SARS-CoV感染布氏田鼠、Lewis大鼠和恒河猴引起的病理变化

目的研究SARS-CoV感染布氏田鼠、Lewis大鼠和恒河猴后引起的病理学、免疫学变化及病毒复制与外排情况变化特点及作为动物模型的可行性。方法SARS病毒感染布氏田鼠、Lewis大鼠和恒河猴,用病毒分离和RT-PCR方法检测病毒复制与外排;用ELISA检测特异性抗体;用光学显微镜对动物的各脏器进行病理观察研究。结果在SARS-CoV感染的3种动物肺组织均出现与人类SARS疾病相似的病理改变,均可检测到病毒核酸和特异性IgG抗体的存在。结论恒河猴、Lewis大鼠和布氏田鼠感染SARS-CoV后,均能在一定程度上再现人类SARS患者的病理特征,其中恒河猴的病理改变更近似于人类患者,故以恒河猴为模型可能对研究SARS发病机制和疫苗评价更为理想。     

EV71型手足口病乳鼠动物模型的建立及免疫、内分泌及病理特征研究

目的建立EV71型手足口病乳鼠动物模型并进行免疫、致病特性研究。方法将临床分离的EV71病毒株经蚀斑纯化,3T3细胞适应,乳鼠驯化,最终获得1株能致死7日龄乳鼠的EV71毒株,命名为BJ09/07(GenBank Accession NO.JQ319054,EV71-BJ)。EV71-BJ感染7日龄ICR乳鼠后,观测临床疾病得分、体质量变化、死亡率并测定病毒载量、免疫分子、内分泌水平、组织病理损伤等病毒、免疫、内分泌、病理指标。结果 EV71-BJ毒株感染7日龄ICR乳鼠,其病毒毒力为150 LD50/ml,感染后不同时期肌肉病毒载量均高于脑中,至第4天达到高峰,后不断下降。至感染后第6天发病达高峰时做病理检测,相对于脑组织,肌肉中有更严重的淋巴细胞浸润,引起更严重的炎性分子升高。肌肉研磨液和血清中MCP-1、IFN-γ、IL-6和TNF-α显著升高,而肾上腺素和皮质醇未见明显变化。结论初步建立了EV71型手足口病乳鼠动物模型,为药物筛选、疫苗研发及免疫机理研究奠定了基础。     

EV71不同接种途径对BALB/c小鼠的感染特点

目的 研究EV71 JN200804株对1日龄BALB/c小鼠的感染特点,建立EV71感染BALB/c小鼠的动物模型,为疫苗和抗病毒药物的研究提供可靠的动物评价工具.方法 EV71JN200804株分别采用口服、颅内注射、肌内注射和腹腔注射感染1日龄BALB/c小鼠,出现后肢麻痹后,做后肢电生理检测;安乐动物,采集脑、脊髓、心脏、肝脏、脾脏、肺脏、肾脏、胸腺、小肠及后肢肌肉,进行动物体内的病毒分离和RT-PCR鉴定,同时对各器官组织进行组织病理学观察.结果 接种EV71后,颅内、肌内和腹腔注射组小鼠体重增长缓慢,4～5d出现后肢麻痹,7d左右死亡.RT-PCR和病毒分离表明颅内、肌内和腹腔注射组的肌肉分离到病毒,颅内注射组脊髓也分离到病毒,经RT-PCR鉴定为EV71感染.肌电图显示颅内、肌内和腹腔注射组的时限显著增加,波幅显著降低,判断小鼠后肢麻痹可能既有神经源性损害又有肌源性损害.组织病理学观察发现,颅内、肌内、腹腔注射组小脑浦肯野细胞及颗粒细胞减少,脊髓前角白质区神经纤维肿胀,后肢肌肉组织大片坏死溶解、炎性细胞浸润,肺组织明显充血,局部心肌细胞肿胀,部分肝组织内可见巨噬样细胞及淋巴样细胞浸润,部分肾皮质中肾小球萎缩、数目减少,而口服组无明显病理变化.结论 EV71 JN200804株通过颅内、肌内和腹腔注射三种途径均能感染并导致1日龄BALB/c小鼠后肢麻痹,此动物模型可用于EV71致病机制和特异性抗病毒药物的研究及疫苗的评价.     

