HMCES蛋白促进猪流行性腹泻病毒在Vero细胞中的复制

【目的】鉴定能够调控猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)复制的关键宿主蛋白。【方法】利用LC-MS/MS技术结合串联质谱标签(tandem mass tag,TMT)，分析PEDV感染Vero细胞36 h后和未感染组的蛋白组学差异。鉴定筛选了114个显著差异表达蛋白，其中宿主胚胎干细胞特异性5-羟甲基胞嘧啶结合蛋白(5-hydroxymethylcytosine binding,ES cell-specific protein,HMCES)显著上调。进一步构建HMCES真核表达质粒，通过蛋白免疫印迹和实时荧光定量PCR检测过表达HMCES对PEDV复制的影响；合成针对HMCES基因的特异性si RNA，利用Western blotting和RT-q PCR检测si RNA对HMCES表达的干扰效果及HMCES被干扰后对PEDV复制的影响。【结果】过表达HMCES能显著促进PEDV在Vero细胞中复制，并且复制水平随着HMCES的剂量递增呈现剂量依赖式增加；si RNA-341下调内源性HMCES表达进而抑制PEDV复制。【结论】H...     

猪流行性腹泻病毒经其附属蛋白抑制细胞凋亡

【背景】orf3位于猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV) s基因与e基因之间,是目前发现的PEDV唯一一个附属基因,编码附属蛋白(ORF3蛋白)。我们前期研究初步发现ORF3蛋白对PEDV诱导的细胞凋亡有影响。【目的】研究ORF3蛋白在PEDV侵染复制过程中的毒力作用机制。【方法】实验用3种PEDV:rDR13att-?ORF3 (orf3基因全部敲除)、DR13-ORF3att (携带有C端截短orf3)、rDR13att-ORF3wt(携带全长orf3基因)感染Vero细胞,观察病变情况,再用活细胞成像仪、流式细胞仪、 DNA断裂的原位末端标记法[terminaldeoxynucleotidyltransferase(TDT)-mediated dUTP nick end labeling,TUNEL]等方法检测不同感染时间点的细胞凋亡情况,然后用蛋白质印迹方法分析PEDV感染宿主细胞中主要凋亡相关蛋白(如Caspase-3)的活化或裂解,最后进行转录组测序研究病毒感染细胞中差异基因的表达情况,再用荧光定量PCR验证转录组结果。【结果】rDR13att-?ORF3引起较多的细胞病变,活细胞成像仪的动态观察结果显示,3种病毒侵染的细胞凋亡水平随着时间的延长均高于正常阴性细胞,但敲除orf3的病毒感染细胞后细胞凋亡率比其他两种病毒更高;敲除orf3病毒感染细胞凋亡率显著高于其他两种病毒;病毒rDR13att-?ORF3感染细胞后TUNEL阳性细胞数比DR13-ORF3att和rDR13att-ORF3wt更多;表达ORF3蛋白的重组PEDV可以抑制Caspase-3的活化;ORF3蛋白对受感染细胞Heat shock 70 kD protein 1B (HSP70)基因转录有促进作用,荧光定量PCR结果表明rDR13att-ORF3wt感染细胞的HSP70表达量高于rDR13att-?ORF3感染细胞。【结论】PEDV通过ORF3蛋白抑制细胞凋亡,而且这种作用可能是通过抑制Caspase-3的活化或增加HSP70的产生来完成的。     

猪流行性腹泻病毒通过病毒蛋白酶激活Caspase-3诱导细胞凋亡

冠状病毒是一大类能够引起呼吸系统疾病,从而威胁人类健康的病毒．目前,对冠状病毒诱导细胞凋亡及其机制研究甚少.本研究以动物冠状病毒 猪流行性腹泻病毒(PEDV) 为模型探讨冠状病毒诱导细胞凋亡效应及其可能作用机制. 通过流式细胞术检测发现感染PEDV病毒后细胞凋亡率明显升高,且PEDV诱导细胞凋亡呈时间和剂量依赖性(P<0.05或P<0.01);进一步研究发现,冠状病毒木瓜样蛋白酶（PLP）在病毒引起凋亡过程中起重要作用.实验发现,转染PEDV-PLP质粒后,caspase-3活化体表达水平明显升高. 提示冠状病毒PLP蛋白酶通过激活caspase-3在病毒诱导细胞凋亡过程中起着关键作用. 以上结果为研究人类冠状病毒PLP蛋白功能及其通过细胞凋亡调节宿主抗病毒天然免疫机制提供重要基础.     

