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。     

猪流行性腹泻病毒感染PK-15细胞miRNA的差异表达分析

为研究猪流行性腹泻病毒(Porcine epidemic diahorrea virus,PEDV)对猪肾传代细胞PK-15miRNA表达谱的影响,将PEDV感染PK-15细胞后,提取总RNA,进行高通量测序,构建感染与未感染PEDV的PK-15细胞的miRNA表达谱,并进行差异性分析,对表达差异显著的miRNA进行heatmap聚类分析及GO(Gene ontology,GO)分子功能(Molecular function,MF)分析,选取10个显著性差异表达的miRNA通过RT-qPCR进行了验证。结果发现,显著性差异表达的miRNA有214个,其中175个miRNA表达上调,39个miRNA表达下调。Heatmap聚类分析表明,病毒组绝大部分差异表达的miRNA较对照组表达量上调,少部分下调。GO分析表明,miRNA广泛参与结合、蛋白结合、蛋白激酶活性、转移酶活性、含磷基团转移、磷酸转移酶活性等生物作用。RT-qPCR验证的各miRNA的表达趋势与高通量测序结果一致。结果表明,猪流行性腹泻病毒感染对PK-15细胞编码的miRNA表达水平有显著影响,从而对进一步研究治疗PEDV的miRNA制剂提供新思路。     

黏蛋白2对猪流行性腹泻病毒感染的拮抗作用研究

肠黏膜屏障的完整性与猪流行性腹泻病毒（Porcine epidemic diarrhea virus,PEDV）的致病性密切相关。黏蛋白2(Mucin 2,Muc2)作为肠黏膜屏障的重要组成蛋白，在阻止病原体、毒素和异物入侵等方面发挥重要作用。本文旨在探究黏蛋白Muc2对PEDV复制的拮抗作用。通过观察PEDV感染仔猪空肠黏膜屏障情况，利用RNA干扰策略和分子病毒学检测方法在细胞水平上研究分析Muc2表达水平与PEDV复制的关系，并进一步探讨猪源Muc2天然蛋白发挥抗PEDV作用的具体阶段。结果显示：PEDV感染仔猪空肠段进行PAS染色发现，病毒感染后肠绒毛表面黏液层变薄，结构被破坏，杯状细胞明显减少；与对照组相比，干扰Muc2基因能够上调PEDV-N的mRNA及蛋白水平；Muc2蛋白添加后显著抑制PEDV-N蛋白的表达，且病毒mRNA水平显著下降（P<0.05）；在病毒感染前使用50μg/mL Muc2蛋白预处理1 h后，PEDV-N mRNA相对表达量极显著下降（P<0.001），而在病毒吸附、入侵和复制阶段无显著差异（P>0.05），表明Muc2具有降低PEDV...     

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nsp1蛋白对Ⅰ型干扰素应答的影响

猪流行性腹泻病毒 (PEDV) 能抑制宿主Ⅰ型干扰素及其诱导的细胞抗病毒免疫应答,但是PEDV抑制Ⅰ型干扰素应答的分子机制尚不明了,尤其是PEDV非结构蛋白 (Nonstructural proteins,nsps) 在Ⅰ型干扰素应答中的调控作用研究不多。为研究PEDV非结构蛋白1 (nsp1) 对细胞Ⅰ型干扰素应答的影响,构建了真核表达载体pCAGGS-nsp1,采用Western blotting和间接免疫荧光试验确定nsp1在细胞中的表达。通过报告基因法、ELISA以及病毒复制抑制试验评估nsp1对Ⅰ型IFN的影响。结果显示,nsp1在转染细胞和病毒感染细胞中均高效表达;双荧光报告基因试验结果表明,nsp1能显著抑制IFN-β启动子活性,且具有剂量依赖性。ELISA结果显示,nsp1能显著抑制IFN-β蛋白的表达。水泡性口炎病毒 (VSV) 复制抑制试验结果显示,nsp1明显抑制poly(I:C)介导的Ⅰ型IFN的抗病毒作用。结果提示,nsp1作为PEDV的保守蛋白,具有拮抗Ⅰ型干扰素启动子活性和应答的功能,为揭示PEDV逃逸宿主天然免疫应答的机制和研发新型高效抗PEDV疫苗奠定基础。     

