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RAPID COMMUNICATION
A novel strain of porcine epidemic diarrhea virus
in Vietnamese pigs
Yong Kwan Kim
1
•Seong-In Lim
1
•Ji-Ae Lim
1
•In-Soo Cho
1
•Eun-Hye Park
2
•
Van Phan Le
3
•Nguyen Ba Hien
3
•Pham Ngoc Thach
3
•Do Hai Quynh
3
•
Tran Quang Vui
4
•Nguyen Trung Tien
5
•Dong-Jun An
1
Received: 23 January 2015 / Accepted: 28 March 2015
Springer-Verlag Wien 2015
Abstract Porcine epidemic diarrhea virus (PEDV) causes
severe diarrhea and dehydration in suckling pigs and has
caused high rates of death among piglets and substantial
economic loss in Vietnam since 2009. To investigate the
genotypes of prevailing PEDVs, intestinal and fecal sam-
ples from piglets from central and northern Vietnam were
collected and analyzed. Phylogenetic analysis of the nu-
cleotide sequences of complete spike genes of PEDVs from
Vietnam resulted in the identification of two divergent
groups. PEDVs (HUA-PED45 and HUA-PED47) belonged
to the G2b group, along with Chinese, US, and Korean
strains occurring at the end of 2010, in May 2013 and in
November 2013, respectively. Six strains from the Quang
Tri region were assigned to the G1b group, along with
Chinese and US strains. The Vietnamese PEDVs detected
in infected piglets had a nationwide distribution and be-
longed to the G2b and G1b genotypes.
Porcine epidemic diarrhea virus (PEDV) belongs to the
family Coronaviridae. It is an enveloped, single-stranded
RNA virus that causes enteritis, vomiting, and watery di-
arrhea [1]. The PEDV genome is approximately 28 kb in
length and is composed of seven open reading frames
(ORFs) encoding four structural proteins (spike, S; en-
velope, E; membrane, M; nucleocapsid, N) and three non-
structural proteins (ORF1a, -1b, and ORF3) [2]. PED was
first reported in Europe (Belgium and the United Kingdom)
in 1971 and has since been recognized in many European
countries (Hungary, Italy, Germany, the Czech Republic,
France and Switzerland) [2–4]. In Asia, PEDV was first
confirmed in China in 1984 and has increasingly become a
problem for the pig industries in Korea [5], Japan [6], and
Thailand [7]. In late 2010 in China, PED outbreaks on
several pig farms had a devastating impact on the pig in-
dustry, because this disease is characterized by high mor-
tality and morbidity among infected suckling piglets [8]. In
April 2013, PEDV emerged in the United States and has
since spread rapidly across almost all states [9]. US-like
PEDV, which causes high mortality among piglets, has a
high degree of nucleotide sequence similarity to strains
reported lately in several countries, including Canada,
Mexico, Japan, Korea, and Taiwan [10–13]. PED first
emerged in Vietnam in 2009 [14]. Complete genome se-
quence analysis of three PEDV isolates from pigs dis-
playing severe diarrhea in northern and southern provinces
of Vietnam revealed the existence of a variant strain [15].
Enteric problems due to PEDV continue to cause huge
economic losses on swine farms in Vietnam. This study
provides new information about the prevalence of PEDV
strains currently circulating in Vietnam.
A total of 160 diarrhea samples from piglets under
35 days old were collected from six provinces in the north
of Vietnam and one province (Quang Tri) in the central
&Van Phan Le
letranphan@gmail.com; letranphan@vnua.edu.vn
&Dong-Jun An
andjun@hanmail.net
1
Animal and Plant Quarantine Agency, Anyang,
Gyeonggi-do 430-824, Republic of Korea
2
The Catholic University of Korea, Seoul,
Gyeonggi-do 420-743, Republic of Korea
3
Department of Microbiology and Infectious Disease, College
of Veterinary Medicine, Vietnam National University of
Agriculture (VNUA), Hanoi, Vietnam
4
Hue University of Agriculture and Forestry, Hue, Vietnam
5
National Center for Veterinary Medicine Control No1, Hanoi,
Vietnam
123
Arch Virol
DOI 10.1007/s00705-015-2411-5
region from November 2013 to April 2014. The number of
samples from each province was as follows: Quang Tri
(n =99), Hung Yen (n =1), Bac Giang (n =50), Hai
Duong (n =1), Thai Nguyen (n =1), Thai Binh (n =3),
and Hai Phong (n =5).
