Jurnal Virologi

MEEGID 2143 No.
of Pages 14, Model 5G

20 November 2014
Infection, Genetics and Evolution xxx (2014) xxx–xxx

1
Contents lists available at ScienceDirect
Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid
5
6
3 Identification of new sub-genotypes of virulent Newcastle disease virus

4 with potential panzootic features
7 Q1 Patti J. Miller a, Ruth Haddas b, Luba Simanov b, Avishay Lublin b, Shafqat Fatima Rehmani c, Abdul Wajid c,
8 Tasra Bibi c, Taseer Ahmad Khan d, Tahir Yaqub c, Surachmi Setiyaningsih e, Claudio L. Afonso a,⇑
9 a
Southeast Poultry Research Laboratory, Agricultural Research Service-United States Department of Agriculture (USDA), Athens, GA 30605, USA
10 b
Kimron Veterinary Institute, Bet Dagan 50250, Israel
11 Q2 c
Quality Operations Laboratory, University of Veterinary and Animal Sciences, Out Fall Road, Lahore, Pakistan
12 d
Poultry Research Laboratory, Department of Physiology, University of Karachi, Karachi, Pakistan
13 e
Department of Infectious Diseases & Veterinary Public Health, Faculty of Veterinary Medicine-Bogor Agricultural University, Jl. Agatis, IPB Dramaga, Bogor 16680, Indonesia
14
15
a r t i c l e i n f o a b s t r a c t
1 1
3 7
18 Article history: Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly 32
19 Received 29 May 2014 spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized 33
20 Received in revised form 25 October 2014 by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, 34
21 Accepted 30 October 2014
which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to 35
22 Available online xxxx
have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, 36
but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. 37
23 Keywords:
Viruses from sub-genotype VIIh were isolated in Indonesia (2009–2010), Malaysia (2011), China 38
24 Newcastle disease
25 NDV
(2011), and Cambodia (2011–2012) and are closely related to the Indonesian NDV isolated in 2007, 39
26 Epidemiology APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related 40
27 Panzootic NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasion- 41
28 Outbreak ally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype 42
29 Poultry VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009–2011, and they 43
30
have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the 44
numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub- 45
genotype are now found more frequently than viruses from the previously predominant sub-genotypes 46
VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to 47
each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to 48
those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of 49
the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) 50
that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a 51
NDV strain from an Indian cockatoo isolated in1982. These data suggest the existence of a new panzootic 52
composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple 53
virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in 54
Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here 55
and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, 56
suggests that identifying wild life reservoirs may help predict new panzootics. 57
Published by Elsevier B.V. 58
59
60
61
62 1. Introduction the raising or keeping of birds (Anonymous, 2011). The etiological 66

