [0.006] [0.008] [0.009] [0.009] [0.008]
VIII (n=4)
0.132 0.151 0.126 0.096 0.125 0.112 0.118
[0.009] [0.01] [0.01] [0.008] [0.008] [0.008] [0.007] [0.009] [0.008]
IX (n=20)
0.099 0.119 0.088 0.077 0.156 0.153 0.15 0.119
[0.008] [0.01] [0.011] [0.01] [0.012] [0.01] [0.009] [0.009]
X (n=19)
0.105 0.109 0.126 0.115 0.183 0.175 0.175 0.148 0.116
[0.011] [0.012] [0.011] [0.012] [0.01] [0.013] [0.012]
XI (n=4)
0.173 0.194 0.165 0.118 0.198 0.199 0.205 0.173 0.154 0.189
[0.011] [0.013] [0.011] [0.012] [0.011]
XII (n=6)
0.167 0.191 0.16 0.136 0.154 0.12 0.109 0.118 0.158 0.176 0.211
[0.006] [0.008] [0.01] [0.008] [0.007]
XIII (n=19)
0.156 0.182 0.156 0.126 0.145 0.121 0.102 0.116 0.146 0.172 0.198 0.096
[0.007] [0.009] [0.007] [0.006]
XIV (n=52)
0.193 0.22 0.195 0.164 0.173 0.152 0.134 0.144 0.191 0.201 0.24 0.125 0.121
[0.01] [0.008] [0.008]
XVI (n=4)
0.155 0.178 0.152 0.119 0.154 0.147 0.154 0.121 0.146 0.166 0.199 0.151 0.146 0.177
[0.01] [0.01]
XVII (n=56)
0.162 0.194 0.167 0.14 0.156 0.138 0.121 0.133 0.157 0.184 0.2 0.114 0.107 0.124 0.165
[0.007]
XVIII (n=15)
0.167 0.189 0.164 0.137 0.154 0.127 0.115 0.127 0.156 0.181 0.201 0.106 0.1 0.124 0.159 0.101
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729
a The number of base substitutions per site from averaging over all sequence pairs between groups are shown (1662 nucleotides). Standard error 730
estimates (500 bootstrap replicates) are shown above the main diagonal between squared brackets. Analyses were conducted using the Maximum 731
Composite Likelihood model. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each 732
sequence pair. 733
b The numbers of sequences analyzed per genotypes are mentioned between round brackets. 734
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Table 4: Fusion protein cleavage site sequences (positions 112-117) and amino acid 118 of 735
all genotype XIV, XVI and XVII strains, as well as ICPI values 736
Genotype Positions 112-117
Position 118
ICPI valuesa
Sub-genotype XIVa (n=17) RRQKR*F I 1.8 (n=1) Sub-genotype XIVb 1.65 (n=1)
28 strains RRRKR*F V
NIE09-1596, NIE09-1599, NIE09-1597 RRQKR*F V
NIE10-034, NIE10-258, NIE10-139, NIE10-325
Genotype XVII
1.51-1.87 (n=8)b
50 strains RRQKR*F I
NIE10-123, NIE10-124, NIE10-304, NIE10-306, NIE10-310, NIE10-335
RRRKR*F I
Sub-genotype XVIIIa 4 strains RRQKR*F I
CIV08-026, CIV08-044, Green Wood Hoopoe/Eastern Hemisphere/5801-22/10
RRQKR*F V
Sub-genotype XVIIIb
1.65-1.7 (n=2)
7 strains RRQKR*F I chicken/Togo/AKO18/2009 RRRKR*F I
a ICPI values from (20, 29); the number of strains tested is indicated between brackets. 737