C亚型SHIV-XJ02170在中国恒河猴体内传代研究

目的 了解SHIV-XJ02170病毒在中国恒河猴体内传代过程中病毒学和免疫学变化特点,构建适用于我国艾滋病疫苗评价的C亚型SHIV/中国恒河猴模型.方法 将SHIV-XJ02170感染性全长质粒转染293T细胞制备病毒,后肢静脉途径在中国恒河猴体内传3～4代.传代过程中采用流式细胞术检测外周血CD4/CD8比值,分析病毒致病能力的变化.实时荧光定量RT-PCR方法 检测SHIV病毒载量,了解病毒学变化特点.同时,利用ELISA和ELISPOT方法 分析病毒传代过程中体液和细胞免疫学变化特点.结果 SHIV-XJ02170病毒传代过程中,CD4/CD8比值未表现出剧烈的下降特点.线路3传代中急性期血浆病毒载量峰值和Setpoint值表现出连续上升的趋势.病毒感染恒河猴后可诱导较强的体液和细胞免疫应答.同时,病毒载量和交叉抗体滴度之间表现出显著的正相关关系.结论 SHIV-XJ02170病毒在中国恒河猴传代后未出现致病性突变株,但是表现出毒力上升的趋势.SHIV-XJ02170/中国恒河猴模型将在我国艾滋病疫苗有效性评价中发挥重要作用.     

EV71型病毒驯化与手足口病动物模型的研究进展

手足口病好发于幼儿,通常是自限性的发热、出疹性疾病,但是肠道病毒71型(EV71)感染引起的手足口病易于出现伴有神经症状的严重病例。EV71的免疫学发病机制较为复杂,从而妨碍了疫苗的研发。建立良好的手足口病动物模型有助于研究其发病机制,而建立动物模型的关键是通过病毒驯化获得EV71的神经细胞适应毒株。本文简要综述EV71的病毒驯化及手足口病动物模型的最新进展,以期推动相关研究。     

恒河猴对多次注射人单克隆抗体的反应

妨碍小鼠单克隆抗体(McAb)用于人体内治疗的主要原因之一,是机体出现抗小鼠McAb反应。作者用2株抗人巨细胞病毒的IgG_1亚型人McAb(EV2-7和EV1-15)在数月内分别多次注射给5只恒河猴,以研究人McAb对灵长类的致免疫性。发现注射EV2-7的3例恒河猴和注射EV1-15的1例恒河猴,McAb在体内均表现为正常的药理动力学过程。仅在注射EV1-15的另一例恒     

重视EV71重症手足口病发病机制及防治对策的研究

人肠道病毒71型（enterovirus71，EV71）感染婴幼儿可导致手足口病（hand—foot-and-mouthdisease，HFMD），少数伴随有神经系统病变、心肺损害等严重并发症甚至死亡。由于缺乏理想的动物模型，影响了对EV7l病毒传播路径、模式特别是神经系统病变致病机制的理解，至今尚无疫苗和有效的病原治疗措施，使得EV71感染成为目前东南亚最严重的公共卫生问题。     

EV71病毒感染动物模型研究进展

手足口病(hand-foot-and-mouth disease,HFMD)是由多种肠道病毒引起的常见传染病,肠道病毒71型(EV71)是其主要病原体[1-2].EV71病毒属于小RNA病毒科、肠道病毒属病毒,为单股正链RNA病毒[3-4].自1974年首次报道从美国加州神经系统疾病患者标本中分离到EV71以来,EV71病毒曾在世界范围内多次引起暴发疫情,主要引起婴幼儿手足口病和无菌性脑膜炎、脑炎等多种严重的神经系统疾病[5].近年来该病在国内流行,并出现重症和死亡病例[6].虽然其致病机制目前尚不十分清楚[7],临床上尚无高效的预防EV71病毒感染的疫苗或药物,部分灭活疫苗、病毒样颗粒疫苗、DNA疫苗等都在研制开发中[8].而合适的动物模型则是评价疫苗免疫及保护效果的重要平台.本文对EV71研究所用动物模型的建立、特点、研究应用等方面进行了综述.     

