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.     

猪流行性腹泻病毒反向遗传操作技术及其应用

猪流行性腹泻病毒(PEDV)是引起猪(特别是新生仔猪)急性、高度传染性消化道疾病的主要病原之一;2010年底以来,猪流行性腹泻(PED)的再次暴发给我国乃至全球养猪业造成了巨大经济损失。最近,研究者已先后建立了基于靶向RNA重组技术、细菌人工染色体(BAC)载体系统和体外连接技术的PEDV反向遗传学操作技术,为PEDV编码的蛋白质的功能、致病机制以及新型疫苗的研发开辟了新的思路。本文对PEDV反向遗传操作技术的研究进展及其在PEDV胰酶依赖性、S蛋白和ORF3蛋白的功能以及新一代疫苗研制等方面的应用现状进行了综述与展望。     

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.     

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] …     

ORF3蛋白促进猪流行性腹泻病毒在Vero细胞上的增殖

【背景】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)感染猪而引起的一种急性肠道传染病,常导致病猪水样腹泻、呕吐、脱水。自2010年起,其大规模的暴发给养猪业造成巨大的经济损失。由于对PEDV免疫机理及侵入机制知之甚少,至今仍缺乏有效的PED防治措施。【目的】研究orf3对PEDV体外增殖的影响。【方法】利用基于RNA同源重组的PEDV反向遗传学操作技术拯救一系列携带不同orf3基因及orf3基因缺失的重组PEDV;将获得的重组PEDV以MOI 0.1感染Vero细胞,分别于感染的第8、16、24、32、40、48 h测定其TCID_(50)并绘制病毒生长曲线;分别在感染25 h和36 h利用全自动细胞计数分析仪对6孔板内的细胞进行计数,并于感染后的第12、24、36、48 h用CCK-8试剂盒对其细胞活力进行测定。【结果】RT-PCR结果及细胞病变观察证明成功拯救到了携带不同orf3基因或orf3基因缺失的重组PEDV;进一步的免疫组化分析结果证实PEDV的ORF3蛋白可以在Vero细胞中合成。SPSS软件分析表明携带orf3基因的重组PEDV的滴度(TCID_(50))显著高于缺失orf3基因的重组PEDV的滴度;带有orf3基因的重组PEDV感染Vero细胞25 h和36 h时的活细胞数显著高于缺失orf3基因的重组病毒感染相同时间时的活细胞数;而且重组PEDV感染Vero细胞24 h后,带有orf3基因的重组PEDV的细胞活性显著高于缺失orf3基因的重组病毒。【结论】ORF3蛋白对于PEDV在Vero细胞中的增殖具有促进作用,该作用是通过延缓或减少感染细胞的死亡实现的。本研究为揭示PEDV orf3基因的功能和PEDV复制机制的研究提供理论基础。     

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。     

与猪流行性腹泻病毒M蛋白互作的宿主蛋白的鉴定

【背景】猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)膜蛋白(M)在病毒粒子的组装、膜融合和病毒复制等方面具有重要的作用,但M蛋白与宿主细胞的互作机制尚不清楚。【目的】利用免疫沉淀技术和液质联用技术筛选细胞内与PEDVM蛋白相互作用的蛋白,为揭示M蛋白在病毒增殖过程中发挥的功能提供研究基础。【方法】将MOI=0.1的PEDV DR13疫苗株接种于长成单层的Vero细胞,感染36 h后,收集细胞并进行裂解。利用抗M的单克隆抗体沉淀与M相互作用蛋白复合物,通过液相色谱串联质谱(LC-MS/MS)进行鉴定并利用细胞功能富集分析(Gene ontology,GO)对感染组鉴定到的细胞蛋白进行分析,确定两个细胞内源性蛋白为候选蛋白,进行免疫共沉淀(Co-IP)验证和共定位分析。【结果】基于鉴定蛋白的肽段数的方法分析显示,感染组与对照组相比,鉴定了218个与M蛋白相互作用的细胞内源性蛋白,分别与蛋白质合成、代谢、细胞信号通路转导等密切相关,选择细胞分裂周期蛋白42 (Cell division cycle 42,CDC42)、真核翻译起始因子3亚基L蛋白(eIF3L)为候选蛋白进行Co-IP(Co-immunoprecipitation)验证和共定位分析,结果证实CDC42、eIF3L蛋白分别与M蛋白在细胞内存在相互作用。【结论】鉴定出PEDV M蛋白能够与宿主细胞CDC42和eIF3L蛋白相互作用,并鉴定出其他可能与M蛋白发生相互作用的宿主蛋白60个,为开展PEDV与宿主细胞蛋白相互作用研究提供了重要理论依据。     

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.     

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。     

Cloning and sequence analysis of the Korean strain of spike gene of porcine epidemic diarrhea virus and expression of its neutralizing epitope in plants

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs and leads to death with a high mortality rate, which has been reported notably in Korea. The spike (S) gene of the PEDV isolated in Korea was cloned and sequenced. The nucleotide sequence encoding the entire S gene open reading frame of Korean strain was 4161 bases long encoding 1387 amino acids. The neutralizing epitope of Korean PEDV (K-COE) was expressed in tobacco plants using Agrobacterium-mediated protein transformation. The recombinant K-COE constituted up to 0.1% of the total soluble protein in the leaves of transgenic tobacco plants. The result of this study opens the way for the development of an edible vaccine against PEDV infection in Korea.     

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

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Evaluation of purified recombinant spike fragments for assessment of the presence of serum neutralizing antibodies against a variant strain of porcine epidemic diarrhea virus

Since 2010, variant strains of porcine epidemic diarrhea virus(PEDV) have caused disasters in the pork industry. The spike(S) protein, as the major immunity-eliciting antigen, has previously been used for serological testing and has been found to correlate significantly with the results of the serum neutralization(SN) test. However, further evaluation of this method is needed as new epidemic strains of PEDV emerge. Hence, the main objective of this study was to assess sow sera and determine the correlation between enzyme-linked immunosorbent assay(ELISA) results(involving a newly isolated GDS01 virus-based ELISA and ELISAs based on seven recombinant fragments comprising overlapping S1 and partial S2 sequences) and SN titers. Furthermore, we determined the reliability of the ELISAs based on receiver operating characteristics(ROC) curve analyses. For the most promising ELISA, i.e., the SP4 ELISA, the correlation coefficient(r) and the area under curve(AUC) were determined to be 0.6113 and 0.8538, respectively. In addition, we analyzed the homology of the SP4 sequences obtained from different strains(including vaccine strains) and found that various strains showed a high degree of homology in this region. Thus, we conclude that SP4 is a promising serological testing protein for use in the field.     

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.