b All 8 strains tested by ICPI cluster in sub-genotype XVIIa. 738
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Côte d’Ivoire
Nigeria
Ghana
BeninTogo
Burkina Faso
Niger
Cameroon
MaliChad
Liberia
Guinea
Central African RepublicNorth West
Bangui
Lagos
Oyo
SokotoYobe
PlateauNasarawa
Benue
1
2 3
4 5
678
9
Sudan
Democratic Republic of
CongoGabon
Equatorial Guinea
Congo
Mauritania
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chicken/Nigeria/NIE09-2087/2009
avian/Nigeria/NIE09-2168/2009
chicken/Nigeria/NIE09-2101/2009
chicken/Nigeria/NIE09-2079/2009
chicken/Nigeria/NIE09-2044/2009
turkey/Nigeria/NIE09-2071/2009
chicken/Nigeria/NIE09-2009/2009
turkey/Nigeria/NIE10-082/2011
JQ039386 chicken/Nigeria/VRD08-36/2008
JN872165 Chicken/Niger/VIR 1377-7/2006
FJ772452 chicken/Niger/1377-8/2006
chicken/Nigeria/NIE08-2150/2009
chicken/Nigeria/NIE08-2194/2009
chicken/Nigeria/NIE08-2117/2009
chicken/Nigeria/NIE08-2362/2009
chicken/Nigeria/NIE08-2280/2009
chicken/Nigeria/NIE07-125/2007
chicken/Nigeria/NIE09-1599/2009
chicken/Nigeria/NIE09-1596/2009
chicken/Nigeria/NIE09-1597/2009
JQ039390 chicken/Nigeria/VRD07-233/2007
chicken/Nigeria/NIE08-2032/2009
chicken/Nigeria/NIE08-0453/2008
chicken/Nigeria/NIE08-2359/2009
chicken/Nigeria/NIE08-2270/2009
chicken/Nigeria/NIE08-2159/2009
chicken/Nigeria/NIE08-2279/2009
JX546245 chicken/Benin/463MT/2009
chicken/Nigeria/NIE10-318/2011
chicken/Nigeria/NIE09-2014/2009
turkey/Nigeria/NIE09-2021/2009
chicken/Nigeria/NIE09-2041/2009
chicken/Nigeria/NIE09-2166/2009
chicken/Nigeria/NIE09-2053/2009
chicken/Nigeria/NIE09-2013/2009
chicken/Nigeria/NIE09-2017/2009
chicken/Nigeria/NIE10-333/2011
chicken/Nigeria/NIE10-032/2011
chicken/Nigeria/NIE10-150/2011
chicken/Nigeria/NIE10-139/2011
chicken/Nigeria/NIE10-034/2011
chicken/Nigeria/NIE10-258/2011
chicken/Nigeria/NIE10-325/2011
chicken/Nigeria/NIE10-122/2011
chicken/Nigeria/NIE10-076/2011
chicken/Nigeria/NIE10-024/2011
chicken/Nigeria/NIE10-409/2011
chicken/Nigeria/NIE10-160/2011
chicken/Nigeria/NIE10-302/2011
chicken/Nigeria/NIE10-041/2011
chicken/Nigeria/NIE10-263/2011
chicken/Nigeria/NIE10-043/2011
XIVa
JF966386 chicken/Mali/ML029_07/2007
chicken/Ivory Coast/CIV08-042/2007
chicken/Ivory Coast/CIV08-069/2007
duck/Ivory Coast/CIV08-062/2006
JN942101 Finch/Eastern Hemisphere/1409-12/2008
FJ772466 chicken/Ivory Coast/2601/2008
JX390609 chicken/Togo/AKO18/2009
chicken/Nigeria/NIE10-171/2011
chicken/Nigeria/NIE11-1286/2011
JF966387 chicken/Mali/ML008_09/2009
JN872157 GWH/Eastern Hemisphere/5801-22/10
chicken/Ivory Coast/CIV08-026/2007
chicken/Ivory Coast/CIV08-044/2007
FJ772455 avian/Mauritania/1532-14/2006
