Phylogenetic analysis of Newcastle disease viruses isolated from waterfowl in the Upper Midwest Region of the United States

  • Naresh Jindal1,

    Affiliated with

    • Yogesh Chander1,

      Affiliated with

      • Ashok K Chockalingam1,

        Affiliated with

        • Martha de Abin1,

          Affiliated with

          • Patrick T Redig1 and

            Affiliated with

            • Sagar M Goyal1Email author

              Affiliated with

              Virology Journal20096:191

              DOI: 10.1186/1743-422X-6-191

              Received: 14 July 2009

              Accepted: 05 November 2009

              Published: 05 November 2009

              Abstract

              Background

              This study was conducted to characterize Newcastle disease virus (NDV) isolates obtained from waterfowl from the Upper Midwest region of the United States. A total of 43 NDVs were isolated by inoculation of cloacal samples in embryonated chicken eggs. These isolates were obtained from 24 mallards, seven American green-winged teals, six northern pintails, four blue-winged teals, and two wood ducks. Partial sequences of fusion gene were analyzed to determine the pathotypes and genotypes involved.

              Results

              Deduced amino acid sequence of the cleavage site of fusion (F) protein revealed that all isolates had avirulent motifs. Of the 43 isolates, 23 exhibited sequence motif of 111GGKQGRL117 at the cleavage site, 19 exhibited 111GEKQGRL117 while one isolate showed 111GERQGRL117. Phylogenetic analysis based on comparison with different classes of NDVs revealed that all 43 isolates clustered with class II NDVs and none with class I NDVs. Within class II, five isolates were phylogenetically close to genotype I NDVs while the remaining 38 were close to genotype II.

              Conclusion

              We conclude that more than one genotype of NDV circulates in waterfowl in the Upper Midwest region of the US. Continuous surveillance may help better understand the epidemiology of NDVs maintained in wild bird populations and their relationship to NDVs in domestic poultry, if any.

              Background

              Avian paramyxoviruses (APMV) belong to genus Avulavirus in the family Paramyxoviridae. The genome of APMV is an approximately 15 kb long, negative-sense, single-stranded RNA molecule. It has six genes that encode for a nucleoprotein (N), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), an attachment protein called hemagglutinin-neuraminidase (HN), and a large polymerase protein (L) [1]. Nine serotypes of avian paramyxoviruses (APMV-1 to APMV-9) have been identified. Of these, APMV-1, also called the Newcastle disease virus (NDV), is the causative agent of Newcastle disease (ND) in poultry. Based on genetic and antigenic analyses of NDV isolates, two major classes (class I and class II) are identified [2, 3] and each class has nine genotypes (1-9 genotypes in class I and I-IX in class II) [4, 5].

              The NDV can cause clinical signs varying from subclinical infections to 100% mortality, depending on the susceptibility of the host and the virulence of the virus. The virus is categorized into velogenic (velogenic neurotropic or velogenic viscerotropic), mesogenic, lentogenic, and asymptomatic enteric strains on the basis of their pathogenesis and virulence. The velogenic strains cause acute fatal infection of chickens of all age groups with clinical findings of nervous signs or extensive hemorrhagic lesions in the gastrointestinal tract. The mesogenic strains are of intermediate virulence and cause moderate respiratory signs with occasional nervous signs while the lentogenic strains cause mild to inapparent infections [1]. The lentogenic strains have been detected in both domestic poultry [68] and wild bird populations [4, 8, 9]. Though velogenic strains are considered exotic (exotic Newcastle disease, END) to US poultry, these strains have been isolated occasionally from different avian species in the US [10, 11]. During 2002-2003, California outbreak of END in backyard fowl and commercial poultry resulted in the destruction of about 3.3 million birds and cost $200 million dollars to control the disease [11, 12]. Outbreaks of ND have been reported in many countries with considerable economic losses [1]. Such outbreaks warrant continuous surveillance for END in commercial poultry and wild birds.

