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.     

Impact of Porcine Epidemic Diarrhea on Performance of Growing Pigs

The impact of porcine epidemic diarrhea virus (PEDv) infection on the US pork industry has mainly been attributed to the mortality that it causes in suckling piglets, and, consequently, much effort has been invested in the quantification of its effect in sow farms. However, no information on the performance of surviving pigs that were exposed to the PEDv as piglets is available. Here, a retrospective cohort study to evaluate the impact of porcine epidemic diarrhea virus (PEDv) infection on growing pigs’ performance, as indicated by mortality, average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) was performed using production records from weaned pigs in nursery and wean-to-finish sites from sow farms that became PEDv-infected between May 2013 and June 2014. Production records from the first batch of growing pigs weaned in infected flows after the PEDv outbreak (“infected batches”) were compared with those from pigs weaned within the previous 14 to 120 days (“control batches”). Performance records from infected and control batches, paired by flow, were compared using non-parametric paired tests. Mortality, ADG and FCR were significantly different in PEDv-positive (infected) compared with PEDv-negative (control) batches, with a mean increase of mortality and FCR of 11% and 0.5, respectively, and a decrease of ADG of 0.16 lb/day. Our results demonstrate a poorer performance of growing pigs weaned after a PEDv outbreak compared with those weaned within the previous 14-120 days, suggesting that in addition to the mortality induced by PEDv in suckling pigs, the disease also impairs the performance of surviving pig. These findings help to quantify the impact of PEDv infection in the US and, ultimately, contribute to efforts to quantify the cost-effectiveness of disease prevention and control measures.     

猪流行性腹泻病毒嵌套式RT—PCR检测方法的建立

Two pairs of primers were designed according to the N gene sequence of PEDV-CV777 strain in Genebank (No. AF353511). PCR amplification product of outer-primers was 1328bp, and the inter-primers amplification product was 538bp, With the primers, a nested PCR assay was established to detect PEDV. This method was sensitive and specific and could be used in PEDV diagnosis and epidemiological investigation.     

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.     

猪流行性腹泻抗原基因在乳酸菌中的表达及优化

目的：优化猪流行性腹泻抗原基因COE在乳酸乳球菌中的表达。方法：将表达猪流行性腹泻抗原基因COE的重组乳酸菌活化,酶切鉴定其稳定性,然后设计实验分别从pH值、温度、Nisin浓度、诱导时间、菌体密度等条件对COE表达进行优化,SDS-PAGE检测表达效果。结果：COE在乳酸乳球菌中的最佳表达条件为pH 7.0、T（温度）=30℃、Nisin=2ng/mLt、（诱导时间）=4h和OD600=0.5,在以上条件下相对表达量分别达到了15.48%、15.05%、15.82%、14.72%和20.47%。在最佳表达条件下得到SOE的相对表达含量达到21%。结论：COE重组质粒稳定,其在乳酸菌中经优化表达后可为今后研制猪流行性腹泻乳酸菌疫苗提供数据。     

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。     

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.     

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.     

A Porcine Epidemic Diarrhea Virus Outbreak in One Geographic Region of the United States: Descriptive Epidemiology and Investigation of the Possibility of Airborne Virus Spread

This study describes a spring 2013 outbreak of porcine epidemic diarrhea virus (PEDv), using data from 222 swine sites in 14 counties area in 4 contiguous states in the United States. During the outbreak, the premises-level incidence of PEDv was 40.5 percent (90/222 sites). One of the three companies from which data were collected had a lower incidence (19.5 percent) than the other two companies (41.1 and 47.2 percent). Sow sites had the highest incidence of PEDv during the outbreak (80.0 percent). Spatial analysis showed that PEDv was clustered rather than randomly distributed, which suggested that sites near a positive site had increased risk of acquiring PEDv infection. Meteorological data were used to investigate the hypothesis that PEDv was spread by air. If airborne dissemination played a role in this outbreak, we would expect the direction of disease spread to correlate with the predominant wind direction. Two methods were used to determine the direction of disease spread—linear direction mean analysis in ArcGIS and the direction test in ClusterSeer. The former method indicated PEDv spread was south to slightly southwest, and the latter indicated spread was to the southeast. The predominant wind direction during the month of the outbreak was toward the south, with some southeast and southwest winds; the strongest wind gusts were toward the southwest. These findings support the hypothesis that PEDv was spread by air. The results, however, should be interpreted cautiously because we did not have information on direct and indirect contacts between sites, such as movement of trucks, feed, pigs or people. These types of contacts should be evaluated before pathogen spread is attributed to airborne mechanisms. Although this study did not provide a definitive assessment of airborne spread of PEDv, we believe the findings justify additional research to investigate this potential mechanism of transmission.     

