Research in Veterinary Science 111 (2017) 49–54

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Research in Veterinary Science

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

Virulence in Newcastle disease virus: A genotyping and molecular

evolution spectrum perspective
Wentao Fan a,1, Yang Wang a,1, Shenghua Wang a,1, Ziqiang Cheng a, Huijun Guo a, Xiaona Zhao b,⁎, Jianzhu Liu a,⁎
a
College of Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China
b
Research Center for Animal Disease Control Engineering Shandong Province, Tai'an 271018, China

a r t i c l e i n f o a b s t r a c t

Article history: In our research, the molecular evolutions of NDV F and HN genes were analyzed. The phylogenetic analyses
Received 25 June 2016 of NDV sequences indicated that NDV could be divided into two genotypes: Class I (lentogenic strains) and
Received in revised form 7 November 2016 Class II (velogenic or mesogenic strains). Each genotype possesses high gene homology. Furthermore, the
Accepted 5 December 2016
selected pressure analysis showed that the dN/dS of velogenic, mesogenic NDV strains F gene was signifi-
cantly high and the ω(dN/dS) is 1.1725 N 1. These results imply that mutations in velogenic, mesogenic
Keywords:
NDV F gene are favored by positive natural selection and it has acted to diversify NDV virulence at the nu-
Newcastle disease virus cleotide and amino acid level. We estimated that the subsequent rapid adaptation of the Newcastle disease
Molecular analyses virus to chickens were likely dependent on a high rate of mutation and the positive selection of mutations in
Virulence the major F gene.
Evolution © 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Newcastle disease (ND) is one of the most severe avian diseases and
usually causes economic catastrophe in poultry industry in the world
(Zhu et al., 2010). Newcastle disease virus (NDV) is the member of
Avulavirus genus in the Paramyxoviridae family (Yue et al., 2009). The
paramyxoviruses have been classified into twelve subtypes that desig-
nated APMV-1 to APMV-12 (Terregino et al., 2013), and NDV belongs
to APMV-1 subtypes (Alexander, 2000). The NDV has been continually
evolving and caused genetic diversity in the world (Ebrahimi et al.,
2012), since the ND first outbreak in 1926. NDV contains six main struc-
ture proteins: nucleocapsidprotein (NP), phosphoprotein (P), matrix
protein (M), fusion protein (F), hemagglutinin–neuraminidase (HN),
and largeprotein (L) (De and Peeters, 1999; Mayo, 2002; Czeglédi et
al., 2006). The envelope of NDV is comprised of two interactive surface
glycoproteins, the HN and the F proteins. The two important genes
make vital sense in virus infection process and determinant of NDV vir-
ulence (Zhu et al., 2010). It has been identified that the potential path-
ogenicity of NDV is related to the F0 cleavage site motif (residues112–
117) of the NDV F protein (‘standard’ method) (de Leeuw et al., 2003).
In our study, we consulted the Genbank information and referenced
the paper of selected strains to identify the genotype of used sequences.
⁎ Corresponding authors.
E-mail addresses: zhaoxn@sdau.edu.cn (X. Zhao), Liujz@sdau.edu.cn (J. Liu).
1
These authors worked equally in this paper.
The phylogenetic method was used to analyze the molecular evolution
process of NDV.
Recent studies state that evolution of viruses primary related to the
evolution of functional proteins in the gene duplication process (Nei et
al., 2008). The most extensively referred model for the protein function
evolution supposed that the selectively neutral mutations provide oppor-
tunities to the protein to get a new function, adventitiously (Horvath,
2005). A variety of methods about detecting a high rate of evolution, rel-
ative to selectively neutral sites (Nielsen, 2005) claim that selection is re-
vealed by comparing the patterns of synonymous and non-synonymous
(amino acid-altering) nucleotide substitution. If the number of synony-
mous substitutions per site (dS) exceeds the number of non-synonymous
substitutions per site (dN), in another word, dN/dS b 1, this is so-called
“purifying” selection. Another opposite pattern is natural selection,
which is the major factor of favoring amino acid changes (Kryazhimskiy
and Plotkin, 2008). In this pattern, the number of non-synonymous sub-
stitutions per site (dN) exceeds the number of synonymous substitutions
per site (dS), that is to say, dN/dS N 1. The strong positive selection usually
led to an accumulation of additional beneficial mutations, resulting in the
emergence of the highly successful virus variants. Another factor of func-
tional protein evolution is a priori to these episodes of positive selection
locations on the phylogenetic tree (Travers et al., 2005) or the pattern
of substitutions on the tree (Guindon et al., 2004).
Currently, the existence of NDV in world chicken population has
been widely reported, and several genotyping and genetic diversity
studies have been studied on NDV based on published complete ge-
nome sequences (Courtney et al., 2013; Fernandes et al., 2014), but no

