猪源脑心肌炎病毒GXLC株对小鼠的致病性试验

为研究猪源脑心肌炎病毒(EMCV)GXLC株对小鼠的致病性,将其接种4周龄健康BALB/c小鼠,观察临床症状,检查病理变化,测定心重/体重比值,检测心脏、脑、脾脏组织及血清中病毒含量,检测心脏、脑、脾脏组织中细胞因子IL-1β、TNF-α、IL-6 mRNA表达水平及血清中IL-1β、TNF-α蛋白含量。结果显示,感染小鼠出现明显的临床症状,感染后第4天开始死亡,第5~7天死亡达到高峰;心脏、脑出现典型的大体病理变化和组织病理学变化,心重/体重比值显著升高、组织病理学炎症分值显著增加;感染后第1天即可在心脏、脑、脾脏及血清中检测到大量病毒,直至第14天仍能检测到病毒;血清中IL-1β、TNF-α蛋白含量显著升高,心脏、脑、脾脏组织中IL-1β、TNF-α、IL-6 mRNA表达水平显著上调,而且细胞因子表达的高峰期与出现明显临床症状、严重病理损害及死亡高峰期密切相关。结果表明,猪源EMCV GXLC株对小鼠具有高度致病性,IL-1β、TNF-α、IL-6等促炎细胞因子在EMCV对小鼠的致病机制中发挥重要作用。     

猪脑心肌炎病毒GXLC株的分离及其3D基因分子特征的分析

采集临床疑似脑心肌炎死亡仔猪的组织作为接种材料,接种于BHK-21细胞系,观察细胞病变(CPE),并用RT-PCR和间接荧光抗体试验(IFA)进行鉴定,证实分离到1株脑心肌炎病毒(encephalomyocarditis virus,EMCV),命名为GXLC株。应用RT-PCR方法扩增GXLC株的3D基因,扩增产物克隆入pMD18-T载体后进行测序,对获得的3D基因序列进行分析。序列分析结果表明,GXLC株3D基因全长1380 nt,编码460个氨基酸,含有7个抗原表位。同源性分析结果表明,GXLC株与国内外其它EMCV分离株3D基因核苷酸序列的同源性在84.7%~99.7%之间,氨基酸序列的同源性在96.1%~99.6%之间。遗传进化分析结果表明,基于3D基因核苷酸序列绘制的系统进化树可将所有EMCV分离株分成2个群:Ⅰ群和Ⅱ群,Ⅰ群可再细分为Ⅰa亚群和Ⅰb亚群,其中猪源EMCV在Ⅰ群和Ⅱ群中均有分布,而鼠源EMCV分布在Ⅰ群,人源EMCV分布在Ⅱ群;GXLC株与其它中国分离株均属于Ⅰa亚群。     

猪脑心肌炎病毒感染对iNOS mRNA转录水平的影响

为研究iNOS在猪脑心肌炎病毒(EMCV)致病机制中的作用,本研究将猪源EMCV GXLC株人工感染小鼠、仔猪,观察临床症状和病理变化,检测心、脑组织中iNOS mRNA转录水平。试验结果表明,小鼠和仔猪感染EMCV后均出现明显的临床症状、剖检及组织病理学变化显示心、脑组织的病理炎症分值显著增加;心、脑组织中的iNOS mRNA转录水平显著上调,并且转录水平上调的峰值与感染小鼠、仔猪出现明显临床症状、严重病理损害及小鼠死亡高峰存在明显的时间相关性,与心、脑组织的病理炎症分值成线性正相关。表明组织中iNOS的过量表达与感染动物的发病和死亡密切相关,iNOS在EMCV致病机制中发挥重要作用。     

猪脑心肌炎病毒抗体间接ELISA方法的建立及应用

应用纯化的EMCV VP1重组蛋白建立了检测EMCV抗体的间接ELISA方法,并确定了间接ELISA的最佳工作条件。结果表明,重组蛋白抗原的最适包被浓度为0.54μg/mL;与间接免疫荧光试验比较,结果表明该方法具有良好的特异性和敏感性。此方法中VP1抗原最佳稀释度为1:100,待检血清的最佳稀释度为1:50,酶标二抗的最佳稀释度为1:800,1%明胶为封闭液,OD450时阴阳性血清的临界值为0.35。对2006年天津7个地区66个猪场295份屠宰猪的血清样本的检测结果显示,天津各地区血清EMCV抗体阳性率在41.67%～93.33%之间,平均84.75%,猪场抗体阳性率在50%～100%,平均93.94%。     

