Infection, Genetics and Evolution 29 (2015) 216–229

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Identification of new sub-genotypes of virulent Newcastle disease virus with potential panzootic features

Patti J. Miller a, Ruth Haddas b, Luba Simanov b, Avishay Lublin b, Shafqat Fatima Rehmani c, Abdul Wajid c, ⇑ Tasra Bibi c, Taseer Ahmad Khan d, Tahir Yaqub c, Surachmi Setiyaningsih e, Claudio L. Afonso a, a Southeast Poultry Research Laboratory, Agricultural Research Service-United States Department of Agriculture (USDA), Athens, GA 30605, USA b Kimron Veterinary Institute, Bet Dagan 50250, Israel c Quality Operations Laboratory, University of Veterinary and Animal Sciences, Out Fall Road, Lahore, Pakistan d Poultry Research Laboratory, Department of Physiology, University of Karachi, Karachi, Pakistan e Department of Infectious Diseases & Veterinary Public Health, Faculty of Veterinary Medicine-Bogor Agricultural University, Jl. Agatis, IPB Dramaga, Bogor 16680, Indonesia article info abstract

Article history: Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly Received 29 May 2014 spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized Received in revised form 25 October 2014 by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, Accepted 30 October 2014 which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to Available online 20 November 2014 have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Keywords: Viruses from sub-genotype VIIh were isolated in Indonesia (2009–2010), Malaysia (2011), China Newcastle disease (2011), and Cambodia (2011–2012) and are closely related to the Indonesian NDV isolated in 2007, NDV Epidemiology APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related Panzootic NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasion- Outbreak ally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype Poultry VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009–2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub- genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics. Published by Elsevier B.V.

1. Introduction the raising or keeping of birds (Anonymous, 2011). The etiological agent of ND, virulent NDV, belongs to the genus Avulavirus of the Newcastle disease virus (NDV) is distributed worldwide and its family Paramyxoviridae (Mayo, 2002). The virus was originally continual presence in multiple avian species presents a constant detected in Java, Indonesia and Newcastle-‘on-Tyne, England threat to all poultry industries and other activities that involve (Doyle, 1927), and since then various genotypes have been respon- sible for different ND panzootics. The virus is enveloped, with a single-stranded, non-segmented, negative sense RNA genome. ⇑ Corresponding author. E-mail address: [email protected] (C.L. Afonso). http://dx.doi.org/10.1016/j.meegid.2014.10.032 1567-1348/Published by Elsevier B.V. P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229 217

Multiple genotypes of NDV have been circulating worldwide panzootic constituted by highly related vNDV isolates from (Miller et al., 2010). NDV isolates may be classified into genotypes Indonesia, Israel and Pakistan. These virus strains belong to a based on either the complete genome sequences or the full fusion new vNDV sub-genotype (VIIi), and together with the existence protein sequences from NDV isolates (Diel et al., 2012a). At this of additional sub-genotypes (VIIh and XIIIa and XIIIb) related to time, ND viruses are grouped into one genotype for class I NDV iso- older strains from wild birds suggest that unknown reservoirs lates, and in eighteen genotypes for class II NDV isolates, some harbor new vNDV isolates capable of additional panzootics. with sub-genotypes (Courtney et al., 2013; de Almeida et al., 2013; Diel et al., 2012a; Snoeck et al., 2013). The 2012 a classifica- tion system of NDV was proposed based on the utilization of the 2. Material and methods complete sequence of the fusion (F) protein gene (Diel et al., 2012a). The system was based on the mean inter-population evo- 2.1. Isolation of NDV virulent viruses lutionary distance between previous existing NDV genetic groups, and differences of 10% (at the nucleotide level) were proposed as All laboratories followed the same protocol to isolate NDV the cutoff value to assign new genotypes. This system grouped strains except that chickens used to produce the 9–11 day old NDV isolates of class I into a single genotype comprised of mainly embryonating chicken eggs needed for virus isolation were specific viruses that have been isolated from waterfowl and shorebirds, and pathogen free (SPF) for those isolated in Israel and The United occasionally from samples collected in live bird markets world- States of America (USA) and were free of NDV antibodies for those wide and captured wild birds (Kim et al., 2007a,b; Miller et al., isolated in Pakistan and Indonesia (Alexander and Swayne, 1998). 2010). Class II viruses were initially grouped into 15 genotypes; NDV strains cockatoo/Indonesia/87-36724-524/1988; lory/Indone- however, four additional genotypes have been added since 2012 sia/88-08989-523/1988 and parrot/Indonesia/C300 (19625)-520/ (Courtney et al., 2013; Diel et al., 2012a; Snoeck et al., 2013). 1976 were obtained and propagated in SPF embryos from the Viruses from class II are present in both wild bird and poultry repository of the United States Department of Agriculture (USDA) species; however, most virulent NDV (vNDV) isolates are obtained Southeast Poultry Research Laboratory (SEPRL) (Alexander and from poultry and are responsible for significant economic losses to Swayne, 1998). These three historical samples were obtained dur- the poultry industry worldwide (Dundon et al., 2012). Viruses of ing the importation and quarantine of exotic birds into the USA and 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 zootic during 1920s to 1960s (Alexander, 2001), whereas the sec- than during the transport process. Pakistani isolates were obtained ond panzootic in Europe during the late 1960s was resulted from from swabs samples from poultry obtained from 16 outbreaks in isolates of genotype V (Lomniczi et al., 1998). Viruses from geno- different regions of the country during the winter of October types III, IV, IX and X are related to those of genotypes of I and II, 2011 through March 2012 and propagated in embryonating but only circulate in limited areas of the world. Viruses of sub- chicken eggs that were free from antibodies against NDV. Repre- genotype VIb originated in the Middle East and were responsible sentative samples of each outbreak were characterized by for the third panzootic in pigeons during the 1980s (Kaleta et al., sequencing of the full fusion (F) protein. Israeli isolates consisted 1985). Genotypes VII and VIII were responsible for ND outbreaks of a total of 33 diagnostic swab samples obtained from dry cloacal in Asia, including Pakistan, and in Europe since 1984 or earlier and tracheal swabs from poultry and pet birds that were sent to (Diel et al., 2012a; Shabbir et al., 2013). Viruses from genotype the Kimron Veterinary Institute (KVI) of Israel for evaluation. All VII are responsible for the fourth panzootic, which continues today, data regarding the origins of the samples, and the health and vac- having spread from Asia, Africa, Europe and has even been isolated cination status of the birds sampled were documented. Swabs in South America (Miller and Koch, 2013; Perozo et al., 2012). The were maintained in À20 °C until processing. 300 ll of phosphate fourth panzootic of ND began around 1985 in Southeast Asia and buffer solution (PBS) were added to each swab and incubated at spread to most countries of Africa and in Venezuela, South America room temperature for 30 min before extraction of RNA. Virus isola- (Herczeg et al., 1999; Perozo et al., 2012; Yu et al., 2001). Geno- tion was carried out by inoculation of embryonating SPF chicken 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 ‘‘late’’ genotypes (Czegledi et al., 2006) and only contain vNDV for 6 days (Senne, 1998). Intracerebral pathogenicity index (ICPI) strains. Currently, viruses from genotype VII are most frequently assays were conducted on hemagglutination (HA) positive allan- associated with outbreaks of ND in the Middle East (Radwan toic fluids (Alexander and Swayne, 1998) following established et al., 2013), and Asia (Yi et al., 2011). These viruses are of partic- procedures (OIE, 2012). Fourteen Indonesia isolates were either ular concern as some have demonstrated higher mortality in vacci- obtained from samples from the repository at the Faculty of nated poultry (Yi et al., 2011), while others may have expanded Veterinary Medicine, Bogor Agricultural University (IPB), or iso- their host range and are now able to cause disease in geese lated from diagnostic swab samples collected from field visits to (Wang et al., 2012). In Israel the first case of genotype VII NDV live bird markets and poultry handling facilities. Samples from was reported in 2000 (data not published). Genotype XIV contains commercial poultry farms were from producers willing to partici- vNDV isolates obtained in West and Central Africa between 2006 pate in the study. While the study area included the islands of and 2008 (Snoeck et al., 2013), which are divided into three sub- Sumatra, Kalimantan (Indonesian Borneo), Java, Bali, and Nusa genotypes XIVa, b and c (de Almeida et al., 2013). Genotype XV Tenggara (The Lesser Sunda Islands) representing the western (Diel et al., 2012a) comprises isolates obtained from chickens and and central areas of Indonesia that includes the top six most pop- geese in China, which have been previously classified into ulated urban areas, NDV strains were only able to be isolated from sub-genotype VIId (isolates XJ-2/97 and FJ-2/99) or VIIe (isolate the samples from the island of Java. Oropharyngeal and cloacal JX-2/99) (Liu et al., 2003). swabs were collected from all birds, and organ tissue samples from The emergence and spread of new genotypes across the dead birds. Sample collection information for each bird was world represents a significant threat to poultry and suggest that included when possible: date of sampling, host species (poultry vNDV is continuously evolving, leading to more diversity (Miller species and breed if known), estimated age (breeder, layer, broiler, et al., 2009). However, little has been done to understand the chicks), environment description (type of facilities), and the geo- mechanisms of maintenance and evolution of new genotypes graphic location (name of city and province or GPS coordinates). (Alexander et al., 2012). Here we have characterized recent vNDV The GPS coordinates, flock size data, and percent mortality were isolates and present evidence that suggest the emergence of a fifth not available for the Indonesian samples. 218 P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229