SIVmac251感染中国恒河猴外周血免疫学及病毒学指标动态变化

目的 观察中国恒河猴感染猴免疫缺陷病毒(simian immunodeficiency virus,SIV)后病毒学和免疫学基本指标的变化规律,探讨中国恒河猴用于艾滋病模型时外周血白细胞(WBC)和CD4+T细胞比例的标准.方法 以SIVmac251感染36只中国恒河猴,于感染前1天及感染后1～8周每周及感染后第10周分别采集外周静脉血,检测血常规、外周血淋巴细胞亚群和病毒载量.结果 除WBC计数变化不显著外,其余检测指标均在感染后第1、2周时变化最为剧烈;WBC计数在(4～10)×106个/ml之间结合外周血CD4'T细胞比例高于25％的可作为艾滋病模型的选猴标准.结论 为应用中国恒河猴进行艾滋病研究提供了有益的信息.     

SIVmac239感染中国恒河猴动物模型组织病理学变化

目的探讨中国恒河猴艾滋病模型的组织病理学变化以及引发这些变化的机制,为艾滋病防治研究提供参考资料。方法用SIVmac239毒株经静脉感染2只中国恒河猴,4个月后处死动物,常规尸检。取淋巴结、心、肝、脾、肺、肾、消化管等组织10%中性福尔马林固定,石蜡包埋后切片,HE染色、免疫组化及特殊染色,光镜观察。结果(1)感染猴的病理组织学改变以免疫系统为主,表现为淋巴结滤泡的增生、萎缩、或二者兼有,部分淋巴结淋巴滤泡内细胞稀疏,纤维组织增生伴有免疫复合物(immune complex,IC)沉着,出现"焚毁"现象。(2)脾脏增大伴脾小结内血管及其内皮细胞增生,并伴有IC沉着。(3)其它部位的黏膜相关淋巴组织亦有不同程度的增生、萎缩。结论SIVmac239感染中国恒河猴动物模型的病理变化与人HIV极为相似,表明该模型是艾滋病防治研究的有用工具。     

An outbreak of enterovirus 71 infection in Taiwan, 1998. II. Laboratory diagnosis and genetic analysis.

BACKGROUND: An epidemic of enterovirus 71 (EV71) occurred in Taiwan from April to December of 1998, with two peaks, one in June and the other in October. Many enteroviruses were isolated in our laboratory from 258 cases during this outbreak. Approximately half of the enteroviruses isolated were EV71 and one fifth were coxsackievirus A16. OBJECTIVES: To analyze laboratory findings in the EV71 epidemic of 1998 in Taiwan, various EV71 specimens in different cell lines were examined. In addition, genetic analysis of 5' non-coding region (NCR) was performed to analyze the strain variation in this outbreak. RESULTS: The cytopathic effect induced by EV71 was observed 2-13 (mean of 4.5) days post-inoculation in Vero cells and 4-15 (mean of 6.6) days in green monkey kidney (GMK) cells inoculated with throat swabs. Of the total positive EV71 cases, virus was most frequently obtained from throat swabs (91.7%), less from stools (64.8%), and none from cerebral spinal fluid (CSF). Molecular analyses of EV71 by sequencing the 5' NCR of 34 strains obtained from different clinical categories and various geographic areas showed that their sequences differed (0-13 bp in 681 bp sequenced) by approximately 0-2%. The sequences of these isolates differed from EV71 prototype BrCr or MS strain by 17.5-19%, with the exception of two samples which exhibited nucleotide variation by only 8.9 and 8.2%, when compared to the MS strain. CONCLUSION: EV71 was most frequently isolated from throat swab specimens in Vero cells. The molecular analyses of the 5' NCR of EV71 revealed that most isolates from this epidemic belonged to a group of closely related clones and only two were in a different group which was clustered with the EV71 MS strain.     