agent of ND, virulent NDV, belongs to the genus Avulavirus of the 67
63 Newcastle disease virus (NDV) is distributed worldwide and its family Paramyxoviridae (Mayo, 2002). The virus was originally 68
64 continual presence in multiple avian species presents a constant detected in Java, Indonesia and Newcastle-‘on-Tyne, England 69
65 threat to all poultry industries and other activities that involve (Doyle, 1927), and since then various genotypes have been respon- 70
sible for different ND panzootics. The virus is enveloped, with a 71
⇑ Corresponding author. single-stranded, non-segmented, negative sense RNA genome. 72
E-mail address: Claudio.Afonso@ars.usda.gov (C.L. Afonso).
http://dx.doi.org/10.1016/j.meegid.2014.10.032
1567-1348/Published by Elsevier B.V.
Please cite this article in press as: Miller, P.J., et al. Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic fea-
tures. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.10.032
MEEGID 2143 No. of Pages 14, Model 5G
20 November 2014
2 P.J. Miller et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx
73 Multiple genotypes of NDV have been circulating worldwide panzootic constituted by highly related vNDV isolates from Indo- 139
74 (Miller et al., 2010). NDV isolates may be classified into genotypes nesia, Israel and Pakistan. These virus strains belong to a new 140
75 based on either the complete genome sequences or the full fusion vNDV sub-genotype (VIIi), and together with the existence of addi- 141
76 protein sequences from NDV isolates (Diel et al., 2012a). At this tional sub-genotypes (VIIh and XIIIa and XIIIb) related to older 142
77 time, ND viruses are grouped into one genotype for class I NDV iso- strains from wild birds suggest that unknown reservoirs harbor 143
78 lates, and in eighteen genotypes for class II NDV isolates, some new vNDV isolates capable of additional panzootics. 144
79 with sub-genotypes (Courtney et al., 2013; de Almeida et al.,
80 2013; Diel et al., 2012a; Snoeck et al., 2013). The 2012 a classifica-
81 tion system of NDV was proposed based on the utilization of the 2. Material and methods 145
82 complete sequence of the fusion (F) protein gene (Diel et al.,
83 2012a). The system was based on the mean inter-population evo- 2.1. Isolation of NDV virulent viruses 146
84 lutionary distance between previous existing NDV genetic groups,
85 and differences of 10% (at the nucleotide level) were proposed as All laboratories followed the same protocol to isolate NDV 147
86 the cutoff value to assign new genotypes. This system grouped strains except that chickens used to produce the 9–11 day old 148
87 NDV isolates of class I into a single genotype comprised of mainly embryonating chicken eggs needed for virus isolation were specific 149
88 viruses that have been isolated from waterfowl and shorebirds, and pathogen free (SPF) for those isolated in Israel and The United 150
89 occasionally from samples collected in live bird markets world- States of America (USA) and were free of NDV antibodies for those 151
90 wide and captured wild birds (Kim et al., 2007a,b; Miller et al., isolated in Pakistan and Indonesia (Alexander and Swayne, 1998). 152
91 2010). Class II viruses were initially grouped into 15 genotypes; NDV strains cockatoo/Indonesia/87-36724-524/1988; lory/Indone- 153
92 however, four additional genotypes have been added since 2012 sia/88-08989-523/1988 and parrot/Indonesia/C300 (19625)-520/ 154
93 (Courtney et al., 2013; Diel et al., 2012a; Snoeck et al., 2013). 1976 were obtained and propagated in SPF embryos from the 155
94 Viruses from class II are present in both wild bird and poultry repository of the United States Department of Agriculture (USDA) 156
95 species; however, most virulent NDV (vNDV) isolates are obtained Southeast Poultry Research Laboratory (SEPRL) (Alexander and 157
96 from poultry and are responsible for significant economic losses to Swayne, 1998). These three historical samples were obtained dur- 158
97 the poultry industry worldwide (Dundon et al., 2012). Viruses of ing the importation and quarantine of exotic birds into the USA and 159
98 genotypes II, III and IV of class II were responsible for the first pan- it is presumed that the birds were infected at their origin rather 160
99 zootic during 1920s to 1960s (Alexander, 2001), whereas the sec- than during the transport process. Pakistani isolates were obtained 161
100 ond panzootic in Europe during the late 1960s was resulted from from swabs samples from poultry obtained from 16 outbreaks in 162
101 isolates of genotype V (Lomniczi et al., 1998). Viruses from geno- different regions of the country during the winter of October 163
102 types III, IV, IX and X are related to those of genotypes of I and II, 2011 through March 2012 and propagated in embryonating 164
103 but only circulate in limited areas of the world. Viruses of sub- chicken eggs that were free from antibodies against NDV. Repre- 165
104 genotype VIb originated in the Middle East and were responsible sentative samples of each outbreak were characterized by 166
105 for the third panzootic in pigeons during the 1980s (Kaleta et al., sequencing of the full fusion (F) protein. Israeli isolates consisted 167
106 1985). Genotypes VII and VIII were responsible for ND outbreaks of a total of 33 diagnostic swab samples obtained from dry cloacal 168
107 in Asia, including Pakistan, and in Europe since 1984 or earlier and tracheal swabs from poultry and pet birds that were sent to 169
108 (Diel et al., 2012a; Shabbir et al., 2013). Viruses from genotype the Kimron Veterinary Institute (KVI) of Israel for evaluation. All 170
109 VII are responsible for the fourth panzootic, which continues today, data regarding the origins of the samples, and the health and vac- 171
110 having spread from Asia, Africa, Europe and has even been isolated cination status of the birds sampled were documented. Swabs 172
111 in South America (Miller and Koch, 2013; Perozo et al., 2012). The were maintained in 20 °C until processing. 300 ll of phosphate 173
112 fourth panzootic of ND began around 1985 in Southeast Asia and buffer solution (PBS) were added to each swab and incubated at 174
113 spread to most countries of Africa and in Venezuela, South America room temperature for 30 min before extraction of RNA. Virus isola- 175
114 (Herczeg et al., 1999; Perozo et al., 2012; Yu et al., 2001). Geno- tion was carried out by inoculation of embryonating SPF chicken 176
115 types V, VI, VII, VIII and XI emerged after 1960’s and are considered eggs that were 11-days old, incubated at 37 °C, and monitored 177
116 ‘‘late’’ genotypes (Czegledi et al., 2006) and only contain vNDV for 6 days (Senne, 1998). Intracerebral pathogenicity index (ICPI) 178
117 strains. Currently, viruses from genotype VII are most frequently assays were conducted on hemagglutination (HA) positive allan- 179
118 associated with outbreaks of ND in the Middle East (Radwan toic fluids (Alexander and Swayne, 1998) following established 180
119 et al., 2013), and Asia (Yi et al., 2011). These viruses are of partic- procedures (OIE, 2012). Fourteen Indonesia isolates were either 181
120 ular concern as some have demonstrated higher mortality in vacci- obtained from samples from the repository at the Faculty of Veter- 182
121 nated poultry (Yi et al., 2011), while others may have expanded inary Medicine, Bogor Agricultural University (IPB), or isolated 183
122 their host range and are now able to cause disease in geese from diagnostic swab samples collected from field visits to live bird 184
123 (Wang et al., 2012). In Israel the first case of genotype VII NDV markets and poultry handling facilities. Samples from commercial 185
124 was reported in 2000 (data not published). Genotype XIV contains poultry farms were from producers willing to participate in the 186
125 vNDV isolates obtained in West and Central Africa between 2006 study. While the study area included the islands of Sumatra, Kali- 187
126 and 2008 (Snoeck et al., 2013), which are divided into three sub- mantan (Indonesian Borneo), Java, Bali, and Nusa Tenggara (The 188
127 genotypes XIVa, b and c (de Almeida et al., 2013). Genotype XV Lesser Sunda Islands) representing the western and central areas 189
128 (Diel et al., 2012a) comprises isolates obtained from chickens and of Indonesia that includes the top six most populated urban areas, 190
129 geese in China, which have been previously classified into sub- NDV strains were only able to be isolated from the samples from 191
130 genotype VIId (isolates XJ-2/97 and FJ-2/99) or VIIe (isolate JX-2/ the island of Java. Oropharyngeal and cloacal swabs were collected 192
131 99) (Liu et al., 2003). from all birds, and organ tissue samples from dead birds. Sample 193
132 The emergence and spread of new genotypes across the world collection information for each bird was included when possible: 194
133 represents a significant threat to poultry and suggest that vNDV date of sampling, host species (poultry species and breed if 195
134 is continuously evolving, leading to more diversity (Miller et al., known), estimated age (breeder, layer, broiler, chicks), environ- 196
135 2009). However, little has been done to understand the mecha- ment description (type of facilities), and the geographic location 197
136 nisms of maintenance and evolution of new genotypes (name of city and province or GPS coordinates). The GPS coordi- 198
137 (Alexander et al., 2012). Here we have characterized recent vNDV nates, flock size data, and percent mortality were not available 199
138 isolates and present evidence that suggest the emergence of a fifth for the Indonesian samples. 200