2.2. RNA isolation and nucleotide sequencing on 104 complete fusion gene sequences was performed to identify the phylogenetic relationship of our isolates in comparison to rep- For the three older Indonesian strains from the SEPRL resentative viruses of different genotypes for which full F gene repository, RNA was extracted from allantoic fluids using Trizol sequences were available (Diel et al., 2012a)(Fig. 1). The phyloge- LS (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s netic analysis was performed with the software MEGA5 (MEGA, instructions. The complete F gene nucleotide sequences were version 5) (Tamura et al., 2011) and the evolutionary history was determined by using a RT-PCR/sequencing approach (Diel et al., inferred by using the Neighbor Joining (data not shown), and the 2012b). Amplification reactions were performed with a one-step Maximum likelihood methods (Tamura and Kumar, 2002), with RT-PCR kit (Qiagen, Valencia, CA, USA) and a set of fusion (F) gene statistical analysis based on 100 bootstraps. specific primers (fwd_upper-f1-ttgcttatagttagttcgcctgtc, and To assess if the results obtained with the analysis of the com- rev_down-f2-acccgtgtattgctctttgg). The PCR amplicons were sub- plete F gene sequences would be reflected at the whole genome jected to electrophoresis in 1% agarose gels and the DNA bands level, we performed a phylogenetic analysis with complete gen- were excised from the gels and purified by using the Quick Clean ome sequences available on GenBank (n = 100) (Supplementary DNA gel extraction kit (Qiagen, Valencia, CA, USA). The purified Fig. S2). For this analysis complete coding sequences for all six PCR products were cloned into the TOPO TA vector (Invitrogen, genes were concatenated and the complete genome analysis was Carlsbad, CA, USA) according to manufacturer’s instructions and bases on the concatenated coding regions only. Analysis of the subjected to DNA sequencing. All sequencing reactions were per- best-fit substitution model was performed using MEGA5, and the formed with fluorescent dideoxy-nucleotide terminators in an goodness-of-fit of each model was measured by Bayesian Informa- ABI 3700 automated sequencer (Applied Bio systems Inc., Foster tion Criterion (BIC) and corrected Akaine Information Criterion City, CA, USA). Sequence editing and assembly were performed (AIC) (Tamura et al., 2011). The General Time Reversible (GTR) with Laser Gene sequence analysis software package (Laser Gene, model with a discrete gamma distribution (+G) and allowing for version 5.07; DNA Star, Inc., Madison, WI, USA). RNA isolation invariant sites (+I) was selected and used in all data analyses and nucleotide sequencing of the Pakistani viruses were performed (Tamura et al., 2011). Codon positions included in the analysis as follows: samples of HA positive allantoic fluids (Alexander and were the 1st, 2nd, 3rd, and non-coding. All positions containing Swayne, 1998) were extracted using QIA ampÒ Viral RNA mini kits, gaps and missing data were eliminated. A total of 1650 positions Qiagen (AA) following the manufacturer’s recommended proce- were included in the analysis of the complete F gene dataset, and dure and confirmed to be NDV using RT-PCR and universal primers 13,735 positions were included in the analysis of the complete for the nucleoprotein (NP) of NDV. A total of 16 viruses, one from genome data set. different outbreaks, were selected for sequencing of the full F The full fusion data set used for the phylogenetic reconstruction protein. Complementary DNA (cDNA) was synthesized from 10 ll was also used to infer the evolutionary distances within and of eluted RNA produced with reverse transcriptase of the First between groups comparing recent epizootic strains to three histor- Strand cDNA Synthesis KitÒ, Thermo Fisher Scientific, (Fermentas, ical samples from Indonesia (Tables 3–5). The evolutionary dis- Waltham, Massachusetts, USA) following manufacturer’s recom- tances were inferred by pair-wise analysis using the MEGA5 mendations. Primers specifically designed for this study (Supple- software (Tamura and Kumar, 2002; Tamura et al., 2011) and the mentary Table 1) were used for the amplification of complete F maximum composite likelihood method was used to calculate gene. Using the SEPRL protocols described above, the PCR ampli- the number of base substitutions per site (Nei and Kumar, 2000). cons were run on 1% agarose gels, purified and sequenced, as The variation rate among sites was modeled with a gamma distri- described above. For Israeli isolates viral RNA was extracted from bution (shape parameter = 1) and the differences in the composi- the swabs using the QIA amp Viral RNA Mini Kit (Qiagen, Santa tion bias among sequences were considered in the evolutionary Clarita, CA, USA) according to the manufacturer’s instructions. comparisons (Tamura et al., 2011). Codon positions included in The real time RT-PCR (RRT-PCR) assay that targeted the matrix the analysis were the 1st, 2nd, 3rd, and non-coding. All positions (M) gene was performed as previously described (Wise et al., containing gaps and missing data were eliminated. The numbers 2004), and sub-typing by RRT-PCR that targeted the fusion protein presented in the phylogenetic trees represent the nine-digit Gen was performed according to Fuller et al. (2009), with modification Info Identifier (GI) sequence identification number, and/or the to the velogenic probe VRP-1 (Aguilar and Lee, 2011) CCTATAAAG- two letter-six digit accession number from GenBank, while the CGTTTCTGTCTCCTTCC [BHQ1], (Haddas et al., 2013). The portion numbers at the bifurcations of the tree nodes represent the boot- targeting a 382-bp fragment encoding the amino terminal end of strap support. the F protein of the 33 viruses was amplified by PCR using primers specifically designed for this study according to Aldous et al, 2003 2.4. Recombination analysis and purified as described above (Aldous et al., 2003). Partial fusion sequencing was performed for 14 representative isolates from Recombination analysis was performed with all class II com- Indonesia. For these samples RNA isolation and viral sequencing plete genome (n = 103) and complete F gene (n = 602) sequences was done using SEPRL protocols for viral RNA extraction and for using the program RDP3 (Martin et al., 2010) (data not shown). cDNA production from RT-PCR, as described above. Four statistical methods (RDP, GENECONV, Maxchi and Chimera) were used to identify putative recombinant sequences. Sequences 2.3. Phylogenetic analyses and inference of the evolutionary distances with recombination events identified by at least two detection methods (p < 0.001) were considered as true recombinants. A preliminary phylogenetic analysis was performed with 81 sequences (available on GenBank as of 03/30/2014: http:// 2.5. Genotype classification criteria www.ncbi.nlm.nih.gov/genbank/) corresponding to the partial coding sequence of the F gene with 481 nucleotides (nt) to identify Genotypes and sub-genotypes were assigned based on the and select viral isolates that closely related to sequenced viruses of phylogenetic topology and on the evolutionary distances between Israel, Indonesia and Pakistan (Supplementary Fig. S1). The S1 phy- different taxonomic groups utilizing the criteria previously estab- logenetic identifies highly related viruses to those studied here, lished (Diel et al., 2012a). Phylogeny was determined using com- and documents epidemiological information from isolates that plete genomes for those sequences were that information was did not have a full fusion sequence available. A similar analysis available. However, the classification and calculation of distances P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229 219

