人轮状病毒的细胞培养分离

用CV-1细胞从急性腹泻病儿7份粪便标本中直接分离出4株人轮状病毒(HRV),并适应传代14代。感染细胞出现特征性细胞病变,经电镜、ELISA、免疫荧光染色及病毒RNA电泳等试验证实HRV毒株在CV-1细胞中的繁殖及抗原特异性。病毒滴度为10~(6.0)TCID_(60)。分离的4株HRV毒株均为RNA电泳型长型,经CV-1细胞传7～14代后,RNA图型与原粪样相比未见变异。     

Enzyme-linked immunosorbent assay employing monoclonal antibodies for direct identification of enteric adenoviruses (Ad40,41) in feces

For detection and identification of enteric adenovirus (Ad) types 40 and 41 in stool specimens, enzyme-linked immunosorbent assay (ELISA) was developed with the use of three monoclonal antibodies: Ad group-specific, Ad40 type-specific, and Ad41 type-specific antibodies. Of 860 fecal samples from patients with acute gastroenteritis, 44 strains of Ad were isolated using Graham 293 cell cultures. Of these isolates, 20 were typed as Ad40, 18 were Ad41, and 6 were other Ads by neutralization tests with cell cultures. Results of the ELISA tests on these 860 fecal samples resulted in good agreement to those with the cell culture method. The ELISA tests using Ad type-specific monoclonal antibodies proved to be a specific and rapid technique for laboratory diagnosis of acute gastroenteritis caused by enteric Ads.     

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。     

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.     

Serotype analysis of group A human rotavirus related to acute gastroenteritis in winter in Gifu city

A total of 348 fecal specimens collected from children with acute gastroenteritis in Gifu city over three consecutive winter seasons (from November 1986 to March 1989) were examined for group A human rotavirus (HRV) by either a commercial test kit or a sandwich enzyme-linked immunosorbent assay (sandwich ELISA). One hundred twelve of the 173 group A HRV-positive specimens were further subjected to serotype determination by ELISA with four serotype-specific monoclonal antibodies to VP7 (ELISA-serotyping). Ninety-one specimens (81.3%) were successfully serotyped: 41 (36.6%) were serotype 1, 13 (11.6%) serotype 2, 27 (24.1%) serotype 3, and 10 (8.9%) serotype 4. The serotypes of the remaining 21 (18.8%) could not be determined. The predominant serotype of HRV that prevailed in Gifu city changed every winter: serotype 3 (63.4%) was most prevalent in the 1st winter, serotype 4 (42.9%) in the 2nd winter, and serotype 1 (64%) in the 3rd winter.     

Identification of nonspecific reactions in laboratory rodent specimens tested by Rotazyme rotavirus ELISA

Fecal specimens from several laboratory animal species were tested for rotavirus antigen by Rotazyme II, a commercially available enzyme-linked immunosorbent assay (ELISA) that is widely used in human diagnostic studies. Fecal samples from rabbits, hamsters, guinea pigs, dogs, and cats tested negative; whereas those from rats and mice yielded a high proportion of positive results. Rats had the highest rate with 82% of the samples being positive. However, the presence of rotavirus in positive rodent samples could not be confirmed by virus isolation, electron microscopy or blocking ELISA using anti-EDIM mouse rotavirus serum. Several lines of evidence indicated that these positive reactions were false positives, apparently due to a non-specifically reacting substance in the diet of rats and mice. All the positive fecal samples were from rats and mice that had been fed nonautoclaved diet. Samples from rodents fed autoclaved diet were consistently negative in the Rotazyme test. When rats fed autoclaved diet were subsequently fed nonautoclaved diet, their stool converted from negative to positive within 6 hours. Conversely, rats with positive stool samples converted to negative within 15 hours when fed autoclaved diet. Similar results were found with mice. Positive fecal specimens and nonautoclaved rodent diet both contained a substance that apparently attached nonspecifically to the antibody coated beads used in the ELISA and reacted directly with the substrate in the absence of the conjugate. This substance was heat labile and trypsin sensitive, suggesting that it was a protein.     

Genotypes of rotavirus associated with acute gastroenteritis in Seoul, Korea

Acute viral gastroenteritis is one of the most common infectious diseases in infants and young children. Rotavirus is mainly important in childhood. The present study determined the detection rate, seasonality and G and P genotypes of rotaviruses in children hospitalized for acute gastroenteritis in Seoul, Korea in 2009. A total of 1,423 stool specimens were screened by ELISA for the presence of rotavirus antigens and the rotavirus-positive stools genotyped by RT-PCR. The G genotype was determined for 90% of samples (242/269) and the P genotype for 93.3% (251/269). During the study, 25 G-P combinations were detected with G1P[8] in 38.3% (n= 103) and G4P[6] in 5.9% (n= 16) cases. These data provided information on rotavirus in patients with acute gastroenteritis in Seoul, Korea and provided baseline data to motivate for the implementation of control measures for rotavirus disease.     

猪和牛轮状病毒的分离与鉴定

应用MA-104细胞、胰蛋白酶处理培养物和旋转培养技术,成功地从患腹泻仔猪和犊牛的粪便样品中,分离到6株猪轮状病毒和4株牛轮状病毒。经形态学、血清学和病毒RNA电泳分析等鉴定,确为典型轮状病毒。经MA-104细胞连续传代,这10株轮状病毒均成为细胞适应毒株,并在该细胞上产生明显的细胞病变。     

Use of monoclonal antibody blends in the identification of enteroviral aseptic meningitis

Monoclonal antibody pools directed against group B coxsackievirus and echovirus antigens (Chemicon, Temecula, California) were evaluated as tools in the identification of enteroviral aseptic meningitis. Cerebrospinal fluid (CSF) and serial dilutions of stock coxsackievirus were inoculated into tube and shell vial Rhesus monkey kidney (RhMk) cell cultures. Positive cellular fluorescence was observed only in conjunction with cytopathic effect (CPE). The time from inoculation to CPE was similar with both tube and shell vial cultures.Direct CSF testing failed to reliably identify positive specimens as fluorescent debris, and a lack of available cells hindered results.Viral components of each antibody blend demonstrated positive cellular fluorescence when appropriately stained. False-positive fluorescence was not observed when cells, infected with other CSF viruses, were stained with these reagents.The findings suggest a role for these reagents (available both as blends and type-specific reagents) in the culture confirmation and identification of many common enteroviral serotypes associated with aseptic meningitis.     