              The surveillance of NDVs in waterfowl is sporadic and often occurs with other monitoring programs such as those for avian influenza viruses (AIV) [13, 14]. Wild birds are considered the natural reservoirs of NDVs and mostly harbor lentogenic strains. Studies on genetic diversity among lentogenic strains of NDVs revealed that some of the NDVs from waterfowl and shorebirds were phylogenetically related with NDVs isolated from live-bird markets in the US [4]. It is recommended that epidemiological studies should be continued to determine the prevalence of lentogenic NDVs in wild bird populations [4]. An epidemiological link between isolates recovered from outbreaks in domestic poultry with those obtained from wild bird populations has also been suggested [8, 9, 15, 16]. Therefore, continuous surveillance of wild bird populations may help better understand the NDVs circulating in the environment. This study was conducted to characterize NDV isolates obtained from waterfowl samples. In this study, the cloacal samples from waterfowl from Upper Midwest region of the US were initially screened for AIV by real time reverse transcription-polymerase chain reaction (rRT-PCR); the AIV positive samples by rRT-PCR were inoculated on to the embryonated eggs for virus isolation that yielded NDV in some of them. The NDV isolates were characterized by sequencing to determine the pathotypes and genotypes involved and the changes at the nucleotide and amino acid levels.

              Results

              Altogether, 159 viral isolations from cloacal samples of AIV rRT-PCR-positive waterfowl (n = 890) were obtained, as shown by hemagglutinating (HA) activity of allantoic fluid in embryonated eggs. Of these, 43 were positive for NDV by reverse transcription-polymerase chain reaction (RT-PCR). BLAST analysis of partial sequences of F gene of NDV isolates confirmed their identity. These isolates were obtained from 24 mallards (MALL; Anas platyrhynchos), seven American green-winged teals (AGWT; Anas crecca), six northern pintails (NOPI; Anas acuta), four blue-winged teals (BWTE; Anas discors), and two wood ducks (WODU; Aix sponsa). Spatial distribution revealed that 28 isolates were obtained from South Dakota, 14 from Minnesota, and 1 from North Dakota.

              Cleavage site analysis

              The F gene portion (333 base pairs) corresponding to nucleotide positions 170-502 of GenBank accession number AF217084 was sequenced. Deduced amino acid sequences of the F gene cleavage site were used to determine the pathotypes involved and are shown in Table 1. The fusion gene of virulent NDVs is characterized by the presence of a pair of dibasic amino acids at the cleavage site while in lentogenic strains it is characterized by the presence of monobasic amino acids. None of the isolates had the sequence motif of 111GR/KRQRK/RF117, a characteristic of the virulent strains. All 43 NDVs had an avirulent motif of monobasic amino acids at their F gene cleavage sites. Of the 43 isolates, 23 exhibited sequence motif of 111GGKQGRL117, 19 exhibited the sequence motif of 111GEKQGRL117, and one isolate exhibited the sequence motif of 111GERQGRL117 at the cleavage site of F gene.
              Table 1

              Details of Newcastle disease viral isolates of this study.

              GenBank

              accession

              number

              Isolate name

              Fusion gene

              cleavage

              site (111-117)