http://dx.doi.org/10.1016/j.rvsc.2016.12.001
0034-5288/© 2016 Elsevier Ltd. All rights reserved.
50 W. Fan et al. / Research in Veterinary Science 111 (2017) 49–54

extensive molecular evolution studies have been conducted on NDV. genotyping, evolution and virulence based on F and the HN genes. Our
Furthermore, although there is a consensus that NDV can be divided data provide important new information to study NDV virulence
into two major genotypes, no research on relationship about based on F gene evolution.

Fig. 1. Evolutive relationships among NDV groups. A: Phylogenetic tree based on the nucleotide sequences of complete genome and the NJ method for the 72 NDV sequences. Numbers
along the branches refer to the percentages of confidence in the NJ analyses by using MEGA6. Only bootstrap support values of N50% are indicated. The regions of red color indicated the
velogen or mesogen isolates, the region of green color indicated the lentogenic NDV strains.
 W. Fan et al. / Research in Veterinary Science 111 (2017) 49–54 51

Table 1 2. Methods and materials

Newcastle disease virus whole cDNA sequences used in present study. (out) = Sequence
formally used as outgroup representatives for the NDV ingroup.
2.1. Statistical methods
Number Country Accession Number Country Accession
number number The open reading frames (ORFs) were identified according to the
1 India KR072665 2 USA NC002617 major criteria of (Ettinger et al., 2012), a minimum length of 100 bp,
3 India JX316216 4 India KM056358 an ACC start codon, and b 60% overlap with adjacent ORFs using DNAstar
5 Clone-30 Y18898 6 Mexico KJ577136
7.1 program. In order to inquiry the phylogenetic inferences and molec-
7 India KP089979 8 Brazil KJ123642
9 India KJ636208 10 India KF740478 ular evolutionary analyses, all available NDV complete genome are ob-
11 India KJ577585 12 India KF727980 tained from GenBank at the NCBI (GenBank). Strains with 97% identity
13 China KC542912 14 China KF306265 were excluded from the dataset. In total of 72 strains were chosen for
15 India KM056348 16 India KC987036 analysis. The APMV-2 (NC028245) sequence was included in the tree
17 China KJ528559 18 China KC844235
19 China KC551967 20 China KC461214
as out-group. Multiple sequence alignments were performed by Clustal
21 China FJ436303 22 China KC246549 X, with the default settings for gap costs (gap opening penalty = 15.00;
23 China FJ754273 24 China JX110635 gap extension penalty = 6.6) (Thompson et al., 1997). The molecular
25 Dominican Republic JX19193 26 Malaysia JX012096 evolutionary genetics analysis (MEGA6) (Lewis et al., 2013) software
27 Italy AB524406 28 France JQ013039
was utilized to determine the degree of diversity among all sequences
29 China JN688864 30 China JN986839
31 China JX193083 32 China JN653340 and analyzing the alignment of the complete genome, F, and HN genes
33 China KC853020 34 China JN618348 through the neighbor-joining (NJ) method. The phylogenetic trees
35 China KF915807 36 China JF950509 were constructed using the neighbor-joining (NJ) method based on
37 Australia JX524203 38 Indonesia HQ697255 the Tamura-Nei model, with standard errors being calculated based on
39 China F361507 40 China HQ008337
1000 bootstrap replicates and expressed based on the number of nucle-
41 China EU167540 42 India GU187941
43 Sweden GU585905 44 Sweden GQ918280 otide substitutions per site. The codon positions included in the analysis
45 South Korea JX401405 46 Egypt FJ919313 were the 1st, 2nd, 3rd. All positions containing gaps and missing data
47 China JN653339 48 China FJ766528 were eliminated from the data set (the “complete deletion” option).
49 China JF795531 50 China FJ751919
The numbers of synonymous (dS) and nonsynonymous (dN) nucleotide
51 China HM125898 52 China FJ436304
53 Takaaki AF375823 54 China FJ436302
substitutions per site was estimated by Nei and Gojobori's method (Nei
55 China DQ097393 56 China FJ938175 and Gojobori, 1986), dN and dS are the nonsynonymous and synony-
57 VG/GA KC906188 58 VG/GA EU289029 mous rates estimated from the molecular evolution codon model, for
59 Mexico HM177201 60 Australia AY935490 example, HyPhy.
61 Germany KJ736742 62 India AY845400
63 Ireland AY562991 64 Japan AB853928
65 Japan AB853926 66 Japan AB524406 2.2. Substitution rate estimation and selection pressure on the NDV
67 Maryland FJ410147 68 Alaska AB524405
69 Pakistan KP776462 70 Herts AY741404
The difference between non-synonymous substitution (dN) and
71 USA AY562990 72 USA GU97877
73 Togo (out) HM159993 synonymous substitution (dS) rates for the aligned H and FN genes
were calculated to estimate the presence of selective pressures on the

