2016 Occurrence and sequence analysis of porcine deltacoronaviruses in southern China

2016·0.96 MB·English

Zhaietal.VirologyJournal (2016) 13:136
DOI10.1186/s12985-016-0591-6
RESEARCH Open Access
Occurrence and sequence analysis of
porcine deltacoronaviruses in southern
China
Shao-Lun Zhai1†, Wen-Kang Wei1†, Xiao-Peng Li1†, Xiao-Hui Wen1, Xia Zhou1, He Zhang1, Dian-Hong Lv1*,
Feng Li2,3 and Dan Wang2*
Abstract
Background: Following theinitial isolation of porcine deltacoronavirus (PDCoV) from pigs withdiarrheal disease in
theUnited States in 2014,thevirus has been detectedon swine farms in some provincesof China. Todate, little is
known about the molecular epidemiology ofPDCoV insouthernChina where major swine production is operated.
Results: ToinvestigatetheprevalenceofPDCoVinthisregionandcompareitsactivitytootherentericdiseaseofswine
causedbyporcineepidemicdiarrheavirus(PEDV),transmissiblegastroenteritiscoronavirus(TGEV),andporcinerotavirus
groupC(RotaC),390fecalsampleswerecollectedfromswineofvariousagesfrom15swinefarmswithreporteddiarrhea.
Fecalsamplesweretestedbyreversetranscription-PCR(RT-PCR)thattargetedPDCoV,PEDV,TGEV,andRotaC,respectively.
PDCoVwasdetectedexclusivelyfromnursingpigletswithanoverallprevalenceofapproximate1.28%(5/390),notin
sucklingandfatteningpiglets.Interestingly,allofPDCoV-positivesampleswerefrom2015ratherthan2012–2014.Despite
alowdetectionrate,PDCoVemergedineachprovince/regionofsouthernChina.Inaddition,comparedtoTGEV(1.54%,
5/390)orRotaC(1.28%,6/390),therewerehighlydetectionratesofPEDV(22.6%,88/390)inthosesamples.Notably,all
fivePDCoV-positivepigletswereco-infectedbyPEDV.Furthermore,phylogeneticanalysisofspike(S)andnucleocapsid
(N)genesequencesofPDCoVsrevealedthatcurrentlycirculatingPDCoVsinsouthernChinaweremorecloselyrelated
tootherChinesestrainsofPDCoVsthantothosereportedinUnitedStates,SouthKoreaandThailand.
Conclusions:ThisstudydemonstratedthatPDCoVwaspresentinsouthernChinadespitethelowprevalence,and
supportedanevolutionarytheoryofgeographicalclusteringofPDCoVs.
Keywords:Porcinedeltacoronavirus,Occurrence,Spikegene,Nucleocapsidgene,Sequenceanalysis,SouthernChina
Background (PEDV), transmissible gastroenteritis virus (TGEV), and
Before 2012, the subfamily Coronavirinae included three porcinerespiratorycoronavirus(PRCV)belongtothegenus
genera (Alphacoronavirus, Betacoronavirus and Gamma- Alphacoronavirus;meanwhile,porcinehemagglutinatingen-
coronavirus).However,in2012,anemerginggenus,Delta- cephalomyelitis virus (PHEV) and Porcine deltacoronavirus
coronavirus, was found in many animal species including (PDCoV) are assigned to the genus Betacoronavirus and
swine from Hong Kong [1]. At present, more than five the genus Deltacoronavirus, respectively [1]. Numerous
differentcoronaviruseshavebeen described in swine pop- studieshaveshownthatmorethanhalfofporcine corona-
ulations. Among them, porcine epidemic diarrhea virus viruses (including PDCoV) were enteropathogenic and
caused acute diarrhea and vomiting in pigs, which resulted
*Correspondence:[email protected];[email protected] inhugeeconomiclossesfortheglobalswineindustry[2–6].
†Equalcontributors Currently, PDCoV has been reported in Hong Kong,
1AnimalDiseaseDiagnosticCenter,InstituteofAnimalHealth,Guangdong
NorthAmerica,Mexico,SouthKorea,Thailandandsome
AcademyofAgriculturalSciences,GuangdongKeyLaboratoryofAnimal
DiseasePrevention,GuangdongOpenLaboratoryofVeterinaryPublicHealth, provinces of China [1, 7–19]. Despite recent progress,
Guangzhou510640,China little is known about the prevalence and epidemiology of
2DepartmentofBiologyandMicrobiology,SouthDakotaStateUniversity,
PDCoV in southern China (including Guangdong
Brookings,SD57007,USA
Fulllistofauthorinformationisavailableattheendofthearticle
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InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
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Zhaietal.VirologyJournal (2016) 13:136 Page2of11
province, Hainan province, and Guangxi autonomous previously published methods [8]. For amplification of the
region), where major swine production is operated. full-length S and N genes, previously reported RT-PCR
Therefore, the aim of this study was to investigate the primers (PDCoV-SF2: 5’-AGCGTTGACACCAACCTA
prevalence and sequence properties of PDCoV in this TT-3’and PDCoV-SR2: 5’-TCGTCGACTACCATTCCT
region. TAAAC-3’; PDCoV-NF1 : 5’-CCATC GCTCCAAG TC
ATTCT-3’ and PDCoV-NR1: 5’-TGGGTGGGTTTAA
Methods CAGACATAG-3’) were used. PCR was carried out at
Sampling 50°C30minand95°Cfor5min,followedby40cycles
A total of 390 fecal samples (Table 1) were collected of98°Cfor10s,55°Cfor15s,and68°Cfor5and2min
from 15 commercial swine farms with reported diarrhea for S and N genes, respectively; final extension was per-
in southern China. Farms A, D, E, J-O were from formed at 68 °C for 15 min. Positive amplicons were
Guangdong province, Farms B, F, G were from Hainan cloned into the pGM-19 T vector (Tiangen Inc. Beijing).
