Characterization of bovine ileal epithelial cell line for lectin binding,susceptibility to enteric pathogens,and TLR mediated immune responses

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.     

鸡流行性腹泻病原的分离鉴定及特性研究

用胰蛋白酶处理发病鸡的粪便和肠内容物，接种Marc145细胞，盲传数代后，从山东不同地区发生流行性腹泻的鸡群中分离到轮状病毒，并对分离的轮状病毒的生物学特性和理化特性进行了部分研究。结果表明病毒粒子的形态呈车轮状、大小为70-80nm；其病毒基因组的电泳图谱为5：1：3：2；病毒在Marc145细胞上传到第9代时的TCID50为10^-4.62／0．1mL；该分离株病毒对氯仿、乙醚有抵抗力；对pH3．0处理60min稳定；50℃ 30min能使其感染力下降10^2；1mol／L MgCl2不能增强其对50℃ 60min的抵抗力。动物回归试验中接种两周龄SPF鸡，24h后陆续发病，表现为持续性水样腹泻，与自然发病相同；剖检可见病鸡脱水、小肠内有大量的液体和气泡、肠粘膜变薄；组织学变化为肠绒毛上皮坏死、脱落，绒毛平均长度减少而隐窝深度增加，固有层中淋巴细胞浸润。其临床症状及病理组织学变化与自然发病相同。因此确定发生在山东鸡流行件腹泻的病原为轮状病毒．     

Rotavirus infection in calves in Bangladesh

Faecal samples from 434 calves under 1 year of age (307 diarrhoeal and 127 normal) were collected from three dairy farms and one village in selected areas of Bangladesh. The samples were tested by an enzyme-linked immunosorbent assay (ELISA) to detect the presence of rotavirus antigen. Of 402 dairy calves tested, 28 (7.0%) were positive, of which 21 (7.2%) were from diarrhoeic calves and 7 (6.3%) from non-diarrhoeic calves. Rotavirus infection varied from farm to farm (2.7–9.2%) and there was no positive response from any of the 32 village calves. Rotavirus was most commonly found in calves of 1 week of age or less (up to 22.2% in one group) but was not found in any calves later than 6 months of age. More than 80% of rotavirus-positive samples from diarrhoeic calves exhibited a titre of 128 or more (geometric mean 345±4.5), whereas non-diarrhoeal calves had titres less than or equal to 128 (geometric mean=29±1.9), suggesting that rotavirus infection in calves in Bangladesh was mostly associated with diarrhoea.     

检测猪流行性腹泻病毒的R-PCR方法的建立

根据猪流行性腹泻病毒 (PEDV)的N基因自行设计和合成了一对可扩增长度为 641bp目的片段的引物 ,成功地建立了检测的猪流行性腹泻病毒的RT PCR方法。对猪轮状病毒 (PRV)、猪传染性胃肠炎病毒 (TGEV)的RT PCR检测结果均呈阴性。对PEDV JS株的RT PCR产物的序列分析表明 ,与CV777株的同源性为 97 3 %。     

