Completion of the Porcine Epidemic Diarrhoea Coronavirus (PEDV) Genome Sequence

PEDV、PoRVA和PDCoV TaqMan三重RT-qPCR检测方法的建立与初步应用胡泽奇;李润成;谭祖明;谢秀艳;王江平;秦乐娟;李荣;葛猛【期刊名称】《畜牧兽医学报》【年(卷),期】2024(55)5【摘要】旨在建立一种可快速同时检测猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)、猪A群轮状病毒(porcine rotavirus type A, PoRVA)和猪丁型冠状病毒(porcine deltacoronavirus, PDCoV)三重荧光定量PCR方法。

针对PEDV-N、PoRVA-VP6和PDCoV-M基因设计了引物和探针,条件优化后进行性能评估,并与商品化试剂盒检测对比。

结果显示:本研究建立的三重RT-qPCR方法具有良好特异性,对PRRSV、PCV_2和PRV等阳性核酸不发生扩增;具有较高的敏感性,PEDV、PoRVA和PDCoV的最低检测限均达1 copies·μL^(-1);重复性良好,组内和组间变异系数均小于1%;样本适应性广,检测不同样本类型时,变异系数均小于1%。

与商品化试剂盒对比,本研究建立的方法PEDV和PoRVA的检测符合率为92.5%和97.5%,并对PDCoV的检测范围更广,对其变异毒株仍有较好的检测效果。

本研究建立的检测方法具有特异性好、敏感性高、稳定性强和样本适应性广等优势,为临床腹泻样本提供了一种好的检测方法。

【总页数】6页(P2267-2272)【作者】胡泽奇;李润成;谭祖明;谢秀艳;王江平;秦乐娟;李荣;葛猛【作者单位】湖南农业大学动物医学院;湖南龙华农牧发展有限公司;茶陵县畜牧水产事务中心【正文语种】中文【中图分类】S858.285.3【相关文献】1.PEDV、TGEV、PDCoV、PRoV多重TaqMan荧光定量RT-PCR检测方法的建立及应用2.基于Taqman探针三重Real-Time RT-PCR检测PEDV、TGEV、PoRV方法的建立与应用3.BVDV、BRV和BCoV TaqMan三重RT-qPCR检测方法的建立4.猪圆环病毒-Ⅱ型、猪瘟病毒和猪繁殖与呼吸综合征病毒三重RT-qPCR 检测方法的建立及初步应用5.猪肉及制品中HEV、PEDV和PDCoV三重qPCR 方法的建立与应用因版权原因，仅展示原文概要，查看原文内容请购买。

猪流行性腹泻(PED)的预防和控制日前，广西大学动物科学技术学院刘芳副教授在广西畜牧兽医学会养猪分会2016年年会学术报告会上介绍了猪流行性腹泻(PED)的预防和控制。

据介绍，PED首先在1971年被发现于英国，而我国于1976年首次报道该病。

猪流行性腹泻病毒(PEDV)目前仍只有一个血清型。

猪群感染PEDV后1-2天即开始排毒，5-6天达到高峰，并可持续排毒35天。

母猪感染后，可通过乳汁把病毒传给初生仔猪。

在2010年之前，PED多发生在每年12月至次年2月，季节性明显，但2010年之后一年四季都有流行，仔猪出生时正常，吃奶后半天-1天开始呕吐，接着很快出现腹泻，发病率最高可达100%，死亡率高达90%-100%，保育猪、中大猪、母猪、公猪少见腹泻，使用抗生素、干扰素、抗病毒药物等治疗，效果不理想。

PED是导致近年产房仔猪高发病率和高死亡率的主要原因。

疫苗免疫、生物安全、猪群的健康水平、猪舍的环境控制是防控PED的重要措施;母猪对PED的免疫力是预防初生仔猪发病的关键;4-13日龄仔猪对PEDV的抵抗能力取决于母猪初乳及乳汁中抗体含量水平。

