Preliminary Study On Herpes Simplex Virus Type 1 Infection Of Human Oral Epithelial Cell In Vitro

Periodontal disease,one of the most common human oral diseases in human race,is a kind of chronic infectious diseases that caused by the invasion of microorganism and resulting in the destruction of periodontal tissue.During the past 100 years,the majority of researchers considered that bacterial plaque was the most important etiological factor of periodontal disease.Since the mid 1990s,many studies have indicated that herpes simplex viruses(HSV)is often detected in periodontitis sites, especially herpes simplex virus type 1(HSV-1).These results indicated that HSV-1 may play a significant role in the development of periodontal disease.Some researchers presumed that the damage to the epithelium barrier of the host may be one of the most important pathogenesis of HSV-1-associated periodontal disease.However,the mechanism has rarely been researched for lack of the model that HSV infects human oral epithelial cells in vitro directly.In this study,we abundantly amplified HSV-1 strains in Vero cells which were used to infect human oral epithelial cells,and established the model that HSV-1 infected human oral epithelial cells in vitro.The present study provides a basis of experimental technique for the further study of the pathogenesis of herpesvirus-associated periodontal diseaseExperiment one:Establishment of the model that HSV-1 infected human oral epithelial cells in vitro.Objective:To abundantly amplify HSV-1 strains and establish the model that HSV-1 infected human oral epithelial cells in vitro.Materials and Methods:HSV-1 strains were abundantly amplified in Vero cells and viral titer was detected by plaque forming experiment.KB cells were infected with HSV-1 at an MOI of 0.5,1.0 or 3.0 PFU/cell.The morphology of the cells was inspected for microscopically detectable alterations through inverted microscope when HSV-1 infected KB cells for 0 h,24 h,48 h respectively.Infected KB cells were collected and then observed through TEM.Nucleic acid of the virus was detected by PCR.Result:HSV-1 strains were abundantly amplified in Vero cells and the titer was standardized to 10~6 PFU/ml.The infected KB cells in three groups didn't display obvious cytopathic effect(CPE)under inverted microscope at either 24 h or 48 h under inverted microscope.There were no mature virus particles in endochylema or nucleus under TEM.The glycoprotein D gene order of HSV-1 could be detected in infected KB cells by PCR. Conclusion:The model that HSV-1 infected human oral epithelial cells in vitro was established.KB cells are the non-permissive cell of HSV-1.HSV-1 infects KB cells leading to transforming infection or virus incubated in the cells causing latent infection.Experiment Two:Transfer infection of HSV-1 to Vero cellsObjective:To checkout indirectly that whether the model of HSV-1 infected human oral epithelial cells in vitro is successful or not.Materials and Methods:After HSV-1 infected KB cells for 48 h,the culture supernatant of infected groups and blank control group were collected.The collected culture supematant were incubated on Vero monolayer.The morphology of the cells was observed through inverted microscope when HSV-1 transfer infected Vero cells for 0 h,24 h,48 h respectively.Infected Vero cells were collected and then observed through TEM.We scored CPE ratio of infected Vero cells in the three groups when transfer infected for 48 h.Result:Compared with control group,Vero cells which infected by the culture supernatant of infected KB cells showed typical CPE at 24 h i.p. under inverted microscope.The amount of CPE was increased at 48 h i.p. Numerous immature virus particles were prominent within the nucleus under TEM.Significant difference of CPE ratio of infected Vero cells in three different groups. Conclusion:The latent viruses in KB cells were activated and produced infectious viral particles.We conclude from this study that:The model that HSV-1 infected human oral epithelial cells in vitro could be established.HSV-1 infects KB cells leading to transforming infection or latent infection.HSV-1 could transfer its DNA genes or fragments of its genome into chromosome of KB cells and steadily express them in progeny virus after cell division.