Viral RNA was extracted from feces using TRIzol LS
b
according to the manufacturer’s instructions. PEDV was
detected in fecal specimens by RT-PCR as described previ-
ously [16,17], with primers specific for the spike and ORF3
regions of PEDV. Products of the expected size were cloned
using pGEM-T Vector System II (Promega, Cat. No. A3610,
USA). The cloned genes were sequenced with T7 and SP6
sequencing primers on an ABI PRISM
3730xl DNA Se-
quencer at the Macrogen Institute (Macrogen Co., Ltd.). The
sequences of all of the positive samples for complete spike
and complete ORF3 genes of PEDV were submitted to
GenBank under accession numbers KP455313-KP455320
and accession numbers KP455967-KP455974.
Of 160 diarrhea samples from piglets, 30 samples were
identified as positive based on the partial sequence of the
spike gene. The regions in which PEDV-positive samples
were identified were Quang Tri (n =19), Hung Yen
(n =1), Hai Duong (n =1), Thai Nguyen (n =1), Thai
Binh (n =3), and Hai Phong (n =5). No positive samples
were identified in Bac Giang province. Complete sequence
analysis of two (spike and ORF3) genes in 30 positive
samples identified eight strains, of which six were from
the Quang Tri region (HUA-PED55, -PED58, -PED60,
-PED63, -PED67, and -PED68) and two were from the
Thai Binh region (HUA-PED45 and -PED47). The spike
gene for strains HUA-PED45 and -PED47 was 4,158 nu-
cleotide (nt) in length, but strains isolated from the Quang
Tri region had a spike gene length of 4,149 nt. All strain
sequences were aligned initially using the CLUSTAL X
alignment program [18]. The nucleotide sequences were
translated, and nucleotide and amino acid sequence iden-
tities among the PEDV strains were calculated using
BIOEDIT 7.053 [19]. A comparison to complete spike
gene sequences available in GenBank demonstrated that
HUA-PED45 has high similarity (99.7 % and 99.4 % at the
nt and amino acid (aa) level, respectively) to HUA-PED47
and 97.6–97.8 % nt sequence similarity to Vietnamese
strains (VN/KCHY-310113, VAP1113-1, and JFP1013-1)
responsible for the 2013 outbreaks [15]. However, HUA-
PED45 has low similarity (94.7–95.1 % nt and
94.0–94.5 % aa) to the six strains from the Quang Tri re-
gion. Interestingly, all strains from Vietnam, including VN/
KCHY-310113, VAP1113-1, and JFP1013-1, showed very
high similarity (99.0–100 % at the nt level) in a compar-
ison of complete ORF3 gene sequences. The complete
ORF3 gene in Vietnam PEDVs has a unique characteristic,
with several amino acid changes in the encoded protein
(
25
L?
25
S,
75
I?
75
V,
107
C?
107
F,
168
D?
168
N) due to
point mutations. In comparison to the CV777 strain, the
spike genes of the HUA-PED45 and -PED47 strains have
two insertions of four (QGVN) and one (N) aa at amino
acid positions 58 to 59 and 135 to 136 and deletions of two
(GK) and one (Y) aa at amino acid positions 158 to 159 and
1194. This result is similar to those of previous studies, in
which insertions and deletions were located in the hyper-
variable domain in the N-terminus of the S1 region [15,
20]. By contrast, the strains from Quang Tri had no dele-
tions or insertions of amino acids in the spike genes.
There are four major epitope regions in the spike glyco-
protein: amino acid positions 504–643 [21],
753
YSNIG
VCK
760
[22],
769
LQDGQVKI
776
[22], and
1373
GPRLQPY
1379
[23]. Amino acid position
753
YSNIGVCK
760
(SS2 region) in
the S1 domain is conserved between the Vietnam PEDV
strains and isolates from other countries. The largest number
of amino acid differences was observed at amino acid posi-
tions 504–643 (core neutralizing epitope: COE region) in the
S1 domain in comparison to Vietnamese PEDV strains and
strains from other countries. Amino acid sequence differences
for all Vietnamese strains (except HUA-PED58) were also
observed in the
769
LQDGQVKI
776
(SS6 region) in the S1
domain, with the substitutions of L ?SandD ?Satamino
acid positions 769 and 771, respectively. In the HUA-PED58
strain, substitutions of L ?S, D ?S, and Q ?Rwere
identified at amino acid positions 769, 771, and 773, respec-
tively. Previous studies also suggested that the COE region
and SS6 epitopes tend to undergo high levels of mutation, but
the SS2 and 2C10 epitopes are well conserved [16,24].