              Class

              Genotype

              Species

              State

              Country

              GQ229531

              NDV-001/US(MN)/2008

              GEKQGRL

              II

              II

              Mallard

              Minnesota

              USA

              GQ229532

              NDV-002/US(MN)/2008

              GGKQGRL

              II

              I

              AGWTA

              Minnesota

              USA

              GQ229533

              NDV-003/US(MN)/2008

              GEKQGRL

              II

              II

              AGWT

              Minnesota

              USA

              GQ229534

              NDV-004/US(MN)/2008

              GEKQGRL

              II

              II

              Mallard

              Minnesota

              USA

              GQ229535

              NDV-006/US(MN)/2008

              GGKQGRL

              II

              II

              Northern pintail

              Minnesota

              USA

              GQ229536

              NDV-007/US(SD)/2008

              GGKQGRL

              II

              I

              Northern pintail

              South Dakota

              USA

              GQ229537

              NDV-009/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229538

              NDV-011/US(SD)/2008

              GGKQGRL

              II

              I

              Mallard

              South Dakota

              USA

              GQ229539

              NDV-012/US(SD)/2008

              GGKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229540

              NDV-013/US(SD)/2008

              GGKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229541

              NDV-015/US(SD)/2008

              GEKQGRL

              II

              II

              Northern pintail

              South Dakota

              USA

              GQ229542

              NDV-016/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229543

              NDV-017/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229544

              NDV-018/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229545

              NDV-019/US(SD)/2008

              GGKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229546

              NDV-020/US(SD)/2008

              GGKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229547

              NDV-021/US(SD)/2008

              GGKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229548

              NDV-022/US(SD)/2008

              GGKQGRL

              II

              II

              AGWT

              South Dakota

              USA

              GQ229549

              NDV-023/US(SD)/2008

              GGKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229550

              NDV-024/US(SD)/2008

              GGKQGRL

              II

              I

              Northern pintail

              South Dakota

              USA

              GQ229551

              NDV-025/US(SD)/2008

              GGKQGRL

              II

              II

              AGWT

              South Dakota

              USA

              GQ229552

              NDV-026/US(SD)/2008

              GEKQGRL

              II

              II

              Northern pintail

              South Dakota

              USA

              GQ229553

              NDV-027/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229554

              NDV-028/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229555

              NDV-029/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229556

              NDV-030/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229557

              NDV-031/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229558

              NDV-032/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229559

              NDV-033/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229560

              NDV-034/US(SD)/2008

              GEKQGRL

              II

              II

              Mallard

              South Dakota

              USA

              GQ229561

              NDV-035/US(SD)/2008

              GEKQGRL

              II

              II

              AGWT

              South Dakota

              USA

              GQ229562

              NDV-036/US(MN)/2008

              GGKQGRL

              II

              II

              Wood duck

              Minnesota

              USA

              GQ229563

              NDV-037/US(MN)/2008

              GGKQGRL

              II

              II

              Mallard

              Minnesota

              USA

              GQ229564

              NDV-038/US(MN)/2008

              GGKQGRL

              II

              II

              Blue-winged teal

              Minnesota

              USA

              GQ229565

              NDV-039/US(MN)/2008

              GGKQGRL

              II

              II

              Blue-winged teal

              Minnesota

              USA

              GQ229566

              NDV-040/US(MN)/2008

              GGKQGRL

              II

              II

              AGWT

              Minnesota

              USA

              GQ229567

              NDV-041/US(MN)/2008

              GGKQGRL

              II

              II

              AGWT

              Minnesota

              USA

              GQ229568

              NDV-042/US(MN)/2008

              GGKQGRL

              II

              II

              Blue-winged teal

              Minnesota

              USA

              GQ229569

              NDV-043/US(MN)/2008

              GGKQGRL

              II

              II

              Wood duck

              Minnesota

              USA

              GQ229570

              NDV-048/US(SD)/2008

              GGKQGRL

              II

              II

              Blue-winged teal

              South Dakota