猪流行性腹泻病毒分子生物学特征

猪流行性腹泻(porcine epidemic diarrhea,PED)是以水泻、呕吐和脱水为特征的一种急性病毒性腹泻.猪流行性腹泻现已成为世界范围内的猪病之一.猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)是PED的致病因子,是导致类似猪传染性胃肠炎(porcine transmissible gastroenteritis,TGE)临床症状的真正病原.迄今为止已发现PEDV与TGEV[1]、PEDV与PCV混合感染猪[2].已有用蛋黄IgY 预防PED效果的报道[3],还有用弱化的PEDV疫苗对仔猪进行免疫的报道[4],但都对其作用机制未作深入探讨.弄清PEDV的分子生物学特征,针对PED进行特异性免疫,必将对PED的诊断、治疗和综合防治产生深远影响.本文仅就PEDV的分子生物学特征作一综述.     

猪流行性腹泻病毒分子生物学特征

猪流行性腹泻 (ｐｏｒｃｉｎｅｅｐｉｄｅｍｉｃｄｉａｒｒｈｅａ ,ＰＥＤ)是以水泻、呕吐和脱水为特征的一种急性病毒性腹泻。猪流行性腹泻现已成为世界范围内的猪病之一。猪流行性腹泻病毒 (Ｐｏｒｃｉｎｅｅｐｉｄｅｍｉｃｄｉａｒｒｈｅａｖｉｒｕｓ ,ＰＥＤＶ)是ＰＥＤ的致病因子 ,是导致类似猪传染性胃肠炎 (ｐｏｒｃｉｎｅｔｒａｎｓｍｉｓｓｉｂｌｅｇａｓｔｒｏｅｎｔｅｒｉｔｉｓ ,ＴＧＥ)临床症状的真正病原。迄今为止已发现ＰＥＤＶ与ＴＧＥＶ[1] 、ＰＥＤＶ与ＰＣＶ混合感染猪[2 ] 。已有用蛋黄ＩｇＹ预防ＰＥＤ效果的报道[3] …     

猪流行性腹泻病毒ORF3蛋白40–91 aa是其细胞质定位的关键结构域

ORF3蛋白是猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)基因组编码的唯一的辅助蛋白,与病毒毒力相关。为确定PEDV ORF3细胞质定位信号,文中构建了系列PEDV DR13wt ORF3蛋白全长或截短肽重组表达载体,转染Vero细胞并利用激光共聚焦显微镜分析与EGFP融合表达的全长ORF3蛋白和其系列截短肽在细胞内的分布。结果表明,全长ORF3蛋白或所有包含2个跨膜域的40–91 aa基序的ORF3截短肽均只定位于细胞质中,而不包含40–91aa基序的ORF3截短肽分布于整个细胞中(细胞质和细胞核均有分布)。这表明40–91 aa是猪流行性腹泻病毒ORF3蛋白细胞质定位的关键结构域。PEDVORF3蛋白细胞质定位结构域的明确为进一步研究其细胞内转运和生物学功能提供了参考。     

猪流行性腹泻病毒功能性受体的鉴定

为研究猪氨基肽酶(Porcine Aminopeptidase N,pAPN)是否作为猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的细胞感染受体,通过转染技术,使PEDV非容许性细胞MDCK表达pAPN,并用PEDV感染转染细胞。结果发现转染的MDCK细胞可以感染PEDV,并且该病毒可以在转染细胞中连续传代。免疫荧光法鉴定存在病毒抗原。进一步实验证实,抗pAPN血清可以抑制PEDV感染转染的MDCK细胞。这些结果展示转染的MDCK细胞、pAPN表达及PEDV病毒复制之间存在直接联系,证明pAPN是PEDV的细胞感染受体之一。     

猪流行性腹泻病毒逃逸宿主天然免疫的研究进展

猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)是一种肠道α冠状病毒,靶向猪小肠上皮细胞,使得小肠上皮组织被破坏,肠道充血,肿胀,引起猪群水样腹泻,导致育肥猪厌食和体型消瘦。其中哺乳仔猪死亡率高。2010年后,随着新的PEDV变异毒株出现,PED再一次在全球暴发,特别是亚洲国家,造成了严重的经济损失。机体的先天免疫并不能完全抵抗PEDV对机体的侵害,因此了解PEDV通过影响干扰素(Interferon,IFN)的产生来逃逸先天性免疫的途径十分必要,同时也为治疗PEDV感染以及研发PEDV疫苗提供了思路。     