猪脑心肌炎病毒2A蛋白的原核表达与纯化

本试验对猪脑心肌炎病毒（encephalomyocarditis virus,EMCV）2A蛋白进行原核表达和纯化。采用大肠杆菌原核表达系统,将EMCV 2A基因插入原核表达载体pET-28a-sumo中构建重组原核表达质粒pET28a-EMCV-2A,经PCR和测序鉴定无误。将重组原核表达质粒转化至大肠杆菌BL21（DE3）感受态细胞中,超声破碎后,2A蛋白的表达主要以包涵体形式存在。通过优化蛋白表达条件,使目的蛋白能够实现可溶性表达。利用磁珠纯化方法对重组蛋白进行纯化,并以SDS-PAGE和Western blotting进行双重鉴定。结果显示,本试验成功构建2A的重组表达质粒;重组蛋白分子质量约为25 ku,与预期大小一致;IPTG浓度1.0 mmol/L、16 ℃低温诱导16 h为最佳诱导条件,约有50%的蛋白呈现可溶性表达;纯化后获得2A重组蛋白。本试验通过对EMCV 2A蛋白的原核表达及纯化,成功获得纯度较高的EMCV 2A蛋白,为进一步阐明EMCV的分子致病机制以及研发基因疫苗和抗病毒药物奠定基础。     

Prokaryotic Expression and Purification of 2A Protein of Porcine Encephalomyocarditis Virus

In this study,encephalomyocarditis virus 2A protein was prokaryotic expressed and purified.E.coli was used to express foreign proteins,the EMCV 2A gene was inserted into the prokaryotic expression vector pET-28a-sumo to obtain the recombinant plasmid pET28a-EMCV-2A and confirmed by PCR and sequencing.The plasmid was transformed into E.coli BL21(DE3).After ultrasound fragmentation,the expression of 2A protein was mainly in the form of inclusion bodies.By optimizing the protein expression conditions,the soluble expression of the target protein could be achieved.The recombinant protein was purified by beads and identified by SDS-PAGE and Western blotting.The results showed that the recombinant plasmid was successfully constructed.The molecular weight of the recombinant 2A protein was about 25 ku.The best condition was 16 ℃ with 1.0 mmol/L IPTG,about 50% of the proteins were soluble.The purified recombinant protein was double-stained by SDS-PAGE and Western blotting,and indicated that the purified protein was the recombinant protein.The study provided the basic theoretical basis for elucidating the molecular pathogenesis of EMCV and preparation of gene vaccines and antiviral drugs.     

猪脑心肌炎病毒RT—LAMP检测方法的建立

针对猪脑心炎病毒（Encephalomyocarditis virus,EMCV）3D基因保守序列的6个特异性部位设计了2对引物,利用BstDNA聚合酶,63℃恒温保持50min即可完成反转录与扩增反应,产物中加入SYBR GreenI染料,在紫外光下可直接观察判定扩增结果,试验成功建立了猪EMCV的RT-LAMP检测方法。结果表明：该方法灵敏度比RT-PCR高100倍,具有良好的重复性和稳定性,而且对其他病毒均无扩增结果,可作为猪EMCV的快速诊断方法。     

猪脑心肌炎病毒和猪繁殖与呼吸综合征病毒二重RT-PCR检测方法的建立

根据猪脑心肌炎病毒(EMCV)3D基因和猪繁殖与呼吸综合征病毒(PRRSV)N蛋白基因序列设计合成了两对特异性引物,经过PCR反应条件的优化,建立了EMCV和PRRSV二重RT-PCR方法,其PCR产物大小分别为286 bp和390 bp,并具有EMCV和PRRSV特征序列。该方法对EMCV和PRRSV的最低检测量分别为10×TC ID50和1×TC ID50;而对乙型脑炎病毒、猪瘟病毒、猪细小病毒、猪圆环病毒2型、猪伪狂犬病毒的PCR检测结果均为阴性;20份临床发病仔猪样品检测结果为PRRSV阳性者15份,EMCV阳性者3份,证明我国存在EMCV感染。该方法敏感、特异,可用于EMCV和PRRSV感染的检测。     

Encephalomyocarditis virus infection of captive elephants.

Four African elephants at each of 2 widely separated zoologic gardens in Florida died following a fulminating illness. Tissue suspensions obtained from an elephant from each of the zoologic gardens were inoculated into newborn mice, 3- to 4-week-old mice, and buffalo green monkey and baby hamster kidney cell cultures. Encephalitis and myocarditis developed in the mice. The cell cultures were destroyed within 24 to 72 hours, and intracytoplasmic viral inclusions were observed in infected cells by electron microscopy. The viral agent was neutralized by known antiserum to encephalomyocarditis virus.     