              USA

              GQ229571

              NDV-049 US(MN)/2008

              GERQGRL

              II

              I

              Mallard

              Minnesota

              USA

              GQ229572

              NDV-050/US(SD)/2008

              GEKQGRL

              II

              II

              Northern pintail

              South Dakota

              USA

              GQ229573

              NDV-051/US(ND)/2008

              GGKQGRL

              II

              II

              Mallard

              North Dakota

              USA

              AAGWT = American Green-winged teal

              Phylogenetic analysis

              Phylogenetic analysis of partial F gene nucleotide sequences of NDV isolates was done by comparing them with already published F gene sequences of both class I and class II NDVs. None of the isolates clustered with class I NDVs (Figure 1); all isolates clustered with class II NDVs (Figure 1). Within class II, all isolates clustered with genotype I or II. Five of the 43 isolates clustered with NDV sequences of genotype I/Ia suggesting them to belong to genotype I (Figure 1). Four of the five isolates clustered together with genotype I NDVs from the US [Mallard/US(MD)/04-483/2004, EF564942; Mallard/US(MD)/04-204/2004, EF564821; and Mallard/US(MD)/04-235/2004, EF564901] and Korea [KR/duck/05/07, EU547755]. The sequence homology among these four isolates was 99.6% to 100% at the nucleotide level. The remaining one isolate was in a different group from these four isolates and was phylogenetically closer to genotype I NDVs from China [Heb02, AY427817], the US [AV 80/97 D813-2, AY175736] and Ireland [AV 963/98 NZ5/97, AY175726]. This isolate had sequence homology of 90.9% to 90.4% at nucleotide level with the other four isolates of genotype I of this study. All five genotype I isolates had sequence homology of 87.9% to 100% with class II genotype I NDVs used for comparison.
              http://static-content.springer.com/image/art%3A10.1186%2F1743-422X-6-191/MediaObjects/12985_2009_Article_705_Fig1_HTML.jpg
              Figure 1

              Phylogenetic tree based on partial nucleotide sequences [corresponding to nucleotide positions 170-502 of GenBank: AF217084] of fusion gene of Newcastle disease virus. The sequences starting with NDV (without accession numbers) are from the present study, and the sequences with virus name (GenBank accession numbers) are previously published sequences of NDVs. The phylogenetic tree was constructed by Neighbor-Joining method, 500 bootstrap replicates (bootstrap values are shown on tree).

              The remaining 38 isolates clustered with genotype II NDVs. These isolates clustered into two groups with 19 isolates in each group. For ease of understanding, we have named these two groups as X and Y (Figure 1). The isolates in group X were phylogenetically close to genotype IIa NDVs from wild birds from different regions of the US [Mallard/US(MD)/03-152/2003, EF564972; Mallard/US(MD)/01-618/2001, EF565012; Mallard/US(MN)/99-397/1999, EF565032; Mallard/US(MN)/98-350/1998, EF565019; and Mallard/US(MD)/03-807/2003, EF564993]. The isolates in group X were also phylogenetically close to a genotype IIa NDV from Argentina [32C/T.98, AY727881], but the latter was in a different group. None of the already reported NDV sequences of class II genotype II used for comparison clustered together with NDV isolates of group Y. Though the isolates in group Y were phylogenetically close to already reported genotype IIa NDVs from wild birds in the US [Blue winged teal/US(LA)/87-190/1987, EF564836; Blue winged teal/US(LA)/87-155/1987, EF564834; Blue winged teal/US(LA)/87-247_b/1987, EF564841], they were not in the same group. The vaccine strains [LaSota, AY845400; B1, M24695] clustered in a different group from isolates of this study. All already published sequences of velogenic strains with in class II were phylogenetically distinct from NDVs of this study (Figure 1). The sequence homology of genotype II isolates of this study ranged from 95.5% to 100% at the nucleotide level, and the homology as compared to already published sequences of class II genotype II ranged from 90.4% to 100%.

              Discussion

              This study was conducted to characterize NDVs isolated from waterfowl in the Upper Midwest region of the US. The initial aim of this study was to isolate and characterize AIV from waterfowl. During the study period, 7458 cloacal samples were collected and of these, 11.9% samples were AIV positive by rRT-PCR. Inoculation of these AIV positive samples in embryonated chicken eggs yielded hemagglutinating viruses and of these, 43 were identified as NDVs by RT-PCR using primer specific for F gene. We were expecting the isolation of AIV rather than NDV on inoculation in embryonated eggs as the samples were initially positive for AIV by rRT-PCR. The possibility of the presence of other hemagglutinating virus(es) in HA positive-AIV negative (by RT-PCR for matrix gene)-NDV negative (by RT-PCR for F gene) allantoic fluid cannot be ruled out and testing of such allantoic fluid is underway in our laboratory. The isolation of NDV from samples that were rRT-PCR positive for AIV indicates that the cloacal sample may have mixed infection with NDV and AIV with concentration of NDV being higher than that of AIV. Hence, the NDV probably overgrew AIV upon inoculation in embryonated chicken eggs. It is to be noted that we tested only AI rRT-PCR positive samples by inoculation in embryonated eggs; testing of more samples might have led to isolation of more NDVs. The isolation of NDV from AIV positive samples indicates the presence of both viruses (AIV and NDV) in waterfowl. The AIV positive allantoic fluid by RT-PCR was not tested for NDV; this testing might provide a better picture of mixed infection of both NDV and AIV. Mixed infection of AIV and NDV in waterfowl has been reported earlier [17, 18].