20 November 2014
P.J. Miller et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx 3
201 2.2. RNA isolation and nucleotide sequencing on 104 complete fusion gene sequences was performed to identify 264
the phylogenetic relationship of our isolates in comparison to rep- 265
202 For the three older Indonesian strains from the SEPRL reposi- resentative viruses of different genotypes for which full F gene 266
203 tory, RNA was extracted from allantoic fluids using Trizol LS (Invit- sequences were available (Diel et al., 2012a) (Fig. 1). The phyloge- 267
204 rogen, Carlsbad, CA, USA) following the manufacturer’s netic analysis was performed with the software MEGA5 (MEGA, 268
205 instructions. The complete F gene nucleotide sequences were version 5) (Tamura et al., 2011) and the evolutionary history was 269
206 determined by using a RT-PCR/sequencing approach (Diel et al., inferred by using the Neighbor Joining (data not shown), and the 270
207 2012b). Amplification reactions were performed with a one-step Maximum likelihood methods (Tamura and Kumar, 2002), with 271
208 RT-PCR kit (Qiagen, Valencia, CA, USA) and a set of fusion (F) statistical analysis based on 100 bootstraps. 272
209 gene specific primers (fwd_upper-f1-ttgcttatagttagttcgcctgtc, and To assess if the results obtained with the analysis of the com- 273
210 rev_down-f2-acccgtgtattgctctttgg). The PCR amplicons were sub- plete F gene sequences would be reflected at the whole genome 274
211 jected to electrophoresis in 1% agarose gels and the DNA bands level, we performed a phylogenetic analysis with complete gen- 275
212 were excised from the gels and purified by using the Quick Clean ome sequences available on GenBank (n = 100) (Supplementary 276
213 DNA gel extraction kit (Qiagen, Valencia, CA, USA). The purified Fig. S2). For this analysis complete coding sequences for all six 277
214 PCR products were cloned into the TOPO TA vector (Invitrogen, genes were concatenated and the complete genome analysis was 278
215 Carlsbad, CA, USA) according to manufacturer’s instructions and bases on the concatenated coding regions only. Analysis of the 279
216 subjected to DNA sequencing. All sequencing reactions were per- best-fit substitution model was performed using MEGA5, and the 280
217 formed with fluorescent dideoxy-nucleotide terminators in an goodness-of-fit of each model was measured by Bayesian Informa- 281
218 ABI 3700 automated sequencer (Applied Bio systems Inc., Foster tion Criterion (BIC) and corrected Akaine Information Criterion 282
219 City, CA, USA). Sequence editing and assembly were performed (AIC) (Tamura et al., 2011). The General Time Reversible (GTR) 283
220 with Laser Gene sequence analysis software package (Laser Gene, model with a discrete gamma distribution (+G) and allowing for 284
221 version 5.07; DNA Star, Inc., Madison, WI, USA). RNA isolation invariant sites (+I) was selected and used in all data analyses 285
222 and nucleotide sequencing of the Pakistani viruses were performed (Tamura et al., 2011). Codon positions included in the analysis 286
223 as follows: samples of HA positive allantoic fluids (Alexander and were the 1st, 2nd, 3rd, and non-coding. All positions containing 287
224 Swayne, 1998) were extracted using QIA ampÒ Viral RNA mini kits, gaps and missing data were eliminated. A total of 1650 positions 288
225 Qiagen (AA) following the manufacturer’s recommended proce- were included in the analysis of the complete F gene dataset, and 289
226 dure and confirmed to be NDV using RT-PCR and universal primers 13,735 positions were included in the analysis of the complete 290
227 for the nucleoprotein (NP) of NDV. A total of 16 viruses, one from genome data set. 291
228 different outbreaks, were selected for sequencing of the full F pro- The full fusion data set used for the phylogenetic reconstruction 292
229 tein. Complementary DNA (cDNA) was synthesized from 10 ll of was also used to infer the evolutionary distances within and 293
230 eluted RNA produced with reverse transcriptase of the First Strand between groups comparing recent epizootic strains to three histor- 294
231 cDNA Synthesis KitÒ, Thermo Fisher Scientific, (Fermentas, ical samples from Indonesia (Tables 3–5). The evolutionary dis- Q3 295
232 Waltham, Massachusetts, USA) following manufacturer’s recom- tances were inferred by pair-wise analysis using the MEGA5 296
233 mendations. Primers specifically designed for this study (Supple- software (Tamura and Kumar, 2002; Tamura et al., 2011) and the 297
234 mentary Table 1) were used for the amplification of complete maximum composite likelihood method was used to calculate 298
235 F gene. Using the SEPRL protocols described above, the PCR ampli- the number of base substitutions per site (Nei and Kumar, 2000). 299
236 cons were run on 1% agarose gels, purified and sequenced, as The variation rate among sites was modeled with a gamma distri- 300
237 described above. For Israeli isolates viral RNA was extracted from bution (shape parameter = 1) and the differences in the composi- 301
238 the swabs using the QIA amp Viral RNA Mini Kit (Qiagen, Santa Cla- tion bias among sequences were considered in the evolutionary 302
239 rita, CA, USA) according to the manufacturer’s instructions. The real comparisons (Tamura et al., 2011). Codon positions included in 303
240 time RT-PCR (RRT-PCR) assay that targeted the matrix (M) gene the analysis were the 1st, 2nd, 3rd, and non-coding. All positions 304
241 was performed as previously described (Wise et al., 2004), and containing gaps and missing data were eliminated. The numbers 305
242 sub-typing by RRT-PCR that targeted the fusion protein was presented in the phylogenetic trees represent the nine-digit Gen 306
243 performed according to Fuller et al. (2009), with modification to Info Identifier (GI) sequence identification number, and/or the 307
244 the velogenic probe VRP-1 (Aguilar and Lee, 2011) CCTATAAAG- two letter-six digit accession number from GenBank, while the 308
245 CGTTTCTGTCTCCTTCC [BHQ1], (Haddas et al., 2013). The portion numbers at the bifurcations of the tree nodes represent the boot- 309
246 targeting a 382-bp fragment encoding the amino terminal end of strap support. 310
247 the F protein of the 33 viruses was amplified by PCR using primers
248 specifically designed for this study according to Aldous et al, 2003 2.4. Recombination analysis 311
249 and purified as described above (Aldous et al., 2003). Partial fusion
250 sequencing was performed for 14 representative isolates from Recombination analysis was performed with all class II com- 312
251 Indonesia. For these samples RNA isolation and viral sequencing plete genome (n = 103) and complete F gene (n = 602) sequences 313
252 was done using SEPRL protocols for viral RNA extraction and for using the program RDP3 (Martin et al., 2010) (data not shown). 314
253 cDNA production from RT-PCR, as described above. Four statistical methods (RDP, GENECONV, Maxchi and Chimera) 315
were used to identify putative recombinant sequences. Sequences 316
254 2.3. Phylogenetic analyses and inference of the evolutionary distances with recombination events identified by at least two detection 317
methods (p < 0.001) were considered as true recombinants. 318
255 A preliminary phylogenetic analysis was performed with 81
256 sequences (available on GenBank as of 03/30/2014: http:// 2.5. Genotype classification criteria 319
257 www.ncbi.nlm.nih.gov/genbank/) corresponding to the partial
258 coding sequence of the F gene with 481 nucleotides (nt) to identify Genotypes and sub-genotypes were assigned based on the 320
259 and select viral isolates that closely related to sequenced viruses of phylogenetic topology and on the evolutionary distances between 321
260 Israel, Indonesia and Pakistan (Supplementary Fig. S1). The S1 phy- different taxonomic groups utilizing the criteria previously estab- 322
261 logenetic identifies highly related viruses to those studied here, lished (Diel et al., 2012a). Phylogeny was determined using 323
262 and documents epidemiological information from isolates that complete genomes for those sequences were that information 324
263 did not have a full fusion sequence available. A similar analysis was available. However, the classification and calculation of dis- 325
20 November 2014
Fig. 1. Phylogenetic analysis of selected NDV isolates using the nucleotide sequence encoding for the full fusion protein. The evolutionary history of 104 selected isolates was
inferred by using the Maximum Likelihood method based on the General Time Reversible model (Nei and Kumar, 2000). The evolutionary history was inferred by using the
Maximum Likelihood method based on the General Time Reversible model (Nei and Kumar, 2000). The tree with the highest log likelihood ( 12579.2213) is shown. The
percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying the
Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. A discrete Gamma distribution was used to
model evolutionary rate differences among sites (5 categories (+G, parameter = 0.5429)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I],
24.2884% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 104 nucleotide sequences. Codon
positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 1650 positions in the final dataset.
Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2011). Genotypes and sub-genotypes are shown in brackets to the right of the tree.
326 tances for most viruses were performed using only the complete (0.1) was used as the criteria to separate genotypes and distances 335
