Cytobiological Characteristics And Preliminary Investigation Of Replication-defective Mechanism Of Highly Attenuated NTV Strain Of Vaccinia Virus Tiantan

Vaccinia virus(VACV)took an essential role in the campaign of eradication of smallpox as the vaccine aiming at smallpox in early time,while since smallpox was eradicated,vaccinia virus was introduced into laboratory as gene expression vector which is widely used in prevention and treatment of multiple kinds of infectious diseases and cancers.Vaccinia virus was modified by researchers to not replicate effectively in human cell lines,called Replication-deficient vaccinia virus,which improved safety of vaccinia virus vector,as immunologic suppression is needed in medical process such as infection of human immunodeficiency virus(HIV)and organ transplantation,which made safety of vaccinia virus vector a challenge.At present,the commonly used replication-deficient vaccinia virus strains in the world are:Modified Vaccinia Ankara(MVA),NYVAC and the derivative strain of Chinese Vaccinia Virus Tiantan(VTT),Replication-deficient Vaccinia Virus Tiantan(NTV).The biological characteristics and replication defect mechanism of MVA and NYVAC were clear,but little was known about NTV,which is to be explored.To clearly understand biological characteristics and replication defect mechanism of NTV,and to provide data support for safely use of NTV as a vaccine vector in the future,this study Studied on the replication ability,cytophilicity and virulence of NTV,and detected viral functional protein expression level,mRNA transcription level,eIF2? phosphorylation level and PARP cleavage.The main results are as follow.1.Preparation of polyantiserum of protein closely related with vaccinia virus replication:Efficiently prokaryotic expressed vaccinia virus early protein E3 and late protein A17,F17 with His-bind affinity chromatography column purification or gel cutting purification.Protein purity of all the three proteins was up to 90%.Mouse polyclonal antiserum was prepared by immunizing mice to provide antibody basis of detection of vaccinia virus protein expression level.2.Cellular biological characteristics of NT V:We detected virus replication in cells and intercellular diffusion ability in CEF,BHK-21,Vero,143,HeLa,2BS,and Hep-2 cell lines,finding out that VTT,the parental strain of NTV can format plaque and product high titer progeny virus in all the cell lines above,while NTV can only format plaque and product high titer progeny virus in CEF and BHK-21 cell.Plaque cannot be formatted in NTV infected143,HeLa,2BS,Hep-2 and Vero cell,it cannot replicate effectively.The ability of replication of NTV in most human and other animal cells is weakened or blocked,and cannot be replicated effectively.3.Exploration of replication-defective mechanism of NTV includes detection of expression level of virus protein,transcription level,eIF2a phosphorylation level and PARP cleavage.In NTV infected BHK-21 cell,early protein E3 and late protein A17,A27,F17 were expressed as normally as VTT,while in NTV infected HeLa cell,protein E3 and A27 were expressed normally but late protein A17 and F17 were only detected faint binds,which shows a marked decrease in expression.Transcription level and eIF2?phosphorylation level in non-permissive cell were detected as well,showing that the amount of A17,A27,F17 mRNA at the same time were not significantly different in VTT and NTV infected cells.Meanwhile,eIF2? phosphorylation level was higher in NTV infected cells,showing that,the blocking of protein expression was in the stage of translation.Detection of PARP cleavage in NTV infected cells:A large number of PARP degradation fragments PARP cleavage was detected in NTV infected cells during the infection period.But there is only a weak PARP cleavage in VTT infected cells,showing that NTV infection induces apoptosis,which directly restrict the replication and proliferation of viruses.This study found out that NTV cannot replicate efficiently in most mammalian cells by detecting replicability,cytotropism,cytopathy of NTV infected cells from different species and tissue sources;expressed and purified vaccinia virus early protein E3 and late protein A17,F17 and A27;detected corresponding protein expression level in NTV infected HeLa cells,and find out that expression of late protein A17 and F17 were inhibited in the translation stage in NTV infected HeLa cell.Meanwhile,further study shows that degradation production of PARP can be detected in late stage of NTV infected cells,indicating that apoptosis occurs in the late stage of NTV infected cells.In this study,we conclude that the mechanism of replication defect of NTV may be related to inhibition of late protein translation and apoptosis,and the block of NTV late protein is associate with the increase of eIF2aphosphorylation level after infection.