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。     

流行性成人腹泻病人排出成人腹泻轮状病毒的持续时间

成人腹泻轮状病毒(Adult Diarrhoea Rotavirus,简称ADRV)是引起青壮年急性胃肠炎的主要病原之一,1983年至1985年在我国许多地区暴发流行,严重危害着人民健康,至今尚无治疗药物。因此,摸清病人排病毒情况或规律,对及时控制传染源,预防ADRV大量传播有着重要意义。现将本文实验结果报告如下。     

成人腹泻轮状病毒的血清流行病学

本文用对流电泳技术进行成人腹泻轮状病毒(ADRV)血清流行病学调查,发现我国大陆、香港及澳大利亚正常人群中,抗ADRV抗体阳性率都在20％以下,而发生过腹泻流行的地区,阳性率高达40％以上。在动物中,抗体反应谱广泛(猪、鸡、鸭、家鼠、大鼠、豚鼠、小鼠)。以猪(36.13％)和家鼠(46.66％)为最高,讨论了成人腹泻轮状病毒动物来源的可能性。     

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。     

Comparison of ELISA with shell vial cell culture method for the detection of human rotavirus in fecal specimens

The aim of the study was to compare an enzyme immunoassay method with shell vial cell culture method for detection of rotavirus in fecal specimens. In addition, the correlation between laboratory results and clinical scores of patients with gastroenteritis was evaluated. A total of 219 fecal specimens from children (ages 3 weeks to 5 years) with acute gastroenteritis submitted to pediatric emergency room were evaluated by both ELISA and shell vial cell culture. A Vesikari score was used for assessing the severity of the illness. Among 219 stool samples tested, 107 (48.9%) were determined to be positive. Two specimens were positive by shell vial cell culture method while they were ELISA negative. According to these results the calculated sensitivity, specificity, PPV, and NPV of ELISA were 98.1%, 100%, 100%, and 98.2%, respectively. The mean severity score for the 107 episodes of rotavirus diarrhoea was 11.0 +/- 3.6 compared to 4.5 +/- 1.9 for the 112 episodes of non-rotavirus diarrhea in the same population. Our study indicates that ELISA, which is easier to perform, faster and cheaper than cell culture methods may be suitable for routine diagnosis of rotavirus infections. The severity of rotavirus positive gastroenteritis was significantly higher than that of rotavirus negative patients.     

Detection of human rotavirus in hospitalized diarrheic children in central India

During the present study, group A human rotaviruses were detected among diarrheic children using polyacrylamide gel electrophoresis (PAGE) technique, with a typical RNA migration pattern of 4:2:3:2, suggestive of group A rotavirus. During the study, a total of 46 fecal samples collected from hospitalized children with acute diarrhea as well as children inhabiting nearby animal farms with history of presence of animal rotaviruses on the farms were processed for detection of human rotavirus. Out of 33 diarrheic children, 12 showed presence of rotavirus infection (36.36%), however, none of the children from animal farm areas showed presence of rotavirus. Female children were more susceptible to rotavirus infection (46.15%) than males (30%). Majority of the cases of rotavirus gastroenteritis belonged up to one year of the age, with an incidence of 40.91%. RNA profile of rotaviruses suggested circulation of 5 different electropherotypes in this geographical locale of the country, indicating existence of genomic diversity among human rotaviruses. Majority of the isolates were of long pattern (66.67%), whereas short pattern was detected only in one third of the viruses. This preliminary study emphasizes for further detailed studies on the molecular characterization of rotaviruses circulating in this part of country and their relationship with other human rotavirus strains and animal strains in the country.     

昆明婴幼儿腹泻的轮状病毒分子基因特征研究

目的了解昆明婴幼儿腹泻中轮状病毒感染情况。方法收集昆明医学院第一附属医院儿科2004年10-12月住院的腹泻的患儿粪便标本60份,采用聚丙烯酰胺凝胶电泳（PAGE）法和RT—PCR法进行轮状病毒分型。结果60份粪便标本中,29份检测到A组轮状病毒RNA基因,阳性检出率为48.3％,未发现B组及C组轮状病毒。其中RNA长型有29份（100％）,未发现短型和混合型。对29份有明确编号的轮状病毒阳性标本来源的患儿分析显示,平均发病年龄10.5个月。29份标本中,有20份可用RT—PCR分型,均为G3型,未发现其他型。结论A组轮状病毒是昆明地区5岁以下婴幼儿腹泻病的主要病原,3月龄-2岁婴幼儿是轮状病毒的易感人群,以冬季10月份至12月份为流行高峰,基因组以长型为主,血清型为G3型。     

快速检测成人腹泻轮状病毒(ADRV)抗原和抗体的研究

本文报道应用反相间接血凝和间接血凝试验技术快速检测ADRV抗原和抗体,一般在60分钟左右作出诊断。用ELISA方法和协同凝集试验作对照,结果一致。本实验与其它肠道病毒不发生交叉凝集反应,与菌痢、肠炎病人粪便和正常人粪便标本也不发生非特异性反应。实验准确可靠。本实验用间接血凝试验技术快速检测了ADRV抗体,观察了流行性腹泻病人血清中ADRV抗体持续时间。发现流行性腹泻患者发病后ADRV抗体存在半年以上,八个月有不同程度的下降,流行地区的健康人的ADRV抗体水平占33％左右,非流行地区正常人的ADRV抗体占11.3％左右。并用血凝抑制试验检测ADRV抗体,与小儿轮状病毒抗体无交叉反应。本试验对流行性腹泻的流行病学研究和临床诊断具有实用意义。     