Fig. 2. F, HN protein sequence percent identities (upper right) and divergence(lower left) among NDV strains by phylogenetic analysis. 1–6 are lentogenic strains, 7–20 are velogenic,
mesogenic NDV strains.
52 W. Fan et al. / Research in Veterinary Science 111 (2017) 49–54

Fig. 3. Evaluation of differences in non-synonymous and synonymous rate ratios (dN/dS) of complete NDV genes (A) and F, HN genes (B) substitution rate between lentogen strains and
velogen or mesogen isolates.

velogenic or mesogenic isolates genome. Pairwise comparisons were
made for each H and FN gene between the 72 sampled strains and Alas-
ka 415–91,which was used as reference. For each codon, estimates of
the numbers of inferred synonymous (s) and nonsynonymous (n) sub-
stitutions are presented along with the numbers of sites that are esti-
mated to be synonymous (S) and nonsynonymous (N). These
estimates are produced using the joint Maximum Likelihood recon-
structions of ancestral states under a Muse-Gaut model (Muse and
Gaut, 1994) of codon substitution and Felsenstein (1981) model of nu-
cleotide substitution. For estimating NJ values, a tree topology was auto-
matically computed. The test statistic dN-dS is used for detecting codons
that have undergone positive selection, where dS is the number of syn-
onymous substitutions per site (s/S) and dN is the number of
nonsynonymous substitutions per site (n/N). A positive value for the
test statistic indicates an overabundance of nonsynonymous substitu-
tions. In this case, the probability of rejecting the null hypothesis of neu-
tral evolution (P-value) is calculated (Suzuki and Gojobori, 1999; Pond
and Frost, 2005). Values of P b 0.05 are considered significant at a 5%
level and are highlighted. Normalized dN-dS for the test statistic is ob-
tained using the total number of substitutions in the tree (measured
in expected substitutions per site). It is useful for making comparisons
across data sets. Maximum Likelihood computations of dN and dS