鉴于PED的发生已无明显季节性，建议对母猪实行普免(4次/年)或跟胎免疫。

1。

广西部分地区猪群PEDV N基因克隆及序列分析许瑞胜;熊毅;马琳;何奇松;李春英;冯淑萍;易春华;韦达有;胡丽萍;黄胜斌;胡巧云【摘要】[目的]了解广西区猪流行性腹泻病毒(PEDV)的感染及病毒的遗传变异特点,为更好防控猪流行性腹泻提供科学依据.[方法]应用RT-PCR方法从2012-2016年广西地区25个猪场收集的PEDV病料中扩增出86株PEDV的N基因,并进行序列比对及遗传进化分析.[结果]86株PEDV的N基因核苷酸同源性为94.2％～100.0％,与参考毒株核苷酸同源性为94.1％～100.0％.N基因遗传进化分析显示广西的PEDV可分为2个群,Cluster2由韩国株DR13、日本株83P-5、中国株CH/S以及本研究的GP35-8、LC37-22、LC107-5、LC107-19和LC107-16毒株组成,其他81株毒株属于Cluster 3,包括中国株DX、LJB-03、HuN、JS-2004-2;Cluster 1和Cluster 4则由其它参考毒株组成.[结论]PEDV是引起广西规模化猪场新生仔猪腹泻的主要病原,广西地区的PEDV流行毒株可分为2个群且其N基因仍然保守.%[Objective] To provide a scientific basis for better prevention and control of PEDV,the infection of PEDV and its current characteristics of genetic evolution in Guangxi were studied.[Method]86 PEDV field strains were isolated from 25 farms in Guangxi during 2012-2016.N gene was amplified from the 86 PEDV field strains by RT-PCR,cloned,sequenced and compared with the other reference strains in GenBank.[Result] Sequence analyses of the N genes were performed,and the results showed that 86 strains had nucleotide homology of 94.2 ％-100.0 ％ and 94.1％-100.0 ％homology with reference strains.Phylogenetic analysis of N gene indicated that Guangxi strain could be divided into two groups.Cluster 2 consisted of the Korean strains DR13,Japanese strain 83P-5 and six strains from ourstudy (GP35-8,LC037-22,LC107-5,LC107-5,LC107-19,LC107-16).Other81 strains and Chinese strain DX,LJB-03,HuN,JS-2004-2 belonged to cluster 3 while cluster 1 and cluster 4 were composed of other reference strains.[Conclusion] PEDV was the main pathogen that caused diarrhea of neonatal piglets in Guangxi.The PEDV-N genes were divided into two series,and the N genes of the current PEDV were still relatively conservative.【期刊名称】《西南农业学报》【年(卷),期】2018(031)002【总页数】6页(P409-414)【关键词】猪流行性腹泻病毒;N基因;序列分析;遗传进化分析【作者】许瑞胜;熊毅;马琳;何奇松;李春英;冯淑萍;易春华;韦达有;胡丽萍;黄胜斌;胡巧云【作者单位】广西动物疫病预防控制中心,广西南宁530001;广西动物疫病预防控制中心,广西南宁530001;广西动物疫病预防控制中心,广西南宁530001;广西动物疫病预防控制中心,广西南宁530001;钦州市动物疫病预防控制中心,广西钦州535000;广西动物疫病预防控制中心,广西南宁530001;桂林市动物疫病预防控制中心,广西桂林541002;桂林市动物疫病预防控制中心,广西桂林541002;广西动物疫病预防控制中心,广西南宁530001;广西动物疫病预防控制中心,广西南宁530001;广西动物疫病预防控制中心,广西南宁530001【正文语种】中文【中图分类】S828【研究意义】猪流行性腹泻(Porcine epidemic diarrhea, PED)可发生在各阶段猪，主要危害哺乳仔猪且具有较高的死亡率，是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)感染引起的一种急性传染性肠道疾病[1]。