成人腹泻轮状病毒蛋白的免疫印迹分析

我们从腹泻病人便样中纯化了病毒,其结构蛋白组分按分子量大小分别为VP1(136K),VP2(113K),VP3(92K),VP4(84K),VP5(64K),VP6(47K),VP7(41K)。所有这些蛋白皆具有抗原性。WP6是B组轮状病毒的共同抗原。B组轮状病毒的每一结构蛋白与A组轮状病毒都无交叉免疫反应。另外注意到二例不同病人对VP6和VP7刺激产生的抗体水平不同。     

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.     

杭州地区腹泻儿童轮状病毒与腺病毒感染调查

目的分析不同季节及不同年龄段腹泻患儿感染轮状病毒(RV)与腺病毒(Eads)情况,为临床提供早期、快速、准确、可靠的依据,以便及时采取相应的治疗使患儿早日康复。方法采用杭州艾博医药有限公司提供的轮状病毒(A组)/腺病毒检测试剂盒(乳胶法),对2012年1月至2015年12月浙江省人民医院门诊和住院就诊的3 368例腹泻儿童粪便标本进行轮状病毒和腺病毒抗原检测。结果 3 368例腹泻儿童粪便中,RV阳性578例占17.16%,Eads阳性106例占3.15%。11月至次年1月为轮状病毒感染的高发月份,1~3岁为儿童感染的高发年龄;腺病毒感染阳性率低,呈散发性。结论杭州地区近年1~3岁婴幼儿的腹泻主要是由轮状病毒和腺病毒引起,其中轮状病毒感染率明显高于腺病毒,且有明显的季节性;而腺病毒感染呈散发流行,未表现出明显的季节性及年龄分布。     

在我国患急性腹泻成人中发现一种新轮状病毒

从3例急性成人腹泻患者粪便中,经电镜观察,成人腹泻轮状病毒—酶联免疫吸附试验(ADRV-ELISA)、普通轮状病毒—酶联免疫吸附试验(Rota-ELISA),猪抗C型(组)轮状病毒和鸡抗D型(组)轮状病毒血清分别与本病毒的免疫电镜试验以及RNA电泳等实验结果,发现了一种新轮状病毒,该病毒的形态结构与普通轮状病毒(Rotavirus)、成人腹泻论状病毒(Adult Diarrhoea Rotavirus,简称ADRV)极为相似,但抗原性与普通轮状病毒(A组),成人腹泻轮状病毒(B组),C型轮状病毒(C组),D型轮状病毒(D组)显然无关。基因分析表明,该病毒基因组由11个双链RNA片段组成,但其RNA电泳图型具有独自的特点,与目前公认的A组、B组、C组、D组论状病毒韵RNA电泳图型均不同,免疫电镜证实,该病毒能被人恢复期血清所凝集,表明该病毒可能是腹泻病人的病因。     

腹泻患儿粪便A群轮状病毒抗原检测的结果分析

目的：了解本地区腹泻婴幼儿的A群轮状病毒感染情况及其流行特点。方法：采用胶体金法对我院2009年10月-2010年9月2104例有腹泻和肠炎特征的婴幼儿粪便进行A群轮状病毒抗原检测。结果：在2104例受检者中,A群轮状病毒感染的总阳性率是24．71％,其中男性感染率24．17％,女性为25．40％。不同年龄组间以1—2岁婴幼儿感染率最高,为32．13％,0-1岁为20．72％,2-5岁为12．03％。感染的季节特征是秋末冬初季（10—12月）阳性率最高,为42．82％,春末夏初季（4．6月）最低,为8．81％。结论：由A群轮状病毒感染引发的急性腹泻主要发生在1-2岁的婴幼儿,各个季节均有发生,以秋末冬初季为高发。     

Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins.

Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.     

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。     

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。     

婴幼儿轮状病毒肠炎48例病原学检测及治疗

轮状病毒是婴幼儿秋冬季腹泻的最常见的病原。我们用ＥＬＩＳＡ试剂盒对５４例大便常规检查无ＷＢＣ或偶见的疑似轮状病毒腹泻患儿进行了轮状病毒病原学检测,从中检出轮状病毒阳性患儿４８例,被确诊为轮状病毒肠炎。结果显示粪便中轮状病毒滴度越高,其临床症状也相应越重。此４８例患儿均采用病毒唑、双歧杆菌活菌制剂、补液纠正脱水等综合治疗,其中３０例辅以消化道粘膜保护剂思密达治疗,治疗组显效率为６０％,而对照组为１１．１％,经ｔ检验两组间显效率及总有效率均有非常显著性差异。表明思密达用于轮状病毒肠炎的治疗是一种较为有效的方法。