大黄体外抗猪流行性腹泻病毒的研究

为研究大黄对猪流行性腹泻病毒(PEDV)感染所引起的细胞病变的抑制作用及其机理,本试验采用形态学观察法测定大黄溶液在Vero细胞上的最大安全浓度,建立体外病毒感染Vero细胞模型并利用该模型进行了大黄对病毒抑制作用的研究。结果显示,本试验成功建立了体外病毒感染模型和大黄的抗病毒试验方法;大黄在Vero细胞上的最大安全浓度为3.28 mg/mL;形态学观察法和MTT法均显示大黄不仅能阻断病毒吸附Vero细胞,抑制病毒的合成复制,还可直接灭活病毒。本研究结果表明,大黄具有抗PEDV的作用,为临床上预防和治疗猪流行性腹泻(PED)提供理论基础。     

Isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate.

Porcine epidemic diarrhea virus (PEDV) was isolated in Vero cell cultures from the small intestine of a piglet experimentally infected with porcine coronavirus 83P-5, that had been isolated during outbreaks of porcine acute diarrhea and passaged in piglets. The isolation of the PEDV was successful only in Vero cells maintained in the maintenance medium (MM) containing trypsin. Infected Vero cell cultures exhibited CPE characterized by cell-fusion and syncytial formation, as well as cytoplasmic fluorescence when examined by the indirect immunofluorescent test using rabbit anti-83P-5 virus serum. The isolate was adapted to serial propagation in Vero cell cultures by adding trypsin to MM. Vero cell-adapted PEDV was successfully propagated in the MA104, CPK and ESK cell lines in the presence of trypsin in MM. Vero cell-adapted PEDV had morphologic and physicochemical characteristics similar to those of other members of the coronaviridae. The isolate differed serologically from porcine transmissible gastroenteritis (TGE) and porcine hemagglutinating encephalomyelitis viruses, and no antigenic relationship between the isolate and TGE virus could be detected by the indirect immunofluorescent test. Attempts to isolate PEDV in 6 types of primary fetal pig cell cultures and 6 of 10 established cell lines resulted in the failure, probably because these cells were damaged by the action of trypsin.     

Oral efficacy of Vero cell attenuated porcine epidemic diarrhea virus DR13 strain

A Vero cell attenuated porcine epidemic diarrhea virus (PEDV) strain, DR13, was distinguished from wild-type PEDV using restriction enzyme fragment length polymorphism (RFLP). Cell attenuated DR13 was orally or intramuscularly (IM) administered to late-term pregnant sows, and mortality resulting from the highly virulent PEDV challenge was investigated in passively immunized suckling piglets of the two different groups. The mortality rate of the oral group (13%) was lower than that of the IM group (60%). In particular, the concentration of IgA against PEDV was higher in piglets of sows in the oral group, compared to the IM group. The attenuated DR13 virus remained safe, even after three backpassages in piglets. The findings of this study support the theory that the Vero cell attenuated DR13 virus may be applied as an oral vaccine for inducing specific immunity in late-term pregnant sows with a high margin of protection against PEDV infection.     

猪流行性腹泻病毒CH-HuN1301株N基因重组表达载体的构建及稳定表达细胞系的建立

本研究旨在建立猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV） CH-HuN1301株N蛋白稳定表达细胞系。采用RT-PCR扩增PEDV新流行毒株CH-HuN1301株N基因,分别克隆至原核表达载体pET28a和真核表达载体pEGFP-C1构建重组表达载体,所测定的序列采用Mega 5.0软件进行进化分析。将原核表达的N蛋白免疫家兔制备多克隆抗体,重组真核表达载体pEGFP-PEDV-N转染HEK293细胞,经用G418筛选获得稳定的表达细胞系。荧光倒置显微镜观察细胞荧光,免疫过氧化物酶单层细胞染色法（IPMA）检测N蛋白的表达。结果表明,PEDV HuN1301-14毒株N基因大小为1 323 bp,原核表达重组N蛋白分子质量约60 ku,其多克隆抗体与PEDV呈特异性反应,荧光倒置显微镜观察和IPMA检测结果显示N蛋白在HEK293细胞中稳定表达。该细胞系的建立为进一步研究PEDV的诊断方法提供了基础材料。     

