Comparative analysis of the biological characteristics of three CV-A10 clones adaptively cultured on Vero cells
Comparative analysis of the biological characteristics of three CV-A10 clones adaptively cultured on Vero cells
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DOI：
10.1002/jmv.27796
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发表时间：
2022-05-09
影响因子：
12.7
通讯作者：
Xie，Zhongping
中科院分区：
文献类型：
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作者：
Zhao，Hong;Yang，Ting;Xie，Zhongping
柯萨奇病毒 A10（CV-A10）是引起手足口病的主要病原体。 CV-A10感染尚无有效治疗药物；因此，应该开发CV-A10疫苗。此前，我们分离出了可在 Vero 细胞上培养的 CV-A10 菌株（N25）。在本研究中，N25菌株从Vero细胞中进行了3次空斑纯化，并选择了3个克隆进行适应性培养。对第5代、第12代和第19代的3个克隆的病毒滴度、噬斑形态、对乳鼠的致病性以及全基因组的核苷酸和氨基酸序列进行了比较和分析。三个克隆(P2-P22)的感染滴度维持在6.5-7.0 lgCCID50/ml。三个克隆在6小时开始增殖，并在36小时达到峰值；相应的CCID50在106.5~106.875/ml范围内，并逐渐降低。攻击组的乳鼠出现四肢瘫痪等临床症状，并逐渐恶化直至死亡。使用这三个克隆制备的灭活疫苗可有效诱导小鼠产生抗原特异性血清抗体。三个克隆中有 8 个核苷酸突变，分别导致 VP3 和 VP1 编码区出现 2 个和 4 个氨基酸替换。三个克隆与N25的核苷酸和氨基酸序列同源性分别为99.92%~100%和99.78%~100%，表明遗传稳定性较高。我们的研究结果为筛选 CV-A10 疫苗候选克隆提供了理论基础。
Coxsackievirus A10 (CV‐A10) is a major pathogen that causes hand, foot, and mouth disease. There are no effective therapeutic drugs for CV‐A10 infection; therefore, CV‐A10 vaccines should be developed. Previously, we isolated a CV‐A10 strain (N25) that can be cultured on Vero cells. In this study, the N25 strain was plaque‐purified three times from Vero cells, and three clones were selected for adaptive culture. The three clones of the 5th, 12th, and 19th generations were compared and analyzed in terms of viral titers, plaque morphology, pathogenicity in suckling mice, and nucleotide and amino acid sequences of the complete genome. The infectivity titers of the three clones (P2–P22) were maintained at 6.5–7.0 lgCCID50/ml. The three clones began to proliferate at 6 h and peaked at 36 h; the corresponding CCID50was in the range of 106.5–106.875/ml, which gradually decreased. The suckling mice in the challenged group exhibited clinical symptoms such as paralysis of the limbs, which gradually worsened until death. The inactivated vaccines prepared using the three clones efficiently induced antigen‐specific serum antibodies in mice. There were eight nucleotide mutations in the three clones, which resulted in two and four amino acid substitutions in the VP3 and VP1 coding regions, respectively. The nucleotide and amino acid sequence homology between the three clones and N25 were 99.92%–100% and 99.78%–100%, respectively, indicating high genetic stability. Our findings provide a theoretical basis for screening CV‐A10 vaccine candidate clones.