Construction and characterization of an infectious cDNA clone of coxsackievirus A 10
Construction and characterization of an infectious cDNA clone of coxsackievirus A 10
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柯萨奇病毒 A 10 感染性 cDNA 克隆的构建和表征
DOI：
10.1186/s12985-019-1201-1
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发表时间：
2019-08-06
期刊：
影响因子：
4.8
通讯作者：
Liu, Hongbo
中科院分区：
文献类型：
--
作者：
Liu, Qiliang;Dan, Hanliang;Liu, Hongbo
柯萨奇病毒A10（Coxsackievirus A10，CA 10）是引起婴幼儿手足口病的四大病原体之一。感染性克隆对于研究病毒基因功能和致病机制具有重要意义。然而，目前还没有关于构建CA 10感染性克隆的报道。方法从临床分离的CA 10的全基因组扩增成两个片段，并连接到线性化的质粒载体中，在一个步骤中通过In-Fusion Cloning。将获得的CA 10 cDNA克隆和编码T7 RNA聚合酶的质粒共转染293 T细胞以拯救CA 10病毒。用SDS-PAGE、Western blotting和透射电镜对病毒进行鉴定。用CA 10病毒脑内接种1日龄ICR小鼠，观察临床症状。结果从构建的cDNA克隆中获得CA 10病毒，在RD细胞中传代1代后，病毒滴度达到最高，达108. 125 TCID 50/mL。该病毒表现出与亲本病毒相似的物理和化学性质。它还表现出高毒力和诱导新生ICR小鼠死亡的能力。感染小鼠出现严重的坏死性肌炎、肠绒毛间质水肿和严重的肺泡萎缩。结论首次成功构建了CA 10感染性克隆，为建立标准化的新生小鼠CA 10感染模型，用于疫苗和抗病毒药物的研究奠定了基础。以及模式菌株的保存和共享。
BackgroundCoxsackievirus A10 (CA10) constitutes one of the four major pathogens causing hand, foot and mouth disease in infants. Infectious clones are of great importance for studying viral gene functions and pathogenic mechanism. However, there is no report on the construction of CA10 infectious clones.MethodsThe whole genome of CA10 derived from a clinical isolate was amplified into two fragments and ligated into a linearized plasmid vector in one step by In-Fusion Cloning. The obtained CA10 cDNA clones and plasmids encoding T7 RNA polymerase were co-transfected into 293 T cells to rescue CA10 virus. The rescued virus was identified by SDS-PAGE, Western blotting and transmission electron microscopic. One-day-old ICR mice were intracerebrally inoculated with the CA10 virus and clinical symptoms were observed. Multiple tissues of moribund mice were harvested for analysis of pathogenic changes and viral distribution by using H&E staining, real-time PCR and immunohistochemical staining.ResultsCA10 viruses were rescued from the constructed cDNA clone and reached a maximum titer of 108.125TCID50/mL after one generation in RD cells. The virus exhibited similar physical and chemical properties to those of the parental virus. It also showed high virulence and the ability to induce death of neonatal ICR mice. Severe necrotizing myositis, intestinal villus interstitial edema and severe alveolar shrinkage were observed in infected mice. The viral antigen and the maximum amount of viral RNA were detected in limb skeletal muscles, which suggested that the limb skeletal muscles were the most likely site of viral replication.ConclusionInfectious clones of CA10 were successfully constructed for the first time, which will facilitate the establishment of standardized neonatal mouse models infected with CA10 for the evaluation of vaccines and antiviral drugs, as well as preservation and sharing of model strains.