间接ELISA检测⽅法ARTICLEDevelopment of an indirect ELISA based on a truncated S protein of the porcine epidemic diarrhea virusYufeng Li,Fangyuan Zheng,Baochao Fan,Hassan Mushtaq Muhammad,Yao Zou,and Ping JiangAbstract:Porcine epidemic diarrhea (PED)is a highly contagious,enteric disease of swine caused by the porcine epidemic diarrhea virus (PEDV).To ?nd a suitable ELISA method to assess the infection of PEDV and the effective-ness of vaccines,we developed and evaluated an indirect enzyme-linked immunosorbent assay (iELISA)based on a truncated recombinant spike (S)protein expressed in Escherichia coli .The parameters of the iELISA were opti-mized,and the cutoff value determined as 0.259by analyzing optical density (OD)values of 80PEDV negative sera con?rmed by westernblot.Repeatability tests revealed that the coef?cients of variation of positive sera within and between runs were both less than 10%.Cross-reactivity assays demonstrated that iELISA was PEDV-speci?c.A virus neutralization test with sera of 7different OD values showed a positive correlation between the OD values and virus neutralization.The results suggest this iELISA is speci?c,sensitive,and repeatable.Further studies should focus on the relationship between OD values of sera and its virus neutralization.Key words:indirect ELISA,PEDV,truncated S protein,virus neutralization.Résumé:La diarrhée épidémique porcine (DEP)est une maladie entérique du porc hautement contagieuse qui estcausée par le virus de la diarrhée épidémique porcine (PEDV).A?n de concevoir une méthode ELISA apte a`évaluer l’infection au PEDV et l’ef?cacitédes vaccins,on a élaboréet mis a`l’essai un test enzymatique indirect par immunoadsorption (iELISA)basésur une forme recombinante et tronquée de la protéine de spicule (S)expriméechez Escherichia coli .On a optimiséles paramètres de l’iELISA et établi la valeur seuil a`0,259en vertu d’une analyse des valeurs de DO de 80sérums a`PEDV négatif con?rmépar immunobuvardage de type western.Les tests de répétabilitéont révéléque les coef?cients de variation des sérums positifs intraessais et interessais se situaient tous deux sous la barre des 10%.Les essais de réactivitécroisée ont démontréque l’iELISA était spéci?que aux PEDV.Un test de neutralisation du virus d’après 7valeurs de DO a mis en évidence une corrélation positive entre les valeurs de DO et la neutralisation virale.Les résultats indiquent que cet iELISA est spéci?que,sensible etreproductible.Les analyses ultérieures devront s’attarder a`la relation entre les valeurs de DO des sérums et la neutralisation virale correspondante.[Traduit par la Rédaction]Mots-clés :ELISA indirect,PEDV,protéine S tronquée,neutralisation virale.IntroductionPorcine epidemic diarrhea virus (PEDV)is an envel-oped virus possessing an approximately 28kb,positive-sense,single-stranded RNA genome with a 5=cap and a 3=polyadenylated tail (Pensaert and Debouck 1978).The virus possesses glycosylated spike (S),Poll (P1),en-velope (E),glycosylated membrane (M),and an unglyco-sylated RNA-binding nucleocapsid (N)proteins (Song and Park 2012).The S protein of PEDV is a type I mem-brane glycoprotein composed of 1383amino acids and is predicted to contain a signal peptide (1–24aa),a large extracellular region,a single transmembrane domain (1334–1356aa),and a short cytoplasmic tail (Lee et al.2010).The S protein plays important roles in induction of neutralizing antibodies,speci?c receptor binding,and cell membrane fusion (Chang et al.2002;Sun et al.2008).The S1-GST protein (S1D,aa 636–789)is essential for in-duction of neutralizing antibodies (Sun et al.2007).The N-terminal region of the S protein is especially impor-tant for the receptor binding,and the 25–225aa region is indispensable to bind with amino peptidase N (receptor of PEDV)(Lee 2011).To date,most of the reported PEDV vaccines belong to attenuated vaccines and subunit vac-cines (Kweon et al.1999;Kang et al.2005;Song et al.2007;Received 8April 2015.Revision received 31July 2015.Accepted 4August 2015.Y.Li,F.Zheng,B.Fan,Y.Zou,and P.Jiang.Key Laboratory of Bacteriology,Ministry of Agriculture,College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,People’s Republic of China.H.M.Muhammad.Department of Epidemiology and Public Health,Faculty of Veterinary Science,University of Veterinary and Animal Sciences,Lahore,Pakistan.Correspondence author:Yufeng Li (e-mail:yufengli@/doc/597e2535d5bbfd0a785673aa.html ).811C a n . J . M i c r o b i o l .D o w n l o a d e d f r o m w w w .n r c r e s e a r c h p r e s s .c o m b y F o s h a n u n i v e r s i t y o n 11/03/15F o r p e r s o n a l u s e o n l y .Meng et al.2013).To date,M protein-based enzyme-linked immunosorbent assay (ELISA)and double anti-body sandwich ELISA have been developed to detect PEDV (Sozzi et al.2010;Ren etal.2011).N protein and viral protein have been used as coating antigens to estab-lish ELISA methods to detect antibodies against PEDV (Oh et al.2005;Hou et al.2007).Viral proteins have been used as a coating antigen of PEDV ELISA,and ELISA has been compared with virus neutralization.An overall agreement of 84.2%was generated between the serum neutralization and ELISA (Oh et al.2005).In the present study,an indirect ELISA (iELISA)based on a truncated recombinant protein S (tSc,N-terminal region 25–225aa of PEDV)was developed and assay conditions were optimized.The relationship between the iELISA results and virus neutralization titers was assessed.Materials and methodsSera and antibodyEighty-six sow sera were obtained from farms with the outbreaks of porcine epidemic diarrhea (PED)and health farms without clinical signs of PED.Among of them,6and 80sera were con?rmed as positive and negative,respectively,with western blot (WB)and immuno?uo-rescence (IFA)and were then stored at –80°C.The blood was drawn from the precaval vein of anesthetized pigs.Animal experiments were approved by the Institutional Animal Care and Ethics Committee of Nanjing Agricul-tural University (Approval No.IACECNAU20100902).Cloning and sequencing of the recombinant tSc protein geneThe tSc gene was ampli?ed from plasmids pFast-S (constructed by our laboratory)using a forward primer (5=-ATAGGATCCAGGTGCCAGTCTACT-3=)and a reverse primer (5=-GCGCTCGAGTTAAGGTTCATAGTAAAT-3=)(positions 20682–21284;DR13:GenBank accession No.JQ023162.1)containing restriction enzyme sites Bam HI and Xho I,underlined).The PCR product was cloned into the prokaryotic expression vector pET-28a(+)on corresponding restriction enzyme sites.This vector was designated as pET-28a/tSc,and its sequence was con-?rmed by sequencing analysis by Invitrogen (Shanghai,China).Expression and puri?cation of tSc proteinThe recombinant plasmid was transformed into Escherichia coli BL-21.The tSc protein was induced by addi-tion of 1mmol/L isopropyl-?