high genetic diversity of newcas tle disease virus in poultry...

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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

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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

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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

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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

[email protected] 28

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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

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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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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




turkey/Nigeria/NIE09-2071/2009



JQ039386 chicken/Nigeria/VRD08-36/2008

JN872165 Chicken/Niger/VIR 1377-7/2006

FJ772452 chicken/Niger/1377-8/2006

















JX546245 chicken/Benin/463MT/2009

























XIVa

JF966386 chicken/Mali/ML029_07/2007

chicken/Ivory Coast/CIV08-042/2007


duck/Ivory Coast/CIV08-062/2006

JN942101 Finch/Eastern Hemisphere/1409-12/2008

FJ772466 chicken/Ivory Coast/2601/2008

JX390609 chicken/Togo/AKO18/2009




JN872157 GWH/Eastern Hemisphere/5801-22/10



FJ772455 avian/Mauritania/1532-14/2006


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


XVIII

chicken/Central African Republic/CAF09-014/2008



avian/Cameroon/CAE08-318/2009





FJ772446 avian/Nigeria/913-1/2006


guinea fowl/Nigeria/NIE08-2004/2009



JX546243 chicken/Benin/373GC/2009

JX546244 chicken/Benin/376GT/2009




FJ772469 chicken/Niger/2602-348/2008

FJ772472 chicken/Niger/2602-468/2008







FJ772463 chicken/Burkina Faso/2415-580/2008


FJ772458 chicken/Burkina Faso/2415-361/2008

JX546247 chicken/Benin/488MT/2009




JQ039392 avian/Nigeria/VRD07-733/2007





FJ772478 chicken/Cameroon/3490-149/2008

FJ772484 chicken/Cameroon/3490-147/2008









FJ772475 chicken/Niger/2602-605/2008

FJ772481 chicken/Niger/2602-625/2008







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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high genetic diversity of newcas tle disease virus in poultry...

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