Complete Genome Sequence of a Variant Porcine Epidemic Diarrhea Virus Strain Isolated in China

Since October 2010, an outbreak of porcine epidemic diarrhea (PED) has been observed in some provinces of China. Here we report the complete genome sequence of porcine epidemic diarrhea virus (PEDV) strain LC, which was recently isolated from sucking piglets that suffered from severe watery diarrhea in Guangdong. It will help in understanding the epidemiological and molecular characteristics of PEDV in China.     

猪流行性腹泻病毒S基因片段的克隆及原核表达

为研究猪流行性腹泻病毒(porcine epidemic diarrhea virus PEDV)S基因片段的原核表达产物是否具有抗原性,分析S基因抗原位点后,用PCR技术扩增S蛋白主要抗原区的核酸序列,经克隆后将目的片段连接到原核表达载体pET-28a(+)中,成功构建了重组质粒pET-28a-PEDV-Sp,其重组菌于37℃、0.5 mmol/L IPTG诱导表达4 h后进行SDS-PAGE分析,在分子质量约为29 kDa处出现一新蛋白带,与预期相符。质谱鉴定表明,已成功表达了目的蛋白。纯化后的重组蛋白免疫兔制备多克隆抗体,抗体效价检测结果显示该蛋白具有良好的抗原性。该研究为猪流行性腹泻基因工程疫苗的研制奠定基础。     

猪流行性腹泻病毒单克隆抗体及其抗原表位的研究进展

猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(PED virus,PEDV)引起的一种严重危害养猪生产的常见疫病。近年来,由于新的PEDV变异毒株的出现,许多国家的养猪业遭受了巨大的经济损失。PEDV也因此受到更多关注,关于PEDV的研究报道也日渐增多。基于国内外有关PEDV的最新研究进展,本文系统归纳和分析了PEDV结构蛋白和非结构蛋白单克隆抗体以及单克隆抗体识别的特异性抗原表位,以期为开发鉴别诊断方法和表位疫苗等提供信息。     

Complete Genome Sequence of a Vero Cell-Adapted Isolate of Porcine Epidemic Diarrhea Virus in Eastern China

In early 2012, a widespread porcine epidemic diarrhea virus (PEDV) occurred in eastern China. A cell-adapted isolate, SD-M, was at the four-passage level of virulent field strain SD, which was isolated from a 2-day-old dead suckling piglet that had suffered from severe diarrhea in Shandong Province, China. We report here the complete genome sequence of SD-M. This sequence will promote a better understanding of the molecular pathogenesis of PEDV.     

Complete Genome Sequence of Porcine Epidemic Diarrhea Virus Strain AJ1102 Isolated from a Suckling Piglet with Acute Diarrhea in China

A diarrhea outbreak caused by porcine epidemic diarrhea virus (PEDV) has been observed in China since December 2010. We report here the complete genome sequence of PEDV strain AJ1102 isolated from a suckling piglet with acute diarrhea, which will help toward understanding the molecular and evolutionary characteristics of the epidemic PEDV in China.     

Evaluation on the Efficacy and Immunogenicity of Recombinant DNA Plasmids Expressing Spike Genes from Porcine Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus

Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PDEV) can cause severe diarrhea in pigs. Development of effective vaccines against TGEV and PEDV is one of important prevention measures. The spike (S) protein is the surface glycoprotein of TGEV and PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In this study, the open reading frames of the TGEV S1 protein and in addition of the S or S1 proteins of PEDV were inserted into the eukaryotic expression vector, pIRES, resulting in recombinant plasmids, pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Subsequently, 6–8 weeks old Kunming mice were inoculated with both DNA plasmids. Lymphocyte proliferation assay, virus neutralization assay, IFN-γ assay and CTL activity assay were performed. TGEV/PEDV specific antibody responses as well as kinetic changes of T lymphocyte subgroups of the immunized mice were analyzed. The results showed that the recombinant DNA plasmids increased the proliferation of T lymphocytes and the number of CD4+ and CD8+ T lymphocyte subgroups. In addition, the DNA vaccines induced a high level of IFN-γ in the immunized mice. The specific CTL activity in the pIRES-(TGEV-S1-PEDV-S) group became significant at 42 days post-immunization. At 35 days post-immunization, the recombinant DNA plasmids bearing full-length S genes of TGEV and PEDV stimulated higher levels of specific antibodies and neutralizing antibodies in immunized mice.