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.     

Species‐specific impact of the autophagy machinery on Chikungunya virus infection

Chikungunya virus (CHIKV) is a recently re‐emerged arbovirus that triggers autophagy. Here, we show that CHIKV interacts with components of the autophagy machinery during its replication cycle, inducing a cytoprotective effect. The autophagy receptor p62 protects cells from death by binding ubiquitinated capsid and targeting it to autophagolysosomes. By contrast, the human autophagy receptor NDP52—but not its mouse orthologue—interacts with the non‐structural protein nsP2, thereby promoting viral replication. These results highlight the distinct roles of p62 and NDP52 in viral infection, and identify NDP52 as a cellular factor that accounts for CHIKV species specificity.     

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.     

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。     

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。     

Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.     

Aminopeptidase N Knockout Pigs Are Not Resistant to Porcine Epidemic Diarrhea Virus Infection

<正>Dear Editor,Porcine epidemic diarrhea virus(PEDV) is the etiologic agent of porcine epidemic diarrhea(PED), which is an acute, highly contagious, and devastating enteric viral disease in pigs(Lee 2015). PEDV is a coronavirus that mainly infects and replicates in villous enterocytes of the small intestine in pigs(Li et al. 2016). PEDV can infect     

Cellular Localization, Expression, and Structure of the Nuclear Dot Protein 52

Nuclear dots containing PML and Sp100 proteins (NDs) play a role in the development of acute promyelocytic leukemia, are modified after infection with various viruses, and are autoimmunogenic in patients with primary biliary cirrhosis (PBC). PML and Sp100 gene expression is strongly enhanced by interferons (IFN). Based on immunostaining with a monoclonal antibody (mAb C8A2), a third protein, nuclear dot protein 52 (NDP52), was recently localized in NDs. Here we analyzed the cellular localization, expression, and structure of NDP52 in more detail. Our NDP52-specific sera revealed mainly cytoplasmic staining but no ND pattern, neither in untreated nor in IFN-treated cells. Cells transfected with NDP52 expression vectors showed exclusively cytoplasmic staining. In subcellular fractionation experiments, NDP52 was found in cytoplasmic and nuclear fractions. Unlike as described for Sp100 and PML, NDP52 mRNA and protein levels were only marginally enhanced by IFN γ and not enhanced at all by IFN β. NDP52 homodimerization but no heterodimerization with Sp100 or PML could be demonstrated. None of the 93 PBC sera tested contained autoantibodies against NDP52. Finally, mAb C8A2 reacted not only with NDP52 but also with a conformation-dependent epitope on the Sp100 protein. These data imply that NDP52 forms homodimers but no heterodimers with Sp100 and PML, lacks autoantigenicity in PBC, localizes mainly in the cytoplasm, and is associated with the nucleus, but not with NDs. Finally, unlike Sp100 and PML, NDP52 expression is neither markedly enhanced nor localization detectably altered by type I and II IFNs.     

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH‐dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC‐mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2‐Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC‐mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.     

Functional order of assembly of herpes simplex virus DNA replication proteins into prereplicative site structures.

Herpes simplex virus replicates its DNA within nuclear structures called replication compartments. In contrast, in cells in which viral DNA replication is inhibited, viral replication proteins localize to punctate structures called prereplicative sites. We have utilized viruses individually mutated in each of the seven essential replication genes to assess the function of each replication protein in the assembly of these proteins into prereplicative sites. We observed that four replication proteins, UL5, UL8 UL52, and UL9, are necessary for the localization of ICP8 (UL29) to prereplicative sites natural infection conditions. Likewise, four of the seven viral DNA replication proteins, UL5, UL52, UL9, and ICP8, are necessary for the localization of the viral DNA polymerase to prereplicative sites. On the basis of these results, we present a model for prereplicative site formation in infected cells in which the helicase-primase components (UL5, UL8, and UL52), the origin-binding protein (UL9), and the viral single-stranded DNA-binding protein (ICP8) assemble together to initiate the process. This is followed by the recruitment of the viral polymerase into the structures, a step facilitated by the polymerase accessory protein, UL42. Host cell factors can apparently substitute for some of these viral proteins under certain conditions, because the viral protein requirements for prereplicative site formation are reduced in transfected cells and in infected cells treated with drugs that inhibit DNA synthesis.     

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。     

Bile acids promote the caveolae-associated entry of swine acute diarrhea syndrome coronavirus in porcine intestinal enteroids

Intestinal microbial metabolites have been increasingly recognized as important regulators of enteric viral infection. However, very little information is available about which specific microbiota-derived metabolites are crucial for swine enteric coronavirus (SECoV) infection in vivo. Using swine acute diarrhea syndrome (SADS)-CoV as a model, we were able to identify a greatly altered bile acid (BA) profile in the small intestine of infected piglets by untargeted metabolomic analysis. Using a newly established ex vivo model–the stem cell-derived porcine intestinal enteroid (PIE) culture–we demonstrated that certain BAs, cholic acid (CA) in particular, enhance SADS-CoV replication by acting on PIEs at the early phase of infection. We ruled out the possibility that CA exerts an augmenting effect on viral replication through classic farnesoid X receptor or Takeda G protein-coupled receptor 5 signaling, innate immune suppression or viral attachment. BA induced multiple cellular responses including rapid changes in caveolae-mediated endocytosis, endosomal acidification and dynamics of the endosomal/lysosomal system that are critical for SADS-CoV replication. Thus, our findings shed light on how SECoVs exploit microbiome-derived metabolite BAs to swiftly establish viral infection and accelerate replication within the intestinal microenvironment.     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