              A large amount of sequence data on NDVs isolated throughout the world has been published over the years and is now available for sequence comparison and phylogenetic analysis which can be used to predict the pathotypes and to determine the origin of NDV outbreaks. It has been well established that cleavage of NDV fusion protein is a major determinant for viral virulence. In this study, the F gene sequence of NDVs was used for pathotyping as well as their characterization into different classes and genotypes. None of the isolates was found to be velogenic on the basis of sequence motif of F gene cleavage site. It has been reported that virulent virus has at least one pair of basic amino acids at residues 115 and 116 plus a phenylalanine at residue 117 and a basic amino acid (R) at 113 at the cleavage site whereas lentogenic strains lack dibasic amino acids [19]. All NDV isolates of this study had lentogenic motif at the cleavage site. These results are in agreement with previous studies reporting the detection of lentogenic NDVs in wild birds and domestic ducks [4, 9, 15, 20, 21]. None of the isolates had the sequence motif of 111GERQE/DRL117 of class I isolates, although the latter have been reported in wild birds and domestic ducks [4, 21]. For example, [4] reported seven of the nine genotypes of class I NDVs in waterfowl and shore birds in the US while [21] reported the presence of class I genotype 2 NDVs in domestic ducks in Korea.

              Of the 43 isolates, 42 had the sequence motif of 111GG/EKQGRL117 at the cleavage site and were phylogenetically similar to either genotype I or genotype II within class II. This sequence motif has been reported earlier in genotypes I and II of class II NDVs [4]. However, a different sequence motif (111GRRQRRF117) was reported in the lentogenic strains from Australia [22]. One of the isolates had the sequence motif of 111GERQGRL117 and this isolate also clustered with class II genotype I strains. This isolate differed from other 42 isolates in the sense that the amino acid lysine was replaced by arginine at position 113.

              Overall genotype II viruses were more predominant than genotype I viruses in this study. This finding has the support of [4] who also observed more genotype IIa viruses than genotype I viruses within class II. The NDV isolates in this study were derived only from rRT-PCR AIV positive samples, the possibility of presence of genotypes of both classes (that were not detected in this study) in rRT-PCR AIV negative samples cannot be ruled out. Within class II, the NDV sequences clustered into two different groups. None of the isolates was phylogenetically close to vaccine strains used for comparison. This indicates that in spite of the regular use of live vaccines in poultry throughout the world, their transmission to wild birds may not be a common phenomenon. In an earlier study, [4] also did not detect any vaccine strains in wild birds in the US. Since wild birds have been reported to be a reservoir of NDV [16, 23], the mixing of different species at stop-overs during migration and the sharing of common wintering and breeding areas may provide opportunity for virus spread within and between countries and may help perpetuate different genotypes and classes of NDVs in these birds. The phylogenetic proximity of our isolates with those from the US, China, Korea, and Ireland points to this likelihood.

              The presence of class II viruses in wild birds is of concern because this class of viruses has been responsible for several panzootics of Newcastle disease in poultry [24, 25]. There are reports suggesting that velogenic NDVs might arise from lentogenic NDVs in nature [23, 26]. Further, studies have also suggested that point mutation, and not gene recombination, may be responsible for generating virulent and avirulent strains. For example, the NDV outbreak in Australian poultry during 1998-2000 was caused by a virulent NDV that originated due to mutation in a class II genotype I virus [26]. These authors were of the opinion that lentogenic viruses have the potential to become virulent with the passage of time. Even passaging of NDVs from one host to another has been reported to increase their virulence [16, 27]. In addition, the selective forces imposed by a new host environment may also play a role in acquisition of virulence [28]. These findings suggest that the lentogenic strains from wild birds may acquire virulence by waterfowl-to-domestic poultry transmission in nature. In such a scenario we may encounter an NDV outbreak in domestic poultry.

