呼吸道合胞病毒的分离鉴定及其致病力的初步评价  

Isolation and identification of a respiratory syncytial virus and preliminary evaluation of its pathogenicity

作　　者：朱萍[1] 谷宏静 谢正德[3] 周娅[1] 杨鹏辉[4] 王希良[2] 

机构地区：[1]宁夏医科大学基础医学院病原生物学教研室,银川750004 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071 [3]北京儿童医院病毒研究室,100045 [4]解放军第302医院肝胆外科,北京100039

出　　处：《免疫学杂志》2015年第4期322-325,共4页Immunological Journal

基　　金：国家“863”项目(SS2012AA020905)

摘　　要：目的分离鉴定临床呼吸道合胞病毒(respiratory syncytial virus,RSV),评价其致病力,为疫苗评价及致病机制的研究提供实验材料。方法收集临床呼吸道感染重症患儿的鼻咽分泌物样本进行RT-PCR初步鉴定,通过病毒培养,空斑纯化,电镜观察及病毒滴度测定,筛选出1株呼吸道合胞病毒候选株,经全基因组测序后命名为BJ016。选用14日龄C57乳鼠,每组26只,采用BJ016为实验组,PBS为对照组,分别对小鼠滴鼻感染,30μl/只。连续14日监测2组小鼠体质量变化及死亡率,肺组织的病毒载量、湿干比,HE染色观察肺病理变化并计数炎性细胞总量,同时采用CBA测定血清中细胞因子的变化。结果 100份临床呼吸道感染重症患儿鼻咽分泌物样本中,RT-PCR鉴定为RSV A亚型阳性共21例,经病毒分离培养、空斑纯化、电镜观察及RT-PCR鉴定后得到16株RSV临床分离株,通过TCID50测定病毒滴度筛选出1株滴度为106.5TCID50/ml的候选株,基因组序列分析显示其为NA1基因型,命名为BJ016。BJ016感染14日龄C57乳鼠后造成肺损伤,在第5日达到发病高峰时测定病载、湿干比、病理检测并计数炎性细胞,实验组较对照组具有明显统计学差异,血清中MCP-1、IL-6、G-CSF及MIP-1α均显著性升高。结论成功分离出1株能致死14日龄C57乳鼠的RSV病毒株并初步评价了其致病力,为疫苗评价及RSV致病机制的研究提供了良好的实验材料。Respiratory syncytial virus（RSV） is a major pathogen which can cause respiratory disease especially bronchopneumonia in infants and elderly. In this study, a RSV strain was isolated from the swabs of human infant patients and identified by the cytopathic effect（CPE） assay, virus plaque assay, 50% tissue culture infectious dose（TCID50） assay, and electron microscopy. On infection, the Hep-2 cell line showed obvious cytopathic effects within48 to 72 hours post-inoculation, and as infected cells became rounded, bright and shrunken, the virus was plaquepurified in Hep-2 cells. The TCID50 of the virus was 106.5/ml. Electron microscopy showed viral particles were150 nm in diameter. According to the identification results, the isolate stain was obtained and named as RSV BJ016, which could make 14-day old suckling mice infection and death. BJ016 was used to infect 14-day old suckling mice, and we found the survival rate of the mice was significantly reduced compared with the PBS infected mice, and body weight losses in BJ016 infected mice were more serious. On day 5 post infection, viral loads of the lung, the pulmonary edema, and pathological damages of the BJ016 infected lungs were greatly serious than those of the PBS infected lungs. Furthermore, the levels of IL-6, G-CSF, MCP-1, and MIP-1α in sera of BJ016 infected suckling mice were higher than those of control from 1 to 5 days post infection. In conclusion, a respiratory syncytialvirus has been isolated and identified, and its pathogenicity has been preliminary evaluated, which will serve medicine screening, vaccines development and immunopathology researches for RSV infection.

关 键 词：RSV 病毒分离 C57乳鼠 致病力 

分 类 号：R392[医药卫生—免疫学]