327 fusion (F) gene coding sequences since those dataset were more of 3–9.99% differences were used to separate sub-genotypes 336
328 complete. Complete F gene sequences of viruses of three new (0.030–0.099). 337
329 sub-genotypes were identified after the phylogenetic analysis
330 was performed and all viruses were used to infer the evolutionary 2.6. Accession numbers 338
331 distances between genetic sub-groups. The mean inter population
332 evolutionary diversity (mean evolutionary distance between geno- All the Pakistani complete F gene sequences (n = 16) used in this 339
333 types) was determined using the maximum composite likelihood study were submitted to GenBank and are available under 340
334 model (Tamura et al., 2004). The cutoff distance value of 10% the accession numbers from KF113338 to KF113353. GenBank 341
20 November 2014
Table 1
Epidemiological and genetic description of vNDV isolates from Pakistan, Indonesia and Israel.
GenBank Virus designation Month Farm type/location Breed Latitude Longitude Flock Age Mortality Cleavage
accession # size (days) % site
KF113338 Chicken/Pak/University Nov. UDL, UVAS, Lahore, Broiler NA NA 28000 32 60 RRQKRF
Diagnostic Lab./12/2010 Punjab
KF113339 Chicken/Pak/Lahore/30/2011 Nov. Mashallah P/F Raiwind, Broiler 31.235531 74.2171 30000 30 >80 RRQKRF
Lahore, Punjab
KF113340 Chicken/Pak/Lahore/32/2011 Nov. Mashallah P/F Raiwind, Broiler 31.235531 74.2171 30000 30 >80 RRQKRF
Lahore,Punjab
KF113353 Chicken/Pak/University Dec. UDL,UVAS, Lahore, Broiler NA NA NA NA 100 RRQKRF
Diagnostic Lab./33/2011 Punjab
KF113341 Chicken/Pak/Lahore /43/2011 Dec. S/S P/F, Barki road, Broiler 31.495 74.487 27000 33 >80 RRQKRF
Lahore, Punjab
KF113342 Chicken/Pak/Lahore/50/2011 Dec. AM P/F, Raiwind, Lahore, Broiler 31.235531 74.2171 20400 41 <60 RRQKRF
Punjab
KF113343 Chicken/Pak/Gujranwala/56/ Dec. Usman Gorya P/F Broiler 31.418 73.07757 55000 40 60 RRQKRF
2011 Gujranwala, Punjab
KF113344 Chicken/Pak/Okara/103/2011 Dec. Rajput P/F, Okara, Punjab Broiler 30.8013 73.4483 26000 28 100 RRQKRF
KF113345 Chicken/Pak/KPK/117/2011 Dec. Asad khan P/F, Nowshera, Broiler 34.006 71.9998 1500 20 10 RRQKRF
KPK
KF113346 Chicken/Pak/Khyber Pukhtun Dec. Kabir P/F, Kohat road Broiler 33.5199 71.5963 2500 20 >80 RRQKRF
Khawa/118/2011 Kohat, KPK
KF113347 Chicken/Pak/Khyber Pukhtun Jan. Haleem P/F, Warsak road, Broiler 34.0264 71.5348 5000 26 90 RRQKRF
Khawa /119/2012 Peshawar KPK
KF113348 Chicken/Pak/Khyber Pukhtun Jan. K&N’s Lab Mansehra, KPK Broiler 34.3333 73.2 0 21 >60 RRQKRF
Khawa /162/2012
KF113349 Chicken/Pak/Kasure/191/2012 Jan. Suye-Hasil, Kasur, Punjab Broiler 31.1176 74.4499 28000 25 80 RRQKRF
KF113350 Chicken/Pak/Lahore/200/2012 Jan. Ahad P/F, Baidian road Broiler 31.4629 74.4356 30000 22 100 RRQKRF
Lahore Punjab
KF113351 Chicken/Pak/University Vet. Jan. QOL,UVAS, Lahore, Layer 31.54505 74.340683 50 350 100 RRQKRF
Animal Sci./211/2012 Punjab
KF113352 Chicken/Pak/University Vet. Feb. QOL, UVAS, Lahore, Layer 31.54505 74.340683 50 84 100 RRQKRF
Animal Sci./212/2012 Punjab
KF792023 Turkey/Isr/Revadim/497/2009 Jun. Turkey breeders 31.48 34.46 NA 305 NA RRQKRF
KF650612 Chicken/Isr/Givat-Chen/1224/ Dec. Broiler 32.16 34.87 20000 30 3 RRQKRF
2010
KF650613 Chicken/Isr/Newe-Yamin/1330/ Dec. Layers 32.16 34.93 1000 300 0.25 RRQKRF
2010
KF792018 Chicken/Isr/1115-818/2011 NA NA NA NA NA NA NA RRQKRF
KF650614 Chicken/Isr/Hod-Hasharon/19/ Jan. Broiler breeders 33.9 35.53 32000 208 NA RRQKRF
2011
KF650615 Turkey/Isrl/Ram-On/21/2011 Jan. Turkey 32.52 35.35 1500 122 NA RRQKRF
JN849575 Chicken/Isr/Melea/30/2011 Jan. Broiler 32.52 35.35 32550 45 NA RRQKRF
JN849578 Chicken/Isr/Baqua-al-Garbia/ Jan. Broiler breeders 32.43 35.04 5500 326 NA RRQKRF
174/2011
JN849576 Chicken/Isr/Mashabei-Sade/ Mar. Broiler 31.42 34.79 15500 16 0.02 RRQKRF
352/2011
JN849577 Chicken/Isr/Argaman/555/2011 Apr. Broiler 32.4 35.3 30000 20 NA RRQKRF
JN638345 Falcon/Isr/Zichron-Yaakov/638/ May Common kestrel 32.1 35.04 6 NA 100 RRQKRF
2011
JN979566 Owl/Isr/Ramat-Gan/645-3/ May Little owl 31.85 34.8 6 NA 80 RRQKRF
2011/
JN979565 Penguin/Isrl/Ramat-Gan/669-2/ May African penguin 31.85 34.8 16 NA 6 RRQKRF
2011
JN638349 Buzzard/Isr/Zichron-Yaakov/ May Buzzard 32.1 35.04 NA NA NA RRQKRF
692-10/2011
JN638358 Owl/Isr/Ratmat-Gan/2011/713 May Barn owl 31.85 34.8 NA NA NA RRQKRF
KC484655 Chicken/Isr/Maaleh- Sep. Broiler breeders 31.81 35.11 5466 287 NA RRQKRF
Hachamisha/998/2011
JX192955 Chicken/Isr/Natur/1004/2011 Nov. Broiler 32.98 35.23 35000 26 NA RRQKRF
JX192956 Turkey/Isr/Avnei-Eytan/1090/ Dec. Turkey 32.98 35.23 13000 43 NA RRQKRF
2011
KF650617 Chicken/Isr/Shaal/235/2012 Feb. Broiler 31.85 35.22 5500 20 NA RRQKRF
KF650618 Chicken/Isr/Ramat-Gan/838/ May Ornamental chicken 31.85 34.8 NA NA NA RRQKRF
2012
KF792020 Parrot/Isr/Beit-Shikma/841- May Parrot 31.63 34.6 NA 60 NA RRQKRF
824/2012
KF650620 Falcon/Isr/Jerusalem/843/2012 May Common kestrel 31.76 35.22 NA NA NA RRQKRF
KF650621 Quail/Isr/Ramat-Gan/844/2012 May Common quail 31.85 34.8 NA NA NA RRQKRF
KF650622 Chicken/Isr/Holon/870/2012 Jun. Ornamental chicken 32.1 34.76 NA NA NA RRQKRF
KF650623 Canary/Isr/Jerusalem/875/2012 Jun. Canary 31.76 35.22 NA NA NA RRQKRF
KF650624 Falcon/Isrl/Rishon-Lezion/911/ Jun. Common kestrel 32.1 34.76 NA NA NA RRQKRF
2012
KF650625 Falcon/Isr/Ashdod/925/2012 Jun. Falcon 31.75 34.66 NA NA NA RRQKRF
KF650626 Falcon/Isr/Ben-Shemen/926/ Jun. Common kestrel 31.59 35.03 NA NA NA RRQKRF
2012
(continued on next page)
20 November 2014
Table 1 (continued)
GenBank Virus designation Month Farm type/location Breed Latitude Longitude Flock Age Mortality Cleavage
accession # size (days) % site
KF650627 Falcon/Isr/Ramat-Gan/939/ Jun. Eurasian hobby 31.85 34.8 NA NA NA RRQKRF
2012
KF650628 Turkey/Isr/Brosh/1301/2012 Oct. Turkey 31.22 34.79 3800 14 NA RRQKRF
NA Chicken/Pal Auth/Jerico/1349/ Dec. Broiler breeders 31.87 35.05 NA 399 NA RRQKRF
2012
KF792019 Chicken/Isr/Kvuzat-Yavne/50- Jan. Broiler breeders 31.81 34.72 NA 213 NA RRQKRF
826/2013
KF792021 Chicken/Isr/Be’er Tuvia/120- Jan. Broiler breeders 31.73 34.72 NA 236 NA RRQKRF
827/2013
KF792022 Pheasant/Isr/Hertzelia/746-16/ Jun. Pheasant 31.85 35.22 40 NA 50 RRQKRF
2013
KF767106 Parrot/Indo/76-19625- Aug Cockatoo NA NA NA NA NA RRQKRF
520(C300)/1976
KF767104 Cockatoo/Indo/87-367224-524/ Jul. Cockatoo NA NA NA NA NA RRQKRF
1987
KF767105 Lory/Indo/88-08989-523/1988 Nov. Lory NA NA NA NA NA RRQKRF
KF767107 Chicken/Indo/Bandung/ks0116/ Jan. Native chicken-backyard NA NA NA 140 NA RRQKRF
2009
KF767113 Chicken/Indo/Subang/bs06310/ Aug. Broiler-commercial farm NA NA NA 21 NA RRRKRF
2009
KF767108 Chicken/Indo/Yogyakarta/ Sep. Native chicken-poultry collector NA NA NA 210 NA RRQKRF
ks13335/2009 facility
KF767114 Chicken/Indo/IndraMayu/ Sep. Native chicken-poultry collector NA NA NA 210 NA RRRKRF
ks10331/2009 facility
KF767120 Duck/Indo/Tangerang/ Nov. Native runner duck-farm NA NA 500 70 NA GKQGRL
is05a789/2009
KF767115 Chicken/Indo/Cianjur/ls14381/ Mar. Spent layer-poultry NA NA NA NA NA RRQKRF
2010 collector facility
KF767109 Chicken/Indo/Sukabumi/ May Native chicken-live bird NA NA NA 140 NA RRQKRF
ks16812/2010 market
KF767118 Chicken/Indo/Bogor/bs18816/ Jun. Broiler-live bird market NA NA NA 21 NA RRRKRF
2010
KF767116 Chicken/Indo/Sukabumi/ Sep. Layer-Farm NA NA 5000 280 NA RRRKRF
ls11759/2010
KF767117 Chicken/Indo/Sukabumi/ Dec. Broiler-live bird market NA NA NA 21 NA RRRKRF
bs15811/2010
KF767110 Chicken/Indo/Tangerang/ Oct. Native chicken-live bird NA NA NA 140 NA RRQKRF
ks22973/2011 market
KF767111 Chicken/Indo/Tangerang/ Oct. Native chicken-live bird NA NA NA 210 NA RRQKRF
ks231017/2011 market
KF767119 Chicken/Indo/Bogor/ks19964/ Nov. Native chicken-backyard NA NA NA 140 NA RRRKRF
2011
KF767112 Chicken/Indo/Tangerang / Apr. Native chicken-live bird NA NA NA 140 NA RRQKRF
ks251217/2012 market
342 accession numbers from the full F gene sequences of Israeli NDV environments, and types of production systems. However, the levels 361
343 strains not previously published are KF792018 to KF792023. The of mortality and morbidity depended on the amount of time 362
344 partial F sequences for the fourteen Indonesia NDV strains are between vaccination with live attenuated vaccines and the onset 363
345 found under accession numbers KF767104 through KF767120. of exposure to the virus, with more time leading to fewer losses. 364
346 The three older Indonesian isolates from the SEPRL repository are Most notable, is the inexplicable occurrence of mortality greater 365
347 available under the accession numbers KF767104 to KF767106. than 60% in broiler production facilities that practice intensive 366
vaccination with flock sizes larger than 20,000 birds. In addition, 367
layers, small flocks, and non-poultry species have also been occa- 368
348 3. Results sionally infected. It has been noted that in the province of Punjab 369
the mortality rate was often higher in flocks maintained in environ- 370
349 3.1. Epidemiological description of the new epizootic viruses mentally controlled houses than those in open-air houses. Most 371
(60–80%) of the reported outbreaks either occurred during the win- 372
350 The 2011–2012 outbreaks in Pakistan were unique in several ter season of the year, or time throughout the year when there is a 373
351 aspects. These were the first documented cases in which wild birds significant variation (10–15 °C) between the day and night ambient 374
352 (peacocks, pheasants, and parrots of different breeds) reared in cap- temperatures, which is usually at the start of winter season and 375
353 tivity in both public and private zoos of Pakistan were affected with before summer. The ND outbreak occurred from November 2011 376