-D -thiogalactopyranoside (IPTG).Four hours after IPTG induction,the cells were harvested and lysed by sonication.According to the man-ufacturer’s instructions,under the native condition,the recombinant protein was puri?ed by af?nity chro-matography with Ni-nitrilotriacetic acid agarose.The expressed protein was analyzed by sodium dodecyl sulfate –polyacrylamide gel electrophoresis (SDS–PAGE)with Coomassie brilliant blue R250and con?rmed by WBanalysis using His Tag monoclonal antibody (Boster,China)and PEDV polyclonal antibody prepared by immu-nizing rabbits with tSc protein that containing tSc.The puri?ed tSc protein was separated by SDS–PAGE using 12%separating gel and 5%stacking gel.Gels were stained with Coomassie brilliant blue R250,followed by immunoblotting.Brie?y,after transferring the proteins to nitrocellulose membrane,the membrane blocking was performed with 10%skimmed milk and incubated overnight at 4°C.The membranes were incubated with His Tag monoclonal antibody and PEDV polyclonal anti-body in blocking solution at 1:5000and 1:500dilution,respectively,for 2h at room temperature.After 1h of incubation,the membrane was extensively washed with PBS containing 0.05%Tween 20(PBST)and incubated for 45min with goat anti-mouse IgG (Boster,Wuhan).After washing,the membranes were incubated with horserad-ish peroxidase (HRP)-tagged goat anti-mouse IgG second-ary antibody (Boster,Wuhan),and the colorimetric reaction was developed using chemiluminescence lumi-nal reagents (Super Signal West Pico TrialKit,Pierce).iELISAA microtiter plate (96-well;Nunc,Nunclon,China)was coated with 100?L of tSc in 0.05mol/L bicarbonate–carbonate buffer (pH 9.6)and incubated for 2h at 37°C.Following 3washes with PBST,the plates were blocked for 2h at 37°C with 5%skimmed milk in PBST and then incubated for 1h at 37°C with 100?L serum samples in 5%skimmed milk in PBST.All samples were analyzed in triplicate.After5washes with PBST,the plates were fur-ther incubated with 100?L of HRP-conjugated protein A in PBST at 37°C.Then,the plates were washed again,and the colorimetric reaction was developed using 100?L of chromogenic substrates (0.4mol/L tetramethylbenzidine and 1mmol/L H 2O 2in 100mmol/L acetate buffer,pH 5.6)at 37°C.Color development was stopped with 2mol/L H 2SO 4,and the optical density at 450nm (OD 450)was recorded using an ELISA plate reader (BioTek,USA).Optimization of tSc iELISA working conditionsBased on the procedure described above,the optimal antigen concentration and sera dilutions were deter-mined through standard checkerboard titration proce-dures with triplicates.Brie?y,the tSc protein was coated on 96-well microtiter plates in serial 2-fold dilutions from 8?g to 250ng/well.Correspondingly,standard swine PEDV-positive serum and PEDV-negative control serum were also diluted in serial 2-fold dilutions from 1:25to 1:800for optimization.After the antigen and anti-serum dilutions were analyzed,HRP-labeled protein A was added to the plate with dilutions from 1:5000to1:20000to determine the optimal conjugate dilution.The conditions that gave the highest OD 450ratio between the positive and negative serum (P/N value)and an OD 450value for positive serum close to 1.0(with the OD 450value812Can.J.Microbiol.Vol.61,2015C a n . J . M i c r o b i o l .D o w n l o a d e d f r o m w w w .n r c r e s e a r c h p r e s s .c o m b y F o s h a n u n i v e r s i t y o n 11/03/15F o r p e r s o n a l u s e o n l y .of negative serum ≤0.2)were scored as optimal working conditions.After the conditions mentioned above were deter-mined,the coating conditions were optimized from at 37°C for 2h to 4°C for overnight.Then,the blocking buffers with 1%BSA,1%gelatin,5%and10%skimmed milk were used and blocked for 1,2,and 3h.The dilu-tions and incubation time of serum sample and HRP-conjugate staphylococal protein A were optimized with 15,30,45,60,90,or120min.The reactions were stopped and optimized by assessing 5,10,15,and 20min.Validation of tSc iELISATo establish a negative–positive cutoff value for this assay,80negative serum samples collected from healthy pig farms without clinical signs of PED (con?rmed by WB)were tested in duplicate using the tSc iELISA.The cutoff value of the ELISA was calculated from the mean OD values of the 80sera negative samples plus 2or 3standard deviations (SDs).This calculation gives 95%or 99%con? dence,respectively,that all negative values will fall within the de?ned range.To determine the speci?city of the tSc iELISA,positive sera against porcine circovirus type 2(PCV2),classical swine fever virus (CSFV),pseudorabies virus (PRV),foot-and-mouth disease virus (FMDV),porcine reproductive and respiratory syndrome virus (PRRSV),and transmissi-ble gastroenteritis virus (TGEV)were tested using the tSc iELISA protocol.Each sample was tested in triplicate,and the mean OD values were calculated.At the same time,6positive and another 30negative sera con?rmed by WB and IFA were detected by tSc iELISA to determine the sensitivity of this assay.Reproducibility experimentsEvaluation of assay reproducibility within and between runs was performed as previously proposed (Jacobson 1998).Six ?eld serum samples (5WB-positive samples and 1WB-negative sample)were selected for reproduc-ibility experiments.For intra-assay reproducibility,3replicates of each serum sample were assigned to the same plate.For inter-assay (between-run)reproducibil-ity,3replicates of each sample were run on different plates.The mean OD values,standard deviation (SD),and coef?cient of variation (CV)were calculated.Neutralization testVero-E6cells (1×105cells/well)were seeded in a 96-well cell culture plate,and plates were incubated overnight at 37°C with 5%CO 2until a con?uent monolayer formed.Seven serum samples with different OD values (1.531,1.294,0.980,0.784,0.661,0.378,and 0.078)were heated at 56°C for 30min and used for testing.Sera were diluted 2-fold and incubated with 200TCID 50of strain DR13at 37°C for 1h.After incubation,100?L of the serum+virus mixture was transferred from each well of the incuba-tion plate to a 96-well cell culture plate containing Vero-E6cells.After 1h of incubation at 37°C,the serum+virus mixtures were replaced by complete Dul-becco’s Modi?ed Eagle Medium with 10%fetal calf se-rum.The plates were incubated at 37°C with 5%CO 2for 5days,and the numbers of wells with cells with cyto-pathic effect were counted under an inverted micro-scope and neutralization titers were evaluated.StatisticsPASW Statistics for Windows,Version 18(SPSS Inc.,Chicago,Illinois,USA),was used to analyze standard de-viation,CV,and correlation analysis.ResultsExpression,puri?cation,and identi?cation of tSc proteinSDS–PAGE and Coomassie brilliant blue staining dem-onstrated the successful expression of the tSc protein in E.coli (Fig.1a ).Compared with the expression of induced E.coli /pET-28a and E.coli culture,tSc with a molecular mass of approximately 28kDa was observed in induced E.coli /pET-28a-Sc (Fig.1a ).The tSc protein was expressed in large amounts and puri?ed using nickel af?nity chro-matography.SDS–PAGE indicated that the protein was of high purity (Fig.1a ).In addition,WB showed that the tSc protein could be detected with an anti-His antibody and PEDV-positive sera (Fig.1b ). Fig.1.Identi?cation of the truncated recombinant protein S (tSc)protein by SDS–PAGE (a )and western blot (b )by His tag monoclonal antibody and porcine epidemic diarrhea virus (PEDV)polyclonal antibody.