Analysis Of The Infection Characteristics Of The Genetically Modified Mutant Strain M3 And Mutant Strain M5 Of Herpes Simplex Virus Type I (HSV-1)

Herpes simplex virus 1(HSV-1)is an important pathogen with a worldwide epidemic trend that affects populations of various ages.HSV-1 infection is primarily characterized cold sores,corneal herpes,herpes encephalitis,and genital herpes.In addition,it may frequently be reactivated from a latent to recurrent infection.Researches on the prevention and treatment of this pathogen have been going on for a long time,and some drugs such as acyclovir and its derivatives have been widely used in clinical practice.However,there is currently no effective vaccine to prevent herpes simplex virus infection.Attenuated vaccines have been considered as effective candidates,mainly because of its capacity to ensure a cellular immune response capable of inducing a cytotoxic effect in the vaccinated individual.In the previous study,we have constructed the attenuated strains M3 and M5 through the CRISPR/Cas9 system.The M3 strain was obtained by sequentially deleting the virulence factor UL7,immunoregulatory protein Vhs(UL41),and the latent associated transcript LAT gene.Based on M3 strain,we then modified the US3 and US5 genes of HSV-1 in turn and obtained the attenuated strain M5.These two genes play an inhibitory role in the process of apoptosis induced by cytotoxic T cells(CTL)and participate in the immune escape process of HSV-1.In the first part of the work,BALB/c mice were infected with the HSV-1 wild-type strain Mckrae and the attenuated strain M3 through corneal,nasal and intramuscular routes,respectively.We then compared the clinical,etiology,and histopathological characteristics of the viral infected BALB/c mice to determine the attenuated phenotype of the M3 strain in acute infection.In the second part of the work,BALB/c mice were immunized with the attenuated strain M5,followed by the viral challenge with wild-type strainson the 28th and 56th days post immunization.The clinical manifestations,etiology,and immune responses of BALB/c mice immunized with M5 immunized group and PBS immunized group were compared to evaluate the safety and protection effect of attenuated strain M5.In the first part of the work,preliminary clinical observations showed that there were differences in the survival rate of mice infected by McKrae strain of different routes.However,the clinical manifestation and mortality rate that occurred in these mice did not appear in mice infected with M3 strains.The q-PCR results also showed that the viral load in the major tissues of mice infected with M3 strain was significantly lower than that of Mckrae-infected mice by different inoculation routes.Similarly,histopathological examinations revealed that the degree of pathological damage in the liver and lung tissues of Mckrae-infected mice was significantly stronger than that of M3 strain.The results of immunohistochemistry also showed that the expression of viral antigen in liver tissue of mice infected with M3 attenuated strain was significantly lower than that of Mckrae infected mice.These results suggest that artificial modification of certain viral proteins does indeed have the potential to alter the pathogenicity of HSV-1 strains.In the second part of the work,we also found that,compared with the PBS negative control group,there is no clinical symptoms in mice infected with HSV-1 wild-type strain(Mckrae,17+)on the 28 days and 56 days after M5 immunization.Serum neutralizing antibody detection and Elispot assay showed that M5-immunized mice could induce strong cellular and humoral immune responses.The results of histopathological examination showed that the degree of pathological damage of the central nervous system tissue of M5-immunized mice infected was significantly lower than that of PBS-immunized group after viral challenge.The viral load in the central nervous system and lung tissues of M5-immunized mice was also significantly lower than that of PBS-immunized group after wild-type virus infection.These results indicate that M5 strains can induce ideal adaptive immune responses in mice model.These immune responses can effectively control the pathogenicity and proliferation of wild-type virus in mice.