猪流行性腹泻病毒地方株LJB/03分离及培养特性

从黑龙江省某猪场疑为病毒性腹泻的发病猪采集腹泻粪便样品,以RT-PCR法扩增出猪流行性腹泻病毒M基因后,采用细胞培养法进行病毒分离。对细胞培养分离物进行间接免疫荧光、免疫电镜观察、RT-PCR及ELISA法检验,其中间接免疫荧光试验可见培养细胞中存在明显的特异性绿色荧光;免疫电镜下可见大小符合预期、有囊膜、花瓣状的典型冠状病毒结构特征;RT-PCR检测证实存在PEDVM基因;间接ELISA检测中平均P/N比值为7.6;从而确认为分离到一株猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV),命名为PEDVLJB/03株。随后,对该分离毒株的培养特性及如何提高病毒滴度进行探索。通过摸索该分离毒株的蚀斑形成条件,建立了PEDV蚀斑形成方法,并采用该方法进行病毒的蚀斑纯化,纯化得到PEDV大蚀斑克隆株和小蚀斑克隆株。对大、小两种蚀斑克隆株的病毒滴度测定结果表明,大小蚀斑克隆株细胞感染滴度相差明显。     

猪流行性腹泻病毒嵌套式RT-PCR检测方法的建立

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)引起的一种以呕吐、腹泻和失水为特征的猪肠道传染病.各种年龄的猪都可发病[1],哺乳仔猪、架子猪或育肥猪的发病率最高可达100%,尤其哺乳仔猪的受害最严重.本病在欧洲许多国家均有报道.上海畜牧兽医研究所等单位证实,我国近年来发生的仔猪腹泻,有很多为PEDV引起.     

Human coronavirus 229E: receptor binding domain and neutralization by soluble receptor at 37 degrees C

Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37 degrees C, but not 4 degrees C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37 degrees C that facilitate virus entry.     

Receptor Usage and Cell Entry of Porcine Epidemic Diarrhea Coronavirus

Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America''s pork industry. Here we investigate the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human and sugar coreceptor N-acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support the idea of bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread.     

普通冠状病毒229E株的分子流行病学分析

目的明确哈尔滨地区普通冠状病毒229E(Human Coronavirus-229E,HCOV-229E)株的流行和变异情况及其与SARS-CoV的异同,为进一步掌握该地区常见上呼吸道病毒的流行规律及预防,乃至疫苗的制备打下基础。方法利用RT—PCR法对2003年上半年采集的部分发热病人血清及血细胞进行筛选,同时采用基因测序、序列分析等手段对扩增的HCOV-229E N gene片断进行蛋白和基因分析。结果55例标本中HCOV-229E RNA阳性病例5例,占9．09％;测序结果看出哈尔滨地区检出229E株N基因的序列与已公布的HCOV-229E Ngene(295—802)序列完全相同,所测片断为HCOV-229E N gene的部分序列。该基因与猪流行性腹泻病毒、犬冠状病毒、TGEV及猫感染性腹膜炎病毒有一定同源性;但与已公布的SARS-CoV N gene序列比较,相似性小于1％。结论(1)该地区发热病人普通冠状病毒229E株阳性率为9．09％;(2)哈尔滨地区流行的HCOVN基因无变异发生;(3)HCOV-229E与猪流行性腹泻病毒、犬冠状病毒、TGEV和猫感染性腹膜炎病毒有一定的同源性;(4)哈尔滨地区HCOV-229E N gene与SARS-CoV N gene相似性小于1％。     

Antiviral Compounds Against Nucleocapsid Protein of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is a severe diarrhea disease in swine that is caused by porcine epidemic diarrhea virus (PEDV). Nucleocapsid (N) protein is the RNA-binding protein of PEDV, which plays an important role for virus life cycle. The aim of this research was to screen and characterize the compounds that could inhibit the activity of PEDV N protein. The gene encoding PEDV N protein obtained from PEDV Thai isolate was cloned and expressed in E. coli. Its amino acid sequence was employed to generate the three dimensional structure by homology modeling. There were 1,286 compounds of FDA-approved drug database that could virtually bind to the RNA-binding region of N protein. Three compounds, trichlormethiazide, D-(+) biotin, and glutathione successfully bound to the N protein, in vitro, with the IC₅₀ at 8.754?mg/mL, 0.925?mg/mL, and 2.722?mg/mL. Antiviral activity in PEDV-infected Vero cells demonstrated that the effective concentration of trichlormethiazide, D-(+) biotin, and glutathione in inhibiting PEDV replication were 0.094, 0.094 and 1.5?mg/mL. This study demonstrated a strategy applied for discovery of antiviral agents capable of inhibiting PEDV N protein and PEDV replication. The compounds identified here exhibited a potential use as therapeutic agents for controlling PEDV infection.     

Protease-mediated entry via the endosome of human coronavirus 229E

Human coronavirus 229E, classified as a group I coronavirus, utilizes human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.     

Development of a porcine epidemic diarrhea virus M protein-based ELISA for virus detection

A membrane (M), protein-based ELISA was developed to detect porcine epidemic diarrhea virus (PEDV). The M gene of PEDV was expressed in Escherichia coli. The purified recombinant M protein was used to immunize rabbits to generate a polyclonal antibody. Immunofluorescence analysis indicated that the anti-PEDV-M antibody reacted with PEDV-infected cells. The antibody was utilized to develop an indirect ELISA to detect PEDV. Other viruses, porcine transmissible gastroenteritis coronavirus, avian infectious bronchitis coronavirus, porcine reproductive and respiratory syndrome virus, classic swine fever virus and porcine pseudorabies virus, were unreactive.     