(a )Lanes:M,unstained protein molecular mass marker;1,pET-28a-Sc induced with IPTG;2,pET-28a-Sc not induced with IPTG;3,supernatant of cell lysates;4,precipitation of cell lysates;5,puri?ed Sc protein.(b )Cell lysates were incubated with His tag monoclonal antibody (lanes 1and 2)and PEDV polyclonal antibody (lanes 3and 4).Lanes:1and 3,pET-28a-Sc induced with IPTG;2and 4,pET-28a-Sc not induced withIPTG.Li et al.813C a n . J . M i c r o b i o l .D o w n l o a d e d f r o m w w w .n r c r e s e a r c h p r e s s .c o m b y F o s h a n u n i v e r s i t y o n 11/03/15F o r p e r s o n a l u s e o n l y .Optimization of iELISA working conditionsUsing a checkerboard ELISA,the optimal antigen concentration and serum sample dilution were set at 1?g/well and 1:100,respectively,based on the following standards:the OD 450value of positive serum was close to 1.0,the OD 450ratio between positive and negative serum (P/N value)was highest,and the background was /doc/597e2535d5bbfd0a785673aa.html ing the same standards,the optimal blocking condi-tion was at 37°C for 2h,and the optimal dilution of the conjugate was de?ned as 1:20000.After the above-mentioned conditions were determined,it was found that 5%skimmed milk in PBST was the optimal block-ing buffer,and the best blocking time was 2h at 37°C.For the optimal exposure time of serum samples and conjugate,it was determined that incubations of 60and 45min for the serum samples and the conjugate,respectively,were suf?cient and time-saving in the tSc ELISA,as the P/N value was relatively stable and high.Finally,the optimal stopping time was determined to be 15min (Fig.2).Cutoff value of iELISAThe OD values of 80negative serum samples varied from a minimum of 0.052to a maximum of 0.201.Thus,samples with OD values of ≤0.223were considered neg-ative,and those with values of≥0.259were considered positive (Supplemental Table 11).After the cutoff was determined,the speci?city of the tSc iELISA was evaluated by testing the reactivity of antibodies against other porcine viruses.The OD values of standard positive sera againstPCV2,CSFV,PRV,FMDV,TGEV,and PRRSV are shown in Table 1.The OD values of all anti-sera were smaller than the cutoff value.These data revealed that there is no cross-reactivity between the PEDV tSc antigen and antibodies against other por-cine viruses,proving that the tSc antigen is speci?c for antibodies against PEDV.Diagnostic sensitivity tests showed that 6positive sera and another 30negative sera identi?ed by WB and IFA were still positive and negative in ELISA results.Repeatability of the tSc iELISAThe repeatability of tSc iELISA was determined by comparing the OD value of each ?eld serum sample.As shown in Table 2,the intra-assay CV of 5positive serum samples ranged from 0.019to0.094,whereas the inter-assay CV of these positive serum samples ranged be-tween 0.038and 0.088.These data indicate that the tSc iELISA is repeatable and yields low and acceptable varia-tion (DeSilva et al.2003).Neutralization testsThe results showed that 6positive sera had different levels of virus neutralization activity.Interestingly,OD values are positively correlated with virus neutralization activity (Fig.3).More positive sera are needed to further con?rm the relationship between OD values and virus neutralization titers.DiscussionIn China,PEDV was ?rst con?rmed in 1984,and since 2010,it has caused enteric disease with a devastat-ing impact on swine industry.This disease is character-ized by high morbidity and mortality in preweaning piglets,causing serious economic losses to the swine industry in China (Gao et al.2013;Li et al.2014).On the basis of the gene sequence of PEDV,many methods had been developed to detect the infection of PEDV,such as RT-PCR (Ishikawa et al.1997),duplex RT-PCR (Kim et al.2001),immunohistochemistry,in situ hy-bridization (Kim and Chae 2002),and RT-LAMP (Ren and Li 2011).Recombinant N protein had been used to establish an ELISA method to detect antibodies against PEDV (Hou et al.2007).To date,the recombi-nant S protein as a coating antigen for the ELISA method has not been reported.1Supplementary data are available with the article through the journal Web site at /doc/597e2535d5bbfd0a785673aa.html /doi/suppl/10.1139/cjm-2015-0213.Table 1.Speci?city of the truncated S pro-tein indirect ELISA to antibodies against some swine viruses.Virus antisera X ¯±SD PEDV1.037±0.012PRV 0.054±0.030PCV20.046±0.016FMDV 0.066±0.028PRRSV 0.055±0.035CSFV 0.047±0.023TGEV0.086±0.013Negative serum0.063±0.008Note:Data are the mean ±standard deviation of 3replicate measurements.PEDV,porcine epi-demic diarrhea virus;PRV,pseudorabies virus;PCV2,positive sera against porcine circovirus type2;FMDV,foot-and-mouth disease virus;PRRSV,porcine reproductive and respiratory syndrome vi-rus;CSFV,classical swine fever virus;TGEV,trans-missible gastroenteritis virus.Table 2.Intra-assay and inter-assay repeatability of the in-direct ELISA.Serum sample Intra-assay variability Inter-assay variability X¯±SD CV X¯±SD CV 1 1.095±0.0210.019 1.087±0.0510.0472 1.066±0.0770.0720.987±0.0560.0573 1.076±0.0750.070 1.100±0.0970.08840.955±0.0560.0590.912±0.0350.03850.730±0.0640.0880.690±0.0660.096Negative0.096±0.0090.0940.100±0.0090.090Note:Data are the mean ±standard deviation of 3replications.Li et al.815C a n . J . M i c r o b i o l .D o w n l o a d e d f r o m w w w .n r c r e s e a r c h p r e s s .c o m b y F o s h a n u n i v e r s i t y o n 11/03/15F o r p e r s o n a l u s e o n l y .Recent studies reported that the N-terminal region (25–225amino acids)of PEDV is associated with the binding of the virus to the receptor aminopeptidase N (Lee 2011).So,we postulated that antibody against this protein fragment may be associated with virus neu-tralization.In this study,the tSc protein (25–225ami-no acids)was expressed and an iELISA method was constructed.Cross-reactivity assays revealed that the tSc iELISA was PEDV-speci?c,and repeatability tests demonstrated that the assay is repeatable.Thus,the tSc iELISA is simpler to produce and perform,time-saving,and may be more suitable for large-scale surveys of PEDV /doc/597e2535d5bbfd0a785673aa.html bining