province, and farms C, H, I were the Guangxi autono- Furthermore, all positive recombinant plasmids were sub-
mous region. Farms A-I derived 30 samples with the mitted to a sequencing company (The Beijing Genomics
following arrangement: ten samples from suckling Institute, BGI) and sequenced at least three times. Five S
piglets (<3 weeks old), ten samples from nursing piglets gene sequences and five N gene sequences were obtained
(between 3 and 9 weeks old), and ten samples from (Additional file 1: Table S1), and have been submitted
fattening piglets (>9 weeks old). The samples of farms to GenBank database (accession numbers KU204694-
A-I were collected between July and August 2015 and KU204701, KX534090-KX534091).
stored at −80 °C until further use. However, the samples
of farms J-O were archived samples from 2012 to 2014. PhylogeneticanalysisoftheSandNgenes
Prior to viral RNA extraction, fecal samples were diluted Sequence alignment analysis was performed using the
one time using Phosphate Buffered Saline (PBS) (pH: Clustal W program implemented in DNAStar software. A
7.4).Thesupernatantswerethencollectedbycentrifuga- phylogenetic tree was then constructed by the neighbor-
tion at 5000×g for 5 min. 200 μl of clarified superna- joining method using the Molecular Evolutionary Genetics
tants was used to extract viral RNA following the Analysis(MEGA)softwareversion5.1with1000bootstrap
manufacturer’s recommendations (Axygen Scientific replications set at 1000. Moreover, the possible recombin-
Inc.). RNA samples were stored at −80 °C until further ation event wasevaluated in the S and Ngenes by recom-
analysis. binationdetectionprogram(RDP)3.34software.
Reversetranscriptionpolymerasechainreaction(RT-PCR) Results
detection PDCoVdetection
To detect PDCoV genome in collected fecal samples, A total of 390 pig fecal samples, collected from 15 swine
the previously reported RT-PCR primers (41 F: 5’-TTT farms with reported diarrhea in southern China, were
CAGGTGCTCAAAGCTCA-3’ and 735R: 5’-GCGAAA assessed for the presence of PDCoV and other viral en-
AGCATTTCCTGAAC-3’) targeting the nucleocapsid teric pathogens (PEDV, TGEV, and Rota C) by RT-PCR.
(N) gene with reaction conditions (50 °C for 30 min As summarized in Table 1, the PDCoV genome was de-
and 95 °C for 15 min for the reverse transcription reac- tected in specimens from 4 of 15 swine farms. Interest-
tion, followed by 40 cycles of PCR amplification at 95 °C ingly, the PDCoV genome was detected only in nursing
for 15 s, 55 °C for 45 s, and 72 °C for 1 min, with a final piglets, and was absent in suckling piglets and fattening
extension at 72 °C for 7 min) were used [15]. In addition, pigs. Although PDCoV was detected in each province/
molecular detection of the three diarrhea-related enteric region of southern China, its overall prevalence in the
viruses (Porcine epidemic diarrhea virus, PEDV; Porcine investigated pigs of various age groups (n=390) was
transmissible gastroenteritis virus, TGEV; Porcine rota- relatively low (5/390, 1.28 %). The positive rate could be
virusgroupC,RotaC)wasperformedinaccordancewith higher if only nursing piglets were included (5/150,
previous methods [20–22] for further evaluation of the 3.33%).Incontrast,theprevalenceofPEDV,anotherpor-
possible co-infection status with PDCoV in investigated cine coronavirus causing epidemic diarrhea, was relatively
pigsamples. higher (22.6 %, 88/390). In addition, PEDV was different
fromPDCoVinthatitdistributedsimilarlybetweennurs-
Amplificationofthespike(S)andNgenes ing (36/150, 24 %) and suckling piglets (47/150, 31.3 %).
