柯萨奇病毒A组16型临床分离株的生物学特性分析

Biological characteristics of clinical isolates of coxsackievirus group A type 16

目的对引起手足口病（hand,foot and mouth disease,HFMD）的重要病原体柯萨奇病毒A组16型（coxsackievirus group A type16,CA16）临床分离株的生物学特性进行分析,为后续的疫苗研发奠定基础。方法从昆明地区HFMD临床疑似患者178份咽拭子及粪便标本中分离并鉴定CA16毒株,并对病毒的生长特性、蚀斑形态以及对乳鼠的致病性等生物学特性进行分析。结果共分离出7株CA16病毒：KM1、KM15、KM154、KM165、KM168、KM263、KM881,均属于B1亚型;病毒在Vero细胞上增殖较快,多数毒株4～6 d即可达到增殖高峰,但感染性滴度差别较大,最低为4.75 lgCCID50/ml,而最高可达7.78 lgCCID50/ml;各毒株在Vero细胞上培养相同时间形成的蚀斑形态不同,其中KM15、KM154、KM168蚀斑呈圆形,针尖样大小,边缘较清晰,KM1、KM881、KM263、KM165蚀斑较大,不规则,边缘较模糊;各毒株毒力均较强,经乳鼠颅内注射后,乳鼠均于3～6 d陆续开始发病,除KM168、KM154、KM15组乳鼠为不同程度的发病及死亡外,其他组乳鼠发病率及病毒致死性死亡率均达100%;各毒株对乳鼠的脑、心肌、肺、肌肉、脊髓和肝脏等组织均有不同程度的损伤,其中损伤较严重的组织为脑和肌肉。结论分离的CA16毒株对Vero细胞均具有良好的适应性,蚀斑清晰,毒力较强,且多数毒株感染性滴度较高,可用于CA16病毒致病机理的研究及疫苗的研发。

Objective To analyze the biological characteristics of clinical isolates of coxsackievirus group A type 16（CA16）,a major pathogen of hand,foot and mouth disease（HFMD）and lay a foundation of vaccine development. Methods CA16 strains were isolated and identified from 178 stool and throat swab specimens of suspected patients with HFMD in Kunming Region,Yunnan Province,China,of which the growth characteristics,plaque morphology and virulence to suckling mice were analyzed. Results Seven CA16 strains,KM1,KM15,KM154,KM165,KM168,KM263 and KM881,were isolated,all of which belonged to B1 subtype. The strains were proliferated rapidly in Vero cells, most of which reached peak values of proliferation 4 ～ 6 d after inoculation. However,the infectious titers of these strains were significantly different,which ranged from 4. 75 to 7. 78 lgCCID50/ ml. The morphologies of plaques formed by the strains cultured in Vero cells for the same days were different,of which those formed by KM15,KM154 and KM168 were round,in needle-like size,and with clear edges,while those formed by KM1,KM881,KM263 and KM165 were large, irregular and with blurry edges. All the strains showed strong virulence,with which the suckling mice injected i. c. were attacked 3 ～ 6 d later. Both the morbidity and mortality of mice challenged with the strains except KM168,KM154, KM15 were 100%. The strains caused lesions at various degrees in brain,heart,lungs,muscles,spinal cord,liver and other tissues of suckling mice,especially in brain and muscle tissues. Conclusion The clinical isolates of CA16 in this study showed good adaptability to Vero cells,clear plaques and strong virulence,most of which showed high infectious titers. The isolates may be used for study on pathogenic mechanism of CA16 and vaccine development.