virus neutral-ization results with 7different OD values sera,we ?nd that the OD values obtained by iELISA established by tSc are positively correlated with virus neutralization.The preliminary results showed that the sensitivity of this assay is 100%;however,more positive sera samples should be used to further con?rm the sensitivity.ConclusionThe tSc iELISA established in the present study was repeatable and speci?c for PEDV antibody detection,simple and economical to produce and perform,and time-saving.The present report may facilitate the devel-opment of a reliable tool for the large-scale detection of PEDV neutralization antibodies and assess the effective-ness of vaccines.AcknowledgementsWe thank Jiang Ping for helpful comments.The au-thors declare no potential con?icts of interest with re-spect to the research,authorship,and (or)publication of this article.This study was supported by public welfare grants from the Ministry of Agriculture,the People’s Re-public of China (201203039),and Priority Academic Pro-gram Development (PAPD)of Jiangsu Higher Education Institutions. ReferencesChang,S.H.,Bae,J.L.,Kang,T.J.,Kim,J.,Chung,G.H.,Lim,C.W.,et al.2002.Identi?cation of the epitope region capable of inducing neutralizing antibodies against the porcine epi-demic diarrheavirus.Mol.Cells,14(2):295–299.PMID:12442904.DeSilva,B.,Smith,W.,Weiner,R.,Kelley,M.,Smolec,J.,Lee,B.,et al.2003.Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacoki-netic assessments of macromolecules.Pharm.Res.20(11):1885–1900.doi:10.1023/B:PHAM.0000003390.51761.3d .PMID:14661937.Gao,Y.,Kou,Q.,Ge,X.,Zhou,L.,Guo,X.,and Yang,H.2013.Phylogenetic analysis of porcine epidemic diarrhea virus ?eld strains prevailing recently in China.Arch.Virol.158(3):711–715.doi:10.1007/s00705-012-1541-2.PMID:23151819.Hou,X.L.,Yu,L.Y.,and Liu,J.2007.Development and evaluation of enzyme-linked immunosorbent assay based on recombi-nant nucleocapsid protein for detection of porcine epidemic diarrhea (PEDV)antibodies.Vet.Microbiol.123(1–3):86–92.doi:10.1016/j.vetmic.2007.02.014.PMID:17368968.Ishikawa,K.,Sekiguchi,H.,Ogino,T.,and Suzuki,S.1997.Direct and rapid detection of porcine epidemic diarrhea virus by RT-PCR.J.Virol.Methods,69(1–2):191–195.doi:10.1016/S0166-0934(97)00157-2.PMID:9504764.Jacobson,R.H.1998.Validation of serological assays for diagno-sis of infectious diseases.Rev.Sci.Tech.17(2):469–526.PMID:9713892.Kang,T.J.,Kim,Y.S.,Jang,Y.S.,and Yang,M.S.2005.Expression of the synthetic neutralizing epitope gene of porcine epi-demic diarrhea virus in tobacco plants without nicotine.Vac-cine,23(17–18):2294–2297.doi:10.1016/j.vaccine.2005.01.027.PMID:15755614.Kim,O.,and Chae,/doc/597e2535d5bbfd0a785673aa.html parison of reverse transcription polymerase chain reaction,immunohistochemistry,and in situ hybridization for the detection of porcine epidemic di-arrhea virus in pigs.Can.J.Vet.Res.66(2):112–116.PMID:11989732.Kim,S.Y.,Song,D.S.,and Park,B.K.2001.Differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex RT-PCR.J.Vet.Diagn.Invest.13(6):516–520.doi:10.1177/104063870101300611.PMID:11724144.Kweon,C.H.,Kwon,B.J.,Lee,J.G.,Kwon,G.O.,and Kang,Y.B.1999.Derivation of attenuated porcine epidemic diarrheaFig.3.Virus neutralization titers and optical density (OD)values of truncated recombinant protein S indirect enzyme-linked immunosorbent assay for 7sera.816Can.J.Microbiol.Vol.61,2015C a n . J . M i c r o b i o l .D o w n l o a d e d f r o m w w w .n r c r e s e a r c h p r e s s .c o m b y F o s h a n u n i v e r s i t y o n 11/03/15F o r p e r s o n a l u s e o n l y .virus (PEDV)as vaccine candidate.Vaccine,17(20–21):2546–2553.doi:10.1016/S0264-410X(99)00059-6.PMID:10418901.Lee,D.K.2011.The N-terminal region of the porcine epidemic diarrhea virus spike protein is important for the receptor binding.Korean J.Microbiol.Biotechnol.39(2):140–145.Lee,D.K.,Park,C.K.,Kim,S.H.,and Lee,C.2010.Heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in Korea.Virus Res.149(2):175–182.doi:10.1016/j.virusres.2010.01.015.PMID:20132850.Li,R.,Qiao,S.,Yang,Y.,Su,Y.,Zhao,P.,Zhou,E.,and Zhang,G.2014.Phylogenetic analysis of porcine epidemic diarrhea vi-rus (PEDV)?eld strains in central China based on the ORF3gene and the main neutralization epitopes.Arch.Virol.159:1057–1065.doi:10.1007/s00705-013-1929-7.PMID:24292967.Meng,F.,Ren,Y.,Suo,S.,Sun,X.,Li,X.,Li,P.,et al.2013.Evalu-ation on the ef?cacy and immunogenicity of recombinant DNA plasmids expressing spike genes from porcine trans-missible gastroenteritis virus and porcine epidemic diar-rhea virus.PLoSOne,8(3):e57468.doi:10.1371/journal.pone.0057468.PMID:23526943.Oh,J.S.,Song,D.S.,Yang,J.S.,Song,J.Y.,Moon,H.J.,Kim,T.Y.,and Park,/doc/597e2535d5bbfd0a785673aa.html parison of an enzyme-linked immu-nosorbent assay with serum neutralization test for serodiag-nosis of porcine epidemic diarrhea virus infection.J.Vet.Sci.6(4):349–352.PMID:16294000.Pensaert,M.,and Debouck,P.1978.A new coronavirus-like par-ticle associated with diarrhea in swine.Arch.Virol.58:243–247.doi:10.1007/BF01317606.PMID:83132.Ren,X.,and Li,P.2011.Development of reverse transcription loop-mediated isothermal ampli?cation for rapid detection of porcine epidemic diarrhea virus.Virus Genes,42(2):229–235.doi:10.1007/s11262-011-0570-3.PMID:21286798. Ren,X.,Suo,S.,and Jang,Y.S.2011.Development of a porcine epidemic diarrhea virus M protein-based ELISA for virus de-tection.Biotechnol.Lett.33(2):215–220.doi:10.1007/s10529-010-0420-8.PMID:20882317.Song,D.,and Park,B.2012.Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology,diagno-sis,and vaccines.Virus Genes,44(2):167–175.doi:10.1007/s11262-012-0713-1.PMID:22270324.Song,D.S.,Oh,J.S.,Kang,B.K.,Yang,J.S.,Moon,H.J.,Yoo,H.S.,et al.2007.Oral ef?cacy of Vero cell attenuated porcine epi-demic diarrhea virus DR13strain.Res.Vet.Sci.82(1):134–140.doi:10.1016/j.rvsc.2006.03.007.PMID:16730762.Sozzi,E.,Luppi,A.,Lelli,D.,Martin,A.M.,Canelli,E.,Brocchi,E.,et /doc/597e2535d5bbfd0a785673aa.html parison of enzyme-linked immunosorbent assay and RT-PCR for the detection of porcine epidemic diar-rhoea virus.Res.Vet.Sci.88(1):166–168.doi:10.1016/j.rvsc.2009.05.009.PMID:19501378.Sun,D.B.,Feng,L.,Shi,H.Y.,Chen,J.F.,Liu,S.W.,Chen,H.Y.,and Wang,Y.F.2007.Spike protein region (aa 636789)of porcine epidemic diarrhea virus is essential for induction of neutral-izing antibodies.Acta Virol.51(3):149–156.PMID:18076304.Sun,D.,Feng,L.,Shi,H.,Chen,J.,Cui,X.,Chen,H.,et al.2008.Identi?cation of 2novel B cell epitopes on porcine epidemic diarrhea virus spike protein.Vet.Microbiol.131(1–2):73–81.doi:10.1016/j.vetmic.2008.02.022.PMID:18400422.Li et al.817C a n . J . M i c r o b i o l .D o w n l o a d e d f r o m w w w .n r c r e s e a r c h p r e s s .c o m b y F o s h a n u n i v e r s i t y o n 11/03/15F o r p e r s o n a l u s e o n l y .。