354 mortality rates of 40–60%. The disease also affected Rock Pigeons to March 2012 and samples from 105 flocks, which were comprised 377
355 (Columba livia) and Indian Peafowl (Pavocristatus) in the province of 1.12 M broilers, 0.0033 M layers, 0.1 M breeder broilers and 378
356 of Sindh, the southern region of the country at the end of 2012 0.0006 M wild birds, were collected to be evaluated for the presence 379
357 and early months of 2013. The peacocks displayed nervous signs, of NDV. The average age of the above flocks and breeds were 380
358 such as tremors, disorientation, and weakness prior to death. Table assessed and found that the range was from 4 weeks old for broilers 381
359 1 and S1 demonstrate that vNDV strains were widely distributed to 25 weeks old for layers. Boiler breeders averaged 50 weeks old 382
360 across Pakistan with a presence at different latitudes, geographic and wild birds were 15 weeks old; however, two samples were from 383
20 November 2014
Table 2 746/2013) with respiratory signs, weakness, cachexia, and mortal- 416
Biological evaluation of virulence from selected isolates. ity within a week. ICPI assays were conducted on selected isolates 417
Virus designationa Genbank acc. ICPIb MDTc and in all cases values higher than 1.7 were observed, confirming 418
Turkey/Isr/Revadim/497/2009 KF792023 1.85 <60 the presence of vNDV strains (Table 2). However, vNDV was also 419
Chicken/Isr/HodHasharon/19/2011 KF650614 1.88 60–90 isolated from vaccinated broiler breeders with no clinical signs 420
Turkey/Isr/Ram-on/21/2011 KF650615 NA <60 (e.g. 1349/2012) in the west bank (Palestinian authority). This 421
Chicken/Melea/Isr/30/2011 JN849575 1.89 <60 sample (e.g. 1349/2012) was collected for routine surveillance to 422
Chicken/Isr/Baqua-al-Garbia/174/2011 JN849578 NA 60–90
Chicken/Isr/Mashabei-Sade/2011/352 JN849576 NA <60
confirm vaccination against infectious bronchitis virus (IBV), avian 423
Chicken/Isr/Argaman/555/2011 JN849577 NA <60 influenza virus (AIV), and NDV. There is also evidence of wide dis- 424
Falcon/Isr/Zichron-Yaakov/638/2011 JN638345 2 <60 tribution of vNDV outside production facilities in captive birds. For 425
Owl/Isr/Ramat-Gan/645-32011 JN979566 1.86 <60 example, one strain (e.g. 838/2012) was obtained from a free-living 426
Penguin/Isr/Ramat-Gan/669-2/2011 JN979565 1.65 >90
rooster found dead at a ‘‘Safari’’ zoo located in an urban area in the 427
Buzzard/Isr/Zichron-Yaakov/692-10/2010 JN638349 NA 60–90
Owl/Isr/Ramat-Gan/713/2011 JN638358 NA 60–90 city of Ramat-Gan, which is in the center of the country, with no 428
Chicken/Isr/Maaleh-Hachamsha/998/2011 KC484655 1.63 <60 other reports of vNDV at this time in this zoo, or from any commer- 429
Chicken/Isr/Natur/1004/2011 JX192955 1.75 <60 cial farms nearby. However, a year earlier vNDV was detected at 430
Turkey/Isr/Avnei-Eytan/1090/2011 JX192956 NA 60–90 that zoo in Little Owls and African Penguins (Haddas et al., 431
Chicken/Israel/Shaal/235/2012 KF650617 NA 60–90
2013), suggesting an in apparent chain of transmission in the 432
Chicken/Isr/Ramat-Gan/838/2012 KF650618 1.74 <60
Parrot/Isr/Beit-Shikam/841/2012 KF792020 NA <60 Zoo. Samples from other infected wild bird species included a Com- 433
Falcon/Isr/Jerusalem/843/2012 KF650620 1.74 <60 mon Quail (e.g. 844/2012) that died without clinical signs of ND, 434
Quail/Isr/Ramat-Gan/844/2012 KF650621 1.88 <60 and a Common Kestrel chick (e.g. 843/2012) at the Jerusalem Bib- 435
Chicken/Isr/Holon/870/2012 KF650622 NA <60
lical zoo that died 3 days after developing symptoms, another (e.g. 436
Canary/Isr/Jerusalem/875/2012 KF650623 1.86 <60
Falcon/Isr/Rishon-Lezion/911/2012 KF650624 1.88 <60 875/2012) isolated from a Atlantic canary chick with respiratory 437
Falcon/Isr/Ashdod/925/2012 KF650625 1.84 <60 distress, anorexia and death after few days from a private breeder 438
Falcon/Isr/Ben-Shemen/926/2012 KF650626 NA <60 in the Jerusalem area, and lastly, one (e.g. 926/2012) isolated from 439
Falcon/Isr/Ramat-Gan/939/2012 KF650627 NA <60 a Common Kestrel that was found dead in an urban Jerusalem area. 440
Turkey/Isr/Brosh/1301/2012 KF650628 NA >90
In Indonesia NDV has been described since 1926 (Miller and 441
Chicken/Isr/Kvuzat-Yavne/50/2013 KF792019 NA 60–90
Chicken/Isr/Be’er Tuvia/120/2013 KF792021 NA 60–90 Koch, 2013) and the disease appears to be endemic with the last 442
Pheasant/Isr/Hertzelia/746-16/2013 KF792022 NA <60 report to the World Organization for Animal Health (OIE) of 443
a infected commercial birds in 2012 (www.OIE.int). The disease 444
Name of the viruses consist of the species it was isolated from/the country
Israel (Isr)/the city/strain number/and year it was collected. causes significant economic impact despite the availability of a 445
b
Values 0.7 or greater are identified as virulent by the O.I.E. range of vaccines. In small family farms (backyard farms; identified 446
c
Mean death times (MDT) less than 60 h are indicative of a virulent NDV strain, by the Indonesian government as ‘‘sector 4’’) chickens are mostly 447
and values between 60 and 90 h suggest a moderately virulent NDV strain. either not vaccinated or inadequately vaccinated, and therefore, 448
outbreaks in young chicks often result in 100% mortality with adult 449
384 peafowl that were 84 weeks of age. As expected, the broiler industry birds also having significant morbidity and mortality. Despite the 450
385 was most affected, as these birds would have lower antibody levels extensive vaccination programs administered to commercial flocks 451
386 from having had fewer vaccines administered over their shorter life- (known as sectors, 1, 2 and 3) frequent outbreaks of ND with mor- 452
387 span. The local broiler breeders also had heavy mortality, ranging talities at lower levels occur with considerable financial losses to 453
388 from 20 to 80%. While virus was recovered from broilers, it was dif- the local poultry industry. 454
389 ficult to isolate vNDV from the grandparent flocks, as well as from
390 the day old chicks, either live or dead. 3.2. Phylogenetic classification, distribution, and possible origin of 455
391 A similar situation has occurred in Israel and despite the fact viruses from this new epizootic 456
392 that vaccination against vNDV in Israel is mandatory ND outbreaks
393 were very severe. Since the end of 2010 through 2013, only with To characterize the molecular epidemiology of recent outbreaks 457
394 some relief in the months of summer (July–August), ongoing ND in Asia characterization of strains of NDV was conducted by 458
395 outbreaks have continued. We present here the evaluation of the biological assessment of ICPI, MDT and by sequencing. Partial and 459
396 virulence of 33 NDV strains with virulent characteristics either full fusion sequences from viruses from Pakistan (n = 16), Israel 460
397 by ICPI (n = 13), mean death time (MDT) (n = 28) or sequencing (n = 34), and Indonesia (n = 17, including three older ancestral 461
398 (n = 33). Virulent NDV infections were reported in very young strains), as described in Table 1, were sequenced and the pathoge- 462
399 chicks, older broiler breeders, zoos and wild birds including nicity of selected isolates was determined (Table 2) to confirm viru- 463
400 pigeons. The outbreak that began at the end of 2010 was vast with lence. The partial and full F gene sequence of NDV isolates were 464
401 vNDV diagnosed in very young chicks and from broiler breeder compared with reference strains belonging to genotypes I–XVIII of 465
402 farms, which are considered to have high biosecurity levels. During class II viruses. The cleavage sites of the fusion protein (Table 1) 466
403 2011–2012, nearly 300 cases of vNDV were diagnosed in commer- demonstrate that the most common configurations of the fusion 467
404 cial chickens of all ages, turkeys, wild birds, pet birds, captive zoo cleavage site were 112RRQKR;F117 (n = 59) and 112RRRKR;F117 468
405 birds, and African penguins. Since June of 2012, additional cases (n = 7), suggesting that all of these strains are virulent with the 469
406 have been observed in broiler breeder farms. The usual presenta- exception of one Indonesian strain isolated from a duck 470
407 tion of respiratory disease was observed in different types of birds (112GKQGR;L117). Phylogenetic trees were generated based on 481 471
408 and isolates representing each are these; in vaccinated layers (e.g. base pairs (bp) of the variable region (Aldous et al., 2003), for max- 472
409 isolate 259/2009), chickens (e.g. 235/2012) or turkeys (e.g. 497/ imum sample representation (Supplementary Fig. S1), or with the 473
410 2009 and 1301/2012) broilers (e.g. 174/2011 and 998/2011). full fusion (F) coding region for distance determination and classifi- 474
411 NDV infection also caused a drop in egg productions in broiler cation into sub-genotypes (Fig. 1 and Tables 3–5) (Diel et al., 2012a). 475
412 breeders (e.g. isolate 50/2013 and 120/2013). In non-poultry spe- Complete genomes of all NDV strains available from GenBank were 476
413 cies ND infection (e.g. parrot 841/2012) manifested with diarrhea used for confirmation of phylogenetic classification (Supplementary 477
414 and mortality, in Falconiformes (e.g. 925/2012) with disorienta- Fig. S2). The phylogeny of full coding sequences (1650 nt) for the F 478
415 tion, weakness and mortality, and in ornamental chickens (e.g. gene sequences available from GenBank, representing ND viruses 479
20 November 2014
Table 3
Estimated pairwise evolutionary divergences among viruses of recent panzootic.
VII-KF7677104 cockatoo/ VIIi-HQ697254 chicken/ VIIh-HQ697255

