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目的通过系统比较观察一个利用CRISPR/Cas9系统构建的单纯疱疹病毒Ⅰ型(herpes simplex virus,HSV-1)基因修饰减毒株M3和野生型毒株Mckrae感染BALB/c小鼠的感染特征,进一步确定M3毒株的减毒表型。方法利用基因修饰的减毒株M3和野生型毒株Mckrae,经角膜、滴鼻、肌内注射途径感染BALB/c小鼠,模拟急性感染模式;经足垫途径感染BALB/c小鼠,模拟慢性感染模式,观察感染小鼠的相应病理学指征。结果实验小鼠的观察结果表明,虽然Mckrae经不同途径感染的小鼠存活率存在差异,但这些小鼠所出现的弓背、倒毛、单侧眼失明、单侧后肢瘫痪等临床特征和一定的死亡比例等现象,在M3减毒株感染的小鼠中均未出现;q-PCR检测表明,M3不同途径感染小鼠的心、脑、肺、肾、肝组织病毒载量的增殖趋势均显著低于Mckrae感染的小鼠;组织病理学检查结果提示,Mckrae感染小鼠的肝、肺组织出现的病理损伤程度明显强于M3减毒株感染的小鼠。免疫组织化学结果提示,M3减毒株感染小鼠的肝组织病毒抗原表达和Mckrae感染小鼠存在明显差异。结论 HSV-1 M3基因突变株相较于Mckrae野生型毒株表现了明显的临床病理意义上的减毒表型。
Objective To compare the infection characteristics of BALB / c mice infected by herpes simplex virus (HSV-1) genetically modified attenuated strain M3 and wild-type strain Mckrae by using CRISPR / Cas9 system. Determine the attenuated phenotype of M3 strains. Methods BALB / c mice were infected by genetically modified attenuated strain M3 and wild-type strain Mckrae via intra-corneal, intranasal and intramuscular injection to simulate the acute infection pattern. BALB / c mice were infected by the footpad approach and simulated Chronic infection patterns were observed in corresponding pathological signs of infected mice. Results The observation of experimental mice showed that although the survival rates of Mckrae infected by different pathways were different, the clinical features such as dorsal bowel, inverted hair, unilateral eye blindness and unilateral hindlimb paralysis appeared in these mice. Of the mice in the M3 attenuated strain did not appear in all the mice infected with the M3 attenuated strain. The q-PCR results showed that the viral load in the hearts, brain, lung, Was significantly lower than Mckrae infected mice; histopathological examination results suggest that Mckrae infected mice liver, lung tissue lesions were significantly greater than the M3 attenuated strain infected mice. Immunohistochemical results suggest that there is a significant difference between the liver tissue viral antigen expression in mice infected with the M3 attenuated strain and Mckrae-infected mice. Conclusion The HSV-1 M3 mutant showed a clinically significant pathogenic attenuated phenotype compared to the wild type strain Mckrae.