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 (À12579.2213) 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. for most viruses were performed using only the complete fusion (F) the criteria to separate genotypes and distances of 3–9.99% differ- gene coding sequences since those dataset were more complete. ences were used to separate sub-genotypes (0.030–0.099). Complete F gene sequences of viruses of three new sub-genotypes were identified after the phylogenetic analysis was performed and 2.6. Accession numbers all viruses were used to infer the evolutionary distances between genetic sub-groups. The mean inter population evolutionary diver- All the Pakistani complete F gene sequences (n = 16) used in this sity (mean evolutionary distance between genotypes) was deter- study were submitted to GenBank and are available under the mined using the maximum composite likelihood model (Tamura accession numbers from KF113338 to KF113353. GenBank acces- et al., 2004). The cutoff distance value of 10% (0.1) was used as sion numbers from the full F gene sequences of Israeli NDV strains 220 P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229

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 P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229 221

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

not previously published are KF792018 to KF792023. The partial F between vaccination with live attenuated vaccines and the onset sequences for the fourteen Indonesia NDV strains are found under of exposure to the virus, with more time leading to fewer losses. accession numbers KF767104 through KF767120. The three older Most notable, is the inexplicable occurrence of mortality greater Indonesian isolates from the SEPRL repository are available under than 60% in broiler production facilities that practice intensive the accession numbers KF767104 to KF767106. vaccination with flock sizes larger than 20,000 birds. In addition, layers, small flocks, and non-poultry species have also been occa- 3. Results sionally infected. It has been noted that in the province of Punjab the mortality rate was often higher in flocks maintained in environ- 3.1. Epidemiological description of the new epizootic viruses mentally controlled houses than those in open-air houses. Most (60–80%) of the reported outbreaks either occurred during the win- The 2011–2012 outbreaks in Pakistan were unique in several ter season of the year, or time throughout the year when there is a aspects. These were the first documented cases in which wild birds significant variation (10–15 °C) between the day and night ambient (peacocks, pheasants, and parrots of different breeds) reared in cap- temperatures, which is usually at the start of winter season and tivity in both public and private zoos of Pakistan were affected with before summer. The ND outbreak occurred from November 2011 mortality rates of 40–60%. The disease also affected Rock Pigeons to March 2012 and samples from 105 flocks, which were comprised (Columba livia) and Indian Peafowl (Pavocristatus) in the province of 1.12 M broilers, 0.0033 M layers, 0.1 M breeder broilers and 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 and early months of 2013. The peacocks displayed nervous signs, of NDV. The average age of the above flocks and breeds were such as tremors, disorientation, and weakness prior to death. Table 1 assessed and found that the range was from 4 weeks old for broilers and S1 demonstrate that vNDV strains were widely distributed to 25 weeks old for layers. Broiler breeders averaged 50 weeks old across Pakistan with a presence at different latitudes, geographic and wild birds were 15 weeks old; however, two samples were from environments, and types of production systems. However, the levels peafowl that were 84 weeks of age. As expected, the broiler industry of mortality and morbidity depended on the amount of time was most affected, as these birds would have lower antibody levels 222 P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229

Table 2 isolates and in all cases values higher than 1.7 were observed, con- Biological evaluation of virulence from selected isolates. firming the presence of vNDV strains (Table 2). However, vNDV Virus designationa Genbank acc. ICPIb MDTc was also isolated from vaccinated broiler breeders with no clinical Turkey/Isr/Revadim/497/2009 KF792023 1.85 <60 signs (e.g. 1349/2012) in the west bank (Palestinian authority). Chicken/Isr/HodHasharon/19/2011 KF650614 1.88 60–90 This sample (e.g. 1349/2012) was collected for routine surveillance Turkey/Isr/Ram-on/21/2011 KF650615 NA <60 to confirm vaccination against infectious bronchitis virus (IBV), Chicken/Melea/Isr/30/2011 JN849575 1.89 <60 avian influenza virus (AIV), and NDV. There is also evidence of wide Chicken/Isr/Baqua-al-Garbia/174/2011 JN849578 NA 60–90 distribution of vNDV outside production facilities in captive birds. Chicken/Isr/Mashabei-Sade/2011/352 JN849576 NA <60 Chicken/Isr/Argaman/555/2011 JN849577 NA <60 For example, one strain (e.g. 838/2012) was obtained from a free- Falcon/Isr/Zichron-Yaakov/638/2011 JN638345 2 <60 living rooster found dead at a ‘‘Safari’’ zoo located in an urban area Owl/Isr/Ramat-Gan/645-32011 JN979566 1.86 <60 in the city of Ramat-Gan, which is in the center of the country, with Penguin/Isr/Ramat-Gan/669-2/2011 JN979565 1.65 >90 no other reports of vNDV at this time in this zoo, or from any com- Buzzard/Isr/Zichron-Yaakov/692-10/2010 JN638349 NA 60–90 Owl/Isr/Ramat-Gan/713/2011 JN638358 NA 60–90 mercial farms nearby. However, a year earlier vNDV was detected Chicken/Isr/Maaleh-Hachamsha/998/2011 KC484655 1.63 <60 at that zoo in Little Owls and African Penguins (Haddas et al., Chicken/Isr/Natur/1004/2011 JX192955 1.75 <60 2013), suggesting an in apparent chain of transmission in the Turkey/Isr/Avnei-Eytan/1090/2011 JX192956 NA 60–90 Zoo. Samples from other infected wild bird species included a Chicken/Israel/Shaal/235/2012 KF650617 NA 60–90 Common Quail (e.g. 844/2012) that died without clinical signs of Chicken/Isr/Ramat-Gan/838/2012 KF650618 1.74 <60 Parrot/Isr/Beit-Shikam/841/2012 KF792020 NA <60 ND, and a Common Kestrel chick (e.g. 843/2012) at the Jerusalem Falcon/Isr/Jerusalem/843/2012 KF650620 1.74 <60 Biblical zoo that died 3 days after developing symptoms, another Quail/Isr/Ramat-Gan/844/2012 KF650621 1.88 <60 (e.g. 875/2012) isolated from a Atlantic canary chick with respira- Chicken/Isr/Holon/870/2012 KF650622 NA <60 tory distress, anorexia and death after few days from a private Canary/Isr/Jerusalem/875/2012 KF650623 1.86 <60 Falcon/Isr/Rishon-Lezion/911/2012 KF650624 1.88 <60 breeder in the Jerusalem area, and lastly, one (e.g. 926/2012) iso- Falcon/Isr/Ashdod/925/2012 KF650625 1.84 <60 lated from a Common Kestrel that was found dead in an urban Falcon/Isr/Ben-Shemen/926/2012 KF650626 NA <60 Jerusalem area. Falcon/Isr/Ramat-Gan/939/2012 KF650627 NA <60 In Indonesia NDV has been described since 1926 (Miller and Turkey/Isr/Brosh/1301/2012 KF650628 NA >90 Koch, 2013) and the disease appears to be endemic with the last Chicken/Isr/Kvuzat-Yavne/50/2013 KF792019 NA 60–90 Chicken/Isr/Be’er Tuvia/120/2013 KF792021 NA 60–90 report to the World Organization for Animal Health (OIE) of Pheasant/Isr/Hertzelia/746-16/2013 KF792022 NA <60 infected commercial birds in 2012 (www.OIE.int). The disease causes significant economic impact despite the availability of a a 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. range of vaccines. In small family farms (backyard farms; identified b Values 0.7 or greater are identified as virulent by the O.I.E. by the Indonesian government as ‘‘sector 4’’) chickens are mostly c Mean death times (MDT) less than 60 h are indicative of a virulent NDV strain, either not vaccinated or inadequately vaccinated, and therefore, and values between 60 and 90 h suggest a moderately virulent NDV strain. outbreaks in young chicks often result in 100% mortality with adult birds also having significant morbidity and mortality. Despite the from having had fewer vaccines administered over their shorter life- extensive vaccination programs administered to commercial flocks span. The local broiler breeders also had heavy mortality, ranging (known as sectors, 1, 2 and 3) frequent outbreaks of ND with mor- from 20 to 80%. While virus was recovered from broilers, it was dif- talities at lower levels occur with considerable financial losses to ficult to isolate vNDV from the grandparent flocks, as well as from the local poultry industry. the day old chicks, either live or dead. A similar situation has occurred in Israel and despite the fact 3.2. Phylogenetic classification, distribution, and possible origin of that vaccination against vNDV in Israel is mandatory ND outbreaks viruses from this new epizootic were very severe. Since the end of 2010 through 2013, only with some relief in the months of summer (July–August), ongoing ND To characterize the molecular epidemiology of recent outbreaks outbreaks have continued. We present here the evaluation of the in Asia characterization of strains of NDV was conducted by biolog- virulence of 33 NDV strains with virulent characteristics either ical assessment of ICPI, MDT and by sequencing. Partial and full by ICPI (n = 13), mean death time (MDT) (n = 28) or sequencing fusion sequences from viruses from Pakistan (n = 16), Israel (n = 33). Virulent NDV infections were reported in very young (n = 34), and Indonesia (n = 17, including three older ancestral chicks, older broiler breeders, zoos and wild birds including strains), as described in Table 1, were sequenced and the pathoge- pigeons. The outbreak that began at the end of 2010 was vast with nicity of selected isolates was determined (Table 2) to confirm viru- vNDV diagnosed in very young chicks and from broiler breeder lence. The partial and full F gene sequence of NDV isolates were farms, which are considered to have high biosecurity levels. During compared with reference strains belonging to genotypes I–XVIII of 2011–2012, nearly 300 cases of vNDV were diagnosed in commer- class II viruses. The cleavage sites of the fusion protein (Table 1) cial chickens of all ages, turkeys, wild birds, pet birds, captive zoo demonstrate that the most common configurations of the fusion birds, and African penguins. Since June of 2012, additional cases cleavage site were 112RRQKR;F117 (n = 59) and 112RRRKR;F117 have been observed in broiler breeder farms. The usual presenta- (n = 7), suggesting that all of these strains are virulent with tion of respiratory disease was observed in different types of birds the exception of one Indonesian strain isolated from a duck and isolates representing each are these; in vaccinated layers (e.g. (112GKQGR;L117). Phylogenetic trees were generated based on 481 isolate 259/2009), chickens (e.g. 235/2012) or turkeys (e.g. 497/ base pairs (bp) of the variable region (Aldous et al., 2003), for 2009 and 1301/2012) broilers (e.g. 174/2011 and 998/2011). maximum sample representation (Supplementary Fig. S1), or with NDV infection also caused a drop in egg productions in broiler the full fusion (F) coding region for distance determination and breeders (e.g. isolate 50/2013 and 120/2013). In non-poultry classification into sub-genotypes (Fig. 1 and Tables 3–5)(Diel species ND infection (e.g. parrot 841/2012) manifested with et al., 2012a). Complete genomes of all NDV strains available from diarrhea and mortality, in Falconiformes (e.g. 925/2012) with dis- GenBank were used for confirmation of phylogenetic classification orientation, weakness and mortality, and in ornamental chickens (Supplementary Fig. S2). The phylogeny of full coding sequences (e.g. 746/2013) with respiratory signs, weakness, cachexia, and (1650 nt) for the F gene sequences available from GenBank, mortality within a week. ICPI assays were conducted on selected representing ND viruses isolated worldwide, was compared to the P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229 223

