人呼吸道合胞病毒适应株筛选及其生物学特性

Screening and biological characteristics of human respiratory syncytial virus

目的建立在Vero细胞上低温传代的人呼吸道合胞病毒(human respiratory syncytial virus,HRSV)适应株,并分析其生物学特性。方法从155份肺炎儿童痰样中经PCR、测序鉴定出14株RSV,将其在Vero细胞上37℃传代第1次收获的病毒记为野毒株(wild-type,WT),通过逐步降低培养温度的方法在Vero细胞传代获得1株适应株(adapted-strain,AS)KM516-AS。将RSV适应株感染A549细胞后,分别至37、25、32和39℃培养,测定其冷适应性和温度敏感性。分别将RSV适应株及其对应的野毒株KM516-WT经鼻腔免疫小鼠,于免疫后第8天,取肺组织制作病理切片,显微镜下观察病变程度;取鼻甲和右肺匀浆组织,制备上、下呼吸道匀浆液,测定上、下呼吸道病毒滴度;于免疫后第13天,经小鼠尾部静脉采血,分离血清,于免疫后第14天,取肺组织制备匀浆液,采用ELISA法测定总抗体滴度,病毒中和试验法测定中和抗体滴度。结果 RSV野毒株KM516-WT的适应温度范围较广,4个不同温度下的病毒滴度变化不大;RSV适应株KM516-AS的适应温度范围较窄,25℃时的病毒滴度最高,随着温度的升高,病毒滴度逐渐降低,且仅有温度敏感性而无冷适应性。野毒株组小鼠肺部呈暗褐色,无严重的实质化,且肺泡和毛细血管中有明显的淋巴浸润;适应株组小鼠肺泡和毛细血管中淋巴浸润较少。适应株在体内的复制水平低于野毒株,并同野毒株一样具有较好的免疫原性。结论获得1株RSV适应株,并具有一定的减毒效果。

Objective To establish the human respiratory syncytial virus（HRSV）adapted strains subcultured at low temperature in Vero cells and analyze their biological characteristics. Methods Fourteen RSV strains were screened from155 sputum specimens of children with pneumonia by PCR and sequencing,and subcultured in Vero cells at 37 ℃ to prepare wild type strain,then further subcultured in Vero cells at gradually lowered temperatures to obtain a adapted strain KM516-AS,with which A549 cells were infected and cultured at 37,25,32 and 39 ℃ respectively to evaluate the cold-adaptability and temperature-sensitivity. Mice were inoculated i. n. with the adapted strains and the corresponding wild strain KM516-WT,of whom the lung tissues were prepared into sections on day 8 after inoculation and observed for pathological change by microscopy. Turbinate and right lung tissue of mice were prepared into homogenates and determined for virus titers in upper and lower respiratory tracts respectively. Venous blood was collected on day 13 after inoculation,from which serum was separated. Lung tissue was prepared into homogenate on day 14 after inoculation.The serum and lung tissue homogenate were determined for total antibody titer by ELISA,and for neutralizing antibody titer by neutralization test. Results The titers of KM516-WT at four temperature showed no significant difference,indicating that the strain was suitable for a wide range of temperature. However,KM516-AS showed only temperaturesensitivity but no cold-adaptability,of which the titer was the highest at 25 ℃ and decreased gradually with the increasing temperature. The lung tissues of mice inoculated with KM516-WT were in dark brown color and showed no severe substantiation,and obvious lymph infiltration was observed in pulmonary alveoli and blood capillary,while little lymph infiltration was observed in pulmonary alveoli or blood capillary of mice inoculated with KM516-AS. The replication level of KM516-AS in vivo was lower than that of KM516-WT. However,both the strains showed high immunogenicity.Conclusion Both domestic and imported MEM were suitable for growth of MRC-5 cells and virus culture.