猪流行性腹泻病毒SD201604株的分离鉴定及致病性研究

本研究旨在获得猪流行性腹泻病毒（PEDV）分离株,并对其致病性进行研究。应用Vero细胞从山东某猪场腹泻病料中进行病毒分离,通过细胞病变、免疫荧光试验、电镜观察和RT-RCR进行鉴定,并对分离株的S基因序列进行分析;应用10^3.5 TCID₅₀/mL分离株口服接种3日龄仔猪并观察临床症状和病理变化;用不同滴度的分离株分别口服接种3日龄仔猪（3 mL/只）,统计各组仔猪的死亡率,确定最小致死量。结果显示,成功分离到1株PEDV,命名为PEDV SD201604株。该分离毒株能在Vero细胞上增殖,产生细胞病变,传代至F6代时病毒滴度可达10^3.5 TCID₅₀/mL,免疫荧光试验和RT-RCR检测均为PEDV阳性。电镜观察可见直径大小约为100 nm的病毒粒子,有明显的囊膜和纤突,具有PEDV病毒粒子典型的形态特征,确定分离株为PEDV。S基因序列分析显示,分离株为国内流行的PEDV变异株。动物回归试验结果显示,口服感染该分离株的5头3日龄仔猪全部出现典型的PED临床症状和病理变化,其中3头死亡。最小致死量试验结果表明,口服感染3 mL病毒含量为10^4.5 TCID₅₀/mL的分离株可使仔猪全部死亡。本试验结果可为PEDV的分离鉴定及生物学研究提供参考与借鉴。     

Construction of Recombinant Expression Vector and Establishment of Stable Expression Cell Line of N Gene of Porcine Epidemic Diarrhea Virus Strain CH-HuN1301

This study was aimed to construct stable cell line expressing N protein coded by porcine epidemic diarrhea virus (PEDV) strain CH-HuN1301. N gene of this virus strain was amplified by RT-PCR, which was then cloned into prokaryotic expression vector pET28a and eukaryotic expression vector pEGFP-C1,respectively. Recombinant plasmids were sequenced and analyzed by software Mega 5.0. To prepare polyclonal antibody of N protein, the prokaryotic expression recombinant N protein was used as antigen to immunize rabbits. HEK293 transfected with recombinant eukaryotic expression plasmids pEGFP-PEDV-N was selected by G418 to produce a stable cell line which then was identified by immunoperoxidase monolayer assay (IPMA) and fluorescence observation. Results showed that, the N gene of PEDV strain CH-HuN1301 was 1 323 bp, molecular weight of recombinant prokaryotic expressing N protein was about 60 ku, prepared polyclonal antibody against N protein showed wonderful reactivity with PEDV propagated in Vero cells, recombinant eukaryotic expressing N protein was positively detected in the established stable cell line by IPMA and fluorescence observation. This stable cell line provided foundation for the research in PEDV diagnosis methods.     

猪流行性腹泻病毒蛋白对Vero E6核蛋白B23.1的影响

为了解猪流行性腹泻病毒(PEDV)核蛋白N对Vero E6细胞核仁蛋白B23.1的影响,将本实验室构建的DsRed-N1/B23.1重组质粒转染Vero E6细胞,转染6h后接种PEDV,分别在转染后12、18h和24h三个时间点,进行病毒感染细胞的间接免疫试验,同时设未感染对照组,而后利用激光共聚焦显微镜分析感染和转染细胞中B23.1蛋白的变化。结果表明,PEDV N蛋白与Vero E6核蛋白B23.1有共定位特征,在PEDV感染Vero E6细胞12h后,B23.1蛋白开始发生变化,随着时间的延长逐渐碎裂、分散。这个结果提示,病毒可能通过破坏核仁的结构和核仁蛋白的功能,进而为病毒的复制提供便利。     