To performin-depth sequence comparison and phylogen- Five fattening pigs from farms A, D, F and I were also
etic analysis with known reference sequences (Additional tested positive for the PEDV genome. We also examined
file 1: Table S1), the complete spike (S) and N genes of whether pigs with diarrhea harbored other enteric viruses
PDCoV-positive samples were amplified according to such as TGEV and Rota C. Our results showed that the
Zhaietal.VirologyJournal (2016) 13:136 Page3of11
Table1SampleinformationandRT-PCRdetectionresultsoffourdiarrhea-associatedvirusesinpigsofvariousagesfrom15swine
farmsinsouthernChina
Farm Pigs(Age) Number(n) PDCoV PEDV TGEV RotaC
A Sucklingpiglets(<3weeksold) 10 0/10 2/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 2/10 2/10 0/10 0/10
Fatteningpig(>9weeksold) 10 0/10 1/10 0/10 0/10
B Sucklingpiglets(<3weeksold) 10 0/10 3/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 1/10 2/10 0/10 0/10
Fatteningpig(>9weeksold) 10 0/10 0/10 0/10 0/10
C Sucklingpiglets(<3weeksold) 10 0/10 3/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 1/10 3/10 0/10 0/10
Fatteningpig(>9weeksold) 10 0/10 0/10 0/10 0/10
D Sucklingpiglets(<3weeksold) 10 0/10 2/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 1/10 0/10 2/10
Fatteningpig(>9weeksold) 10 0/10 1/10 1/10 0/10
E Sucklingpiglets(<3weeksold) 10 0/10 1/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 1/10 2/10 0/10 0/10
Fatteningpig(>9weeksold) 10 0/10 0/10 0/10 0/10
F Sucklingpiglets(<3weeksold) 10 0/10 3/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 4/10 0/10 0/10
Fatteningpig(>9weeksold) 10 0/10 1/10 2/10 0/10
G Sucklingpiglets(<3weeksold) 10 0/10 5/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 3/10 0/10 0/10
Fatteningpig(>9weeksold) 10 0/10 0/10 0/10 0/10
H Sucklingpiglets(<3weeksold) 10 0/10 7/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 2/10 0/10 0/10
Fatteningpig(>9weeksold) 10 0/10 0/10 0/10 0/10
I Sucklingpiglets(<3weeksold) 10 0/10 4/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 2/10 0/10 0/10
Fatteningpig(>9weeksold) 10 0/10 2/10 0/10 0/10
J Sucklingpiglets(<3weeksold) 10 0/10 4/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 3/10 0/10 0/10
K Sucklingpiglets(<3weeksold) 10 0/10 5/10 0/10 2/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 5/10 2/10 0/10
L Sucklingpiglets(<3weeksold) 10 0/10 2/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 0/10 0/10 0/10
M Sucklingpiglets(<3weeksold) 10 0/10 2/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 3/10 0/10 0/10
N Sucklingpiglets(<3weeksold) 10 0/10 2/10 0/10 1/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 1/10 0/10 0/10
O Sucklingpiglets(<3weeksold) 10 0/10 2/10 0/10 0/10
Nurserypigs(>3weeksold,<9weeksold) 10 0/10 3/10 1/10 0/10
Total 390 5/390 88/390 6/390 5/390
low detection rates (1.54 %, 5/390 for TGEV vs 1.28 %, PDCoV and PEDV was observed (Table 1). All PDCoV
6/390 for Rota C) of the two pathogens were present in positivenursingpigletswerealsotestedpositiveforPEDV,
those pig samples. Intriguingly, co-infection of pigs by therebyindicatinga100%co-infectionrate.
Zhaietal.VirologyJournal (2016) 13:136 Page4of11
SequencecomparisonandphylogeneticanalysisoftheS possible recombinant events occurring at the N gene of
geneofPDCoVs thosePDCoVstrains.
The full-length sequences of S genes in five PDCoV-
positive samples from the four different farms were Discussion
amplified and designated provisionally CH/GD01/2015, EpidemiologyofPDCoVs
CH/GD02/2015, CH/GD03/2015, CH/HN01/2015, and PDCoV was first identified by Deltacoronavirus specific-
CH/GX01/2015, respectively. Sequencing results showed PCR in rectal swabs of pigs (10.1 %, 17/169) with un-
that they were composed of 3480 nucleotides (nt). Com- known healthy status in Hong Kong [1]. Then, PDCoV
pared to all published American and individual Asian emerged in United States, China, South Korea and
strains (including HKU15-44, CHN-AH-2004, KNU14- Thailand [5, 17, 18]. In most of studies, excluding PEDV,
04, PDCoV/Swine/Thailand/S5015L/2015 and PDCoV/ TGEV and porcine rotavirus, PDCoV, as an important
Swine/Thailand/S5011/2015), a 3-nt deletion in the S enteric pathogen, was detected in clinical samples from
gene was identified in the five current PDCoV strains, six pigs with diarrhea [9–12, 23]. In addition, it was con-
otherChineseviralstrainsandoneThaiviralstrainreported firmed experimentally that less than two-week old piglets
previously(Additionalfile1:TableS1)[5,17,18].Sequence were susceptible to PDCoV, which caused mild to moder-
alignmentresultsrevealedthatCH/GD01/2015,CH/GD02/ atediarrheaaswellasmacroscopicandmicroscopiclesions
2015, CH/GD03/2015, CH/HN01/2015 and CH/GX01/ in small intestines of conventional piglets (5-day-old), and
2015 had >99.9 % homology in the S gene nucleotide severe diarrhea, vomiting, fecal shedding of virus, and se-
sequence,indicatingthesefiveviralstrainsevolvedfromthe vere atrophic enteritis in gnotobiotic pigs (11- to 14-day-
same ancestor (Table 2). Further analysis showed that the old) [2, 3]. The data further confirmed that PDCoV were
fivevirusesreportedinthisstudyhadthehighestnucleotide enteropathogenic in pigs. Meanwhile, PEDV or rotavirus
identity (98.9 to 99.5 %) with a Jiangxi strain (PDCoV/ showed higher detection rates in PDCoV-positive samples
CHJXNI2/2015) isolated from Jiangxi province bordering compared with other TGEV and rotavirus [8, 13–15, 24].
with the northern region of Guangdong province, and As shown above, co-infection of PDCoV and PEDV oc-
possessed the lowest nucleotide similarity (95.4 to 95.9 %) curred in nursing piglets (Table 1), indicating that the
with PDCoV/Swine/Thailand/S5011/2015 and PDCoV/ diarrhea-related pathogens were quite complex clinically
Swine/Thailand/S5015L/2015, isolated from Thailand and not easy to control in the field. Moreover, in the two
(Table2).Fromtheabovedata,phylogeneticanalysisofthe recent studies, PDCoV was shown to have higher infectiv-
S gene showed that the current PDCoVs circulating in ity in sows with diarrhea (81.0 %, 34/42) than nonclinical
southern China were most closely related to other Chinese counterparts(23.5%,4/17)[8,15],whichmightimplythat
PDCoV isolates than to those isolated previously from sowsoftencarryPDCoV.Andfurther,itcouldresultinthe
USA,SouthKoreaandThailand(Fig.1).Inaddition,inthe transmission of PDCoVfrom sows to the foetus and even
S gene, any possible recombinant eventswere not detected newborn piglets, although the pathogenesis mechanism of
amongthosePDCoVstrains. PDCoVremainsunclear.