Fig. 4. The partly comparison of the F proteins between the NDV isolates base by hase. The residues different from the Alaska 415-91 are marked. 1–7 were velogenic or mesogenic
isolates, 8–20 were the lentogenic NDV strains.
 W. Fan et al. / Research in Veterinary Science 111 (2017) 49–54
were conducted using HyPhy software package (Pond and Muse, 2005).
Codon positions included were 1st + 2nd + 3rd. All positions contain-
ing gaps and missing data were eliminated. There were a total of 10,920
positions in the final dataset.
3. Results
3.1. The status of ancestor
The phylogenetic tree constructed by NJ method indicates that the
same topology (Fig. 1) among the assayed NDV complete sequences
(Table 1). Two main monophyletic genotypes supported by high boot-
strap values were differentiated: one branch for velogenic or mesogenic
strains, another branch for lentogenic strains. Fig. 3 is the matrix graph
of partial NDV isolates percent identities, the average homology of NDV
gene analyzed here was 90%+ of lentogenic strains, 80%+ of velogenic
or mesogenic strains(Fig. 2). These results suggested a common origin
for NDV.
3.2. Substitution rate and selective pressure
The non-synonymous and synonymous substitution rates (dN/dS)
for the F and HN genes and the complete NDV genome were calculated
for both velogenic and mesogenic isolates (Fig. 3). The differences be-
tween non-synonymous and synonymous substitution rates (dN/dS)
for F genes were almost always positive (Fig. 4) and the mean of
ω(dN/dS) was estimated to be 1.1725 for velogenic or mesogenic iso-
lates, indicating that the NDV genome was under positive selection. In-
cluding the F genes increased the strength of positive selection
measured for the complete NDV gene. Our figure showed that a part
of the NDV complete genomes undergoes purifying selection (ω(dN/
dS) b 1), this part is HN gene.
4. Discussion
Genetic analysis of NDV isolates has revealed the existence of at least
eighteen genotypes associated with spatio-temporal and host (Czeglédi
et al., 2006). Although certain genogroups have been identified, there is
a phenomenon that more and more NDV variants come out. Why so
many high virulent NDV strains to infect chickens, ducks, house spar-
rows? To better solve these problems and better control the Newcastle
disease outbreak, we must figure out the probable NDV evolution pro-
cess and illuminate the conceivable mechanisms. Previous studies
have proved that the F, HN proteins are important regions for functional
specificity of NDV (Fishbein et al., 2015). It is reasonable to study the
NDV virulence evolution based on F, HN gene evolution.
Therefore, in order to examine the phylogenetic analyses of NDV
strains some molecular features, the selection pressure approaches
were used base on NDV F, HN genes. In our study, an elaborate phyloge-
netic analysis of the NDV complete genome sequences (Table 1) clearly
manifested that NDV isolates can be divided into two major genotypes
(velogenic or mesogenic and lentogenic strains), each one with several
clusters(Fig. 1). The average homology of NDV sequences analyzed
showed that percent identities between velogenic and mesogenic is
80%+, percent identities between lentogenic is 90%+ (Fig. 2). Then, un-
derstanding the genetic basis of viral adaptation to host species is crucial
for the evolution, and a high ω(dN/dS) is usually taken as evidence for a
selective pressure on sequence divergence, if dN was significantly
exceeded dS seems to be characteristic of positive selection leading to
diversification of genes (Padhi and Ma, 2015). Besides, due to the posi-
tion nuclear transport and assembly, the frequency the substitutions in
per site maybe severely related to limited number of possible mutations
(Umali et al., 2014). Thus, the mean of ω(dN/dS) for velogenic or
mesogenic F genes is 1.1725 N 1, a higher non-synonymous rate than
the synonymous rate is strong evidence for adaptive protein evolution
(Fig. 4). It maybe the strong positive selection led to an accumulation
53
of additional beneficial mutations of F gene, resulting in the emergence
of the high virulent NDV variants. However, the HN gene undergoes pu-
rifying selection (ω(dN/dS) b 1). It is probably due to the HN protein is
primarily responsible for attachment to host cell-surface receptors
(Scheid et al., 1972; Zhu et al., 2010). This gene was related to host re-
ceptors gene thus it has lower dN/dS rates. Fig. 4 shows more the fre-
quency substitutions in per site of velogenic or mesogenic isolates in F
genes. Although several complex influences such as saturation of muta-
tions among nucleotide bias, calculated ω(dN/dS) may not accurately
reflect the factual selective pressure, with the possible exception of
very closely amino acid homology (Yokoyama et al., 2008) or very dis-
tantly related genes, high ω(dN/dS) appear to be liable indicator of pos-
itive selection (Shen et al., 2010). In our research, average gene
homology of NDV gene analyzed indicated that percent identities be-
tween velogenic or mesogenic NDV F and HN genes are 80%+, thus,
the ω(dN/dS) N 1 obtained for velogenic or mesogenic NDV F and HN
genes more strongly reflect positive natural selection on NDV during
the NDV virulence evolution process(Hussain et al., 2009).
5. Conclusion
The present study demonstrated a common origin for two NDV ge-
notypes with each genotype possessing high gene homology. The find-
ing of mostly positive selected sites on the velogen or mesogen isolates F
and HN genes may indicate successful viral adaptations of NDV to a
wide verity of poultry species. Nevertheless, due to the lack of associa-
tion between the statistically significant sites under positive selection
and the amino acid residues that play crucial roles in specific biological
functions, further experimental studies are needed to assess the biolog-
ical significance of the positively selected sites in the F, HN protein re-
gions of the NDV.
Acknowledgments
The study was supported by the National Key R&D Program
(2016YFD0501208), the Shandong Modern Agricultural Technology &
Industry System (no. SDAIT-11-04).
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