us3基因的缺失对单纯疱疹病毒1型毒力和抗凋亡功能的影响

利用CRISPR/Cas9系统使单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1) ul7、ul41、LAT 基因缺失构建M3减毒株(M3株),在M3株基础上通过缺失 us3 得到M4突变株(M4株)。本研究旨在分析野毒株(McKrae株)、M3株与M4株在毒力和抗细胞凋亡方面的差异。结果表明,McKrae组出现明显的临床症状,且100%死亡(P<0.001),而M3、M4组未出现临床症状。M4组小鼠组织中病毒载量明显低于McKrae组和M3组;病理学检测表明,McKrae组出现蛛网膜出血、胶质小结等现象,而M3、M4组未见病理损伤,M4组炎性因子表达与McKrae、M3组相比也显著下降(P<0.01);免疫后M4组较M3组出现高水平的中和抗体、γ干扰素(interferon γ,IFN-γ)和白细胞介素4(interleukin 4,IL-4)抗原特异性T细胞;McKrae株再次感染时,M4组小鼠组织中病毒载量明显低于对照组和M3组;在人急性T细胞淋巴瘤细胞中,M4株相比McKrae株和M3株可明显诱导细胞凋亡。

Impacts of us3 gene deletion on virulence and anti-apoptotic function of herpes simplex virus type 1

M3 strain with deletion of ul7, ul41 and LAT genes of herpes simplex virus 1 type (HSV-1) was constructed by CRISPR/Cas9 system. M4 strain was obtained by deletion of us3 on the basis of M3 mutant. The purpose of this study was to analyze the differences between McKrae, M3 and M4 strains in attenuation and anti-apoptosis. The results showed that there were obvious clinical symptoms in McKrae group, and 100% of the mice died (P<0.001). No clinical symptoms were found in M3 and M4 groups. The viral load in M4 group was significantly lower than that in McKrae and M3 groups. Pathological examination showed that there were some phenomena such as arachnoid hemorrhage and glial nodules in McKrae group, but no pathological damage was found in M3 and M4 groups. The expression of inflammatory factors in M4 group was significantly lower than that in McKrae and M3 groups (P<0.01). High levels of neutralizing antibodies, interferon γ (IFN-γ) and interleukin 4 (IL-4) antigen-specific T cells were observed in M4 group compared with M3 group after immunization. M4 group had lower viral load than the control and M3 groups when McKrae was re-infected. In Jurkat cells, M4 strains could significantly induce apoptosis compared with McKrae and M3 strains.