JF966388 chicken/Mali/ML225_08/2008
JF966389 guinea fowl/Mali/ML038_07/2007
XVIIIb
XVII
99
100
77
90
63
98
72
86
78
99
78
60
100
90
87
100
66
60
64
77
100
99
99
100
100
100
97
100
73
67
62
61
100
77
100
100
98
87
100
99
99
99
97
63
92
XVIIIa
XIVb
XIV
JF966386 chicken/Mali/ML029_07/2007
XVIII
chicken/Central African Republic/CAF09-014/2008
chicken/Central African Republic/CAF09-015/2008
chicken/Central African Republic/CAF09-016/2008
avian/Cameroon/CAE08-318/2009
chicken/Nigeria/NIE08-2199/2009
chicken/Nigeria/NIE08-2261/2009
avian/Nigeria/NIE07-216/2007
chicken/Nigeria/NIE10-182/2011
FJ772446 avian/Nigeria/913-1/2006
chicken/Nigeria/NIE08-2208/2009
guinea fowl/Nigeria/NIE08-2004/2009
chicken/Nigeria/NIE08-2224/2009
chicken/Nigeria/NIE08-2042/2009
JX546243 chicken/Benin/373GC/2009
JX546244 chicken/Benin/376GT/2009
chicken/Nigeria/NIE08-2119/2009
chicken/Nigeria/NIE08-2187/2009
FJ772486 avian/Nigeria/3724-6/2008
FJ772469 chicken/Niger/2602-348/2008
FJ772472 chicken/Niger/2602-468/2008
chicken/Nigeria/NIE08-2247/2009
chicken/Nigeria/NIE08-2168/2009
chicken/Nigeria/NIE08-2267/2009
chicken/Nigeria/NIE08-2340/2009
chicken/Ivory Coast/CIV08-103/2007
JF966385 chicken/Mali/ML007_08/2008
FJ772463 chicken/Burkina Faso/2415-580/2008
chicken/Ivory Coast/CIV08-104/2007
FJ772458 chicken/Burkina Faso/2415-361/2008
JX546247 chicken/Benin/488MT/2009
chicken/Nigeria/NIE08-2149/2009
chicken/Nigeria/NIE08-2349/2009
FJ772449 avian/Nigeria/913-33/2006
JQ039392 avian/Nigeria/VRD07-733/2007
JQ039394 chicken/Nigeria/VRD07-410/2007
chicken/Nigeria/NIE08-1365/2008
chicken/Nigeria/NIE08-1366/2008
chicken/Nigeria/NIE08-1363/2008
FJ772478 chicken/Cameroon/3490-149/2008
FJ772484 chicken/Cameroon/3490-147/2008
JQ039393 chicken/Nigeria/VRD07-141/2007
avian/Nigeria/NIE09-2167/2009
chicken/Nigeria/NIE09-2034/2009
chicken/Nigeria/NIE09-2072/2009
chicken/Nigeria/NIE09-2083/2009
chicken/Nigeria/NIE09-2028/2009
chicken/Nigeria/NIE09-2031/2009
chicken/Nigeria/NIE09-2128/2009
FJ772475 chicken/Niger/2602-605/2008
FJ772481 chicken/Niger/2602-625/2008
chicken/Nigeria/NIE10-335/2011
chicken/Nigeria/NIE10-304/2011
chicken/Nigeria/NIE10-306/2011
chicken/Nigeria/NIE10-310/2011
chicken/Nigeria/NIE10-123/2011
chicken/Nigeria/NIE10-124/2011
XVIIa
100
100
100
83
100
99
94
99
93
69
82
68
100
89
100
97
91
99
94
88
100
99
69
95
90
64
99
100
92
70
95
99
99
99
97
63
92
0,05
XVIIb
XVII
A B
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Côte d’Ivoire
NigeriaBurkina Faso
Niger
Cameroon
Mali
Central African Republic
Mauritania
TogoBenin
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