              Similar to low pathogenic AIV, the lentogenic NDVs in wild bird populations invariably do not cause obvious disease. Even virulent strains of NDVs that are lethal to chickens, have been isolated from apparently healthy domestic ducks [14, 29, 30]. Though virulent strains of NDVs were not detected in this study, their presence in the population cannot be ruled out in view of the potential created by the comingling nature and migration patterns of wild birds within and across continents. Thus, continuous surveillance for NDV in wild birds is essential for better understanding of its epidemiology. In conclusion, the present study reveals the circulation of class II (genotypes I and II) lentogenic strains of NDVs in wild birds in the Upper Midwest region of the US. Further studies are needed to determine the true prevalence and implications of various genotypes of NDV within wild bird population.

              Conclusion

              This study indicates the circulation of class II genotypes I and II NDVs in waterfowl in the Upper Midwest region of the US with an avirulent motif of monobasic amino acids at their F gene cleavage sites. Phylogenetically distant relationship of NDVs of this study with vaccine strains indicates that in spite of the regular use of live vaccines in poultry, their transmission to wild birds may not be a common phenomenon.

              Methods

              Sample collection

              Under an NIH funded surveillance program on avian influenza, cloacal and oropharyngeal (OP) swabs were collected from various waterfowl species in Minnesota, South Dakota, and North Dakota from April 2008 to October 2008. The swabs were placed in brain heart infusion broth containing antibiotics (penicillin 500 IU/mL, streptomycin 500 μg/mL, neomycin 0.15 mg/mL, fungizone 1.5 μg/mL, and gentamicin 50 μg/mL) and were transported on ice to the laboratory. The initial aim of the project was to test cloacal samples (n = 7458) from waterfowl species for the detection of AIV for which five samples each were pooled and the pools were tested for AIV using rRT-PCR [31]. Individual samples in positive pools were then tested for the detection of AIV by rRT-PCR.

              Virus isolation

              Individual samples positive for AIV by rRT-PCR (n = 890) were inoculated in 9-day-old specific pathogen free embryonated chicken eggs for virus isolation (VI). Allantoic fluid from inoculated eggs was harvested four days post inoculation and subsequently tested for hemagglutination (HA) using 0.5% turkey erythrocytes. The HA positive allantoic fluids (n = 159) were tested by RT-PCR for the confirmation of AIV as described below.

              Total RNA extraction and RT-PCR

              Total RNA was extracted from allantoic fluids and a known AIV isolate using QIAamp Viral RNA extraction kit (Qiagen, Valencia, CA). Extracted RNAs were subjected to RT-PCR using primers targeting the matrix gene of AIV [32]. A band of 1027 base pairs was observed in 52 cases indicating them to be AIV. The HA positive allantoic fluids that were negative for AIV (n = 107) were then tested for NDV by RT-PCR. Total RNA extracted from a known APMV-1 was used as a positive control. The RNA was amplified using primers specific to the F gene of NDV [33]. PCR amplification was carried out using Qiagen OneStep RT-PCR kit (Qiagen, Valencia, CA). Amplified PCR products were electrophoresed on 1.2% agarose gel. A band of 356 base pairs was observed in 43 cases indicating them to be NDVs. Further studies are underway to determine the identity of the remaining HA positive allantoic fluids (n = 64). The NDV positive PCR products were purified using a PCR purification kit (Qiagen, Valencia, CA) and were then sequenced in both directions at the BioMedical Genomic Center, University of Minnesota.