猪流行性腹泻新毒株的分离鉴定和致病性研究

为探明2011年我国猪群仔猪腹泻的病因,采用巢式RT-PCR方法对河南和辽宁省2个规模猪场采集43份出现腹泻的粪便进行猪流行性腹泻病毒（PEDV）、猪传染性胃肠炎病毒（TGEV）和轮状病毒（PRoV）的检测,结果表明：43份腹泻样品均为PEDV,没有检测出TGEV和PRoV。对扩增的PEDV进行测序和分析显示,这些PEDV流行毒株均属于基因G2.3亚型,彼此之间的同源性高达99.2%～100%,与2011年韩国和2008年泰国流行的PEDV毒株同属于一个进化分支。将其中3份病料在Vero细胞传代后接种2日龄仔猪,结果发现接种后13～57h内仔猪全部死亡,接种仔猪呈现典型的腹泻症状和病理变化特征,提示了这些PEDV流行毒株具有较强的致病性。     

猪流行性腹泻病毒分离鉴定及其灭活疫苗免疫保护效果研究

为筛选能用于猪流行性腹泻病毒(PEDV)疫苗研发的流行毒株,收集PEDV阳性病料,以Vero细胞进行病毒分离试验,并对分离毒株进行外源病毒检测、病毒培养特性研究、仔猪毒力试验及免疫效力试验等。9份PEDV阳性病料共分离到3株病毒,分别命名为PEDV-SC12、PEDV-JS12、PEDV-JX12。其中PEDV-SC12株存在支原体污染;PEDV-JS12株与PEDV-JX12株均能被PEDV特异性阳性血清中和;PEDV-JS12株能在Vero细胞上增殖并产生细胞病变,但适应细胞45代以后病毒培养效价仍然偏低;PEDV-JX12滴度能维持在106.0 TCID50/mL以上。PEDV-JX12株毒力试验显示,该分离株能够通过人工感染复制出腹泻病例,并能从发病动物体内检测到感染的病毒。免疫攻毒保护试验显示,以PEDV-JX12株制备的灭活疫苗免疫母猪,对所产仔猪攻毒后免疫组6.25%(1/16)发病,对照组100%(8/8)发病。说明PEDV-JX12株能够适应细胞培养,免疫接种母猪能给仔猪提供有效保护,可以用于PEDV流行毒株的疫苗研发。     

猪流行性腹泻病毒JS2017株的分离鉴定及S基因序列分析

为了解江苏省某规模化猪场猪流行性腹泻病毒(PEDV)遗传变异情况,本试验采集腹泻仔猪小肠组织及肠道内容物,通过RT-PCR进行PEDV鉴定。将检测为阳性的病料组织经20μg/mL胰酶处理后,接种到Vero细胞中进行培养,将细胞培养物进行反复冻融后,收获病毒液,进行RT-PCR鉴定。采用分段重叠策略对其S基因进行全基因序列扩增,并在此基础上分析该毒株S基因的核苷酸同源性及遗传进化关系。结果显示,经RT-PCR鉴定,病料组织呈PEDV阳性,阳性病料接种Vero细胞盲传4代后,出现明显的细胞病变,表现为细胞合胞体病变,空泡化,最终裂解脱落。收获的病毒液经RT-PCR鉴定后,确认分离到1株PEDV,命名为JS2017株。核苷酸序列分析表明,JS2017株S基因序列与美国变异株OH1414、加拿大变异株ON-007、中国变异株PEDV-LYG和CH/PDS/2015位于同一进化分支,其核苷酸同源性高达99.0%以上;其中与中国变异株YC2014亲缘关系最近,核苷酸同源性为100.0%;与经典株CV777、DR13株同源性较低,亲缘关系较远。结果表明,本研究分离获得的JS2017毒株是一株PEDV地方流行变异毒株,与2014年中国江苏分离株(YC2014株)亲缘关系最密切,与当前猪流行性腹泻疫苗株亲缘关系较远。     