Neonatal rhesus monkey is a potential animal model for studying pathogenesis of EV71 infection

Data from limited autopsies of human patients demonstrate that pathological changes in EV71-infected fatal cases are principally characterized by clear inflammatory lesions in different parts of the CNS; nearly identical changes were found in murine, cynomolgus and rhesus monkey studies which provide evidence of using animal models to investigate the mechanisms of EV71 pathogenesis. Our work uses neonatal rhesus monkeys to investigate a possible model of EV71 pathogenesis and concludes that this model could be applied to provide objective indicators which include clinical manifestations, virus dynamic distribution and pathological changes for observation and evaluation in interpreting the complete process of EV71 infection. This induced systemic infection and other collected indicators in neonatal monkeys could be repeated; the transmission appears to involve infecting new monkeys by contact with feces of infected animals. All data presented suggest that the neonatal rhesus monkey model could shed light on EV71 infection process and pathogenesis.     

利巴韦林注射液在细胞水平上抑制EV71病毒复制的初步研究

目的 通过体外实验,观察利巴韦林注射液对肠道病毒71型(EV71)病毒在横纹肌肉瘤传代细胞(RD-A细胞)复制的抑制作用和体外灭活作用.方法 使用在2008年4月安徽阜阳疫情中分离到的EV71病毒,通过三种体外细胞实验:药物在细胞中抑制EV71病毒复制的药效测定实验;药物对细胞的保护作用实验;药物体外对EV71病毒的灭活作用的药效测定实验.观察利巴韦林注射液对EV71病毒感染横纹肌肉瘤传代细胞(RD-A细胞)中所起的作用.结果 在利巴韦林注射液在细胞中抑制EV71病毒复制的药效测定实验中,1:640利巴韦林注射液稀释度组较病毒对照组延迟病变出现2 d,细胞生长未受到影响.在药物对细胞的保护作用实验中,1:8利巴韦林稀释度组较病毒对照组延迟病变出现4 d.在利巴韦林注射液药物体外对EV71病毒的灭活作用的药效测定研究中,1:40稀释度组较病毒对照组延迟病变出现2 d,而细胞生长未受到影响.结论 利巴韦林注射液在体外有抑制EV71病毒复制和部分灭活病毒的作用;并有一定的保护细胞抑制EV71病毒感染的作用.本研究对于临床上EV71感染的早期预防和治疗具有一定的指导意义.     

Pathogenesis study of enterovirus 71 infection in rhesus monkeys

Enterovirus 71 (EV71), a major pathogen that is responsible for causing hand-foot-and-mouth disease (HFMD) worldwide, is a member of the Human Enterovirus species A, family Picornaviridae. HFMD that is caused by EV71 is usually characterized by vesicular lesions on the skin and oral mucosa and high morbidity rates in children; additionally, occasional fatal cases have been reported involving brainstem encephalitis and myelitis associated with cardiopulmonary collapse. Although viral pathogenesis in humans is unclear, previous animal studies have indicated that EV71, inoculated via various routes, is capable of targeting and injuring the central nervous system (CNS). We report here the pathogenic process of systemic EV71 infection in rhesus monkeys after inoculation via intracerebral, intravenous, respiratory and digestive routes. Infection with EV71 via these routes resulted in different rates of targeting to and injury of the CNS. Intracerebral inoculation resulted in pulmonary edema and hemorrhage, along with impairment of neurons. However, intravenous and respiratory inoculations resulted in a direct infection of the CNS, accompanied by obvious inflammation of lung tissue, as shown by impairment of the alveoli structure and massive cellular infiltration around the terminal bronchioles and small vessels. These pathological changes were associated with a peak of viremia and dynamic viral distribution in organs over time in the infected monkeys. Our results suggest that the rhesus monkey model may be used to study not only the basic pathogenesis of EV71 viral infections, but also to examine clinical features, such as neurological lesions, in the CNS and pathological changes in associated organs.     

EV71所致手足口病患儿及其密切接触儿童带毒状况的追踪观察

目的 调查EV71所致手足口病患儿及其密切接触儿童的病毒携带状况,探讨肠道病毒传播规律,为科学防控手足口病提供参考.方法 现场追踪观察18名聚集性患儿及其50名密切接触儿童并采集标本,利用RD细胞分离肠道病毒,实时荧光RT-PCR方法检测肠道病毒核酸.结果 在发病后3～6周内,仍有38.89％(7/18)的确诊患儿粪便...     