猪流行性腹泻的诊断及治疗猪流行性腹泻（PED）是由猪流感冠状病毒（PEDV）引起的一种急性肠道传染病。

该病的主要症状是腹泻和脱水，严重者可导致死亡。

PEDV是一种无囊膜的RNA病毒，具有高的传染性和致死率，已经造成了全球猪业的巨大损失。

本文将介绍PED的诊断和治疗方法。

诊断1.病史和临床症状PEDV感染后猪只会出现腹泻、脱水、食欲不振等症状，症状通常在7-10天内表现出来。

如果一组猪只同时出现上述症状，特别是年龄较小的猪只，应该考虑PEDV感染。

2.实验室检测PEDV的检测主要包括病毒分离、PCR检测和血清学检测。

病毒分离通常使用病毒原代细胞培养，通过检测细胞培养液的特异性病毒抗原来确认PEDV感染。

PCR检测是检测PEDV 基因组的最常见方法。

在PCR检测中，可以从粪便或小肠组织中提取PEDV的RNA并进行扩增，证实PEDV的存在。

血清学检测主要是检测猪群中的PEDV抗体水平。

治疗1.预防有效的预防措施是阻止PED传播的最好方法。

PEDV可以通过直接和间接接触传播。

因此，应实施有效的禁止猪只的移动并且消毒各种器具和设备的防止PEDV的传播。

2.支持性治疗PEDV感染的猪只需要得到适当的液体和营养支持,以避免脱水和营养不良的发生。

猪只要遵循适当的饲料和水的摄入，情况通常会在一周到十天内改善。

3.药物治疗目前没有特定的药物来治疗PEDV感染。

对于PEDV的感染，可使用抗菌素抗生素治疗相关的二次感染以及使用补充酶和维生素以促进肠道健康。

总结PEDV感染的主要症状是腹泻和脱水，预防是控制PEDV传播的最好方法。

对PEDV的治疗主要是支持性治疗和适当的液体和营养支持，同时也需要使用抗生素治疗相关的二次感染。

虽然目前没有特定的药物来治疗PEDV，但是积极的治疗和预防措施可以有效地降低PED的影响。

·综述·Chinese Journal of Animal Infectious Diseases中国动物传染病学报摘 要：猪δ冠状病毒（PDCoV ）是一种新发现的猪肠道冠状病毒，可引起新生仔猪的腹泻、呕吐、脱水、死亡，而且具有跨种感染潜能，给养猪业和公共卫生构成了巨大威胁。

冠状病毒的辅助蛋白（accessory protein ）是一类功能特殊的蛋白，不同属甚至同属不同种的冠状病毒编码的辅助蛋白数量、同源性均存在较大差异，具有属或种特异性。