猪肠道冠状病毒与入侵受体氨基肽酶N的相互作用

猪肠道冠状病毒是目前危害养猪产业的重要病原。目前已发现能够感染猪肠道的致病性冠状病毒有4种：猪传染性胃肠炎病毒、猪流行性腹泻病毒、猪丁型冠状病毒和猪肠道甲型冠状病毒。冠状病毒感染宿主的第一步是识别宿主细胞膜受体分子并与之结合,随后启动入侵及膜融合进而使病毒基因组进入宿主细胞内部。因此,冠状病毒受体是决定其宿主范围及组织嗜性的关键因素。确定冠状病毒受体及病毒与受体的结合机制对预防新发病毒及开发冠状病毒治疗性药物具有重要意义。猪传染性胃肠炎病毒利用猪氨基肽酶N（aminopeptidase N,APN）作为感染宿主的功能性受体,并利用唾液酸作为辅助结合因子。猪APN最初也被鉴定为猪流行性腹泻病毒的功能性受体,但近年的研究结果与前面的报道存在较大的差异,产生了较大的争议。最近的研究认为,猪丁型冠状病毒的功能性受体也是APN,并且猪丁型冠状病毒能够利用多个物种的APN作为功能性受体,这与其跨物种传播具有密切关系。最新发现的猪肠道甲型冠状病毒则不使用APN作为其入侵受体。本文综述了前面3种猪肠道病毒感染宿主细胞的受体及结合机制的研究进展,并比较分析了猪APN及唾液酸在不同猪肠道冠状病毒入侵宿主过程中结合方式的异同,为进一步研究新发猪肠道冠状病毒受体提供参考。     

Human coronavirus 229E infects polarized airway epithelia from the apical surface

Gene transfer to differentiated airway epithelia with existing viral vectors is very inefficient when they are applied to the apical surface. This largely reflects the polarized distribution of receptors on the basolateral surface. To identify new receptor-ligand interactions that might be used to redirect vectors to the apical surface, we investigated the process of infection of airway epithelial cells by human coronavirus 229E (HCoV-229E), a common cause of respiratory tract infections. Using immunohistochemistry, we found the receptor for HCoV-229E (CD13 or aminopeptidase N) localized mainly to the apical surface of airway epithelia. When HCoV-229E was applied to the apical or basolateral surface of well-differentiated primary cultures of human airway epithelia, infection primarily occurred from the apical side. Similar results were noted when the virus was applied to cultured human tracheal explants. Newly synthesized virions were released mainly to the apical side. Thus, HCoV-229E preferentially infects human airway epithelia from the apical surface. The spike glycoprotein that mediates HCoV-229E binding and fusion to CD13 is a candidate for pseudotyping retroviral envelopes or modifying other viral vectors.     

Protective Effects of Cell-Free Supernatant and Live Lactic Acid Bacteria Isolated from Thai Pigs Against a Pandemic Strain of Porcine Epidemic Diarrhea Virus

Probiotics and Antimicrobial Proteins - Porcine epidemic diarrhea virus (PEDV) is a coronavirus which causes severe diarrhea and fatal dehydration in piglets. In general, probiotic supplements...     

Involvement of Aminopeptidase N (CD13) in Infection of Human Neural Cells by Human Coronavirus 229E

Attachment to a cell surface receptor can be a major determinant of virus tropism. Previous studies have shown that human respiratory coronavirus HCV-229E uses human aminopeptidase N (hAPN [CD13]) as its cellular receptor for infection of lung fibroblasts. Although human coronaviruses are recognized respiratory pathogens, occasional reports have suggested their possible neurotropism. We have previously shown that human neural cells, including glial cells in primary cultures, are susceptible to human coronavirus infection in vitro (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800–806, 1997). However, the only reported expression of hAPN in the nervous system is at the level of nerve synapses. Therefore, we asked whether hAPN is utilized as a cellular receptor for infection of these human neural cell lines. Using flow cytometry, we were able to show the expression of hAPN on the surfaces of various human neuronal and glial cell lines that are susceptible to HCV-229E infection. An hAPN-specific monoclonal antibody (WM15), but not control antibody, inhibited the attachment of radiolabeled HCV-229E to astrocytic, neuronal, and oligodendrocytic cell lines. A correlation between the apparent amount of cell surface hAPN and the level of virus attachment was observed. Furthermore, the presence of WM15 inhibited virus infection of these cell lines, as detected by indirect immunofluorescence. These results indicate that hAPN (CD13) is expressed on neuronal and glial cell lines in vitro and serves as the receptor for infection by HCV-229E. This further strengthens the neurotropic potential of this human respiratory virus.