人呼吸道合胞病毒减毒活疫苗培养的初步探索

目的对比人呼吸道合胞病毒（human respiratory syncytial virus，HRSV）经人二倍体KMB17细胞低温传代前后对动物的病理损伤，分析毒力变化情况。方法将HRSVA2株病毒接种到人二倍体KMB17细胞进行低温培养传代，接种原代病毒（5．37LgCCID50／ml）至乳鼠脑内，14d处死解剖，取脑、心、肺作病理切片检查；选择原代、第6代、第11代低温传代病毒接种豚鼠鼻腔，于第7d、第14d分2批分别处死，取心、肝、脾、肺、肾组织作病理切片检查，并取肺组织分离病毒、进行感染性滴度测定、用间接免疫荧光法对其进行鉴定。结果经脑内接种的乳鼠均未死亡，且乳鼠的脑、肺HE染色切片与对照组相比均无明显著差异。豚鼠经鼻腔接种，接种原代病毒组第14d处死时体重明显较对照组轻，6代组和11代组与对照组接近；3组病毒均引发豚鼠间质性肺炎；3组豚鼠肺组织分离的病毒滴度差异不大，平均为4．0Lg。结论乳鼠脑内接种HRSV不能导致死亡，脑组织不是HRSV的病变靶器官；HRSV经鼻腔接种豚鼠能引起强烈的肺部感染，豚鼠可作为HRSV毒力评价的动物模型；HRSV在KMB17细胞上经低温传11代，接种豚鼠仍能引起间质性肺炎，未出现明显的减毒株特征，还需要进一步减毒。

Preliminary Study on Culturing Cold-passaged Human Respiratory Syncytial Virus

Objective To analyze the virulence alteration of RSV A2 after being cold-passage cultured in animal model.Methods The HRSV A2 strain was cultured and passaged in the human diploid cell KMB17 at low temperature.The primary virus(5.37 Lg CCID50/ml) was inoculated into the brain of sulking mouse,and the pathology of heart,brain,and lung tissue was examined on the 14th day.The primal,the 6th and the 11th passaged viruses were used to inoculate into guinea-pigs.The pathology of heart,liver,spleen,lung,and kidney tissue was examined on the 7th and the 14th days.Virus from the lung tissue of guinea-pigs was isolated and detected with IDIF and their titer about CCID50.The lung pathology of guinea-pig samples was examined under electron microscope.Results All sulking mice survived after inoculation,and no inflammation was observed in the brain and lung.In the group of inoculated guinea-pig,the body weight of the primal virus group was significantly lower than that of the control group on the 14th day,the 6th and 11th days groups were similar with the control group.Interstitial pneumonia emerged in all the guinea-pigs.The average titer of isolated virus was 4.0 Lg,with no significant difference.Conclusions No death cause results in inoculating the cultured HRSV to the brain of sulking mouse,and the brain is not the target organ of the A2 strain.Nasal inoculating into guinea-pig can induce lung infection,thus guinea-pig might be the candidate animal model of toxicity assessment.The A2 strain has been cold-passaged 11 passages,and still induces interstitial pneumonia with no obvious character of attenuated strain.It is necessary for continuing to passage HRSV at low temperature.