TofurtherunderstandtheoriginofPDCoV,someretro-
spectivestudiesweremadeusingPCRandenzyme-linked
SequencecomparisonandphylogeneticanalysisoftheN immunosorbentassay(ELISA)[24–26].InDongetal.[24]
geneofPDCoVs study, 2 of 6 samples collected from Anhui Province of
Similarly,NgenesequencesofallfivePDCoV-positivesam- Chinain2004werepositiveforPDCoV,uptonow,itwas
pleswereidentifiedas1029nt insize. Sequencealignment the most ancient time for the detection of PDCoV in
resultssuggested that therewasno deletion or insertionin China. Meanwhile, PDCoV could date back to August
Ngeneregions(Additionalfile1:TableS1).Consistentwith 2013inUnitedStates,whereonly3PDCoV-sampleswere
the data of S gene, multiple sequence alignment results of detected using PCR in archived samples [25]. As for
NgeneshowedthatCH/GD01/2015,CH/GD02/2015,CH/ serology of PDCoV, anti-PDCoV IgG antibodies could
GD03/2015, CH/HN01/2015 and CH/GX01/2015 had the date back to 2010 using an indirect anti-PDCoV IgG
highestnucleotidehomology(99.1to99.7%)withPDCoV/ ELISA based on the putative S1 portion of the spike pro-
CHJXNI2/2015, and the lowest nucleotide homology (96.9 tein [26]. The above studies indicated that PDCoVcould
to 97.1 %) with PDCoV/Swine/Thailand/S5011/2015 have circulated in China at least since 2004 and in United
and PDCoV/Swine/Thailand/ S5015L/2015 (Table 2). In States since 2010. Maybe, due to limted samples in the
addition,inthephylogenetictreebasedonNgene,PDCoVs presentstudy,wedidnotdetectPDCoVinpigsamplescol-
were divided into three main branches (Chinese branch, lected from Guangdong province between 2012 and 2014.
American branch and Thai branch). The five viral isolates Although Asian leopard cat coronavirus (GenBank
reportedfrom thisworkwere clusteredtogetherwithinthe accessionno.EF584908)wasclosetoPDCoVinthephylo-
Chinese branch (Fig. 2). Moreover, there were no any genetic trees (Figs. 1 and 2), in the future, more
Table2Nucleotidesimilarities(%)ofSandNgenesofourfivePDCoVisolatesandotherreportedPDCoVsandcoronaviruses Z
h
a
Sgene Ngene i
e
t
GenBankNo. KU204694 KU204695 KU204696 KU204697 KX534090 KU204698 KU204699 KU204700 KU204701 KX534091 a
l.
V
Strain CH/GD01/ CH/GD02/ CH/HN01/ CH/GX01/ CH/GD03/ CH/GD01/ CH/GD02/ CH/HN01/ CH/GX01/ CH/GD03/ iro
2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 lo
g
y
KR131621 PDCoV/CHJXNI2/2015 99.5 98.9 98.9 99.1 99.2 99.7 99.1 99.1 99.3 99.4 Jo
u
KP757892 CHN-JS-2014 99.1 98.5 98.5 98.7 98.7 99.3 99.3 99.3 99.5 99.0 rn
a
l
KP757891 CHN-HB-2014 98.9 98.4 98.4 98.7 98.5 98.8 98.6 98.6 98.8 98.5 (2
0
JQ065042 HKU15-44 98.6 98.2 98.2 98.5 98.3 98.7 98.5 98.5 98.7 98.4 16
) 1
JQ065043 HKU15-155 98.5 98.2 98.2 98.4 98.2 98.7 98.5 98.5 98.7 98.4 3
:1
3
KT021234 CH/SXD1/2015 98.4 97.9 97.9 98.1 98.0 99.0 98.8 98.8 99.0 98.7 6

KT266822 CH/Sichuan/S27/2012 98.4 98.0 98.0 98.2 98.0 99.2 99.0 99.0 99.2 98.9
KM820765 KNU14-04 98.4 97.9 97.9 98.2 98.1 98.8 98.6 98.6 98.8 98.5
KJ620016 MI6148 98.4 97.9 97.9 98.1 98.0 98.8 98.6 98.6 98.8 98.5
KJ584360 MN3092 -a -a -a -a -a 98.8 98.6 98.6 98.8 98.5
KJ584358 PA3148 98.4 98.0 98.0 98.2 98.0 98.8 98.6 98.6 98.8 98.5
KJ584357 KY4813 98.4 97.9 97.9 98.1 98.0 98.8 98.6 98.6 98.8 98.5
KJ584355 IL2768 98.4 97.9 97.9 98.1 98.0 98.8 98.6 98.6 98.8 98.5
KT381613 OH11846 98.4 97.9 97.9 98.1 98.0 98.8 98.6 98.6 98.8 98.5
KJ601779 PDCoV/USA/Illinois 98.4 97.9 97.9 98.1 98.0 98.5 98.3 98.3 98.5 98.3
136/2014
KJ481931 PDCoV/USA/Illinois 98.4 97.9 97.9 98.1 98.0 98.5 98.3 98.3 98.5 98.3
121/2014
KJ769231 OhioCVM1/2014 98.4 97.8 97.8 98.1 98.0 98.3 98.2 98.2 98.3 98.1
KJ601777 PDCoV/USA/Illinois 98.4 97.8 97.8 98.1 98.0 98.7 98.5 98.5 98.7 98.4
133/2014
KJ584359 NE3579 98.4 97.8 97.8 98.1 98.0 98.7 98.5 98.5 98.7 98.4
KJ584356 SD3424 98.4 97.8 97.8 98.1 98.0 98.5 98.3 98.4 98.5 98.3
KJ462462 OH1987 98.4 97.8 97.8 98.1 98.0 98.7 98.5 98.5 98.7 98.4
KJ601778 PDCoV/USA/Illinois 98.3 97.8 97.8 98.0 98.0 98.7 98.5 98.5 98.7 98.4
134/2014
KP995358 OH-FD22 98.3 97.8 97.8 98.0 98.0 -b -b -b -b -b
KJ601780 PDCoV/USA/Ohio 98.3 97.8 97.8 98.0 98.0 98.7 98.5 98.5 98.7 98.4
137/2014
P
KJ569769 IN2847 98.3 97.8 97.8 98.1 98.0 98.8 98.6 98.6 98.8 98.5 a
g
e
KJ567050 8734/USA-IA/2014 98.3 97.9 97.9 98.1 98.0 98.7 98.5 98.5 98.7 98.4 5
o
KP981395 98.3 97.8 97.8 98.0 97.9 98.7 98.5 98.5 98.7 98.4 f1
1
Table2Nucleotidesimilarities(%)ofSandNgenesofourfivePDCoVisolatesandotherreportedPDCoVsandcoronaviruses(Continued) Z
h
a
i
USA/IL/2014/026 e
t
PDV_P11 a
l.