              Phylogenetic analysis

              The forward and reverse nucleotide sequences of all 43 isolates were curated, edited and aligned using a "Sequencher" software http://​www.​msi.​umn.​edu. The aligned sequences were analyzed on NCBI website http://​www.​ncbi.​nlm.​nih.​gov using BLAST to confirm their identity. The nucleotide sequences were then aligned using MEGA 4.0 software by Clustal W method. The evolutionary distances were computed by Pairwise Distance method using the Maximum Composite Likelihood Model. A phylogenetic tree of aligned sequences was constructed by Neighbor-Joining method (500 replicates for bootstrap). The F gene nucleotide sequences [corresponding to nucleotide positions 170-502 of GenBank: AF217084] were translated to deduced amino acid sequences to determine the pathotype involved. The nucleotide sequences were also compared with NDV sequences available in the GenBank. The virus types and their GenBank accession numbers used for comparison are given in Tables 2 and 3. These included F gene sequences of different genotypes of class I and class II NDVs. To maintain uniformity and consistency, class I genotypes are indicated using Arabic numerals (1-9) while class II genotypes are indicated using Roman numerals (I-IX).
              Table 2

              Previously published F gene sequences of class I Newcastle disease virus used for phylogenetic analysis.

              GenBank

              accession

              number

              Strain name

              Fusion

              cleavage

              site

              Genotype

              Country

              EF565077

              Blue winged teal/US(LA)/88-304/1988

              GERQERL

              1

              USA

              EF565074

              Green winged teal/US(LA)/88-35/1988

              GERQERL

              1

              USA

              EF565014

              Chicken/US(NY)/13828/1995

              GERQERL

              1

              USA

              EF564958

              Mallard/US(MD)/02-224/2002

              GERQERL

              2

              USA

              EF565017

              Mallard/US(MN)/98-49/1998

              GERQERL

              2

              USA

              EF564962

              Wood duck/US(OH)/02-677/2002

              GERQERL

              2

              USA

              EF564955

              Mallard/US(MD)/02-195/2002

              GERQERL

              2

              USA

              EU547751

              KR/duck/01/06

              GERQERL

              2

              Korea

              EF027144

              Poultry/Hong Kong/1252.8/2005

              GERQERL

              3

              Hong Kong

              EF027142

              Chicken/Hong Kong/1250.2/2005

              GERQERL

              3

              Hong Kong

              EF564960

              Mallard/US(MD)/02-308/2002

              GERQERL

              4

              USA

              EF564966

              Mallard/US(MD)/02-868/2002

              GERQERL

              4

              USA

              EF564994

              Black duck/US(MD)/01-431/2001

              GERQERL

              5

              USA

              EF565023

              Mallard/US(MN)/00-470/2000

              GERQERL

              5

              USA

              EF565079

              Mallard/US(MN)/99-348/1999

              GERQERL

              5

              USA

              EF565065

              Environment/US(NJ)/378106-4/2005

              GERQERL

              6

              USA

              EF565035

              Mallard/US(MN)/00-66/2000

              GERQERL

              7

              USA

              EF564895

              Mallard/US(MD)/04-118/2004

              GERQERL

              7

              USA

              EF564892

              Ruddy turnstone/US(DE)/1485/2002

              GERQERL

              8

              USA

              EF565031

              Blue winged teal/US(TX)/02-40/2002

              GERQERL

              9

              USA

              EF565022

              Mallard/US(MN)/00-185/2000

              GERQERL

              9

              USA

              Table 3

              Previously published F gene sequences of class II Newcastle disease virus used for phylogenetic analysis.