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-616914933/KF113346-chicken/Pak/Khyber Pukhtun Khawa/118/2011 0.045 0.001 0.058 VIIi-616914935/KF113347-chicken/Pak/Khyber Pukhtun Khawa/119/2012 0.046 0.001 0,059 VIIi-616914937/KF113348-chicken/Pak/Khyber Pukhtun Khawa/162/2012 0.045 0.001 0.058 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).

Pakistani, Israeli and Indonesian strains. Phylogenetic analysis of 1% and distances between individual strains of genotype VIIi and the complete F coding regions suggests that the new sub-genotype h is also close to 5% indicating that those two groups are separate VIIi circulating in Indonesia is 99% similar to strains present in Israel sub-genotypes. Comparison of nucleotide sequences of NDV strains and Pakistan (Fig. 1(bold) and Table 3). A different sub-genotype included in this study with other published local and foreign iso- (VIIh), also related to viruses previously circulating in Indonesia lates revealed that all of the new Pakistani strains, except (1998–2010), was also isolated from poultry in Malaysia (2004– chicken/University Diagnostic Laboratory (UDL)/Pakistan (Pak)/ 2006), China (2011 and 2012) and Cambodia (2012) (Supplemen- 12/2010 and chicken/Pak/Lahore/33/2011, have >99% homology tary Fig. S2). The third newly identified sub-genotype, XIIIb, was with other Indonesian strains, such as chicken/Banjarmasin/010/ represented by viruses from Iran, Russia, Europe, India and South 2010, chicken/Kudus/018/2010, chicken/Sragen/014/2010 and America is related to previously described isolates from Pakistan. chicken/Gianyar/013/2010. The NDV strains characterized in this The data show that previously predominant genotypes (VIIb, and study have lower identity (87–93%) with other previously charac- VIId in Israel and XIIIa in Pakistan) have been replaced by the new terized Pakistani strains; chicken/backyard poultry (BYP)/Pak/ sub-genotypes VIIi and VIIh during 2012–2013, although VIId con- 2010, chicken-commercial poultry (CP)/Pak/Islamabad3/2010, tinues to circulate in Israel. Isolates from genotype VIIi were chicken/CP/Pak/Rawalpindi2/2010, chicken/Pak/Sindh Poultry Vac- obtained from poultry production facilities throughout Pakistan cine Center (SPVC)/Karachi/1/1974, chicken/Pak/SPVC/Karachi/43/ and Israel, an occasionally from pet birds are also highly related to 2008, chicken/Pak/SPVC/Karachi/26/2005, chicken/Pak/SPVC/Kar- current Indonesian isolates. achi/33/2007, chicken/CP/Pak/2010, chicken/BYP/Pak/Lahore/2010 The phylogenetic analysis for the current NDV isolates (Figs. 1, and chicken/CP/Pak/Islamabad1/2010. While viruses from geno- S1 and S2, and Table 3) demonstrate that NDV strains currently cir- type VIIi were predominantly responsible for the ND outbreaks culating in Pakistan and Israel are nearly identical, however, they during the winter session of October 2011 through March 2012 have separate genetic origins from NDV isolates previously in Pakistan, our previous studies conducted on NDV strains from reported in the country. Table 3 documents the nucleotide dis- this country showed the predominance of genotype XIII in Pakistan tances of the F protein among individual viruses of highly related (Khan et al., 2010). All of the newer NDV strains from Pakistan, strains of sub genotypes VIIi and h. Distances of approximately except chicken/Pak/UDL/12/2010, also clustered together with 5% between the ancestral Cockatoo/Indonesia/1987 strain and Indonesian and Israeli strains, suggesting rapid spread and epizo- viruses of new sub-genotypes VIIi and h are demonstrated otic characteristics for this sub-genotype. Outbreaks in Pakistan (Table 3). However, distances within sub-genotypes is lower than have occurred mainly in poultry, however, the role of wild bird 224 P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229