V
KM012168 Michigan/8977/2014 98.3 97.8 97.8 98.0 97.9 98.7 98.5 98.5 98.7 98.4 iro
lo
KP757890 CHN-AH-2004 98.0 97.5 97.5 97.8 97.7 98.1 97.9 97.9 98.1 97.8 g
y
KU051641 PDCoV/Swine/Thailand/ 95.8 95.6 95.6 95.9 95.4 97.1 96.9 96.9 97.1 96.8 Jo
u
S5011/2015 rn
a
l
KU051649 PDCoV/Swine/Thailand/ 95.8 95.5 95.5 95.9 95.4 97.1 96.9 96.9 97.1 96.8 (2
S5015L/2015 0
1
6
KU984334 P23_15_TT_1115 95.9 95.8 95.6 95.9 95.5 97.8 97.8 97.8 98.0 97.5 ) 1
3
EF584908 Guangxi/F230/2006 97.5 97.1 97.1 97.3 97.0 98.0 97.8 97.8 98.0 97.7 :13
6

JQ065045 HKU17-6124 40.9 40.8 40.8 40.7 40.7 92.9 92.6 92.8 92.9 92.6
FJ376621 HKU12-600 40.7 41.1 41.1 41.0 40.6 76.4 75.7 76.2 76.3 76.3
JQ065044 HKU16-6847 57.0 56.9 56.9 57.0 57.2 75.1 75.3 75.2 75.4 75.1
FJ376619 HKU11-934 65.9 68.9 68.9 66.1 65.8 74.4 74.3 74.5 74.4 74.2
FJ376620 HKU11-796 65.3 65.6 65.7 65.6 65.1 74.4 74.6 74.5 74.4 74.2
KJ408801 OH1414(PEDV) 38.2 38.3 38.3 38.1 37.8 3.6 3.5 3.5 3.6 3.6
FJ755618 H16(TGEV) 38.1 38.1 38.1 38.0 37.9 2.8 4.9 7.5 5.0 2.8
DQ811787 ISU-1(PRCV) 37.4 25.7 25.7 25.7 37.3 7.3 7.4 7.5 7.4 7.4
BCU00735 Mebus(bovine) 12.4 12.5 12.5 12.4 7.1 2.1 2.1 2.1 2.1 2.1
AY654624 TJF(SARS) 6.8 6.9 6.9 6.8 6.9 8.8 8.8 2.4 8.8 8.1
DQ011855 VW572(PHEV) 21.9 21.9 21.9 21.9 21.8 2.1 2.1 2.1 2.1 2.1
JF893452 YN(CIBV) 22.0 22.1 22.1 22.3 22.0 12.5 12.5 12.5 12.6 12.4
NC_010800 MG10(Turkey) 24.1 23.5 23.5 23.3 24.0 14.0 14.0 14.0 14.1 14.1
NC_010646 SW1(whale) 15.0 13.7 13.7 13.8 14.8 7.7 7.7 7.7 7.7 7.7
aSgeneofMN3092wasnotcomplete;bSgeneofOH-FD22wasnotavailable
P
a
g
e
6
o
f
1
1
Zhaietal.VirologyJournal (2016) 13:136 Page7of11
Strains/GenBank No.s Year Genus
Mebus (BCU00735) Betacoronavirus
VW572 (DQ011855) 1972
YN (JF893452) 2005 Gammacoronavirus
MG10 (NC 010800)
TJF (AY654624) 2003 Alphacoronavirus
SW1 (NC 010646) Gammacoronavirus
OH1414 (KJ408801) 2014
ISU-1 (DQ811787) Alphacoronavirus
H16 (FJ755618) 1973
HKU12-600 (FJ376621) 2007
HKU17-6124 (JQ065045) 2007
HKU16-6847 (JQ065044) 2007
2007
HKU11-934 (FJ376619)
2007
HKU11-796 (FJ376620)
Guangxi/F230/2006 (EF584908) 2006
2015
P23 15 TT 1115 (KU984334)
2015
PDCoV/Swine/Thailand/S5011/2015 (KU051641)
2015
PDCoV/Swine/Thailand/S5015L/2015 (KU051649)
2004
CHN-AH-2004 (KP757890)
2010
HKU15-155 (JQ065043)
2009
HKU15-44 (JQ065042)
2014
CHN-HB-2014 (KP757891)
2015
CH/SXD1/2015 (KT021234)
2015
CH/GD01/2015 (KU204694)
2015
CH/GD03/2015 (KX534090)
2015
PDCoV/CHJXNI2/2015 (KR131621)
2015
CH/GX01/2015 (KU204697)
CH/GD02/2015 (KU204695) 2015
2015
CH/HN01/2015 (KU204696)
2014
CHN-JS-2014 (KP757892) Deltacoronavirus
2012
CH/Sichuan/S27/2012 (KT266822)
2014
SD3424 (KJ584356)
2014
OhioCVM1/2014 (KJ769231)
2014
OH1987 (KJ462462)
2014
OH-FD22 (KP995358)
2014
PA3148 (KJ584358)
2014
MI6148 (KJ620016)
2014
8734/USA-IA/2014 (KJ567050)
2014
PDCoV/USA/Ohio137/2014 (KJ601780)
2014
IL2768 (KJ584355)
2014
NE3579 (KJ584359)
2014
PDCoV/USA/Illinois133/2014 (KJ601777)
2014
PDCoV/USA/Illinois134/2014 (KJ601778)
2014
PDCoV/USA/Illinois121/2014 (KJ481931)
2014
PDCoV/USA/Illinois136/2014 (KJ601779)
2014