The amino acid positions
1373
GPRLQPY
1379
(2C10 re-
gion) in the cytoplasmic domain of Vietnamese PEDV
strains were generally conserved, although HUA-PED45
had a G ?S substitutions at amino acid position 1373.
The spike gene is considered the most useful for de-
termining the genetic diversity of PEDV strains. It plays
an important role in molecular epidemiology and in
assessing the genetic variation of PEDV field strains [7,
25]. The calculation method employing the neighbor-
joining (NJ) method of the MEGA 6.06 program [26]was
used to further investigate genetic diversity of Vietnamese
strains. The NJ tree was visualized using TreeView 1.6.1
[27]. Phylogenetic analysis based on a complete spike
gene fragment of Vietnamese field strains, together with
other PEDV reference strains from GenBank, divided the
sequences into four groups (Fig. 1). One group (G1a)
comprised the CV777, DR13, and vaccine strains. The
second group (G1b) consisted of CH6 and JS-2004-2 from
China, strains recently isolated in the US (Minnesota 52,
Ohio 126, Iowa 106, and Iowa 107), and Vietnamese
strains (HUA-PED55, -PED58, -PED60, -PED63,
-PED67, and -PED68). The third group (G2a) was made
up of 15 Korean PEDV strains and two Japanese strains
(NK and KH). The last group (G2b) included four strains
Y. K. Kim et al.
123
from Thailand, Chinese strains of high pathogenicity from
the 2010 outbreak, and US and Korean strains that caused
severe piglet morbidity and mortality in 2013. Five
Vietnamese strains (HUA-PED45, -PED47, VN/KCHY-
310113, VAP1113-1, and JFP1013-1) also belonged to the
G2b group.
K13JA12-3 (KJ539152)
K13JA11-4 (KJ539153)
K13JA12-1 (KJ539151)
K14JB01 (KJ539154)
IA2 (KF468754)
Iowa/18984 (KF804028)
MN (KF468752)
Indiana/17846 (KF452323)
Iowa/16465 (KF452322)
Colorado (KF272920)
IA1 (KF468753)
13-019349 (KF267450)
Ch/ZMDZY/11 (KC196276 )
AH2012 (KC210145)
BJ-2011-1 (JN825712)
GD-B (JX088695)
JS-HZ2002 (KC210147)
FJZZ-9 (KC140102)
ZJCZ4 (JX524137)
HUA-PED45 (KP455313)
HUA-PED47 (KP455314)
AJ1102 (JX188454 )
LC (JX489155)
CH1 (JQ239429)
GD-A (JX112709)
CHGD-01 (JN980698)
VN/JFP1013-1 (KJ960178)
VN/KCHY-310113 (KJ960180)
VN/VAP1113-1 (KJ960179)
FJND-3 (JN381492)
CNU-091222-02 (JN184635)
CNU-091222-01 (JN184634)
KNU-0902 ( GU180145)
NPPED2008-2 (KC764952)
6-56ST0413 (KF724938)
SBPED0211-1 (KC764956)
SPPED0212-02 (KC764960)
PED0212-1 ( KC764954)
AD03 (KC879282)
AD02 (KC879281)
AD01 (KC879280)
KNU-0802 (GU180143)
KNU-0801 (GU180142)
Chinju99 (AY167585)
NK (AB548623)
KNU-0901 (GU180144 )
KH (AB548622)
KNU-0905 (GU180148)
KNU-0903 (GU180146)
NJ02 (KC879279)
NJ01 (KC879278)
KNU-0904 (GU180147)
AS01 (KC879275)
AS03 (KC879277)
AS02 (KC879276)
SM98 (GU937797)
CV777 ( AF353511)
DR13 (DQ862099)
Attenuated DR13 (DQ462404)
MK (AB548624)
HUA-PED60 (KP455317)
HUA-PED68 (KP455320)
HUA-PED58 (KP455316)
HUA-PED55 (KP455315)
HUA-PED63 (KP455318)
HUA-PED67 (KP455319)
CH6 (JQ239434)
JS-2004-2 (AY653204)
Minnesota52 (KJ645704)
Ohio126 (KJ645702)
IOWA106 (KJ645695)
IOWA107 (KJ645696)
100
100
100
100
100
88
100
89
100
99
100
100
97
84
97
100
100
100
73
96
100
100
