沙市婴幼儿腹泻中检出3株C群轮状病毒

1987～1988年在沙市165份婴幼儿急性腹泻标本中,用PAGE法检出轮状病毒37株(22.4％),其中3株为少见的轮状病毒,此种病毒经电镜观察,具有典型轮状病毒的形态结构,ELISA证实该病毒不具有A群和B群轮状病毒的群特异性抗原。RNA电泳分析表明,其基因组由11个双链RNA片段组成,电泳图型特殊,呈4:3:2:2的排列模式。上述试验表明,该病毒为世界上罕见的C群轮状病毒。免疫电镜证实,该病毒能被病人恢复期血清所凝集,提示该病毒是腹泻病儿的致病因子。     

从绵羊羔和山羊羔腹泻粪样中检出B组轮状病毒

用聚丙烯酰胺凝胶电泳法,从新疆巴里坤县的22份绵、山羊羔腹泻粪样中,检出11份具有B组轮状病毒的RNA电泳图型,联合电泳表明,绵羊羔与山羊羔的B组轮状病毒电泳图型完全相同,电镜观察表明,该病毒与典型轮状病毒(A组)具有相同的形态特征,但易降解破碎,口服接种剥夺初乳的绵羊羔和山羊羔,经11～13小时羔羊发生急性水样腹泻,并排出大量病毒,经A组轮状病毒组抗原ELISA检测,不具有共同组特异抗原,但经对流免疫电泳检测,它与成人腹泻轮状病毒(B组)具有共同组特异抗原,从而证实我国存在与国外报道相同的羊B组轮状病毒,既感染绵羊羔也感染山羊羔。是引起绵,山羊羔急性腹泻的不可忽视的病原之一。     

成人腹泻轮状病毒的提纯及其高价免疫血清的制备

本文采用交叉免疫电泳技术,首先提纯了成人腹泻轮状病毒(Adult DiarrhoeaRotavirus简称:ADRV),并经电镜确认,用作免疫抗原,首次制备了高价兔与豚鼠抗ADRV血清和小鼠抗ADRV腹水,其特异性经交叉免疫电泳和对流免疫电泳鉴定,其效价经酶联免疫吸附试验(ELISA)检测均在1:2,000以上。     

仔猪副轮状病毒(C群)的检出

利用聚丙烯酰胺凝胶电泳法,从某猪场的20份仔猪腹泻标本中检出具有副轮状病毒(C群)RNA电泳图型者11份,其检出率高达55％。该病毒经口服接种未吮初乳的新生仔猪,可致急性水样腹泻,并排出大量病毒。经轮状病毒(A群)ELISA和副轮状病毒(B群)对流免疫电泳检测证明,它与轮状病毒(A群)和副轮状病毒(B群)均无血清学交叉。本文首次证实在我国猪群中存在有与国外报道的相同的猪副轮状病毒(C群),且可引起仔猪爆发流行,是仔猪腹泻不可忽视的病原之一。     

成人腹泻轮状病毒ELISA方法的建立和应用

本文通过特异性试验、阻断试验、交叉试验、敏感度试验和重复性试验,建立了成人腹泻轮状病毒一酶联免疫吸附试验法(ADRV—ELISA)。应用此法检测了全国20多个省区202份病人腹泻标本,检出率为91％。采用本ELISA、核酸电泳、电镜三种方法对48份病人腹泻标本进行了双盲法检测比较,结果三种方法的阳性检出率分别为100％、85.4％、56.25％(P<0.05)。实验结果表明,本ELISA应用于检测成人腹泻轮状病毒(ADRV),具有敏感度高。特异性强等优点。     

小儿副轮状病毒的检出

用聚丙烯酰胺凝胶电泳法,从婴幼儿急性腹泻粪便中检出轮状病毒RNA电泳阳性293份,发现一株副轮状病毒(青-27株),此株病毒经电泳观察,呈典型的轮状病毒形态,但易破碎。ELISA检测表明不具有一般轮状病毒的特异性群抗原,病毒RNA基因组由11个片段组成,但电泳图型特殊,吴4:3:2:2排列模式,本文证实,此一小儿副轮状病毒与国外报道的,散发罕见的小儿副轮状病毒RNA电泳图型相同,提示此病毒的重要意义。     