虽然辅助蛋白是冠状病毒增殖非必需的，但在调控宿主天然免疫、病毒复制与致病性中发挥重要作用。

目前，已证实PDCoV 至少编码3个辅助蛋白，而且近年来在PDCoV 辅助蛋白的功能研究方面取得了显著进展。

本文简要介绍了PDCoV 辅助蛋白的鉴定及其在免疫调控和致病性中的作用，并对今后的研究进行了展望。

关键词：猪δ冠状病毒；辅助蛋白；感染；免疫抑制；致病性中图分类号： S852.651文献标志码： A文章编号：1674-6422（2023）01-0213-09Research Progress on A ccessory Proteins of Porcine DeltacoronavirusZHANG Huichang 1,2, FANG Puxian 1,2, FANG Liurong 1,2, XIAO Shaobo 1,2(1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070,China; 2. The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China)收稿日期：2020-09-03基金项目：国家自然科学基金重点项目（31730095）作者简介：张蕙畅，女，硕士研究生，预防兽医学专业通信作者：肖少波，E-mail：*************猪δ冠状病毒辅助蛋白研究进展张蕙畅1,2，方谱县1,2，方六荣1,2，肖少波1,2（1.华中农业大学动物医学院 农业微生物学国家重点实验室，武汉430070；2.湖北省生猪健康养殖协同创新中心，武汉430070）2023,31(1):213-221Abstract: Porcine deltacoronavirus (PDCoV) is a newly discovered Porcine intestinal coronavirus, which causes diarrhea, vomit, dehydration and death in newborn piglets. Importantly, PDCoV has the potential of cross-species infection, posing a huge threat to public health. Accessory proteins encoded by Coronavirus are a unique set of proteins with special functions. These accessory proteins encoded by different genera, or even by the same genus and different species of Coronavirus vary in number and homology, making them genus or species specifi c. Though accessory proteins are dispensable for replication of Coronavirus in vitro , they play important roles in regulating innate immunity, viral proliferation and pathogenicity. PDCoV has been confi rmed to encode three accessory proteins, and remarkable progress has been achieved on their functions during the past few years. In this review, we briefl y introduced the research progress on PDCoV accessory proteins, including their identifi cation, functions on regulating antiviral innate immune and pathogenicity. We also discussed the future research on PDCoV accessory proteins.Key words: Porcine deltacoronavirus; accessory protein; infection; immunosuppression; pathogenicity· 214 ·中国动物传染病学报2023年2月猪δ冠状病毒（Porcine deltacoronavirus，PDCoV）是一种有囊膜、不分节段的单股正链RN A 病毒，属于套式病毒目冠状病毒科δ冠状病毒属成员，病毒粒子呈球形，直径约为120~180 nm，表面分布有放射状的纤突[1]。

pedv灭活方法
PEDV的灭活方法主要包括以下几个方面：
1.病原学：PEDV是冠状病毒科冠状病毒属的一种病毒，其对外界环境的抵抗力较弱。

例如，病毒在低温下可以长期存活，但在4℃～50℃的温度范围内，其活性相对稳定。

在4℃条件下，pH3～10的细胞培养基中孵育6小时后，PEDV表现出低至中等的残留感染性，而在37℃条件下6小时，其仅可在pH5～8.5的范围内保持其感染性。

当溶液pH超出4.0～9.0之间时，PEDV可以被完全灭活。

紫外线能使病毒迅速灭活。

另外，高温、福尔马林、氢氧化钠、含氯或碘的消毒剂、季胺盐类化合物等都可以灭活PEDV。

2.流行病学：PEDV主要感染猪，各年龄段的猪都可能被感染。

病毒可以通过呼吸道感染，从而使传染病得到更广范围的更快传播。

292015年32卷第8期 SWINE INDUSTRY SCIENCE 猪业科学海外文摘OVERSEAS ABSTRACTS集约化饲养对母猪福利的影响来自澳大利亚和丹麦的研究人员发现，母猪的攻击行为基本都是来自于对食物的竞争，在遗传育种种群中也是同样的情况。

文章指出，我们对这个项目的认知在很多方面是不完全的。

来自澳大利亚墨尔本大学的P.H. Hemsworth 和来自阿德雷德大学、丹麦的哥本哈根大学 、丹麦食品和农业委员会的合作作者共同阐述了集约化饲养对动物福利的影响因素。

研究者们发表在《Journal of Animal Science》杂志上的文章中指出，平均饲养面积显著影响着动物福利。

另外，整体饲养面积的大小及质量是很重要的，可以设置视觉及物理屏障对母猪进行空间隔离，有报道指出1.4 m 2的饲养面积是不够的，后续将详细研究1.8～2.4 m 2的饲养面积对动物福利的影响。

母猪混群后立即就会出现侵略和攻击行为，因此，设计更好的混养圈舍以减少攻击、伤害和饲养压力的发生，促进饲养圈舍中母猪社会等级的快速形成。

由于饥饿会导致对食物和空间的竞争，所以应该采取提高饲养水平、膳食纤维和改变饲料等策略来减少母猪两餐之间的饥饿感。

然而，研究人员发现，饲喂系统，比如电子饲喂系统，通过提供饲养保护同样会影响攻击行为和饲养压力。

由于研究直接比较饲养面积、饲槽及电子饲喂系统等尚未进行，这个研究项目的结论难以定论。

熟悉的母猪之间的攻击更少，所以混群前妊娠母猪饲养在一起可以减少攻击。

尽管在其他种群中，一些研究表明早饲可能会影响其后续的社会技能，但很少有关于早期“社会化”对成年母猪攻击行为影响的研究。

遗传选择具有减少攻击的潜力，因此，后续对遗传选择对抗攻击行为及其广泛影响的研究是必要的。

Hemsworth 及其合作者强调说，目前大多数的研究都针对妊娠母猪的混养，但是关于断奶母猪混养的研究很少，对这方面的研究状况有待改变。