不同CA16流行株的相关生物学和VP1基因的分子生物学研究  

Comparison of biological and genetic background of CAI6 between different epidemic strains

作　　者：郝春生[1] 杨永娟 宋衍燕[3] 李懿[1] 张中洋[1] 郭会杰[1] 赵敏[1] 支惠[1] 罗凤基[3] 李秀玲[1] 

机构地区：[1]北京生物制品研究所第二研究室,100024 [2]北京微谷生物医药有限公司,100024 [3]北京市朝阳区疾病预防控制中心

出　　处：《中华微生物学和免疫学杂志》2012年第3期199-204,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的对柯萨奇病毒A组16型（CA16）病毒株进行生物学特征与基因背景研究，为CA16病原和疫苗的进一步研究奠定基础。方法从咽拭子标本中分离出CA16病毒；通过中和鉴别试验和RT—PCR对病毒进行分子生物学鉴定；病毒的VPl基因序列测定后，与其他CA16序列进行比较，绘制种系发育树，确定病毒基因型；测定病毒滴度，观察病毒在Vero细胞上的噬斑形态；攻击乳鼠分析病毒的致病性。结果从手足口病患者咽拭子标本中分离到6株病毒，采用肠道病毒通用引物可以从病毒感染细胞中扩增出约150bp大小的目的产物带；采用CA16特异性引物可以从病毒感染细胞中扩增出210bp大小的目的产物带。而采用EV71特异性引物未见特异性扩增条带。噬斑分析发现，6株病毒在Vero细胞上接种96h后均能出现清晰的噬斑，其中BJ-3噬斑直径为4—5mm，BJ-5噬斑直径约为3mm，BJ-1、BJ-2、BJ4、BJ-6噬斑直径为1．5～2mm；除BJ-3噬斑边缘模糊外，其余5株噬斑边缘整齐。该6株病毒均可被人CA16抗体阳性血清中和。在Vero细胞上连续传5代，滴度均在7．0LgcCID50／ml左右。基于VPI基因序列的种系发育分析结果显示，BJ-2、BJ-4、BJ-5属于C1亚型，BJ-1、BJ-3、BJ-6属于C3亚型，该6株病毒核苷酸同源性达90％以上，氨基酸同源性达99％以上。乳鼠攻击试验结果显示，BJ-3和BJ-5攻击后第4～5天乳鼠开始发病，后肢出现明显的麻痹、瘫痪症状，至第6～7天全部死亡，正常对照组和其他病毒组直至攻击后第14天均无异常。结论不同CA16病毒株的生物学特性有所不同，基因背景相近，致病性明显不同，需进一步了解中国流行的CA16病毒株的致病特点，为疫苗的研发提供直接依据。Objective To study the biological characterization and the genetic background of circulating CA16 strains in mainland of China for the purpose of CA16 vaccine development in the future. Methods CA16 strains were isolated from throat swabs of patients with hand-foot-mouth disease and identified by neutralization assay and RT-PCR. The genotype of these isolates were determined by sequence alignment and phylogenetic analysis of VP1 gene. The proliferation dynamics and the plaque morphology were observed when propagated in Vero cells. The pathogenicity of these CA16 isolates was evaluated by challenging newborn mice. Results In this study, six CA16 circulating isolates, BJ-1-6 were obtained. The RT-PCR products were 150 bp amplified with the general enterovirus primers and 210 bp with CA16 primers respectively, which cannot be amplified by EV71 primers. Additionally, these isolates were identified to display some obvious proliferation dynamics and plaque morphology when propagated for 96 h in Vero cells. The di- ameter of plaques were about 1.5 to 2 mm for BJ-1, BJ-2, BJ-d, BJ-6, 4-5 mm for BJ-3 and 3 mm for BJ- 5, the plaques were regular except BJ-3. All the six isolates can be neutralized by the convalescent serum of patient infected with CA16. The virus titer of different isolates propagated for five passages in Vero cells was 7.0LgCCIDS0/ml. The sequence alignment of VP1 gene demonstrated that the genotypes of B J-2, BJ-d, B J- 5 were C1 and BJ-1, BJ-3, BJ-6 were C3 comparatively. The genetic distance of the VP1 gene from these isolates suggested that they were highly genetic identity with the homology of 90% in nucleotide and 99% in dedicated amino acid respectively. However, a distinctive difference in pathogenic ability in neonatal mice was found that the suckling mice challenged with B J-3 ＆ B J-5 were paralyzed 4-5 d and dead 6-7 d postchallenge, compared with the control group without any abnormality in the during of 14 d. Conclusion The circulating CA16 isolates in China have different biological charac

关 键 词：柯萨奇病毒A组16型(CA16) 流行株 手足口病 

分 类 号：R373[医药卫生—病原生物学]