婴幼儿轮状病毒腹泻的研究进展

1973年澳大利亚学者R．F．Bishop在电镜下于急性腹泻患儿十二指肠黏膜活检标本中,发现上皮细胞内存在大量球形病毒颗粒,根据其类似车轮状形态,1975年命名为“轮状病毒”（Rotavirus,RV）。该病毒是引起婴幼儿腹泻的主要病原之一,发病高峰在秋季,故又名“婴幼儿秋季腹泻”。     

聚丙烯酰胺凝胶电泳(PAGE)法诊断轮状病毒腹泻的研究

轮状病毒是致人及动物腹泻的一个重要病原,从Bishop等用电镜从粪便中发现了该病毒以来,相继出现了许多有效的检测方法。由于轮状病毒的基因组是具有11节段的双股RNA,因此可用聚丙烯酰胺凝胶电泳(PAGE)法进行检测。轮状病毒的RNA在电泳中形成了11个片段的迁移区带图,称为电泳型(electropherotype),不同种型的轮状病毒其电     

2010-2013年上海长宁地区成人轮状病毒急性胃肠炎的分子特征

为研究上海长宁地区成人轮状病毒急性胃肠炎的分子特征,收集2010年6月-2013年12月长宁区定点医院成人急性胃肠炎患者的粪便标本1 554份,采用酶联免疫吸附试验(enzyme-linked immunosorbentassay,ELISA)进行A组轮状病毒检测,多重聚合酶链反应(polymerase chain reaction,PCR)进行B组和C组轮状病毒检测.结果显示,1 554例患者的平均年龄为(46.19±15.59)岁,其中男性691例、女性863例.共检出轮状病毒200株,检出率为12.87％.18～39岁组的轮状病毒检出率为9.15％,40～59岁组为15.58％,60～79岁组为15.16％,≥80岁组为18.57％.A组轮状病毒基因型检测结果显示,G型以G9亚型最多(29.82％),P型以P[8]亚型最多(77.19％),GP组合以G9P[8]最多(25.73％).结果提示,2010-2013年上海长宁地区成人轮状病毒急性胃肠炎在就诊患者中以中老年多见,基因型以G9P[8]最多.     

哈尔滨地区婴幼儿腹泻轮状病毒优势株的变化

本文对1988—1989年哈尔滨地区婴幼儿腹泻进行了轮状病毒核酸电泳型调查,并于1984—1985年资料比较,以探明这一地区轮状病毒流行优势株的变化。 标本:婴幼儿腹泻粪便标本采自哈尔滨医科大学附属二院儿科及哈尔滨红十字儿童医院。病毒RNA提取:参考Herring方法。聚丙烯酰胺电泳(PAGE):参考Uaemmli方法。 结果:123份急性婴幼儿腹泻粪便标本PAGE检测,47例呈现轮状病毒RNA图型,阳性率为38.2％。电泳图型的基本模式为4:2:3:2,表明均为A组轮状病毒。根据各片段的迁移位置差异,共可见9种不同的电泳型,其中短型3例,占6.4％,含2个电泳型;长型44例,     

Four nucleotides are the minimal requirement for RNA recognition by rotavirus non-structural protein NSP3.

The interaction of the group A rotavirus non-structural protein NSP3 (NSP3A) with RNA has been studied in vitro. Using semi-purified NSP3A protein expressed by a recombinant baculovirus and in vitro synthesized RNA, we determined by UV cross-linking and gel retardation assays that NSP3A binds, in a sequence-specific manner, the consensus sequence (AUGUGACC) present on the 3' ends of all group A rotavirus mRNAs. Using short oligoribonucleotides, we established that the minimal RNA sequence required for binding of NSP3A is GACC. Modifications of the UGACC oligonucleotide sequence impaired binding of the protein to the RNA. Furthermore, the recombinant NSP3 protein from rotavirus group C showed specificity for the 3' end consensus sequence (AUGUGGCU) of only group C mRNAs. Sequence analysis of the NSP3 proteins did not reveal significant homologies with other RNA binding proteins, thus the NSP3 proteins of rotaviruses are the prototypes of a new kind of sequence-specific RNA binding protein.     

Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction

The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a "bristled" outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a "core" region contains a third, nonspherical shell of density at a mean radius of 225 A that encapsidates the double-stranded RNA genome; and (f) the space between the outer and inner capsids forms an open aqueous network that may provide pathways for the diffusion of ions and small regulatory molecules as well as the extrusion of RNA. The assignment of different viral structural proteins to specific features of the reconstruction has been tentatively made on the basis of excluded volume estimates and previous biochemical characterizations of rotavirus.     