猪流行性腹泻病毒与宿主抗病毒天然免疫抑制

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)能引起猪腹泻等肠道疾病,属于α属冠状病毒,它的爆发给很多国家养猪业造成了严重的经济损失。2010年以来,PEDV感染在中国出现大规模爆发,一种突变型PEDV也于2013年在美国出现并迅速传播。 RNA病毒能够通过Toll样受体通路3（TLR3）和RIG-I样受体通路（RLR）诱导I型干扰素的产生。但以往的研究表明,PEDV感染能抑制I型干扰素的合成。近年来有关PEDV调节宿主天然免疫应答的研究取得了很大进展。PEDV主要通过编码作为干扰素拮抗剂的病毒蛋白以及隐藏病毒自身病原相关分子模式（PAMP）等两种方式逃逸宿主天然免疫应答。目前已报道,PEDV非结构蛋白1可通过降解CBP阻碍干扰素调节因子3(IRF-3)组装成增强子复合体;木瓜蛋白酶样蛋白酶可通过其去泛素化酶活性阻断天然免疫信号通路传递;3C样蛋白酶可通过剪切NEMO发挥干扰素拮抗剂活性;核衣壳蛋白通过结合TBK1抑制I型干扰素产生。PEDV也可通过合成加帽酶隐藏其病原相关分子dsRNA来避免激活天然免疫通路。PEDV抗病毒天然免疫机制阐明为研究PEDV感染免疫和致病机制提供了重要的理论依据,为研发抗PEDV新型疫苗和药物提供了基础。     

Proteome analysis of porcine epidemic diarrhea virus (PEDV)‐infected Vero cells

Porcine epidemic diarrhea virus (PEDV) causes an acute, highly contagious, and devastating viral enteric disease with a high mortality rate in suckling pigs. A large‐scale outbreak of PED occurred in China in 2010, with PEDV emerging in the United States in 2013 and spreading rapidly, posing significant economic and public health concerns. In this study, LC–MS/MS coupled to iTRAQ labeling was used to quantitatively identify differentially expressed cellular proteins in PEDV‐infected Vero cells. We identified 49 differentially expressed cellular proteins, of which 8 were upregulated and 41 downregulated. These differentially expressed proteins were involved in apoptosis, signal transduction, and stress responses. Based on these differentially expressed proteins, we propose that PEDV might utilize apoptosis and extracellular signal regulated kinases pathways for maximum viral replication. Our study is the first attempt to analyze the protein profile of PEDV‐infected cells by quantitative proteomics, and we believe our findings provide valuable information with respect to better understanding the host response to PEDV infection.     

猪流行性腹泻病毒S蛋白主要抗原表位区的原核表达及间接ELISA检测方法的建立

【目的】建立一种快速、特异、敏感的检测血清中猪流行性腹泻病毒(PEDV)抗体的方法。【方法】利用生物学软件对PEDV S蛋白进行抗原位点分析,选择S蛋白的主要抗原表位区进行原核表达。采用SDS-PAGE和Western-blot对重组蛋白进行鉴定及抗原性分析。用纯化的重组蛋白作为包被抗原,经过条件优化、特异性和重复性试验,建立一种针对血清中PEDV抗体的间接ELISA检测方法。【结果】表达了重组S蛋白,重组的S蛋白能与PEDV阳性血清发生特异性反应,并建立一种基于重组S蛋白的间接ELISA检测方法。组内及组间变异系数均小于10%,重复性较好。建立的间接ELISA检测方法分别与商品化PEDV抗体检测试剂盒和Western-blot鉴定结果相比,两者符合率分别为86.67%和88.89%。【结论】建立的间接ELISA方法可以用于PEDV抗体的检测。     

Genome-wide transcriptome analysis of porcine epidemic diarrhea virus virulent or avirulent strain-infected porcine small intestinal epithelial cells