反向遗传技术在牛轮状病毒研究中的应用

本文综述了牛轮状病毒的生物学特性、反向遗传学系统的建立及其研究进展,并对反向遗传技术应用于牛轮状病毒进行展望。     

三种融合标签对大熊猫轮状病毒结构蛋白VP6-VP7可溶性表达的影响

大熊猫轮状病毒(giant panda rotavirus,GPRV)是引起幼龄大熊猫腹泻的主要病原,对圈养大熊猫产生了较大的危害。轮状病毒结构蛋白VP6是一种载体蛋白,可介导黏膜免疫反应。VP7是轮状病毒结构蛋白中主要的中和抗原。因此,VP6-VP7的融合表达作为候选抗原对该病的防治具有重要的意义。传统大肠埃希菌原核表达存在表达量低、可溶性差以及纯度低等弊端。本研究使用醛缩酶(EDA)、谷胱甘肽S-转移酶(GST)、麦芽糖结合蛋白(MBP)3种融合标签,以实现获得表达量高和纯度高的GPRV-VP6-VP7重组表达蛋白。将扩增的VP6、VP7基因片段利用同源重组酶构建到含3种融合标签的表达载体pET21b上,将重组质粒转化至大肠埃希菌Rosetta(DE3)感受态细胞中进行低温诱导表达。用Ni-柱亲和层析法纯化目的蛋白,SDS-PAGE和Image J分析蛋白表达量和可溶性,Western blot分析得到表达的重组表达蛋白正确且具有蛋白活性。实验结果证明,EDA标签能显著促进VP6-VP7蛋白的原核可溶性表达,提高VP6-VP7蛋白表达量。     

Sequence Analysis and Prokaryotic Expression of NSP1 Gene of Bovine Rotavirus UK Strain

Rotavirus is a major cause of acute diarrhea in both many kinds of young animals and children under 5 years old.Rotavirus NSP1, a 55 ku RNA binding protein, is the product of gene 5, which can subvert innate immune responses and be one of virulent determinant factors.According to the sequence in GenBank, specific primers targeting to NSP1 gene were designed and the gene was amplified by RT-PCR, following by being cloned into the pET-28a(+) vector.It showed that the full length of NSP1 gene was 1 473 bp, encoding 491 amino acids.The NSP1 shared the highest identity with WC3 strain.The recombinant protein was induced in E.coli Rosetta(DE3) by IPTG and was analyzed by SDS-PAGE and Western blotting.The results revealed that NSP1 recombinant protein existed in the form of inclusion body with the molecular weight of 55 ku.The purified recombinant protein could be recognized by His-tag antibody.This study laid the foundation for further research on the relationship between the intracytoplasmic location of NSP1 protein and its activity.     

Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.     

An Outbreak of Diarrhoea in One-week-old Piglets Caused by Group A Rotavirus Genotypes P[7],G3 and P[7],G5

Thirty-two group A isolates of rotavirus detected in faecal samples from diarrhoeic piglets, were selected for P and G genotyping using a Multiplex RT-PCR. Ten isolates, from animals less than 8 days old, characterized an outbreak of diarrhoea caused by group A rotavirus in animals. P[7],G3 (CRW8-like) and P[7],G5 (OSU-like) genotypes were detected in 5 animals each. Isolates of a group A rotavirus of genotypes compatible with the OSU prototype were those most frequently identified in single infections in older animals (20/32 strains). In addition to these, 20 isolates from piglets with diarrhoea caused by group A rotavirus, collected between May 1998 and June 1999, but not from the outbreak month, were analysed. These isolates were used to compare the types observed on the farm outside the outbreak in May 1999 and the CRW8-like genotype was found in none of these faecal samples. P[7],G5 was the most frequent genotype (10/20 strains). No outbreak of diarrhoea caused by rotavirus in 1-week-old piglets was found in any other period during the 13 months of this study.     

A群猪轮状病毒JS株vp7基因序列分析

根据已发表的猪轮状病毒OSU毒株vp7基因核苷酸序列ORF两端保守区序列,设计一对特异引物,以猪轮状病毒JS毒株反转录cDNA为模板,通过PCR方法扩增出长约1000 bp目的片段。将其进行T-A克隆、序列测定和分析。结果表明,vp7基因全长1062 bp,含有一个981 bp的开放阅读框,编码326个氨基酸。与已知的15个毒株vp7全长基因的核苷酸及推导的氨基酸序列比较,同源性分别为74.5%～78.5%和75.2%～83.1%,核苷酸系统发育进化树结果表明,JS毒株与轮状病毒G9型参考毒株ICB2185、O-1亲缘关系较近,分为一个群,表明JS毒株血清型为G9型。目前,我国尚未见猪及其它动物轮状病毒G9型流行株的报道。