猪流行性腹泻病毒JX16株的分离鉴定及体外传代对其毒力的影响

2010年中国新出现了以新生仔猪高发病率、高死亡率为主要特征的变异株。为了明确山东地区猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）变异株特征，本试验利用Vero细胞对莒县某猪场的腹泻病料进行了病毒分离和体外传代培养。通过光学显微镜和细胞超薄切片观察、RT-PCR检测、间接免疫荧光试验、S基因序列进化分析、滴度测定和致病力试验进行病毒序列及特征分析，并探究接毒后细胞脱落时间的变化。光学显微镜观察发现，该病毒感染Vero细胞后能引起典型的合胞体病变；细胞超薄切片观察发现，病毒粒子多呈致密核芯，能引起宿主细胞线粒体肿胀，溶解；RT-PCR检测和间接免疫荧光试验结果证实其为PEDV，最终通过测序证实该毒株为变异毒株。体外传代培养至100代过程中，随着病毒代次升高，其对细胞适应性不断增强。接毒后细胞脱落时间由48 h逐渐缩短至17 h，病毒滴度由6代时的10^5.3TCID₅₀/mL逐渐升高至100代的10^7.5TCID₅₀/mL。致病力试验结果显示，病毒毒力随代次升高逐渐下降，40代时对仔猪已无致病力。本试验结果为PEDV突变株致病基因的确定及疫苗的研发奠定了基础。     

一株变异型猪流行性腹泻病毒的分离鉴定及其灭活疫苗的免疫效力试验

本研究旨在分离猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）变异株,通过悬浮培养工艺制备成高效价的PEDV灭活疫苗。2017年从中国多个规模化猪场采集腹泻病死仔猪的小肠及其内容物200份,通过RT-PCR方法进行PEDV检测并测序,筛选一株PEDV变异株,将其在2 L反应器里悬浮培养的Vero细胞上进行病毒分离与传代培养,收获的病毒液鉴定后测定TCID₅₀,经甲醛灭活后加入氢氧化铝胶佐剂配制成PEDV灭活疫苗,对其物理性状、稳定性、黏度、无菌等进行检验,检验合格后免疫妊娠母猪及所产仔猪,对其安全性和免疫效力进行研究。结果显示,200份病料中有86份为PEDV阳性,将筛选的PEDV变异株病料在Vero细胞上传至第5代时出现细胞病变,传至第10代收获病毒液,经鉴定后确定为PEDV变异毒株,并命名为PEDV-GF10株。收获的病毒液浓缩后测得病毒滴度可达1×10^8.0 TCID₅₀/mL。疫苗检验合格后在母猪产前40和25 d时试验组后海穴肌内注射4 mL疫苗,空白组不免疫,结果显示试验组与空白组母猪的生产情况无明显差异,所产3日龄仔猪分别免疫不同剂量后体温无显著差异,表明该疫苗对母猪和仔猪均安全性良好。随机挑选试验组与空白组母猪所产3日龄仔猪各20头,分别口服4 mL PEDV-F10病毒培养物,空白组母猪所产仔猪在攻毒24 h后PEDV发病率为100%,抗体均为阴性;试验组母猪所产仔猪只有10%出现了轻微的腹泻症状,仔猪获得了高达90%保护率,且仔猪被动免疫后抗体能持续至35 d以上。以上结果表明,PEDV-GF10变异株通过悬浮细胞培养后病毒滴度显著提高,研制的PEDV-GF10株灭活疫苗安全有效,能够对中国的PEDV变异株达到有效防控,为国内PED防控提供了理论依据。     

Isolation and Identification of a Variant Porcine Epidemic Diarrhea Virus and the Immune Efficacy of Its Inactivated Vaccine