OH11846 (KT381613)
2014
IN2847 (KJ569769)
2014
KNU14-04 (KM820765)
2014
KY4813 (KJ584357)
2014
Michigan/8977/2014 (KM012168)
2014
0.05 USA/IL/2014/026PDV P11 (KP981395)
Fig.1PhylogeneticanalysisbasedontheSgeneofPDCoVsandothercoronaviruses.Note:ThosePDCoVstrainsfromChina,America,South
KoreaandThilandwerelabelledusing“ ”,“ ”,“ ”and“ ”,respectively.Moreover,PDCoVstrainsinthisstudywerelabelledusingleftarrows.
Thecollectiontimewasnotavailableforthosecoronavirusstrainslabelledusingstarsymbols
Zhaietal.VirologyJournal (2016) 13:136 Page8of11
Strains/GenBank No.s Year Genus
PDCoV/USA/Illinois133/2014 (KJ601777) 2014
PDCoV/USA/Illinois134/2014 (KJ601778) 2014
2014
KY4813 (KJ584357)
IL2768 (KJ584355) 2014
PDCoV/USA/Ohio137/2014 (KJ601780) 2014
OH-FD22 (KJ584358) 2014
2014
OH1987 (KJ462462)
2014
NE3579 (KJ584359)
8734/USA-IA/2014 (KJ567050) 2014
2014
SD3424 (KJ584356)
2014
Michigan/8977/2014 (KM012168)
2014
USA/IL/2014/026PDV_P11 (KP981395)
2014
OhioCVM1/2014 (KJ769231)
2014
IN2847 (KJ569769)
2014
OH11846 (KT381613)
2014
MN3092 (KJ584360)
2014
MI6148 (KJ620016)
2014
PDCoV/USA/Illinois121/2014 (KJ481931)
2014
PDCoV/USA/Illinois136/2014 (KJ601779)
KNU14-04 (KM820765) 2014
HKU15-44 (JQ065042) 2009 Deltacoronavirus
CHN-HB-2014 (KP757891) 2014
CH/SXD1/2015 (KT021234) 2015
CH/Sichuan/S27/2012 (KT266822) 2012
CH/GD01/2015 (KU204694) 2015
CH/GD03/2015 (KX534091) 2015
PDCoV/CHJXNI2/2015 (KR131621) 2015
CHN-JS-2014 (KP757892) 2014
CH/GD02/2015 (KU204699) 2015
CH/GX01/2015 (KU204701) 2015
CH/HN01/2015 (KU204700) 2015
Guangxi/F230/2006 (EF584908) 2006
HKU15-155 (JQ065043) 2010
CHN-AH-2004 (KP757890) 2004
P23_15_TT_1115 (KU984334) 2015
PDCoV/Swine/Thailand/S5011/2015 (KU051641) 2015
PDCoV/Swine/Thailand/S5015L/2015 (KU051649) 2015
HKU17-6124 (JQ065045) 2007
HKU11-934 (FJ376619) 2007
HKU11-796 (FJ376620) 2007
HKU12-600 (FJ376621) 2007
2007
HKU16-6847 (JQ065044)
SW1 (NC_010646)
Gammacoronavirus
YN (JF893452) 2005
MG10 (NC_010800)
OH1414 (KJ408801) 2014
ISU-1 (DQ811787) Alphacoronavirus
H16 (FJ755618) 1973
2003
TJF (AY654624)
Mebus (BCU00735) Betacoronavirus
0.5
VW572 (DQ011855) 1972
Fig.2PhylogeneticanalysisbasedontheNgeneofPDCoVsandothercoronaviruses.Note:ThosePDCoVstrainsfromChina,America,South
KoreaandThilandwerelabelledusing“ ”,“ ”,“ ”and“ ”,respectively.Moreover,PDCoVstrainsinthisstudywerelabelledusingleftarrows.
Thecollectiontimewasnotavailableforthosecoronavirusstrainslabelledusingstarsymbols
Zhaietal.VirologyJournal (2016) 13:136 Page9of11
epidemiological surveys should be warranted to uncover found in CH/Sichuan/S27/2012 [16]. However, for Thai
theoriginofPDCoV. viralisolates,theyownedoneadditionaluniquenucleotide
At the territory of China, Southern China mainly (C)insertioninthe3’UTR[17,18].Thebiologicalsignifi-
includes Guangdong province, Hainan province and the cance of these naturally occurring deletions or insertions
Guangxi autonomous region. Although molecular in PDCoV biology and pathogenesis warrants further
detection of PDCoV was performed in these regions investigations.