Porcine epidemic diarrhea virus (PEDV) is the main cause of diarrhea, vomiting, and mortality in pigs, which results in devastating economic loss to the pig industry around the globe. In recent years, the advent of RNA-sequencing technologies has led to delineate host responses at late stages of PEDV infection; however, the comparative analysis of host responses to early-stage infection of virulent and avirulent PEDV strains is currently unknown. Here, using the BGI DNBSEQ RNA-sequencing, we performed global gene expression profiles of pig intestinal epithelial cells infected with virulent (GDS01) or avirulent (HX) PEDV strains for 3, 6, and 12 h. It was observed that over half of all significantly dysregulated genes in both infection groups exhibited a down-regulated expression pattern. Functional enrichment analyses indicated that the differentially expressed genes (DEGs) in the GDS01 group were predominantly related to autophagy and apoptosis, whereas the genes showing the differential expression in the HX group were strongly enriched in immune responses/inflammation. Among the DEGs, the functional association of TLR3 and IFIT2 genes with the HX and GDS01 strains replication was experimentally validated by TLR3 inhibition and IFIT2 overexpression systems in cultured cells. TLR3 expression was found to inhibit HX strain, but not GDS01 strain, replication by enhancing the IFIT2 expression in infected cells. In conclusion, our study highlights similarities and differences in gene expression patterns and cellular processes/pathways altered at the early-stage infection of PEDV virulent and avirulent strains. These findings may provide a foundation for establishing novel therapies to control PEDV infection.     

Structure and immune recognition of the porcine epidemic diarrhea virus spike protein

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猪流行性腹泻病毒免疫及疫苗研制

猪流行性腹泻(Porcine epidemic diarrhea,PED)是严重危害我国和世界养猪业的重要动物疫病,其致病原为猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV),属冠状病毒科α冠状病毒属.文中综述分为5个部分.前两部分在介绍该病病原及其流行病学的基础上先概括了初...     

Expression of neutralizing epitope of porcine epidemic diarrhea virus in potato plants

Transgenic plants expressing recombinant proteins from pathogenic microorganisms provide an inexpensive edible vaccine for induction of local immunity. A neutralizing epitope of porcine epidemic diarrhea virus (PEDV) gene containing SEKDEL was expressed in potato using Agrobacterium-mediated transformation system. Putative transgenic plants were regenerated, and genomic PCR confirmed the presence of PEDV epitope gene in the potato plants. Based on the ELISA results, epitope of PEDV protein made up approximately 0.1% of the total soluble tuber protein.     

猪流行性腹泻病毒核衣壳蛋白与感染细胞核磷蛋白的共定位分析

【目的】阐明猪流行性腹泻病毒(PEDV)核衣壳蛋白与病毒感染细胞核仁成分B23.1蛋白的共定位特征。【方法】分别参照GenBank中PEDV CV777株的N基因序列(AF353511)和编码人细胞核仁蛋白B23.1基因序列(BC050628.1),设计、合成扩增N基因和B23.1基因的引物,利用RT-PCR技术扩增了N基因和Vero E6细胞的B23.1基因的cDNA,分别克隆到真核表达载体pAcGFP1-C1和pDsRed2-N1,获得重组质粒pAcGFP1-C1/N和pDsRed2-N1/B23.1,共转染Vero E6细胞。【结果】Western blots分析表明这些融合蛋白在转染的Vero E6细胞中表达;共聚焦显微镜技术分析表明在共转染Vero E6细胞中猪流行性腹泻病毒N蛋白与Vero E6细胞核磷蛋白B23.1发生共定位。【结论】为进一步鉴定PEDV N蛋白中核仁定位信号和N蛋白核仁定位机制提供可靠依据。     

Surface displayed expression of a neutralizing epitope of spike protein from a Korean strain of porcine epidemic diarrhea virus

The neutralizing epitope (K-COE) of the spike protein from a Korean strain of porcine epidemic diarrhea virus (PEDV) has been shown to prevent and foster an immune response to PED, when orally adjusted. The cell surface of the budding yeast,Saccharomyces cerevisiae, was engineered to anchor the K-COE on the outer layer of the cell, and consequently, the altered yeast was applied as a dietary complement for animal feed, with immunogenic functions. In this study, the K-COE gene (K-COE) of the Korean strain of PEDV with the signal peptide of rice amylase 1A (Ramy 1A), was fused with the gene encoding the carboxyterminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a mating associated protein that is anchored covalently to the cell wall. The glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was selected in order to direct the expression of the fusion construct, and the resulting recombinant plasmid was then introduced intoS. cerevisiae. The surface display of K-COE was visualized via confocal microscopy using a polyclonal antibody against K-COE as the primary antibody, and FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse IgG as the secondary antibody. The display of the K-COE on the cell surface was further verified via Western blot analysis using the cell wall fraction after the administration of α-1,3-glucanase/PNGase F/β-mannosidase treatment.