This study was aimed to isolate a mutant strain of porcine epidemic diarrhea virus and prepare a PEDV inactivated vaccine with high valence by suspension culture process for immunizing against PEDV effectively in China.200 small intestines and theirs contents of diarrhea piglets died of diarrhea,collected from many large-scale pig farms in China,were detected by RT-PCR and sequenced,a mutant strain of porcine epidemic diarrhea virus was selected and put on the suspension-cultured Vero cells in a 2 L reactor for virus isolation and continuous cell culture,the harvested virus suspension,which was identified and determined TCID₅₀,was inactivated by formaldehyde and mixed with aluminum hydroxide gel adjuvant to prepare PEDV inactivated vaccine.After its physical behavior,stability viscosity,sterility test were checked out,the safety and immune efficacy were studied by immunizing the pregnant pigs and theirs piglets.The results were as follows:86 samples were detected positive in 200 samples,cytopathy occurred after the mutant strain samples screened were passaged to 5th generation,the virus suspension was harvested in 10th generation and identified as a mutant strain of PEDV,named PEDV-GF10 strain.The virus titer of harvested virus suspension was measured up to 1×10^8.0 TCID₅₀/mL after concentrated.After the vaccine was checked out,the sows,40 and 25 days before delivery in experimental groups,were injected into Xuehai acupoint with 4 mL vaccine and the pigs in blank group were free of immunifications.The results showed that there were no obvious differences in the production status of the sows in experimental groups and blank group and the temperature of theirs 3-day-old healthy piglets injected different doses of vaccine,and the vaccine was safe to the sows and piglets.Forty 3-day-old piglets producted by pregnant sows in experimental groups and blank group were randomly selected and taken 4 mL 1×10^8.0TCID₅₀/mL F10 virus culture.The PEDV morbidity of piglets in blank group was 100% after injection and the antibodies were negative;10% piglets in blank group had mild diarrhea symptoms,the protection rate was up to 90%,antibody of passive immunity in piglets lasted for more than 35 days.Virus titer of mutant strain of PEDV-GF10 improved a lot by suspension cell culture,the PEDV-GF10 inactivated vaccine was safe,and could effectively prevent and control the variation strain of PEDV in China.     

猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析

本研究旨在获得可在细胞培养中稳定、有效生长增殖的猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）分离毒株,并对其全基因组序列进行测定分析。应用Vero细胞从广西腹泻仔猪肠道内容物中进行病毒分离,通过细胞病变和RT-PCR对细胞培养物进行鉴定,应用下一代测序技术对分离毒株全基因组序列进行测定。结果显示,成功分离到1株PEDV,命名为CH/GX/2015/750A。该毒株可稳定有效地在Vero细胞生长增殖,并引起典型的细胞病变;已在Vero细胞连续传代25代,病毒滴度随着传代次数的增加逐渐提高并稳定在10^7.50TCID₅₀/mL。该毒株全基因组序列长28 038 bp;与22个参考毒株的全基因序列比对显示,核苷酸同源性为96.8%~99.8%,其中与YC2014株同源性最高,为99.8%。全基因组和S基因系统进化分析显示,PEDV CH/GX/2015/750A分离毒株属于Ⅱa亚群,与YC2014、PEDV-WS等变异毒株亲缘关系密切。结果表明,本研究分离获得的CH/GX/2015/750A毒株是PEDV地方流行变异毒株。     

猪流行性腹泻病毒抗体ELISA检测方法的建立

为检测猪血清中猪流行性腹泻病毒(PEDV)抗体水平,以纯化的PEDV作为包被抗原,优化ELISA反应条件,建立了检测PEDV血清IgG抗体的诊断方法:当血清OD_(450nm)值0.31时,判定阳性;当OD_(450nm)值小于0.26时,判定阴性;当OD_(450nm)值介于两者之间判定可疑。结果表明,试验建立的ELISA抗体检测方法可用于检测猪血清中猪流行腹泻病毒抗体、监测猪流行腹泻病的流行情况和评价相关疫苗的免疫效果。     