Indo/97-36724-544/ Indo/Banjarmasin/010/ chicken/Indo/Sukorejo/
1988 2010 019/2010
VII-cockatoo/Indonesia/87-36724-524 (ancestral strain)/1988
VIIi-359358667/HQ69254-chicken/Indo/Banjarmasin/010/2010 0.045
VIIi-359358674/HQ697255-chicken/Indo/Sukorego/019/2010 0.049 0.058
VIIi-359358684/HQ697257-chicken/Indo/Gianyar/013/2010 0.044 0 0.057
VIIi-359358687/HQ697258-chicken/Indo/Sragen/014/2010 0.044 0 0.057
VIIi-359358690/HQ697259-chicken/Indo/Kudus/017/2010 0.044 0 0.057
VIIi-359358693/HQ697260-chicken/Indo/Kudus/018/2010 0.044 0 0.057
VIIi-410994794/JX532092-peacock/Pak/Lahore/MM19/2012 0.061 0.014 0.074
VIIi-426273158/JX854452-pheasant/Pak/Lahore/MM20/2011 0.056 0.1 0.067
VIIi-578004049/KF792021-chicken/Isr/Bé er Tuvia/120-827/2013 0.045 0.002 0.057
VIIi-578004043/KF792018-chicken/Isr/1115-818/2011 0.045 0 0.058
VIIi-578004045/KF792019-chicken/Isr/Kvuzat-Yavne/50-826/2013 0.047 0.001 0.059
VIIi-616914919/KF113339-chicken/Pak/Lahore/30/2011 0.045 0 0.057
VIIi-616914921/FK113340-chicken/Pak/Lahore/32/2011 0.045 0 0.057
VIIi-616914923/KF113341-chicken/Pak/Lahore/43/2011 0.045 0 0.058
VIIi-616914925/KF113342-chicken/Pak/Lahore/50/2011 0.046 0.001 0.059
VIIi-616914927/KF113343-chicken/Pak/Gujranwala/56/2011 0.045 0 0.057
VIIi-616914929/KF113344-chicken/Pak/Okara/103/2011 0.047 0.001 0.06
VIIi-616914931/KF113345-chicken/Pak/Khyber Pukhtun Khawa/117/2011 0.045 0.001 0.058
VIIi-616914935/KF113347-chicken/Pak/Khyber Pukhtun Khawa/119/2012 0.046 0.001 0,059
VIIi-616914939/KF113349-chicken/Pak/Kasure/191/2012 0.047 0.002 0.059
VIIi-616914941/KF113350-chicken/Pak/Lahore/200/2012 0.047 0.001 0.06
VIIi-616914943/KF113351-chicken/Pak/University Vet. Animal Sci./211/2012 0.048 0.003 0.059
VIIi-616914945/KF113352-chicken/Pak/University Vet. Animal Sci./212/2012 0.047 0.001 0.06
VIIi-578004047/KF792020-parrot/Isr/Beit-Shikma/841-824/2012 0.047 0.001 0.059
VIIh-359358681/HQ697256-chicken/Indo/Makassar/003/2009 0.049 0.058 0.004
VIIh-359358696/HQ697261-chicken/Indo/Bali/020/2010 0.051 0.061 0.007
VIIh-402235510/JX193074-egret/China/Guangxi/2011 0.056 0.065 0.013
VIIh-486356757/JX870617-chicken/Cambodia/Phnom Penh/KHcmb06-01/2012 0.053 0.06 0.01
VIIh-486356759/JX870618-chicken/Cambodia/Phnom Penh/KHcmb06-02/2012 0.053 0.06 0.01
VIIh-486356761/JX870619-chicken/Cambodia/Kampong Cham/KHcmb082/2011 0.054 0.062 0.011
The numbers of base substitutions per site from between sequences are shown. The rate variation among sites was modeled with a gamma distribution (shape parame-
ter = 1). The differences in the composition bias among sequences were considered in evolutionary comparisons (Tamura et al., 2011). The analysis involved 35 nucleotide
sequences compared to three historical strains from Indonesia. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data
were eliminated. There were a total of 1598 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).
480 isolated worldwide, was compared to the Pakistani, Israeli and Indo- 3). However, distances within sub-genotypes is lower than 1% 507
481 nesian strains. Phylogenetic analysis of the complete F coding and distances between individual strains of genotype VIIi and h 508
482 regions suggests that the new sub-genotype VIIi circulating in Indo- is also close to 5% indicating that those two groups are separate 509
483 nesia is 99% similar to strains present in Israel and Pakistan sub-genotypes. Comparison of nucleotide sequences of NDV strains 510
484 (Fig. 1(bold) and Table 3). A different sub-genotype (VIIh), also included in this study with other published local and foreign iso- 511
485 related to viruses previously circulating in Indonesia (1998–2010), lates revealed that all of the new Pakistani strains, except 512
486 was also isolated from poultry in Malaysia (2004–2006), China chicken/University Diagnostic Laboratory (UDL)/Pakistan (Pak)/ 513
487 (2011 and 2012) and Cambodia (2012) (Supplementary Fig. S2). 12/2010 and chicken/Pak/Lahore/33/2011, have >99% homology 514
488 The third newly identified sub-genotype, XIIIb, was represented with other Indonesian strains, such as chicken/Banjarmasin/010/ 515
489 by viruses from Iran, Russia, Europe, India and South America is 2010, chicken/Kudus/018/2010, chicken/Sragen/014/2010 and 516
490 related to previously described isolates from Pakistan. The data chicken/Gianyar/013/2010. The NDV strains characterized in this 517
491 show that previously predominant genotypes (VIIb, and VIId in study have lower identity (87–93%) with other previously charac- 518
492 Israel and XIIIa in Pakistan) have been replaced by the new sub- terized Pakistani strains; chicken/backyard poultry (BYP)/Pak/ 519
493 genotypes VIIi and VIIh during 2012–2013, although VIId continues 2010, chicken-commercial poultry (CP)/Pak/Islamabad3/2010, 520
494 to circulate in Israel. Isolates from genotype VIIi were obtained from chicken/CP/Pak/Rawalpindi2/2010, chicken/Pak/Sindh Poultry Vac- 521
495 poultry production facilities throughout Pakistan and Israel, an cine Center (SPVC)/Karachi/1/1974, chicken/Pak/SPVC/Karachi/43/ 522
496 occasionally from pet birds are also highly related to current Indo- 2008, chicken/Pak/SPVC/Karachi/26/2005, chicken/Pak/SPVC/Kar- 523
497 nesian isolates. achi/33/2007, chicken/CP/Pak/2010, chicken/BYP/Pak/Lahore/2010 524
498 The phylogenetic analysis for the current NDV isolates (Figs. 1, and chicken/CP/Pak/Islamabad1/2010. While viruses from geno- 525
499 S1 and S2, and Table 3) demonstrate that NDV strains currently type VIIi were predominantly responsible for the ND outbreaks 526
500 circulating in Pakistan and Israel are nearly identical, however, during the winter session of October 2011 through March 2012 527
501 they have separate genetic origins from NDV isolates previously in Pakistan, our previous studies conducted on NDV strains from 528
502 reported in the country. Table 3 documents the nucleotide dis- this country showed the predominance of genotype XIII in Pakistan 529
503 tances of the F protein among individual viruses of highly related (Khan et al., 2010). All of the newer NDV strains from Pakistan, 530
504 strains of sub genotypes VIIi and h. Distances of approximately except chicken/Pak/UDL/12/2010, also clustered together with 531
505 5% between the ancestral Cockatoo/Indonesia/1987 strain and Indonesian and Israeli strains, suggesting rapid spread and epizo- 532
506 viruses of new sub-genotypes VIIi and h are demonstrated (Table otic characteristics for this sub-genotype. Outbreaks in Pakistan 533
20 November 2014
Table 4
Q5 Estimates of net evolutionary divergence between groups of sequences.
Anc. (n=6) GI G II G III G IV GV G VI G VII G VIII G IX GX G XI G XII G XIII G XIV G XV G XVI G XVII G XVIII G VIIi G VIIh G XIIIb
Ancestral (n=6) 0.011 0.016 0.011 0.010 0.010 0.006 0.004 0.008 0.011 0.013 0.017 0.008 0.006 0.008 0.007 0.008 0.008 0.006 0.004 0.004 0.004
Genotype I (n=69) 0.112 0.009 0.009 0.008 0.014 0.011 0.012 0.010 0.008 0.007 0.013 0.013 0.013 0.016 0.008 0.009 0.013 0.013 0.012 0.012 0.012
Genotype II (n=100) 0.173 0.096 0.014 0.011 0.016 0.015 0.017 0.015 0.012 0.009 0.017 0.017 0.017 0.021 0.008 0.011 0.018 0.017 0.018 0.017 0.016
Genotype III (n=10) 0.125 0.084 0.137 0.008 0.012 0.012 0.012 0.011 0.009 0.012 0.013 0.014 0.013 0.018 0.009 0.011 0.014 0.014 0.012 0.012 0.012
Genotype IV (n=6) 0.091 0.068 0.123 0.071 0.011 0.009 0.011 0.008 0.008 0.009 0.010 0.013 0.011 0.014 0.007 0.007 0.012 0.012 0.011 0.011 0.010
Genotype V (n=36) 0.093 0.135 0.178 0.144 0.112 0.009 0.011 0.010 0.012 0.014 0.017 0.013 0.011 0.014 0.010 0.009 0.012 0.012 0.013 0.012 0.011
Genotype VI (n=55) 0.051 0.117 0.166 0.135 0.093 0.095 0.008 0.006 0.011 0.011 0.015 0.010 0.008 0.011 0.008 0.007 0.010 0.008 0.008 0.008 0.007
Genotype VII (n=227) 0.031 0.137 0.195 0.147 0.117 0.113 0.079 0.009 0.013 0.015 0.019 0.010 0.008 0.010 0.007 0.009 0.009 0.008 0.007 0.006 0.006
Genotype VIII (n=3) 0.074 0.111 0.160 0.124 0.086 0.092 0.071 0.103 0.010 0.012 0.015 0.010 0.009 0.012 0.010 0.007 0.010 0.010 0.010 0.010 0.009
Genotype IX (n=17) 0.128 0.080 0.127 0.089 0.074 0.143 0.134 0.151 0.123 0.011 0.014 0.014 0.013 0.016 0.006 0.010 0.013 0.013 0.013 0.013 0.012
Genotype X (n=18) 0.145 0.073 0.100 0.123 0.106 0.165 0.151 0.172 0.145 0.113 0.015 0.014 0.014 0.017 0.010 0.010 0.015 0.015 0.014 0.014 0.013
Genotype XI (n=4) 0.189 0.162 0.215 0.172 0.113 0.191 0.189 0.218 0.183 0.165 0.200 0.020 0.018 0.021 0.016 0.014 0.018 0.018 0.018 0.018 0.018
Genotype XII (n=2) 0.057 0.153 0.206 0.163 0.135 0.134 0.088 0.094 0.107 0.165 0.178 0.236 0.009 0.010 0.011 0.010 0.011 0.010 0.011 0.010 0.008
Genotype XIII (n=15) 0.055 0.145 0.208 0.164 0.129 0.129 0.095 0.095 0.113 0.161 0.177 0.213 0.096 0.010 0.010 0.009 0.009 0.008 0.009 0.008 0.005
Genotype XIV (n=8) 0.090 0.188 0.256 0.210 0.172 0.157 0.128 0.121 0.138 0.216 0.217 0.265 0.120 0.123 0.012 0.012 0.010 0.010 0.011 0.011 0.009
Genotype XV (n=5) 0.062 0.084 0.082 0.095 0.068 0.106 0.082 0.068 0.094 0.067 0.110 0.170 0.114 0.108 0.149 0.008 0.011 0.010 0.009 0.009 0.008
Genotype XVI (n=3) 0.069 0.093 0.148 0.108 0.067 0.089 0.072 0.100 0.067 0.108 0.122 0.169 0.105 0.104 0.139 0.085 0.011 0.010 0.010 0.009 0.008
Genotype XVII (n=8) 0.062 0.137 0.209 0.162 0.131 0.128 0.100 0.100 0.118 0.158 0.182 0.210 0.101 0.097 0.111 0.117 0.115 0.007 0.011 0.011 0.008
Genotype XVIII (n=6) 0.050 0.142 0.194 0.155 0.122 0.118 0.080 0.085 0.106 0.151 0.173 0.203 0.083 0.086 0.103 0.097 0.104 0.069 0.009 0.009 0.006
Genotype VIIi (n=22) 0.036 0.139 0.210 0.152 0.126 0.134 0.092 0.068 0.111 0.166 0.179 0.222 0.102 0.098 0.138 0.101 0.108 0.110 0.099 0.007 0.007
Genotype VIIh (n=6) 0.038 0.143 0.201 0.146 0.119 0.132 0.093 0.061 0.109 0.155 0.174 0.221 0.104 0.101 0.132 0.091 0.108 0.109 0.099 0.070 0.007
Genotype XIIIb (n=15) 0.030 0.127 0.189 0.145 0.110 0.109 0.071 0.070 0.094 0.141 0.165 0.207 0.070 0.051 0.103 0.091 0.087 0.078 0.061 0.074 0.077
The numbers of base substitutions per site from averaging over all sequence pairs between groups are shown. Standard error estimate(s) are shown above the diagonal.
Analyses were conducted using the Maximum Composite Likelihood model (Tamura and Nei, 1993; Tamura et al., 2004). The rate variation among sites was modeled with a
gamma distribution (shape parameter = 1). The differences in the composition bias among sequences were considered in evolutionary comparisons (Tamura and Kumar,
2002; Tamura et al., 2011). The analysis involved 642 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and
missing data were eliminated. There were a total of 1581 positions in the final dataset. Evolutionary analyses were conducted in MEGA 5 (Tamura et al., 2011).
Table 5
Comparative evolutionary distances among new and previously defined sub-genotypes based complete full fusion coding sequences.
Ancestral VIIb VIId VIIe VIIf VIIg VIIh VIIi XIIIa XIIIb
(n=6) (n=9) (n=13) (n=7) (n=7) (n=4) (n=6) (n=22) (n=7) (n=15)
Ancestral (n=6) 0.007 0.007 0.006 0.005 0.013 0.008 0.007 0.006 0.01
VIIb (n=9) 0.037 0.002 0.003 0.005 0.011 0.012 0.013 0.012 0.017
VIId (n=13) 0.034 0.01 0.003 0.005 0.01 0.011 0.012 0.012 0.017
VIIe (n=7) 0.031 0.017 0.014 0.004 0.011 0.011 0.012 0.011 0.015
VIIf (n=7) 0.024 0.027 0.024 0.02 0.012 0.01 0.011 0.01 0.015
VIIg (n=4) 0.082 0.067 0.059 0.07 0.073 0.018 0.019 0.016 0.022
VIIh (n=6) 0.044 0.072 0.068 0.063 0.056 0.111 0.013 0.014 0.018
VIIi (n=22) 0.038 0.073 0.071 0.067 0.062 0.115 0.076 0.013 0.017
XIIIa (n=7) 0.032 0.076 0.072 0.064 0.06 0.104 0.08 0.073 0.008
XIIIb (n=15) 0.058 0.101 0.098 0.091 0.085 0.131 0.104 0.099 0.044
The numbers of base substitutions per site from averaging over all sequence pairs between groups are shown. Standard error estimate(s) are shown above the diagonal.
Analyses were conducted using the Maximum Composite Likelihood model (Tamura and Nei, 1993; Tamura et al., 2004). The rate variation among sites was modeled with a
gamma distribution (shape parameter = 1). The differences in the composition bias among sequences were considered in evolutionary comparisons (Tamura and Kumar,
2002; Tamura et al., 2011). The analysis involved 96 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and
missing data were eliminated. There were a total of 1581 positions in the final dataset. Evolutionary analyses were conducted in MEGA 5 (Tamura et al., 2011). Shaded areas
highlight differences between sub-genotypes mentioned in the results section.
534 have occurred mainly in poultry, however, the role of wild bird sub-genotypes are different from other recent strains circulating 544
535 populations cannot be ignored as NDV infected wild birds have in West Africa (genotypes XIV, XVII and XVIII), Madagascar (XI), 545