              GenBank

              accession

              number

              Strain name

              Fusion

              cleavage

              site

              Genotype

              Country

              AY175726

              AV 963/98 NZ5/97 (GNZDK98025)

              GGKQGRL

              I

              Ireland

              AY562991

              Chicken/N. Ireland/Ulster/67

              GGKQGRL

              I

              Ireland

              DQ439947

              NDV05-095

              GGKQGRL

              I

              China

              EF564821

              Mallard/US(MD)/04-204/2004

              GGKQGRL

              I

              USA

              EF564901

              Mallard/US(MD)/04-235/2004

              GGKQGRL

              I

              USA

              EF564942

              Mallard/US(MD)/04-483/2004

              GGKQGRL

              I

              USA

              AY130861

              Chicken/U.S.(PA)/31003/92

              GGKQGRL

              I

              USA

              AF217084

              Queensland V4

              GGKQGRL

              I

              Australia

              AY175736

              AV 80/97 D813-2 (HTWDK95193)

              GGKQGRL

              Ia

              USA

              AY427817

              Heb02

              GGKQGRL

              Ia

              China

              AY935489

              01-1108

              GRRQGRL

              Ia

              Australia

              AY289000

              Chicken/USA/Roakin/48

              GRRQKRF

              II

              USA

              AY845400

              LaSota

              GGRQGRL

              II

              China

              M24695

              BI/47

              GGRQGRL

              II

              USA

              AY727881

              32C/T.98

              GGKQGRL

              IIa

              Argentina

              EF564834

              Blue winged teal/US(LA)/87-155/1987

              GGKQGRL

              IIa

              USA

              EF564836

              Blue winged teal/US(LA)/87-190/1987

              GGKQGRL

              IIa

              USA

              EF564841

              Blue winged teal/US(LA)/87-247_b/1987

              GGKQGRL

              IIa

              USA

              EF564972

              Mallard/US(MD)/03-152/2003

              GEKQGRL

              IIa

              USA

              EF564993

              Mallard/US(MD)/03-807/2003

              GEKQGRL

              IIa

              USA

              EF565012

              Mallard/US(MD)/01-618/2001

              GEKQGRL

              IIa

              USA

              EF565019

              Mallard/US(MN)/98-350/1998

              GEKQGRL

              IIa

              USA

              EF565032

              Mallard/US(MN)/99-397/1999

              GEKQGRL

              IIa

              USA

              EU547752

              KR/duck/02/06

              GGKQGRL

              II

              Korea

              EU547755

              KR/duck/05/07

              GGKQGRL

              II

              Korea

              EU547757

              KR/duck/07/07

              GGKQGRL

              II

              Korea

              M24700

              AUS/32

              GRRQKRF

              III

              Australia

              AY741404

              Herts/33

              GRRQRRF

              IV

              USA

              AY288999

              Chicken/Mexico/37821/96

              GRRQKRF

              V

              Mexico

              AY562987

              Gamefowl/U.S.(CA)/211472/02

              GRRQKRF

              V

              USA

              AY288996

              Pigeon/Italy/1166/00

              GRRQKRF

              VI

              Italy

              AF358786

              TW/2000

              GRRQKRF

              VII

              Taiwan

              AF456442

              JS/5/01/Go

              GRRQKRF

              VII

              China

              AF048763

              AF2240

              GRRQKRF

              VIII

              Malaysia

              AY734534

              Chicken/Trenque Lauquen

              GRRQKRF

              VIII

              Argentina

              AF456435

              JS/1/97/Go

              GRRQKRF

              IX

              China

              GenBank accession numbers

              The NDV sequence data were submitted to the GenBank database; the accession numbers and other details are shown in Table 1.

              Abbreviations

              AGWT: 

              American green-winged teal

              AIV: 

              avian influenza virus

              APMV: 

              avian paramyxovirus

              END: 

              exotic Newcastle disease

              HA: 

              hemagglutination

              MALL: 

              mallard

              ND: 

              Newcastle disease

              NDV: 

              Newcastle disease virus

              NOPI: 

              northern pintail

              rRT-PCR: 

              real time reverse-transcription polymerase chain reaction

              RT-PCR: 

              reverse-transcription polymerase chain reaction

              VI: 

              virus isolation

              WODU: 

              wood duck

              Declarations

              Acknowledgements

              This work has been funded in whole or in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institute of Health, Department of Health and Human Services, under Contract No. HHSN266200700007C. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

              Authors’ Affiliations

              (1)
              Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota

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              Copyright

              © Jindal et al. 2009

              This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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