猪病毒性脑心肌炎流行病学特点与防治

猪病毒性脑心肌炎是由脑心肌炎病毒感染引发的一种病毒性肠炎,不同年龄的猪受到病毒侵染后,所表现的临床症状存在一定差异性,其中对仔猪造成的易感性最强,具有很高传染性和致死率。妊娠阶段母猪受到病毒侵染后,表现为突然流产并会影响妊娠母猪的繁殖机能,导致繁殖母猪发情无规律,不能正常发情,多次配种不能正常受孕。防范该种疾病发生流行,需要掌握猪病毒性脑心肌炎的流行病学,指导养殖户强化养殖管理,转变传统养殖方法,提高猪群抗病能力,降低发病率,保护易感群体。该文主要分析猪病毒性脑心肌炎的流行病学和防治措施。     

猪脑心肌炎病毒NJ08株基因组序列测定与分析

为测定猪源脑心肌炎病毒(EMCV)NJ08分离株全基因序列,本研究应用RT-PCR方法分5个基因片段扩增NJ08分离株的基因组,同时应用3'-RACE和5'-RACE技术扩增3'-UTR和5'-UTR,分别克隆于pMD18-T载体中进行测序,获得EMCV NJ08株全基因组cDNA序列,并将全基因组及其推导的氨基酸序列与GenBank中登录的国内外参考病毒株进行同源性比较及系统进化分析.结果表明:EMCV NJ08分离株基因组全长7 724bp;属于Ia亚群;与GXLC、GX0602、BJC3、HB1等国内分离株以及BEL-2887A、CBNU、K3、K11、EMCV-R、PV21等国外分离株同源性达99%以上;EMCV流行株的非结构蛋白中3D最为保守,2A变异最大,结构蛋白中VP2最为保守,VP1变异最大.本研究丰富了我国EMCV分子流行病学资料.     

猪脑心肌炎病毒非结构蛋白3AB基因在重组杆状病毒中的表达与鉴定

利用PCR方法扩增获得EMCV非结构蛋白3AB基因,并将其克隆至杆状病毒转座载体pFastBacTM中,提取阳性克隆质粒转化至DH10Bac感受态细菌,通过蓝白斑筛选和PCR鉴定,得到重组杆状病毒穿梭载体Bacm id-3AB,经脂质体介导转染sf9细胞获得重组杆状病毒,并IFA和W estern b lotting鉴定其抗原性。结果表明,EMCV 3AB基因在昆虫细胞中获得成功表达,分子量约16 ku,具有良好的EMCV抗原性。因此利用杆状病毒表达系统成功表达了EMCV 3AB基因,为该病毒抗原表位和诊断方法研究奠定了重要基础。     

Human respiratory syncytial virus infection in the pre-clinical calf model

Human respiratory syncytial virus (hRSV) is the most important respiratory pathogen in young children worldwide. Experimental modelling of hRSV disease by bovine RSV (bRSV) infection in calves provides an important tool for developing new strategies for prevention and treatment. Depending on the scientific hypothesis under investigation, this cognate host-virus model might have the disadvantage of using a highly related but not genetically identical virus. In this study, we aim to describe viral kinetics and (clinical) disease characteristics in calves inoculated with hRSV. Our results show that hRSV infects the upper and, to a lesser extent, the lower respiratory tract of calves. Infection causes upper airway clinical disease symptoms and neutrophilic infiltration of the lower airways. We conclude that a hRSV model in calves may aid future research involving distinct scientific questions related to hRSV disease in children.     

Molecular characterization of bovine foamy virus and its association with bovine leukemia virus infection in Vietnamese cattle

The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This study provides information regarding BFV infection and confirms the existence of two BFV clades among Vietnamese cattle for the first time.     

规模化猪场脑心肌炎病毒感染的血清学调查

脑心肌炎病毒(EMCV)可引起猪的脑炎、心肌炎和母猪繁殖障碍,该病已在世界上多个国家暴发和流行。我国对EMCV及其所致疾病的研究工作还处于起步阶段,为了揭示EMCV在我国规模化猪场的感染情况,我们应用酶联免疫吸附试验(ELISA)对2005-2006年间采集自全国13个省市46个规模化猪场的3250份血清样本进行了EMCV抗体检测,结果显示,各省阳性率在39.64%~90%之间,平均抗体阳性率为72%,监测的46个猪场都存在EMCV感染,猪场感染阳性率为100%;对不同生长阶段猪群检测结果表明,EMCV抗体阳性率随猪龄的增长而升高,且不同生长阶段猪抗体阳性率差异极显著;成年公猪的抗体阳性率高于成年母猪。本项调查表明,我国规模化猪场已经普遍感染EMCV,这应引起我国养猪业警惕,并做好该病的综合防控。