[23, 24], little information was available on PDCoV In this study, five S and five N gene sequences, respect-
prevalence. In a study by Chen et al. [23], an overall ively, were obtained to evaluate wherever genetic diversity
positive-PDCoV rate of 23.4 % (15/64) was obtained in of PDCoVs existed in southern China. Our results showed
all samples collected from Guangdong, Shanxi and that these five S and five N gene sequences were more
Hubei provinces. However, more detailed data of closelyrelatedtoChinesestrains,andallclusteredtogether
PDCoV was not available in Guangdong province. in the phylogenetic tree (Table 2, Figs. 1 and 2). However,
Meanwhile, in the study from Dong et al. [24], only CH/GD01/2015 and CH/GD02/2015, reported in this
four archived samples from the Guangxi autonomous study, originated from the same pig farm in Guangdong
region were examined, but all negative for PDCoV. In province, but had 48 nt and 12 nt differences in the S and
this study, we demonstrated that PDCoVcirculated and N genes, respectively. The observed 48 nucleotide changes
was co-infected by PEDV on those swine farms in in the S gene made these viruses differ by 25 amino acid
Guangdong province, Hainanprovince and theGuangxi residues (Additional file 2: Table S2). For the N gene, the
autonomous region, which further contributed to the 12 nucleotide changes among these viruses resulted in 3
epidemiology of PDCoV in these regions despite the amino acid substitutions (Additional file 2: Table S2).
relatively low prevalence. Among them, 18 of 25 amino acid differences occurred at
the first two-third parts of S gene. Interestingly, in spite of
GeneticdiversityofPDCoVs aminoacidmutation,bothSandNproteinretainedalmost
The first two reported full-length PDCoV genome se- consistent amino acid properties (especially pH value)
quences (HKU15-44 and HKU15-155) were 25, 437 nt (Additional file 2: Table S2). Future study will address im-
and 25, 432 nt in length, respectively [1], and they had portant roles of these polymorphisms in viral replication
99.1 % nucleotide similarity with each other. Moreover, and pathogenesis. In addition, they were divided into two
further sequence alignment showed a 3-nt (TAA) inser- distinct small branches (Figs. 1and 2). Thesefindings sug-
tion in the S gene and a 3-nt (TTA) insertion in the 3’ gestedthatPDCoVsinsouthernChinahavedivergedfrom
untranslated region (UTR) of HKU15-44 [1, 5]. During acommonancestor.Despitetheemerginggeneticdiversity,
the past3years,PDCoV-associated swine entericdisease overall, PDCoV prevalence is still largely restricted by the
was paid great attention in the major pig producing territoryasdemonstratedinFigs.1and2.
countries, especilly United States and China. Up to May For the two enteric coronaviruses (PEDVand TGEV) in
2016, morethan30 complete PDCoVgenome sequences pigs, the recombination events were often detected. How-
were published in the GenBank database. All were ever,mostofthemwerefromintra-recombination[27–30].
generated in China and United States except for one Recently, only one emerging recombinant/chimeric virus
sequence from South Korea and three sequences from (named swine enteric coronavirus, SeCoV) was discovered
Thailand [8–19]. The Korean strain, KNU14-04, had 25, in swine feces and resulted from inter-recombination of
422 nt in length, with similar genome features (a 3-nt PEDV and TGEV, which had a TGEV backbone and a
insertion in the S gene with 3, 483 nt and a 3-nt inser- PEDVspike gene [31, 32]. In this study, there were no any
tion in the 3’ UTR, respectively) to all American strains possible recombinant events occurring in PDCoV strains.
and the Chinese strain (HKU15-44) [9]. Comparing Maybe,thenumberandlengthofourobtainedPDCoVse-
complete genomes of the remaining Chinese strains to the quenceswerelimited.Inthefollowingstudy,therecombin-
American, Thai and Korean counterparts, CHN-HB-2014, ationeventofPDCoVwarrantsfurtherattentions.
CHN-JS-2014, PDCoV/CHJXNI2/2015, CH/Sichuan/S27/
2012, CH/SXD1/2015 and P23_15_TT_1115 only had the
3-nt(AAT)deletionin the S gene (3, 480 nt) [14, 23, 24], Conclusion
while CHN-AH-2004 only had the 3-nt (TAA) deletion This study reported the prevalence of PDCoV on swine
in the 3’ UTR [5, 24]. In the present study, 3-nt inser- farms in southern China. Phylogenetic analysis of cur-
tion was not found in UTR for our five obtained rently circulating PDCoVstrains in this region and other
PDCoV (data not shown). Moreover, two additional previously reported strains supported the theory of geo-
unique features including a 6-nt (TTTGAA) deletion in graphical clustering of PDCoV infection landscape. The
the nonstructural protein (nsp) 2 region and a 9-nt origin of various PDCoVs in different countries and re-
(GCCGGTTGG) deletion in the nsp 3 region were also gionsshouldbefurther studied.
Zhaietal.VirologyJournal (2016) 13:136 Page10of11
Additional files 5. WangL,HayesJ,SarverC,ByrumB,ZhangY.Porcinedeltacoronavirus:
histologicallesionsandgeneticcharacterization.ArchVirol.2016;161:171–5.
6. ZhangQ,HuR,TangX,WuC,HeQ,ZhaoZ,ChenH,WuB.Occurrence
Additionalfile1:TableS1.SequenceinformationofPDCoVsand
andinvestigationofentericviralinfectionsinpigswithdiarrheainChina.
othercoronavirusesusedinthepresentstudy.(DOC106kb)
ArchVirol.2013;158:1631–6.
