柯萨奇病毒 A10 感染性 cDNA 克隆的构建与鉴定。
Construction and characterization of an infectious cDNA clone of coxsackievirus A 10.
机构信息
Department of Laboratory Medicine, The Second Affiliated Hospital of Guilin Medical University, Guilin, Guangxi, China.
College of Bio-technology, Guilin Medical University, Guilin, Guangxi, China.
出版信息
Virol J. 2019 Aug 6;16(1):98. doi: 10.1186/s12985-019-1201-1.
BACKGROUND
Coxsackievirus 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.
METHODS
The 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.
RESULTS
CA10 viruses were rescued from the constructed cDNA clone and reached a maximum titer of 10TCID/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.
CONCLUSION
Infectious 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.
背景
柯萨奇病毒 A10（CA10）是导致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非常重要。然而，目前尚无关于 CA10 感染性克隆构建的报道。
方法
通过 In-Fusion 克隆技术，将来源于临床分离株的 CA10 全基因组一步扩增为两个片段并连接到线性化的质粒载体上。将获得的 CA10 cDNA 克隆和编码 T7 RNA 聚合酶的质粒共转染 293T 细胞以拯救 CA10 病毒。通过 SDS-PAGE、Western blot 和透射电镜观察鉴定拯救的病毒。将 CA10 病毒通过脑内途径接种于 1 日龄 ICR 小鼠，观察临床症状。对濒死小鼠的多个组织进行 H&E 染色、实时 PCR 和免疫组化染色，分析致病变化和病毒分布。
结果
从构建的 cDNA 克隆中拯救出了 CA10 病毒，在 RD 细胞中传代一代后达到了 10TCID/mL 的最大滴度。该病毒具有与亲本病毒相似的理化性质，也具有高毒力和诱导新生 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收缩。在感染小鼠中检测到病毒抗原和最大量的病毒 RNA，提示肢体骨骼肌可能是病毒复制的最可能部位。
结论
首次成功构建了 CA10 的感染性克隆，这将有助于建立标准化的感染 CA10 的新生小鼠模型，用于疫苗和抗病毒药物的评估，以及模型株的保存和共享。
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