536 been found in unvaccinated wild birds in Pakistan, in Israel and China (VII, XII and XV), North America (genotypes V), the Carib- 546
537 in a public zoo in Mexico (Cardenas Garcia et al., 2013). All three bean (XVI), or Rock Pigeon viruses (VI). These newer isolates 547
538 of the new sub-genotypes described here have international distri- appear to be most distantly related to viruses old genotypes 548
539 bution, suggesting great mobility of the virus despite high host viruses (I, II, III, IV, X), suggesting that they have an independent 549
540 mortality from high virulence. Thus, at least three new groups of origin from these viruses. 550
541 epizootic strains appear to have evolved independently from Although some of the strains of this new panzootic are still 551
542 different ancestors, and are very similar to viruses circulating related to the viruses from genotype VII and others to genotype 552
543 simultaneously in different geographic locations. All three new XIII, phylogenetic evidence (Figs. 1 and S2) and distance analysis 553
20 November 2014
554 (Tables 4 and 5) suggest that the viruses causative of this pandemic (3) easy and sustainable spread among animals. The viruses 615
555 did not originate directly from of genetic shift from previously described of genotype VIIi, circulating and causing ND outbreaks 616
556 described strains of genotypes VII and XIII, respectively, but rather in Asia, belong to a new sub-genotype. These strains readily infect 617
557 are the result of independent evolution from older viruses circulat- chickens and other bird species, and have been able to rapidly 618
558 ing in Indonesia in Parrots, Cockatoos and wild birds. Table 4 illus- spread and persist in multiple countries. These above-mentioned 619
559 trates the comparative distances among strains previously grouped characteristics and the devastating effects from ND outbreaks from 620
560 into genotypes by Diel and others. It is clear that the distance infections with the vNDV strains from these new sub-genotypes in 621
561 between the new strains from genotypes reported here (VIIi, VIIh Pakistan, Israel and Indonesia suggest that viruses of genotype VIIi 622
562 and XIIIb) fulfill the distance criterion for new sub-genotypes (dis- may have the potential to start a new panzootic. A virus genetically 623
563 tance is 3% or greater from any know genotype) (Table 4). Dis- similar to the ones studied here has also been reported in chicken 624
564 tances found between sub-genotypes VIIi and VIIh and genotype in Bulgaria in 2013, further suggesting the rapid worldwide expan- 625
565 VII range between 6.1% and 7% and distances found between the sion of this new sub-genotype in poultry (Dr. Kiril Dimitrov, 626
566 two new sub-genotypes VIIi and VIIh and sub-genotype XIIIb range National Diagnostic and Research Veterinary Medical Institute, 627
567 from 7.4% to 7.7% (Table 5). Bulgaria, personal communication). Additional biological and path- 628
568 A short phylogenetic distance is observed between current and ological characterization of these viruses should be documented, as 629
569 older Indonesia ancestor strains (Fig. 1 and Table 4). Distances more of these strains are isolated. Viruses of sub-genotype VIIh and 630
570 from the ancestral strains are 3.6, 3.8, and 3%, respectively, for XIIIa, also recently isolated in several countries, appear to be have a 631
571 sub-genotypes VIIi, VIIh and XIIIb. Viruses of sub-genotype VIIh more limited distribution, but should also be followed. 632
572 circulating from 2009 to 2012 in Indonesia, China, and Cambodia The factors that allow a virus to become capable of creating a 633
573 are most closely related to the Indonesia/Bali/01/2007 strain (Adi panzootic are unknown. Often there is not enough epidemiological 634
574 et al., 2010) as show in Fig. 1. All available viruses of sub-genotype data available to understand how a panzootic originated, or the 635
575 VIIi isolated recently in Indonesia, Pakistan and Israel (between data with this type of information are produced retrospectively 636
576 2010 and 2012) and viruses of sub-genotype VIIh appear to be (Yang et al., 1999). However, based on past experiences, human Q4 637
577 most closely related to the ancestral strains lory/Indonesia/88- factors such as the movement of live birds without quarantining 638
578 08989-523/1988 (KF67105) and cockatoo/Indonesia/87-36724- (Walker et al., 1973), lack of testing to ensure a disease free status, 639
579 524/1988 (KF67104) than they are to currently circulating viruses or the inability to contain and eradicate a disease after an outbreak 640
580 from genotype VII. Strains from Indonesia from the late 1980s and is identified, are likely to increase the risk of creating a panzootic. 641
581 early 1990s, such as lory/Indonesia/1988 and cockatoo/Indonesia/ Other human factors that may play a role are the lack of efficient 642
582 1990, appear to represent the most closely known ancestors of veterinary and diagnostics services (as in some African countries), 643
583 vNDV of sub-genotype VIIi (Fig. 1). Viruses of genotype XIII are or cultural and socio-economic factors (e.g. backyard poultry and 644
584 most closely related to the ancestor strain, culex pool/Indonesia/ unvaccinated fighting gamecocks, or lack of economic stimulus to 645
585 JKT/1979 (JX393313) (Forrester et al., 2013). Other strains such conduct culling and or reporting). Biological factors are likely also 646
586 as Cockatoo Indonesia (KF767104) and KF767106 Parrot/Indone- important, but often these are not well understood. An increased 647
587 sia/1976 (KF767106) also appear to be earlier ancestors of the capacity of the virus to replicate in a host without producing clin- 648
588 entire genotype. Similarly, the ancestor strains most closely related ical signs of disease, or the existence of highly mobile hosts are fac- 649
589 to the viruses of genotype XIIIa from Iran and Pakistan appear to be tors that should be considered as they have the potential to create 650
590 a cockatoo/India/7847/1982 (JN942041). In summary, the evidence natural reservoirs in wild birds (Pearson and McCann, 1975; 651
591 here shows that while viruses of sub-genotypes VIIb and VIId con- Takakuwa et al., 1998). It is suspected that when the ability of vir- 652
592 tinue to exist in world, other new sub-genotypes that are closely ulent NDV to replicate in a new host improves, as seen with geno- 653
593 related to strains isolated decades earlier from wild birds are type VIId NDV strains commonly isolated from geese (Wu et al., 654
594 now moving out of the Southeast Asian area and into the Middle 2010), or when it has the ability to replicate in a host unnoticed 655
595 East (as revealed by the presence of isolates Turkey/Israel/Reva- (e.g. chickens infected with pigeon genotype VI strains often do 656
596 dim/497-832/2009 and Pheasant/Israel/Hertzelia/746-828/2013). not show any signs of clinical disease) (Dortmans et al., 2011), 657
the virus may be able to propagate and spread unchecked. Other 658
viral characteristics may also give viruses additional advantages 659
597 3.3. Recombination
such as thermostability, UV resistance, or the ability of the viruses 660
to move into environments that favor survival in the environment 661
598 The recombination analyses were performed with the available
for longer periods of time (such a cold water bodies). As seen with 662
599 complete genomes and full F gene sequences in GenBank and there
the genotype I strains, longer survival time may increase the ability 663
600 was no evidence of sequences created as a result of recombination
of NDV to infect more hosts before becoming inactivated (Ezeifeka 664
601 events among NDV strains of different genotypes (data not shown).
and Onunkwo, 2004). In this instance, the identification of new 665
602 While evidence of recombination in NDV isolates has been
sub-genotype VIIi in three different countries that are not sharing 666
603 reported previously, the biological significance of those recombi-
borders suggested that the VIIi strains could potentially become 667
604 nation events remain to be demonstrated as recombinant
panzootic if not contained. 668
605 sequences can potentially be generated during passage in eggs
The epidemiological information available shows that new 669
606 from samples with mixed infections (Afonso, 2008).
viruses of diverse geographic origin are capable of replacing preva- 670
lent strains in vulnerable countries. The distances highlighted 671
607 4. Discussion (Table 4.) and clear phylogenetic differences (Fig. 1.) using criteria 672
previously described document how these sub-genotype differ 673
608 Here, we present evidence that viruses from at least one new from previously described sub-genotypes (Diel et al., 2012). The 674
609 NDV sub-genotype fulfill the definition of panzootic (VIIi), while three countries reported here are not geographically linked by 675
610 two others (VIIh and XIIIb) that are spreading among several coun- land; however, vNDV infections of poultry in these countries with 676
611 tries have the potential to develop into panzootic viruses and strains that are nearly identical (>99%) indicate a common origin. 677
612 should be further monitored. The definition of a panzootic requires Viruses from the new sub-genotype VIIi may be rapidly replacing 678
613 the fulfillment of three criteria: (1) the emergence of a disease to a vNDV isolates from predominant sub-genotypes in certain loca- 679
614 population, (2) the presence of serious illness caused by the agent, tions as is happening with Israeli isolates of sub-genotypes VIIb 680
20 November 2014
681 and VIId, Chinese isolates of genotype VIId, and Pakistani isolates under the current production practices. The two most common 747
682 of genotype XIIIa. In addition, these vNDV isolates that are poten- vaccine strains used in Pakistan are LaSota and Mukteswar 748
683 tially causing a new panzootic in Asia do not appear to have orig- (Shabbir et al., 2013). The LaSota vaccine is formulated from a len- 749
684 inated from the prevalent poultry vNDV strains from genotype VII, togenic (low virulence) clone of the LaSota strain and is manufac- 750
685 but rather from older strains that were isolated in the 1980s from tured in different countries of the world and imported and 751
686 wild birds. While genotype VII is the most diverse among all of the administered to commercial poultry in Pakistan. However, the 752
687 NDV genotypes, worldwide with its diversity leading to classifica- Mukteswar strain (a virulent strain by OIE definition and a strain 753
688 tion first into ten sub-genotypes and then re-classification into five of moderate virulence from genotype III) continues to be manufac- 754
689 sub-genotypes (VIIb, VIId, VIIe, VIIf, and VIIg) (Diel et al., 2012a), tured by the local vaccine companies and administered to backyard 755
690 none of these sub-genotypes appear to be directly the origin of poultry. This strain which was molecularly characterize in 2008 756
691 the strains of the new panzootic (Xiao et al., 2012; Siddique et al., (Khan, 2009) does not appear to be related to the source of current 757
692 2013; Choi et al., 2013, 2014). outbreaks (Supplementary Fig. S2). These current live NDV vac- 758
693 Southeast Asia represents one of the largest concentrations of cines that protect very well against disease in specific pathogen 759
694 commercial and backyard poultry in the world and their poultry free chickens, may not be effective in preventing the replication 760
695 production system displays some common characteristics that of the strains from these newer sub-genotypes. 761
696 may favor the emergence of vNDV isolates diverse enough to be In Israel, intensive vaccination and lower risk production prac- 762
697 considered new sub-genotypes. Indonesia and Pakistan, as well tices were also insufficient to prevent this new panzootic, thus sug- 763
698 as other Asian countries, have mixed poultry production systems gesting the existence of additional epidemiological factors that 764
699 with a large portion of the birds raised in backyard farms, and favor the spread of the virus. Despite of the existence of a highly 765
700 abundance of live birds markets selling birds of multiple species developed veterinary structure, ND has become a major threat to 766
701 and ages. Historically backyard poultry and live bird markets have the Israeli poultry industry in the last 4 years. Israel has large 767