Table 4 Estimates of net evolutionary divergence of the complete fusion coding sequences between groups.

Anc. (n=6) G I G II G III G IV G V G VI G VII G VIII G IX G X 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 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). The grey-highlighted areas with white numbers compare the newly defined sub-genotypes VIIi, VIIh and XIIIb with the ancestral viruses. The 3 grey shaded areas on the bottom right with black numbers are the distances comparing the 3 new sub-genotypes to each other.

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 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.

populations cannot be ignored as NDV infected wild birds have different ancestors, and are very similar to viruses circulating been found in unvaccinated wild birds in Pakistan, in Israel and simultaneously in different geographic locations. All three new in a public zoo in Mexico (Cardenas Garcia et al., 2013). All three sub-genotypes are different from other recent strains circulating of the new sub-genotypes described here have international distri- in West Africa (genotypes XIV, XVII and XVIII), Madagascar (XI), bution, suggesting great mobility of the virus despite high host China (VII, XII and XV), North America (genotypes V), the Carib- mortality from high virulence. Thus, at least three new groups of bean (XVI), or Rock Pigeon viruses (VI). These newer isolates epizootic strains appear to have evolved independently from appear to be most distantly related to viruses old genotypes P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229 225 viruses (I, II, III, IV, X), suggesting that they have an independent two others (VIIh and XIIIb) that are spreading among several coun- origin from these viruses. tries have the potential to develop into panzootic viruses and Although some of the strains of this new panzootic are still should be further monitored. The definition of a panzootic requires related to the viruses from genotype VII and others to genotype the fulfillment of three criteria: (1) the emergence of a disease to a XIII, phylogenetic evidence (Figs. 1 and S2) and distance analysis population, (2) the presence of serious illness caused by the agent, (Tables 4 and 5) suggest that the viruses causative of this pandemic (3) easy and sustainable spread among animals. The viruses did not originate directly from of genetic shift from previously described of genotype VIIi, circulating and causing ND outbreaks described strains of genotypes VII and XIII, respectively, but rather in Asia, belong to a new sub-genotype. These strains readily infect are the result of independent evolution from older viruses circulat- chickens and other bird species, and have been able to rapidly ing in Indonesia in Parrots, Cockatoos and wild birds. Table 4 illus- spread and persist in multiple countries. These above-mentioned trates the comparative distances among strains previously grouped characteristics and the devastating effects from ND outbreaks from into genotypes by Diel and others. It is clear that the distance infections with the vNDV strains from these new sub-genotypes in between the new strains from genotypes reported here (VIIi, VIIh Pakistan, Israel and Indonesia suggest that viruses of genotype VIIi and XIIIb) fulfill the distance criterion for new sub-genotypes may have the potential to start a new panzootic. A virus genetically (distance is 3% or greater from any know genotype) (Table 4). Dis- similar to the ones studied here has also been reported in chicken tances found between sub-genotypes VIIi and VIIh and genotype in Bulgaria in 2013, further suggesting the rapid worldwide expan- VII range between 6.1% and 7% and distances found between the sion of this new sub-genotype in poultry (Dr. Kiril Dimitrov, two new sub-genotypes VIIi and VIIh and sub-genotype XIIIb range National Diagnostic and Research Veterinary Medical Institute, from 7.4% to 7.7% (Table 5). Bulgaria, personal communication). Additional biological and path- A short phylogenetic distance is observed between current and ological characterization of these viruses should be documented, as older Indonesia ancestor strains (Fig. 1 and Table 4). Distances more of these strains are isolated. Viruses of sub-genotype VIIh and 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 sub-genotypes VIIi, VIIh and XIIIb. Viruses of sub-genotype VIIh more limited distribution, but should also be followed. circulating from 2009 to 2012 in Indonesia, China, and Cambodia The factors that allow a virus to become capable of creating a are most closely related to the Indonesia/Bali/01/2007 strain (Adi panzootic are unknown. Often there is not enough epidemiological et al., 2010) as show in Fig. 1. All available viruses of sub-genotype data available to understand how a panzootic originated, or the VIIi isolated recently in Indonesia, Pakistan and Israel (between data with this type of information are produced retrospectively 2010 and 2012) and viruses of sub-genotype VIIh appear to be (Yang et al., 1999). However, based on past experiences, human most closely related to the ancestral strains lory/Indonesia/88- factors such as the movement of live birds without quarantining 08989-523/1988 (KF67105) and cockatoo/Indonesia/87-36724- (Walker et al., 1973), lack of testing to ensure a disease free status, 524/1988 (KF67104) than they are to currently circulating viruses or the inability to contain and eradicate a disease after an outbreak from genotype VII. Strains from Indonesia from the late 1980s and is identified, are likely to increase the risk of creating a panzootic. early 1990s, such as lory/Indonesia/1988 and cockatoo/Indonesia/ Other human factors that may play a role are the lack of efficient 1990, appear to represent the most closely known ancestors of veterinary and diagnostics services (as in some African countries), vNDV of sub-genotype VIIi (Fig. 1). Viruses of genotype XIII are or cultural and socio-economic factors (e.g. backyard poultry and most closely related to the ancestor strain, culex pool/Indonesia/ unvaccinated fighting gamecocks, or lack of economic stimulus to JKT/1979 (JX393313) (Forrester et al., 2013). Other strains such conduct culling and or reporting). Biological factors are likely also as Cockatoo Indonesia (KF767104) and KF767106 Parrot/Indone- important, but often these are not well understood. An increased sia/1976 (KF767106) also appear to be earlier ancestors of the capacity of the virus to replicate in a host without producing entire genotype. Similarly, the ancestor strains most closely related clinical signs of disease, or the existence of highly mobile hosts to the viruses of genotype XIIIa from Iran and Pakistan appear to be are factors that should be considered as they have the potential a cockatoo/India/7847/1982 (JN942041). In summary, the evidence to create natural reservoirs in wild birds (Pearson and McCann, here shows that while viruses of sub-genotypes VIIb and VIId con- 1975; Takakuwa et al., 1998). It is suspected that when the ability tinue to exist in world, other new sub-genotypes that are closely of virulent NDV to replicate in a new host improves, as seen with related to strains isolated decades earlier from wild birds are genotype VIId NDV strains commonly isolated from geese (Wu now moving out of the Southeast Asian area and into the Middle et al., 2010), or when it has the ability to replicate in a host unno- East (as revealed by the presence of isolates Turkey/Israel/Reva- ticed (e.g. chickens infected with pigeon genotype VI strains often dim/497-832/2009 and Pheasant/Israel/Hertzelia/746-828/2013). do not show any signs of clinical disease) (Dortmans et al., 2011), the virus may be able to propagate and spread unchecked. Other viral characteristics may also give viruses additional advantages 3.3. Recombination such as thermostability, UV resistance, or the ability of the viruses to move into environments that favor survival in the environment The recombination analyses were performed with the available for longer periods of time (such a cold water bodies). As seen with complete genomes and full F gene sequences in GenBank and there the genotype I strains, longer survival time may increase the ability was no evidence of sequences created as a result of recombination of NDV to infect more hosts before becoming inactivated (Ezeifeka events among NDV strains of different genotypes (data not shown). and Onunkwo, 2004). In this instance, the identification of new While evidence of recombination in NDV isolates has been sub-genotype VIIi in three different countries that are not sharing reported previously, the biological significance of those recombi- borders suggested that the VIIi strains could potentially become nation events remain to be demonstrated as recombinant panzootic if not contained. sequences can potentially be generated during passage in eggs The