100
85
99
100
86
76
97
97
94
97
G2b
G2a
G1a
G1b
G1
G2
0.005
Korea
USA
China
Vietnam
Vietnam
China
China
Thailand
Korea
China
Korea
Japan
Korea
Japan
Korea
Belgium
Korea
Belgium
Korea
Japan
Vietnam
China
Fig. 1 Phylogenetic analysis of
PEDV strains based on the
nucleotide sequences of the
complete spike glycoprotein
genes. The tree was constructed
using the neighbor-joining
method in the MEGA 6.06
software, using 1,000 bootstrap
replicates. Bootstrap values
[70 % are shown at the branch
points
A novel strain of porcine epidemic diarrhea…
123
Vietnamese PEDV strains have previously been identi-
fied as belonging to the G2b group in the phylogenetic tree
[14,15]. This study revealed a novel genotype (G1b) in
Vietnam. Interestingly, in the NJ tree for complete ORF3
genes, all strains from Vietnam were closely related to one
cluster (Fig. 2). It will be important to investigate further
Korea
USA
China
Vietna
m
Korea
China
China
Korea
China
Korea
Belgium
Belgium
Ohio126 (KJ645702)
Missouri101 (KJ645692)
Minnesota84 (KJ645707)
Colorado (KF272920)
Iowa107 (KJ645696)
Texas128 (KJ645697)
Illinois87 (KJ645680)
K14JB01 (KJ623926)
KUIDL (KJ588064)
KNU-1305 (KJ662670)
CH/JL/09 (GU372741)
CH/GSJI/07 (GU372737)
CH/HLJH/06 (GU372732)
CH/GSJII/07 (GU372742)
CH/HNCH/06 (GU372738)
CH/IMT/06 (GU372739)
CH/HLJM/07 (GU372735)
PFF514 (HQ537453)
BIF256 (HQ537447)
CPF193 (HQ537446)
CPF299 (HQ537450)
CH/JL/08 (GU372734)
CH/HNHJ/08 (GU372736)
CH/SHH/06 (GU372740)
Chinju99 (EU792474)
CH/S (GU372733)
BI1108 (HQ537435)
Parent DR13 (EU054929)
M1763 (HQ537438)
BI976 (HQ537433)
e1642 (HQ537437)
MF78 (HQ537442)
V2501 (HQ537441)
BIF118 (HQ537443)
M1595 (HQ537436)
Attenuated DR13 (EU054930)
VN/VAP1113-1 (KJ960179)
VN/JFP1013-1 (KJ960178)
VN/KCHY-310113 (KJ960180)
HUA-PED45 (KP455967)
HUA-PED47 (KP455968)
HUA-PED60 (KP455971)
HUA-PED68 (KP455974)
HUA-PED58 (KP455970)
HUA-PED67 (KP455973)
HUA-PED55 (KP455969)
HUA-PED63 (KP455972)
CV777 (NC_003436)
LZC (EF185992)
Br1/87 (Z24733)
1
88
100
80
100
82
0.005
Fig. 2 Neighbor-joining tree
based on complete ORF3
nucleotide sequences of PEDV
strains. The bootstrap
percentages (supported by at
least 70 % of the 1,000
replicates) are shown above the
nodes. The scale bar indicates
the number of nucleotide
substitutions per site
Y. K. Kim et al.
123
whether this is an inherent property of Vietnamese PEDV
strains.
PEDV strains from northern Vietnam, bordering south-
ern China, were identified as the prevailing G2b genotype,
while strains circulating in the central region of Vietnam
were the G1b genotype. Therefore, these data will promote
further investigation of genetic evolution and the selection
of PEDV variants for vaccines for control of PED.
Acknowledgments This work was supported by the Vietnam Na-
tional Foundation for Science and Technology Development
(NAFOSTED) under grant number 106-NN.04-2014.16 and by a
grant (Project Code No. 313014-03-1-HD030) from the Korea Insti-
tute of Planning & Evaluation for Technology in Food, Agriculture,
Forestry & Fisheries, 2013.
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