猪流行性腹泻病毒(PEDV)与抗病毒天然免疫

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)是引起猪流行性腹泻病等肠道疾病的一种动物冠状病毒.PEDV与宿主系统相互作用,特别是其对宿主抗病毒天然免疫调节作用和机制是目前动物冠状病毒研究的基础科学问题之一.基于作者近几年来对人类重要冠状病毒对宿主抗病毒天然免疫系统调节作用的研究,本文对PEDV基因组与编码蛋白主要功能以及PEDV调节宿主抗病毒天然免疫反应及其可能机制的进展和现状进行了分析.与人类冠状病毒相似,PEDV编码的木瓜样蛋白酶(papain like protease,PLP)是一个多功能蛋白酶,除了蛋白酶活性外,还具有去泛素化酶(DUB)活性和宿主干扰素拮抗活性,是PEDV编码的一种新型病毒来源DUB和宿主干扰素拮抗蛋白.这些研究为阐明PEDV对宿主抗病毒天然免疫反应调节作用和其致病机制提供了重要的理论依据,为研制新型PEDV免疫防治措施提供了重要理论基础.     

Molecular Characterization and Phylogenetic Analysis of Porcine Epidemic Diarrhea Viruses Associated with Outbreaks of Severe Diarrhea in Piglets in Jiangxi,China 2013

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious, acute enteric viral disease of swine characterized by vomiting, watery diarrhea, dehydration and death. To identify and characterize the field PEDVs associated with the outbreaks of severe diarrhea in piglets in Jiangxi, 2013, the complete genome sequences of two representative strains of PEDV, designated CH/JX-1/2013 and CH/JX-2/2013, were determined and analyzed. The genome sequences of both emergent Jiangxi PEDV strains, CH/JX-1/2013 and CH/JX-2/2013, were 28,038 nucleotides in length excluding 3’ poly (A) tail. Compared to the PEDV CV777 strain, CH/JX-1/2013 and CH/JX-2/2013 had some unique genetic characteristics in the proximal region of the 5´-UTRs. Phylogenetic analysis of the complete genomes and the structural proteins revealed that CH/JX-1/2013 and CH/JX-2/2013 had a close relationship with post-2010 Chinese PEDV strains and US strains identified in 2013. The nucleotide identity between the two Jiangxi strains (CH/JX-1/2013 and CH/JX-2/2013) and 30 strains of PEDV identified ante-2010 and post-2010 ranged from 96.3–97.0% and 97.3–99.7%, respectively. Multiple nucleotide and deduced amino acid mutations were observed in the ORF1a/b, S, ORF3, E, M and N genes among the current field PEDV strains when compared to the CV777 strain. Some of the mutations altered the amino acid charge and hydrophilicity, and notably, there was an amino acid substitution in the middle of one neutralizing epitope (L1371I) of the S gene of both CH/JX-1/2013 and CH/JX-2/2013. Taken together, the accumulated genetic variations of the current field PEDV strains might have led to antigenic changes of the viruses, which might confer the less effectiveness or failure of the CV777-based vaccines currently being widely used in Jiangxi, China.     

Complete genome sequence of a porcine epidemic diarrhea virus variant

In 2011, outbreaks of viral diarrhea were observed on most swine-breeding farms in most of the provinces of China. The disease is characterized by vomiting, severe diarrhea, and a high mortality rate (82.3%) in newborn piglets. The clinical appearance was similar to that of porcine epidemic diarrhea virus (PEDV) infection. PEDVs were detected in samples (feces or small intestines) from most farms. In order to investigate whether there is a PEDV variant circulating in China, we sequenced and analyzed the complete genome of the recently identified field strain, CH/FJND-3/2011. The sequence data indicate that this PEDV variant prevails in China.     

自噬蛋白NDP52与细菌感染的研究进展

自噬（autophagy）是哺乳动物清除入侵细菌的主要途径,可保卫宿主细胞免受细菌的损伤。核点蛋白52（nuclear dot protein 52,NDP52）——核点家族成员之一,是除p62/SQSTM1和NBR1等之外最新发现的自噬关键蛋白。它连接自噬体表面的微管相关蛋白1轻链3（microtubule associ-ated protein 1 light chain 3,LC3）,将披上＂泛素大衣＂的病原菌（如沙门氏菌和化脓性链球菌）递送至自噬体内加以清除。这一发现有助于人们深入了解自噬抵抗病原微生物感染的具体分子机制,为预防和治疗细菌感染提供了新靶点。     

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...