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。     

微生态制剂联合小儿康治疗儿童迁延性腹泻机制研究

目的:探讨微生态制剂思连康联合小儿康治疗儿童迁延性腹泻的疗效,并进一步探讨其机制.方法:我院共收治60例腹泻患儿,随机分为两组,均在常规治疗基础上治疗组加用思连康联合小儿康,对照组加用杜拉宝治疗儿童腹泻.观察两组的疗效,以及两组临床症状的好转情况及住院时间的比较.酶联免疫吸附双抗体夹心法(ELISA法)检测两组患者血清和粪便白介素6和8(IL-6和IL-8)的变化.结果:治疗组总有效率为90％,高于对照组(P＜0.05);临床症状的好转情况及住院时间的比较优于对照组(P＜0.01).治疗后血清及大便细胞因子IL-6和IL-8均有降低,并优于对照组(P＜0.05).结论:思连康联合小儿康治疗儿童迁延性腹泻,疗效显著,值得广泛推广.其机制可能与下调炎性细胞因子有关.     

猪流行性腹泻病毒诱导的猪肠道-乳腺-sIgA轴以及免疫机制探讨

猪的“肠道-乳腺-sIgA轴”免疫通路是指侵染猪的胃肠道病原通过胃肠道免疫可以激发乳腺产生sIgA;sIgA被初生仔猪摄取可以获得针对胃肠道病原的被动免疫保护。该免疫通路的反应动力模式涉及病原侵染、抗原提呈、淋巴细胞活化、淋巴细胞的肠道和乳腺归巢以及抗体分泌等诸多环节,受到病原毒力、母猪的妊娠阶段及免疫生理状态等众多因素影响。目前,猪流行性腹泻病毒（Porcine Epidemic Diarrhea Virus,PEDV）诱发的“肠道-乳腺-sIgA轴”的理论可以解释自然感染状态下哺乳仔猪获得的被动免疫保护,但根据这一概念所设计的疫苗和免疫方案并未取得满意效果。本文综述了PEDV感染和宿主免疫各个环节的研究现状,分析了影响PEDV免疫和肠道-乳腺-sIgA轴系反应的关键病原和宿主因素,提出了在轴系理论基础上应重视PEDV灭活疫苗以及特异IgG作用的建议。     

Genomic characterization of antibiotic resistance-encoding genes in clinical isolates of Vibrio cholerae non-O1/non-O139 strains from Kolkata,India: generation of novel types of genomic islands containing plural antibiotic resistance genes

Non-O1/non-O139 nontoxigenic Vibrio cholerae associated with cholera-like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non-O1/non-O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole-genome sequences of these strains were determined. From the analysis of these sequences, it becomes clear that all quinolone resistance isolates have mutations in quinolone resistance-determining regions. Further research on the genome sequence showed that four strains possess Class 1 integrons in their genomes, and that three of the four integrons are found to be located in their genomic islands. These genomic islands are novel types. This indicates that various integrons containing drug resistance genes are spreading among V. cholerae non-O1/non-O139 strains through the action of newly generated genomic islands.     

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.     

Identification and Characterization of Heat-Stable Enterotoxin II-Producing Escherichia coli from Patients with Diarrhea

Stock strains of Eschericia coli isolated from patients with traveller's diarrhea were examined for production of heat-stable enterotoxin II (STII). Of 400 strains examined, 3 were found to produce STII. The nucleotide sequence of the STII gene of these human strains was shown to be identical to that of porcine strains. Cultured cells of these strains induced fluid accumulation in ligated mouse intestinal loops and the activity was neutralized by anti-STII antiserum. These results suggest that STII-produciing enterotoxigenic E. coli can cause human diarrhea.     

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。