Rhesus monkey responses to multiple injections of human monoclonal antibodies

Five rhesus monkeys were injected multiple times over several months with two different human IgG1 monoclonal antibodies, both directed against human cytomegalovirus. Three monkeys each injected four times with monoclonal antibody EV2-7 for over 200 days showed no response other than a normal decay in antibody level. The in vivo half life of this antibody was substantially longer when measured with an idiotype-specific two site immunoassay than with radiolabeled antibody, indicating that the iodination procedure greatly affected the stability of the antibody. Although there was considerable individual variation in the half-life of EV2-7, from 8.9 to 30.5 days, the half-life was fairly long, especially considering the size of the monkeys. Two monkeys were injected with monoclonal antibody EV1-15. One monkey has responded in a similar manner to the EV2-7-injected monkeys. However, the other monkey has produced an anti-idiotypic antibody (or antibodies) of high affinity. It is possible that this response was triggered by the unusual physical nature of antibody EV1-15 or the effect of the species difference between human and rhesus monkey. In any case, the results from these five monkeys indicate that human monoclonal antibodies should have a significant advantage over mouse monoclonal antibodies for in vivo therapeutic applications.     

IgM抗体检测对肠道病毒71型感染者诊断价值的探讨

目的 探讨IgM抗体检测对肠道病毒71型(EV71)感染者的诊断价值,并与核酸检测和中和试验方法进行比较分析.方法采用EV71 IgM抗体酶免检测试剂盒(EV71 -IgM试剂)、EV71核酸检测试剂盒(EV71-PCR试剂)和中和试验方法,检测115例EV71感染者的EV71 RNA、中和抗体和IgM抗体的水平,并进行比较性研究分析.结果 对115例EV71感染者,EV71 -IgM试剂对患者首份血清的检出率为80.9％ (93/115,95％ CI:72.5～87.6),对228份健康儿童血清,EV71 -IgM试剂的检出率为2.6％.同时,对病发1～2d采集的54份血清检出率为70.4％(38/54),显示了较好的早期诊断价值.EV71-PCR试剂对患者咽拭子标本的检出率为82.6％(95/115,95％ CI:74.4-89.0),与EV71-IgM试剂相比,差异无统计学意义.此外,EV71-IgM试剂与EV71-PCR试剂具有很好的互补性,两种方法联合使用可提高检测灵敏度约10个百分点达到92.2％.结论 EV71 -IgM试剂具有良好的早期诊断价值,与EV71-PCR试剂联用可提高诊断率.     

肠道病毒71型IgM抗体检测在手足口病早期诊断中的价值

目的评估人肠道病毒7l型（EV71）IgM抗体在手足口病早期诊断中的价值。方法自发病后连续每日采集2010年我院收治的38例手足口病患儿血清及咽拭子标本,分别检测EV 71 IgM抗体、肠道病毒核酸、EV71特异核酸。结果38例手足口病患儿IgM抗体,按发病天数累加阳性率分别为：第1天60．5％、第2天71．1％、第3～4天81．5％、第5天92．1％、第6天92．1％;肠道病毒核酸阳性率为73．6％;EV71特异核酸阳性率为60．5％。结论EV 7l IgM抗体在手足口病发病的第1天即可出现,至第5天阳性率达到峰值,可作为手足口病早期诊断指标之一。     

肠道病毒71型IgM抗体检测在手足口病早期诊断中的价值

目的 评估人肠道病毒71型(EV 71)IgM抗体在手足口病早期诊断中的价值.方法 自发病后连续每日采集2010年我院收治的38例手足口病患儿血清及咽拭子标本,分别检测EV 71IgM抗体、肠道病毒核酸、EV 71特异核酸.结果 38例手足口病患儿IgM抗体,按发病天数累加阳性率分别为:第1天60.5%、第2天71.1%、第3～4天81.5%、第5天92.1%、第6天92.1%;肠道病毒核酸阳性率为73.6%;EV 71特异核酸阳性率为60.5%.结论 EV 71 IgM抗体在手足口病发病的第1天即可出现,至第5天阳性率达到峰值,可作为手足口病早期诊断指标之一.