702 been considered as possible culprits in the maintenance and evolu- and well-secured commercial poultry facilities that have become 768
703 tion of vNDV strains because of the difficulties in vaccinating affected with vNDV isolates from this epizootic. The disease 769
704 newly hatched backyard birds. It is suspected that poorly vacci- affected primarily chickens, but also turkeys, and in smaller num- 770
705 nated commercial poultry, non-vaccinated domestic poultry, and bers, wild birds. Vaccination against NDV in Israel is obligatory and 771
706 wild birds may all act as reservoirs of NDV strains, transmitting the vaccine commonly used is a local LaSota (genotype II) strain 772
707 vNDV isolates to susceptible birds. Live bird markets are also a known as the VH strain, available in both live and killed formula- 773
708 prime concern because different species or breeds of birds originat- tions. Vaccines are routinely checked for efficacy at the State Lab- 774
709 ing from various commercial poultry producers, as well as from oratory for Vaccine Control in the Kimron Veterinary Institute 775
710 backyard farms, from different areas are brought together and (KVI). The efficacy is tested by a challenge procedure using a chal- 776
711 housed in small areas, allowing intermingling of the birds and pos- lenge vNDV from genotype VI isolated during 1967. Chickens are 777
712 sible transmission through contact with contaminated cages, food, vaccinated at 2 week of age and then challenged 3 weeks after vac- 778
713 water and feces. Regardless of the existence of these high-risk pro- cination with a dose of EID50 105.3 per bird according to the phar- 779
714 duction and marketing practices in Asia, our data suggest that macopeia protocols. Israeli vaccination protocols for birds up to 780
715 other factors may add epidemiological risk to the emergence of 21 days old are similar for all types of commercial poultry, how- 781
716 new epizootics in Asia. ever, after 21 days of age the protocols are adjusted for the differ- 782
717 Despite considerable efforts, the sanitary measures to control ent commercial sectors (e.g., broilers, layers, broiler breeders). 783
718 avian influenza (AI) in Indonesia and Pakistan have not been suffi- However, Israel is a very small country with great density of farms, 784
719 cient to prevent new NDV outbreaks. Virulent NDV isolates have with different types of birds of different ages living in close prox- 785
720 been endemic in all areas of Pakistan since 1963 and have been imity and the existence of pet and zoo birds. Many of Israeli (and 786
721 reported in commercial poultry, domestic chickens, turkeys, gui- Pakistani) infections have occurred in zoos and in commercially 787
722 nea fowls, parrots, partridges, and wild crows (Khan and Huq, raised exotic pet species. In Israel significant numbers of pet and 788
723 1963). However, new control measures were implemented in zoo birds are in close proximity to commercial birds, raising the 789
724 1990 through 2005 as a result of outbreaks of three serotypes of possibility that short distances among the commercial poultry 790
725 avian influenza (H9N2, H7N3 and H5N1). However, the collection facilities and exotic and wild birds may have played a role in the 791
726 of surveillance samples for AI has helped elucidate the epidemio- spread of the disease. 792
727 logical situation of ND. The epidemiological work described here Spillover of vNDV isolates from wild birds into poultry should 793
728 represents recent ND outbreaks, which emerged in the northern be further studied. Israel is a route for various species of migratory 794
729 region of Pakistan from November 2011 until March 2012, causing birds that pass over the country on their migratory seasonal jour- 795
730 losses in the broiler industry worth more than USD 6 million. This neys and they can be a source of infection, especially migratory 796
731 ND outbreak also affected wild and exotic birds that died in public waterfowl. Furthermore, birds such as Passeriformes, which are 797
732 zoos and domestic poultry in backyard farms, however, no official widespread in Israel and are common in open spaces, may be shed- 798
733 figures on the numbers birds lost in the field have been released. ding vNDV in their feces without showing clinical signs (Haddas 799
734 The incidence of ND has continued at all levels of production facil- et al., 2013). Similarly, in Pakistan during the winter migratory 800
735 ities despite the use of full time veterinary services developed to birds from Siberia harbor in the coastal areas, marshlands, and 801
736 control avian influenza in the most advanced commercial produc- open regions around the Arabian Sea. The problem in Israel may 802
737 tion facilities. Ironically, in 2009 in Pakistan 60% of the commercial be replicated in other nearby countries in the Middle East that have 803
738 broilers initially reported as having died from AI were later con- not yet conducted large epidemiological studies on vNDV (Iran, 804
739 firmed dead due to ND (Khan, 2009). The emergence of AI has facil- Saudi Arabia, Kuwait (Anonymous, 2011). In the Americas a ND 805
740 itated increased surveillance and improved bio-security; outbreak in turkeys in 1972 was caused by a strain of sub-geno- 806
741 surprisingly, the focus on AI has not been enough to prevent the type Va that subsequently became established in wild cormorants. 807
742 spread of vNDV (Naeem and Hussain, 1995; Naeem et al., 1999; Isolates of vNDV of genotype V were first commonly isolated from 808
743 Wasilenko et al., 2012). birds in European countries (Czegledi et al., 2002; Wehmann et al., 809
744 The Pakistani ND outbreaks suggest that the use of live and 2003), but now are mainly distributed in the Americas with most 810
745 inactivated vaccines that typically are able to induce sufficient frequent isolations in Mexico, Canada and the USA (Cardenas 811
746 antibody levels may not be sufficient to control ND outbreaks Garcia et al., 2013; Nolen, 2002; Weingartl et al., 2003). Sub-geno- 812
20 November 2014
813 type Va is comprised of vNDV isolates obtained in North American 6612-32000-064 and from Department of State grants entitled 877
814 cormorants where the virus seems to be maintained year to year Molecular Epidemiology of Newcastle Disease Viruses in Indonesia 878
815 with periodic outbreaks in newly hatched birds (Rue et al., 2010), and Development of Assays for Identification Newly Evolved Viru- 879
816 but has spilled over into other wild bird populations (Diel et al., lent Strains of Newcastle Disease Virus from Pakistan. 880
817 2012b). Viruses of genotype VI also have frequently spilled into
818 poultry and caused outbreaks of economic importance in Europe
819 and Japan. Viruses of sub-genotype VIb are mainly isolated from Appendix A. Supplementary data 881
820 pigeons (Kim et al., 2008), occasionally spill over into chickens or
821 other species (Abolnik et al., 2008, 2004; Irvine et al., 2009) and Supplementary data associated with this article can be found, in 882
822 have some genome variations that are found less frequently the online version, at http://dx.doi.org/10.1016/j.meegid.2014. 883
823 (Pchelkina et al., 2013). 10.032. 884
824 Non-poultry reservoirs that may support the origin and spread
825 of new vNDV isolates may be a factor in this epizootic. Despite the References 885
826 reported existence of 19 genotypes containing vNDV strains, only
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Adi, A.A., Astawa, N.M., Putra, K.S., Hayashi, Y., Matsumoto, Y., 2010. Isolation and 892
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848 2008 outbreak in Peru, but have also been isolated from geese in Cardenas Garcia, S., Navarro Lopez, R., Morales, R., Olvera, M.A., Marquez, M.A., 914
849 live bird markets in China in 2011 (Diel et al., 2012a,c). However, Merino, R., Miller, P.J., Afonso, C.L., 2013. Molecular epidemiology of Newcastle 915
850 the origin of genotype XII is also unknown and its occurrence on disease in Mexico and the potential spillover of viruses from poultry into wild 916
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851 two different continents without a clear epidemiological link sug- Cavill, J.P., 1974. Newcastle disease in imported psittacine birds. Vet. Rec. 94, 226– 918
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853 vNDV strains (Courtney et al., 2013; Diel et al., 2012a). In one- Choi, K.S., Kye, S.J., Kim, J.Y., Damasco, V.R., Sorn, S., Lee, Y.J., Choi, J.G., Kang, H.M., 920
Kim, K.I., Song, B.M., Lee, H.S., 2013. Molecular epidemiological investigation of 921
854 instance NDV isolates appear to have evolved unnoticed over a velogenic Newcastle disease viruses from village chickens in Cambodia. Virus 922
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859 psittacines as reservoirs or origin of new strains has been previ- Afonso, C.L., 2013. Highly divergent virulent isolates of Newcastle disease virus 928
from the Dominican Republic are members of a new genotype that may have 929
860 ously suggested (Lomniczi et al., 1998). There are reports of vNDV 930
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861 strains being isolated from apparently healthy psittacines during Czegledi, A., Herczeg, J., Hadjiev, G., Doumanova, L., Wehmann, E., Lomniczi, B., 931
862 outbreaks and in quarantine stations (Cavill, 1974; Roy et al., 2002. The occurrence of five major Newcastle disease virus genotypes (II, IV, V, 932
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863 1998) and some parrots were able to shed vNDV for over a year
688. 934
864 after they were infected (Erickson et al., 1977). Psittacines were Czegledi, A., Ujvari, D., Somogyi, E., Wehmann, E., Werner, O., Lomniczi, B., 2006. 935
865 suggested to be the cause of the second ND pandemic (Eaves and Third genome size category of avian paramyxovirus serotype 1 (Newcastle 936
866 Grimes, 1978; Hanson, 1974). Genotype XIII NDV isolates have disease virus) and evolutionary implications. Virus Res. 120, 36–48. 937
de Almeida, R.S., Hammoumi, S., Gil, P., Briand, F.X., Molia, S., Gaidet, N., Cappelle, J., 938
867 been reported to have an ancestral connection to strains from Chevalier, V., Balanca, G., Traore, A., Grillet, C., Maminiaina, O.F., Guendouz, S., 939
868 Indonesia (Forrester et al., 2013). Our data support those previous Dakouo, M., Samake, K., Bezeid Oel, M., Diarra, A., Chaka, H., Goutard, F., 940
869 observations. Conclusively, the circulation of vNDV isolates from Thompson, P., Martinez, D., Jestin, V., Albina, E., 2013. New avian 941
paramyxoviruses type I strains identified in Africa provide new outcomes for 942
870 three new sub-genotypes in three different regions of the world phylogeny reconstruction and genotype classification. PLoS ONE 8, e76413. 943
871 indicates that there are very few geographic boundaries for vNDV Diel, D.G., da Silva, L.H., Liu, H., Wang, Z., Miller, P.J., Afonso, C.L., 2012a. Genetic 944
872 in existence within its susceptible hosts. diversity of avian paramyxovirus type 1: proposal for a unified nomenclature 945
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873 Acknowledgments Diel, D.G., Miller, P.J., Wolf, P.C., Mickley, R.M., Musante, A.R., Emanueli, D.C., 948
Shively, K.J., Pedersen, K., Afonso, C.L., 2012b. Characterization of Newcastle 949
disease viruses isolated from cormorant and gull species in the United States in 950
874 We would like to acknowledge Tim Olivier, and Dawn Wil-
2010. Avian Dis. 56, 128–133. 951
875 liams-Coplin for technical assistance with the animal experiment Diel, D.G., Susta, L., Cardenas Garcia, S., Killian, M.L., Brown, C.C., Miller, P.J., Afonso, 952
876 and sequencing. This research was supported by DA-ARS CRIS C.L., 2012c. Complete genome and clinicopathological characterization of a 953
20 November 2014
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MEEGID 2143 No.

of Pages 14, Model 5G

Infection, Genetics and Evolution xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Infection, Genetics and Evolution

3 Identiﬁcation of new sub-genotypes of virulent Newcastle disease virus

62 1. Introduction the raising or keeping of birds (Anonymous, 2011). The etiological 66

⇑ Corresponding author. single-stranded, non-segmented, negative sense RNA genome. 72

E-mail address: Claudio.Afonso@ars.usda.gov (C.L. Afonso).

(continued on next page)

VII-KF7677104 cockatoo/ VIIi-HQ697254 chicken/ VIIh-HQ697255

823 (Pchelkina et al., 2013). 10.032. 884

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