1999～2005年我国婴幼儿人杯状病毒腹泻研究

人杯状病毒(HuCV)是世界范围内急性腹泻的重要病原。此文收集我国长春、北京、河北卢龙、兰州、昆明等13个地区1999-2005年5岁以下腹泻患儿检测轮状病毒为阴性的粪便标本4426份,用ELISA和RT-PCR方法检测HuCV,结果表明HuCV检测阳性率为19%(8.3%-38.6%)。对其中151株HuCV PCR产物测序进行核苷酸序列比对构建的系统发生树表明,我国流行的HuCV以诺如病毒(NV)为主(146/151,占96.7%),其中绝大多数为GGⅡ/4基因型(99/146),其它依次为GGⅡ/3(22/146)、GGⅡ/5(8/146)、GGⅡ/6(2/146)、GGⅡ/7(2/146)、GGⅡ/8(2/146)和GGⅠ/2(2/146),札如病毒(SV)共5株,均属SGⅡ。此外有9株NV GGⅡ遗传组毒株,尚不能归入现有基因型,可能为我国特有的新基因型,表明我国流行的HuCV毒株存在很高的遗传多样性。我国婴幼儿HuCV腹泻全年都有发生,但秋冬季(10月-次年1月)有一个发病高峰,病例以6-17月龄组最多,至3岁时超过96%的儿童都感染过HuCV。对我国流行的HuCV不同毒株的地区分布,不同年度HuCV检测阳性率的变化趋势进行了讨论。     

Inhibitory effects of bile acids and synthetic farnesoid X receptor agonists on rotavirus replication

Rotaviruses (group A rotaviruses) are the most important cause of severe gastroenteritis in infants and children worldwide. Currently, an antiviral drug is not available and information on therapeutic targets for antiviral development is limited for rotavirus infection. Previously, it was shown that lipid homeostasis is important in rotavirus replication. Since farnesoid X receptor (FXR) and its natural ligands bile acids (such as chenodeoxycholic acid [CDCA]) play major roles in cholesterol and lipid homeostasis, we examined the effects of bile acids and synthetic FXR agonists on rotavirus replication in association with cellular lipid levels. In a mouse model of rotavirus infection, effects of oral administration of CDCA on fecal rotavirus shedding were investigated. The results demonstrate the following. First, the intracellular contents of triglycerides were significantly increased by rotavirus infection. Second, CDCA, deoxycholic acid (DCA), and other synthetic FXR agonists, such as GW4064, significantly reduced rotavirus replication in cell culture in a dose-dependent manner. The reduction of virus replication correlated positively with activation of the FXR pathway and reduction of cellular triglyceride contents (r(2) = 0.95). Third, oral administration of CDCA significantly reduced fecal virus shedding in mice (P < 0.05). We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication. The inhibition mechanism is proposed to be the downregulation of lipid synthesis induced by rotavirus infection.     

In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments

Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains.     

猪和牛轮状病毒抗原和抗体的检测

应用ELISA直接双抗体夹心法检查轮状病毒抗原,24份仔猪和29份犊牛的腹泻粪样,分别有12和16份阳性。用病毒RNA电泳分析检查阳性粪样,各出现两种病毒RNA电泳型,用中和试验检查17份成年牛和16份成年猪血清,分别有16和15份病毒抗体阳性。将其与ELISA间接法和结合法进行了比较。     

An Occurrence of Diarrheal Cases Associated with Group C Rotavirus in Adults

Six of the 23 college students who joined a group trip in February of 1991 developed acute nonbacterial gastroenteritis with severe diarrhea. The causal agent was identified as group C rotaviruses by electron microscopy (EM), immune-EM (IEM) and the molecular examinations including polyacrylamide gel electrophoresis (PAGE) and polymerase chain reaction (PCR) on virus particles detected in the extract of watery fecal specimens of the patients. The patients positive for virus isolation showed significant increase in IEM antibody to the isolated virus in their paired sera. These findings suggest that the group C rotavirus is an important etiological agent of diarrhea and may also cause serious food-borne diarrheal disease in adults.     

Adult diarrhea rotavirus in Jilin, China

In May 1986, an outbreak of epidemic acute diarrhea occurred in one city and three counties in Yanbian area. The diarrheal cases were seen in all age groups, the majority of the cases were seen in adult age group. No bacterial pathogens were isolated in 22 fecal samples examined, however rotavirus like particles of 52-68 nm in diameter were found in 50% (11/22) of fecal samples by the immunoelectron microscopy using convalescent sera. Examination by the adult diarrhea rotavirus (ADRV) ELISA kit showed positive reaction in 86% (6/7) of fecal extracts, however in all 22 fecal extracts examination using ELISA kit for detection of group A rotavirus showed negative reaction. The PAGE patterns of viral RNAs similar to those of ADRV were seen in 13 out of 22 fecal extracts. The detection rate by PAGE analysis was 59%. Based on the above data, the etiological agent of the epidemic acute diarrhea, which occurred in Yanbian area, was identified to be ADRV.