Susceptibility Of Primary Tree Shrew Dermis Fibroblasts To HCMV And Apoptosis Induced By Virus Infection

Human cytomegalovirus?HCMV?is widespread and can lead to serious effect.Due to its strict species-specific limitations,animal models are lacking,which seriously hinders the research on its pathogenic mechanism and the development of drugs and vaccines.Based on the close relationship between Tupaia belangeri and primates,the biological characteristics,genomic structural features and encoded proteins of Tupaia herpesvirus?TuHV?have high similarity with primate CMV.Therefore,this study selected primary tree shrew dermis fibroblasts?TSDF?for HCMV susceptibility study,simultaneous selection of macaque skin fibroblasts and human foreskin fibroblasts as parallel controls.The strain used in the infection experiment was the HCMV-Towne_BAC strain with the green fluorescent gene gfp.The cytopathic effect was first observed,and the GFP positive rate was counted.Secondly,the expression of viral proteins IE1,UL44 and PP28 in the early,middle and late stages was detected by immunoblotting.Then the q-PCR was performed on the copy number of the virus pp65 gene of infected cells.Finally,the virus titer of the infected supernatant was detected by the plaque assay.The results showed that infected TSDF can appear pathological effect of cell swelling and rounding which is similar to HFF.GFP expression was delayed at first,However,the GFP positive rate of the two cells was not significantly different between 48 and 96 hpi.TSDF can support the viral protein expression and genomic replication of different phases of HCMV.TSDF also supported the production of a small number of infectious HCMV progeny viruses.Due to partial death of TSDF during the infection,we also explored the phenomenon of cell death.In this study,we have initially explored the death of TSDF that infected with HCMV-Towne.Besides,we used CCK?cell counting kit?to measure cell viability.Similarly vital,we used qRT-PCR to detect the transcriptional differences between apoptosis-related factors bax,bcl-2 and an unfolded protein response relevant factors chop,atf4,xbp1s.Ultimately,western blot was also used to detect the main viral proteins which is IE1,UL44,and apoptosis related factors that was Bax,Caspase-9,Caspase-3 and PARP.Additionally,apoptosis was also detected by AV-PI double staining.All the results showed the cytopathic effect and cellular death gradually worsen as the infection progresses,accompanying significant decrease of cell viability?P<0.001?.Moreover infection caused up-regulates of the transcription levels of bax,bcl-2,chop,atf4,xbp1s,the bax and bcl-2 transcription levels showed significant antagonistic trend.Meanwhile,protein expression and progressive shear activation of Bax,Caspase-9,Caspase-3,PARP,which also performed up-regulation.Nevertheless viral proteins IE1 and UL44 can up-regulation to the end of a complete virus replication cycle.In summary,our study indicates that TSDF is a semi-permissive cell of HCMV,which supports the production of a small number of infectious HCMV progeny viruses.At the same time,cross-species infection of HCMV induces TSDF apoptosis and is closely related to endoplasmic reticulum and mitochondria.TSDF is expected to become a cellular model of HCMV cross-species infection that is beneficial for further exploration of the species-specific mechanisms of HCMV.