猪流行性腹泻病毒变异株CH/GX/750A/2015在Vero细胞上转瓶培养条件的优化试验

本研究旨在确定猪流行性腹泻病毒（PEDV）变异株CH/GX/750A/2015株在Vero细胞上转瓶培养的最佳条件，为大规模生产高效PEDV变异株疫苗提供技术支撑。试验以10 L转瓶培养病毒，对接毒时细胞维持液中胰酶浓度（0、2、4、6、8和10 μg/mL）、细胞密度（40%、60%、80%、100%、200%）、接毒剂量（MOI分别为1、0.1、0.01和0.001）、吸附时间（0、30、60、90、120、240 min）、收毒时间（接毒后12、24、36、48、60、72 h）、维持培养温度（35、36、37和38 ℃）和转瓶转速（6、8、10、12、14、16 r/h）7个条件进行优化。结果显示，PEDV CH/GX/750A/2015株在10 L转瓶中的最佳培养条件为：细胞刚铺满单层时接毒，接毒量为MOI=0.01，维持液（无血清DMEM培养液）中胰酶质量浓度为6 μg/mL，不需吸附，维持培养温度为37 ℃，12 r/h 旋转培养48 h后收获病毒液可获得较高效价的病毒液，效价可稳定达到10^6.50 TCID₅₀/0.1 mL。本试验为 PEDV 变异株大量培养提供了参考，为变异株疫苗研发奠定了基础。     

流行性腹泻病毒分子生物学研究进展

猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起猪的高度接触性肠道传染病,感染PEDV的猪群主要表现为腹泻、呕吐、厌食、脱水等临床症状。PEDV可感染各个阶段的猪群,临床上PEDV主要造成7日龄以内的仔猪高发病率及高死亡率(可达100%)。PEDV的迅速传播给养猪行业造成了巨大的经济损失。本文针对PEDV的病原学、病毒编码蛋白及其生物学功能、感染细胞机制进行综述,以期为深入研究PEDV的致病机制提供参考。     

猪两种腹泻病毒混合感染的病原诊断

对我省流行的疑似猪病毒性腹泻进行了病原诊断，通过细菌培养、ＲＮＡ电泳和电镜形态观察等辅助诊断和免疫萤光特异性诊断，确诊该病病原为猪传染性胃肠炎病毒（ＴＧＥＶ）与猪流行性腹泻病毒（ＰＥＤＶ）的混合感染，混合毒代号为ＴＰＶ３。     

猪流行性腹泻病毒N蛋白与Vero E6核仁蛋白Fibrillarin的亚细胞定位分析

为鉴定猪流行性腹泻病毒(PEDV)核衣壳蛋白(N)与Vero E6细胞核仁fibrillarin蛋白的亚细胞定位的特征.本实验参照GenBank中人细胞fibrillarin基因(M59849.1)序列,设计并合成特异性引物,利用RT-PCR方法扩增了Vero E6细胞的fibrillarin基因,并将其克隆于真核表达载体pDsRed2-N1中,构建重组质粒pDsRed-Fib.将pDsRed-Fib与本实验室构建的含有PEDV N基因的重组质粒pAcGFP-N,共转染Vero E6细胞.Western blot结果表明fibrillarin及N融合蛋白在转染的Vero E6细胞中均获得表达;利用共聚焦显微镜观察显示在共转染Vero E6细胞中PEDV N蛋白与Vero E6细胞核仁中的fibrillarin发生共定位.该结果为进一步研究PEDV和核仁蛋白相互作用奠定基础.     

猪流行性腹泻病毒的分子生物学特性以及胰酶对其作用机理

猪流行性腹泻病毒(PEDV)在体外分离培养需依赖胰酶,目前胰酶对猪流行性腹泻病毒的作用机理尚未完全清晰,文中从分子生物学方面阐述胰酶对猪流行性腹泻病毒的作用。