Comparison of enzyme-linked immunosorbent assay, electron microscopy, and reversed passive haemagglutination for detection of human rotavirus in stool specimens

An enzyme-linked immunosorbent assay (ELISA) using microplates as solid phase, rabbit antiserum against human rotavirus Wa strain as catching antibody, and the same reagent labeled with beta-D-galactosidase as conjugate, has been developed for detection of human rotavirus antigen(s) in stool specimens from patients with acute gastroenteritis. The limit of detection of purified human rotavirus by ELISA was 15.6 ng/ml (1.56 ng/well) of viral protein. The sensitivities of ELISA, electron microscopy, and the reversed passive haemagglutination method (ROTA-CELL) were compared. ELISA was more sensitive than electron microscopy and the reversed passive haemagglutination method. The ELISA blocking assay was useful for detection of an antibody response to human rotavirus in paired sera from children in two institutions during outbreaks of rotavirus gastroenteritis.     

Diagnosis of rotavirus, adenovirus, and herpesvirus infections by immune electron microscopy using a serum-in-agar diffusion method

The sensitivity of immune electron microscopy (IEM) for the detection and identification of bovine rotavirus, infectious bovine rhinotracheitis virus (IBR), and canine adenovirus has been studied by using the serum-in-agar (SIA) method in which a specific antiserum has been incorporated in agar.     

Localization of the 3' end of Escherichia coli 16 S RNA by electron microscopy of antibody-labelled subunits.

The 3′ end of 16 S RNA is localized on the 30 S subunit of Escherichia coli ribosomes by immune electron microscopy. It is located in the groove between the side “ledge” and the “head” of the subunit on the level of the ledge top. Thus, we have localized the 30 S subunit functional site which is believed to be responsible for binding of the specific messenger RNA sequence preceding the initiation codon. The localization of the 3′ end of 16 S RNA has been done by a new approach in immune electron microscopy. It is based on the covalent binding of low molecular weight ligands, containing the residue of phenyl-β-d-lactoside hapten, to certain points of RNA and the localization of the binding site of the antibody specific to this hapten by electron microscopy. The advantages of this approach in comparison with conventional methods of immune electron microscopy are discussed.     

轮状病毒基因重配LH9株(G4型)传代稳定性研究

对兰州生物制品研究所自行构建的轮状病毒基因重配株G4型LH9株在Vero细胞中传代的稳定性进行研究。将基因重配LH9毒株接种于Vero细胞上从7代传至17代,经电镜检查、病毒滴度测定、病毒基因组RNA电泳图谱分析、基因序列测定分析、轮状病毒型别鉴定及其它相关检定,结果均符合药典要求。证实该毒株为A群G4P〔12〕型轮状病毒,在Vero细胞传代中具有良好的稳定性,为疫苗研制提供安全有效的依据。     

Serial observations of chronic rotavirus infection in an immunodeficient child.

Chronic rotavirus infection of an infant with severe combined immunodeficiency (SCID) was studied by virological examinations in association with long-term observation of his symptoms and immune status. During eleven months of hospitalization, the patient was suffering from incurable severe diarrhea with persisting excretion of rotaviruses detected by electron microscopy and the reversed-passive hemagglutination (R-PHA) test and had transient hepatitis symptom despite multiple administrations of human gammaglobulin and high calorie fluids. The detected viruses were morphologically recognized as rotavirus with double capsid structure. Polyacrylamide gel electrophoretic (PAGE) analysis of their genomic RNAs showed the long electropherotype of group A virus with abnormal migration profiles changing considerably from the early to the late phase of illness: (1) The 11th segment became undetectable; (2) the molecular weight of the 6th segment slightly increased; (3) seven to fourteen extra segments appeared; and (4) PAGE patterns of viral genomic RNAs changed every three or four months. These findings suggest that chronic infection with rotavirus accompanied the generation of extra viral genomic segments and their unusual assortments in an immunodeficient host.     

Thiazolides,a New Class of Antiviral Agents Effective against Rotavirus Infection,Target Viral Morphogenesis,Inhibiting Viroplasm Formation

Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC₅₀s) ranging from 0.3 to 2 μg/ml and 50% cytotoxic concentrations (CC₅₀s) higher than 50 μg/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis.