Modified Mutation Of HSV-1 Us3 Gene Encoding Apoptotic Suppressor Promotes Attenuated Phenotype Of The Virus In Mouse Models

Herpes simplex virus type 1(HSV-1)is a double-stranded DNA virus with complex gene structure and transcriptional regulation mechanism,which can cause diseases such as herpes labialis and even nervous system infections,and hide in nerve tissues.At present,HSV-1 has a very high infection rate among different populations around the world,and the therapeutic drugs are relatively simple.Which has not a effective vaccine to prevent infection.Therefore,live attenuated vaccine is one of the options to effectively prevent and control HSV-1 infection,and the research on attenuated gene mutation has important significance in the development of live attenuated HSV-1 vaccine.The tyrosine/threonine protein kinase-encoding gene us3 is an important gene in the HSV-1 genome and has a regulatory effect on apoptosis.In our laboratory,based on the comprehensive exploration of HSV-1 encoded proteins,we prepared M3 attenuated strains with a clear attenuated phenotype.Now,we use the CRISPR/Cas9 system to construct the us3 gene deletion modified mutant M4.However,the biological characteristics and attenuated phenotype of M4 are still unclear,so we conducted preliminary exploration on the BALB/c mouse model.First,we compared the proliferative capacity of M4 and M3 on KMB17 cells.M4 maintained a relatively consistent low proliferative capacity with M3,which suggest that the deletion of us3 gene does not affect the proliferation of M4 in vitro.At the same time,whether M4 lacking us3 gene also has anti-host cell apoptosis function.So we performed in vitro flow cytometry and morphological detection,and the results showed that M4 can induce clear apoptosis compared with wild type strains McKrae and M3,which may be us3 caused by functional inhibition.In further animal experiments,we infected mice with McKrae wild,M3 and M4 mutants at a dose of 1×104 CCID50,and observed the clinical symptoms of the infected mice in the acute phase.The results showed that the M4 attenuated group had no clinical significance with stable weight gain,and good living conditions.The pathological examination of the brain and spinal cord of the nerve tissue showed that the mice in the wild-type group had pathological damage such as hemorrhage,while the mice in the M3 and M4 groups had no pathological damage.The detection of viral proliferation in the vital organs of the M4 group showed that the M4 group was significantly lower than that of the McKrae group,slightly lower than the M3 group,suggesting that the deletion of the us3 gene maintains the low toxicity of M3.In addition,the expression level of some signals related to inflammatory injury was detected.It was found that the expression level of inflammatory factors in multiple tissues of M4 infected mice was significantly lower than that of McKrae and M3 infected mice.We doubt that may be the function of us3 gene inhibit the down-regulation of the body's inflammatory response.Analysis of the immune response and clinical protection rate of the three strains in mice showed that M4 induced stronger neutralizing antibody levels and cellular immune responses than M3,and was important for McKrae challenge M4 infected mice.The viral load in the tissue is lower than that in the M3 group,and the mutation of the us3 gene may enhance the immune protection of the body.The immune protection promotes the effective removal and control of the virus.The above experimental results suggest that the modified mutant strain M4 encoding the HSV-1 us3 gene which inhibits the apoptotic molecule exhibits a more attenuated phenotype than the M3 in the mouse model.The results of this work provide a basis for the in-depth study of HSV attenuation mechanism,and have some clinical transformation significance for the development of live attenuated vaccine of herpes simplex virus type I.