epidemiological information available shows that new from samples with mixed infections (Afonso, 2008). viruses of diverse geographic origin are capable of replacing preva- lent strains in vulnerable countries. The distances highlighted 4. Discussion (Table 4.) and clear phylogenetic differences (Fig. 1.) using criteria previously described document how these sub-genotype differ Here, we present evidence that viruses from at least one new from previously described sub-genotypes (Diel et al., 2012). The NDV sub-genotype fulfill the definition of panzootic (VIIi), while three countries reported here are not geographically linked by 226 P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229 land; however, vNDV infections of poultry in these countries with spread of vNDV (Naeem and Hussain, 1995; Naeem et al., 1999; strains that are nearly identical (>99%) indicate a common origin. Wasilenko et al., 2012). Viruses from the new sub-genotype VIIi may be rapidly replacing The Pakistani ND outbreaks suggest that the use of live and vNDV isolates from predominant sub-genotypes in certain loca- inactivated vaccines that typically are able to induce sufficient tions as is happening with Israeli isolates of sub-genotypes VIIb antibody levels may not be sufficient to control ND outbreaks and VIId, Chinese isolates of genotype VIId, and Pakistani isolates under the current production practices. The two most common of genotype XIIIa. In addition, these vNDV isolates that are poten- vaccine strains used in Pakistan are LaSota and Mukteswar 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- inated from the prevalent poultry vNDV strains from genotype VII, togenic (low virulence) clone of the LaSota strain and is manufac- but rather from older strains that were isolated in the 1980s from tured in different countries of the world and imported and wild birds. While genotype VII is the most diverse among all of the administered to commercial poultry in Pakistan. However, the NDV genotypes, worldwide with its diversity leading to classifica- Mukteswar strain (a virulent strain by OIE definition and a strain tion first into ten sub-genotypes and then re-classification into five of moderate virulence from genotype III) continues to be manufac- sub-genotypes (VIIb, VIId, VIIe, VIIf, and VIIg) (Diel et al., 2012a), tured by the local vaccine companies and administered to backyard none of these sub-genotypes appear to be directly the origin of poultry. This strain which was molecularly characterize in 2008 the strains of the new panzootic (Xiao et al., 2012; Siddique (Khan, 2009) does not appear to be related to the source of current et al., 2013; Choi et al., 2013, 2014). outbreaks (Supplementary Fig. S2). These current live NDV vac- Southeast Asia represents one of the largest concentrations of cines that protect very well against disease in specific pathogen commercial and backyard poultry in the world and their poultry free chickens, may not be effective in preventing the replication production system displays some common characteristics that of the strains from these newer sub-genotypes. may favor the emergence of vNDV isolates diverse enough to be In Israel, intensive vaccination and lower risk production prac- considered new sub-genotypes. Indonesia and Pakistan, as well tices were also insufficient to prevent this new panzootic, thus sug- as other Asian countries, have mixed poultry production systems gesting the existence of additional epidemiological factors that 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 abundance of live birds markets selling birds of multiple species developed veterinary structure, ND has become a major threat to and ages. Historically backyard poultry and live bird markets have the Israeli poultry industry in the last 4 years. Israel has large been considered as possible culprits in the maintenance and evolu- and well-secured commercial poultry facilities that have become tion of vNDV strains because of the difficulties in vaccinating affected with vNDV isolates from this epizootic. The disease newly hatched backyard birds. It is suspected that poorly vacci- affected primarily chickens, but also turkeys, and in smaller num- nated commercial poultry, non-vaccinated domestic poultry, and bers, wild birds. Vaccination against NDV in Israel is obligatory and wild birds may all act as reservoirs of NDV strains, transmitting the vaccine commonly used is a local LaSota (genotype II) strain vNDV isolates to susceptible birds. Live bird markets are also a known as the VH strain, available in both live and killed formula- prime concern because different species or breeds of birds originat- tions. Vaccines are routinely checked for efficacy at the State Lab- ing from various commercial poultry producers, as well as from oratory for Vaccine Control in the Kimron Veterinary Institute backyard farms, from different areas are brought together and (KVI). The efficacy is tested by a challenge procedure using a chal- housed in small areas, allowing intermingling of the birds and pos- lenge vNDV from genotype VI isolated during 1967. Chickens are sible transmission through contact with contaminated cages, food, vaccinated at 2 week of age and then challenged 3 weeks after vac- 5.3 water and feces. Regardless of the existence of these high-risk pro- cination with a dose of EID50 10 per bird according to the phar- duction and marketing practices in Asia, our data suggest that macopeia protocols. Israeli vaccination protocols for birds up to other factors may add epidemiological risk to the emergence of 21 days old are similar for all types of commercial poultry, how- new epizootics in Asia. ever, after 21 days of age the protocols are adjusted for the differ- Despite considerable efforts, the sanitary measures to control ent commercial sectors (e.g., broilers, layers, broiler breeders). avian influenza (AI) in Indonesia and Pakistan have not been suffi- However, Israel is a very small country with great density of farms, cient to prevent new NDV outbreaks. Virulent NDV isolates have with different types of birds of different ages living in close prox- 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 reported in commercial poultry, domestic chickens, turkeys, gui- Pakistani) infections have occurred in zoos and in commercially nea fowls, parrots, partridges, and wild crows (Khan and Huq, raised exotic pet species. In Israel significant numbers of pet and 1963). However, new control measures were implemented in zoo birds are in close proximity to commercial birds, raising the 1990 through 2005 as a result of outbreaks of three serotypes of possibility that short distances among the commercial poultry avian influenza (H9N2, H7N3 and H5N1). However, the collection facilities and exotic and wild birds may have played a role in the of surveillance samples for AI has helped elucidate the epidemio- spread of the disease. logical situation of ND. The epidemiological work described here Spillover of vNDV isolates from wild birds into poultry should represents recent ND outbreaks, which emerged in the northern be further studied. Israel is a route for various species of migratory region of Pakistan from November 2011 until March 2012, causing birds that pass over the country on their migratory seasonal jour- losses in the broiler industry worth more than USD 6 million. This neys and they can be a source of infection, especially migratory ND outbreak also affected wild and exotic birds that died in public waterfowl. Furthermore, birds such as Passeriformes, which are zoos and domestic poultry in backyard farms, however, no official widespread in Israel and are common in open spaces, may be shed- figures on the numbers birds lost in the field have been released. ding vNDV in their feces without showing clinical signs (Haddas The incidence of ND has continued at all levels of production facil- et al., 2013). Similarly, in Pakistan during the winter migratory ities despite the use of full time veterinary services developed to birds from Siberia harbor in the coastal areas, marshlands, and control avian influenza in the most advanced commercial produc- open regions around the Arabian Sea. The problem in Israel may tion facilities. Ironically, in 2009 in Pakistan 60% of the commercial be replicated in other nearby countries in the Middle East that have broilers initially reported as having died from AI were later not yet conducted large epidemiological studies on vNDV (Iran, confirmed dead due to ND (Khan, 2009). The emergence of AI has Saudi Arabia, Kuwait (Anonymous, 2011). In the Americas a ND facilitated increased surveillance and improved bio-security; sur- outbreak in turkeys in 1972 was caused by a strain of sub-geno- prisingly, the focus on AI has not been enough to prevent the type Va that subsequently became established in wild cormorants. P.J. Miller et al. / Infection, Genetics and Evolution 29 (2015) 216–229 227