Additionalfile2:TableS2.AAdifferencesofSpike(S)andNucleocapsid 7. HomwongN,JarvisMC,LamHC,DiazA,RoviraA,NelsonM,MarthalerD.
(N)proteinbetweenCH/GD01/2015andCH/GD02/2015.(DOC42kb) CharacterizationandevolutionofporcinedeltacoronavirusintheUnited
States.PrevVetMed.2016;123:168–74.
8. HuH,JungK,VlasovaAN,ChepngenoJ,LuZ,WangQ,SaifLJ.Isolationand
Abbreviations
characterizationofporcinedeltacoronavirusfrompigswithdiarrheainthe
ELISA,enzyme-linkedimmunosorbentassay;MEGA,molecularevolutionary UnitedStates.JClinMicrobiol.2015;53:1537–48.
geneticsanalysis;N,nucleocapsid;PBS,phosphatebufferedsaline;PDCoV,
9. LeeS,LeeC.CompleteGenomeCharacterizationofKoreanPorcine
porcinedeltacoronavirus;PEDV,porcineepidemicdiarrheavirus;PHEV,porcine
DeltacoronavirusStrainKOR/KNU14-04/2014.GenomeAnnounc.2014;2:
hemagglutinatingencephalomyelitisvirus;PRCV,porcinerespiratorycoronavirus;
e01191–14.
RDP,recombinationdetectionprogram;RotaC,porcinerotavirusgroupC;RT-PCR,
10. LiG,ChenQ,HarmonKM,YoonKJ,SchwartzKJ,HooglandMJ,GaugerPC,
reversetranscriptionpolymerasechainreaction;S,Spike;SeCoV,swineenteric
MainRG,ZhangJ.Full-LengthGenomeSequenceofPorcine
coronavirus;TGEV,transmissiblegastroenteritisvirus;UTR,untranslatedregion
DeltacoronavirusStrainUSA/IA/2014/8734.GenomeAnnounc.2014;2:
e00278–14.
Funding
11. MaY,ZhangY,LiangX,LouF,OglesbeeM,KrakowkaS,LiJ.Origin,evolution,
Thisstudywassupportedbythegrants(No.2013B060500063,No.
andvirulenceofporcinedeltacoronavirusesintheUnitedStates.MBio.
2014B040404061,No.2016A040401012andNo.201508020055)from
2015;6:e00064.
GuangdongProvincialDepartmentofScienceandTechnologyand
12. MarthalerD,JiangY,CollinsJ,RossowK.CompleteGenomeSequenceof
GuangzhouScienceTechnologyandInnovationCommission,respectively.
StrainSDCV/USA/Illinois121/2014,aPorcineDeltacoronavirusfromthe
Moreover,thisstudywasalsoinpartsupportedbyUSDA/NIFA2016-67016-24949 UnitedStates.GenomeAnnounc.2014;2:e00218–14.
(toD.W.).Thefundingbodywasnotinvolvedintothedesignofthestudy,and
13. Marthaler D,RaymondL,JiangY,CollinsJ,RossowK,RoviraA.Rapid
collection,analysis,andinterpretationofdatainthemanuscript.
detection,completegenomesequencing,andphylogenetic analysisof
porcinedeltacoronavirus.EmergInfectDis.2014;20:1347–50.
Availabilityofdataandmaterials 14. SongD,ZhouX,PengQ,ChenY,ZhangF,HuangT,ZhangT,LiA,HuangD,
Thedatasetssupportingtheconclusionsofthisarticleareincludedwithin WuQ,HeH,TangY.Newlyemergedporcinedeltacoronavirusassociatedwith
thearticleandtwoadditionalfiles(Additionalfile1:TableS1andAdditional diarrhoeainswineinChina:identification,prevalenceandfull-lengthgenome
file2:TableS2). sequenceanalysis.TransboundEmergDis.2015;62:575–80.
15. WangL,ByrumB,ZhangY.Detectionandgeneticcharacterizationof
Authors’contributions deltacoronavirusinpigs,Ohio,USA,2014.EmergInfectDis.
Shao-Lun Zhai and Wen-Kang Wei conceived the project; Shao-Lun Zhai 2014;20:1227–30.
designed the experiments; Shao-Lun Zhai and Xiao-Peng Li performed 16. WangYW,YueH,FangW,HuangYW.CompleteGenomeSequenceof
most of the experiments; Xiao-Hui Wen and Dian-Hong Lv contributed PorcineDeltacoronavirusStrainCH/Sichuan/S27/2012fromMainlandChina.
materials and participated in discussion; Xia Zhou and He Zhang added GenomeAnnounc.2015;3:e00945–15.
the data in the revision version; Shao-Lun Zhai wrote the manuscript; 17. MadapongA,Saeng-ChutoK,LorsirigoolA,TemeeyasenG,SrijangwadA,
Feng Li edited the manuscript; Shao-Lun Zhai and Dan Wang supervised TripipatT,WegnerM,NilubolD.CompleteGenomeSequenceofPorcine
thework.Thefinalversionofthemanuscriptwasapprovedbyallauthors. DeltacoronavirusIsolatedinThailandin2015.GenomeAnnounc.2016;4:
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Competinginterests 18. JanetanakitT,LumyaiM,BunpapongN,BoonyapisitsopaS,ChaiyawongS,
Theauthorsdeclarethattheyhavenocompetinginterests. NonthabenjawanN,KesdaengsakonwutS,AmonsinA.Porcine
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1AnimalDiseaseDiagnosticCenter,InstituteofAnimalHealth,Guangdong states,2014.EmergInfectDis.2014;20:1594–5.
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