The Observation Of Autophagy In The Human Neuroblastoma Cells Induced By Varicella Zoster Virus

BackgrandVaricella-zoster virus(VZV)is an alphaherpesviral pathogen that causes chickenpox or implicit infection status during primary infection.After the initial infection,the viral progeny migrate into the dorsal root ganglia or brain nerve ganglia via sensory neurons,where they establish latency.When the body immunity is low,the virus is re-activated and causes herpes zoster.VZV is a neurotropic alphaherpesvirus that causes postherpetic neuralgia(PHN).PHN seriously affects the quality of life of patients and its treatment is a difficult case in clinical practice.Autophagy is a highly conserved cellular degradation pathway,which can degrade damaged Intracellular components via the lysosomal pathway to maintain cellular homeostasis.Autophagy has also recently been shown to be involved in both the innate and adaptive immunity by eliminating the invading pathogens.Recent studies show that host cells can activate autophagic machinery to control the Human Herpes virus infections.However,human herpes virus have evolved diverse defense strategies to escape,suppress,destroy even exploit autophagy.In this artical,we studied the incubation and proliferation characteristics of VZV in SHSY5Y cells,as well as to observe the biological effects in VZV-infected cells.Furthermore,we also investigate the regularity and characteristic of autophagy induced by the VZV,hoping to offer new ideas for the intervene and treatment of VZV Infection.ObjectiveTo study the incubation and proliferation characteristics of VZV in SHSY5Y cells,as well as to observe the biological effects in VZV-infected cells.Furthermore,we also investigate the regularity and characteristic of autophagy induced by the VZV,hoping to offer new ideas for the intervene and treatment of VZV Infection.Method1.The morphological changes in infected and uninfected cells were observed with inverted and electron microscopy.Cell activity was detected by CCK-8 assay.To compare the different MOI and different serum concentrationson the virus titer.2.The formation of autophagosomes was detected by monodansylcadaverine(MDC)staining.The expression of microtubule-associated protein 1 light chain 3B(LC3B)and SQSTM1/p62 at different time points(24h,48h,72h,96h)was determined by flow cytometry.3.DNA was extracted from VZV virus and DNA copy number at different time points(24h,48h,72h,96h)was detected by Quantitative Real-time PCR.Result1.The cytopathic effect of infected cells increased from 24 to 96 h post-infection compared to the uninfected group.Electron micrographs of infected cells show condensed chromatin and pycnosis within the nucleus.And the mitochondrias were become hypertrophy and hyperplasia,followed by disorder within some mitochondrial cristaes.Varying degrees of vacuolization and autophagosome appeared in the cytoplasm.In addition,mature viral particles scattered in the cytoplasm were also observed.The CCK-8 assay indicated that value of OD after 24h post infection were statistically different compared with control group(P<0.05).Virus titer of MOI of 1 and 10 was significantly higher than the other two groups after infected with different MOI(P<0.05),while there were no significant difference in viral titer between MOI of 1 and 10(P>0.05).The virus titer of 2%FCS group was obviously higher than the other three groups.2.Results of MDC staining showed that the number of autophagosomes increased in the VZV-infected SHSY5Y cells.Furthermore,the expression of LC3B-II protein was increased and the expression of p62 protein was decreased after VZV infection.These results were statistically different compared with control group(P<0.05).3.The results of Quantitative Real-time PCR showed that the DNA copy number of VZV after 72h post infection was significantly higher than that of the other three groups(24h?48h?96h)(P<0.05).Conclusion1.Here we established the VZV-infected SHSY5Y cell model in vetro and use it to observe biological effects in VZV-infected cells.The results provided foundations for studying the mechanisms of pathogenesis of VZV.2.The autophagy was induced after VZV infection of SHSY5Y cells,resulting in the increase of autophagosome and autophagy-related protein.And the process of autophagic flux is unobstructed.Thus,our results indicate that activity of autophagy increased in VZV-infected cells.In addition,enhanced autophagy activity of SHSY5Y cells may also promote VZV replication and increase viral titers.