Isolates of vNDV of genotype V were first commonly isolated from Acknowledgments birds in European countries (Czegledi et al., 2002; Wehmann et al., 2003), but now are mainly distributed in the Americas with most We would like to acknowledge Tim Olivier, and Dawn Wil- frequent isolations in Mexico, Canada and the USA (Cardenas liams-Coplin for technical assistance with the animal experiment Garcia et al., 2013; Nolen, 2002; Weingartl et al., 2003). Sub- and sequencing. This research was supported by DA-ARS CRIS genotype Va is comprised of vNDV isolates obtained in North 6612-32000-064 and from Department of State grants entitled American cormorants where the virus seems to be maintained year Molecular Epidemiology of Newcastle Disease Viruses in Indonesia to year with periodic outbreaks in newly hatched birds (Rue et al., and Development of Assays for Identification Newly Evolved 2010), but has spilled over into other wild bird populations (Diel Virulent Strains of Newcastle Disease Virus from Pakistan. et al., 2012b). Viruses of genotype VI also have frequently spilled into poultry and caused outbreaks of economic importance in Appendix A. Supplementary data Europe and Japan. Viruses of sub-genotype VIb are mainly isolated from pigeons (Kim et al., 2008), occasionally spill over into chick- Supplementary data associated with this article can be found, in ens or other species (Abolnik et al., 2008, 2004; Irvine et al., the online version, at http://dx.doi.org/10.1016/j.meegid.2014. 2009) and have some genome variations that are found less fre- 10.032. quently (Pchelkina et al., 2013). Non-poultry reservoirs that may support the origin and spread of new vNDV isolates may be a factor in this epizootic. Despite References the reported existence of 19 genotypes containing vNDV strains, Abolnik, C., Gerdes, G.H., Kitching, J., Swanepoel, S., Romito, M., Bisschop, S.P., 2008. only two natural reservoirs where virus is maintained and shed Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated from apparently healthy birds have been regularly documented in South Africa from 2001 to 2006. Onderstepoort J. Vet. Res. 75, 147–152. over time. 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