AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis ...AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.展开更多
The function of the herpes simplex virus type 1 （HSV-1） UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy wa...The function of the herpes simplex virus type 1 （HSV-1） UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein （EYFP）, the nuclear export signals （NES） of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition, the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattem of UL4. Furthermore, the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1 （CRM1）-dependent manner involving RanGTP hydrolysis展开更多
Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exte...Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exten-sively;however,further genetic engineering of HSV-1 is hindered by its complex genome structure.In the present study,we designed and constructed a synthetic platform for HSV-1 based on H129-G4.The complete genome was constructed from 10 fragments through 3 rounds of synthesis using transformation-associated recombination(TAR)in yeast,and was named H129-Syn-G2.The H129-Syn-G2 genome contained two copies of the gfp gene and was transfected into cells to rescue the virus.According to growth curve assay and electron microscopy results,the synthetic viruses exhibited more optimized growth properties and similar morphogenesis compared to the parental virus.This synthetic platform will facilitate further manipulation of the HSV-1 genome for the devel-opment of neuronal circuit tracers,oncolytic viruses,and vaccines.展开更多
Objective:To study the immune response of CD8+T cell from HLA-A2+ patients with Nasoparygeal cancer(NPC), induced by autologous dendritic cells(DC) loaded with HLA-A2-restricted epitope peptides from EBV latent membra...Objective:To study the immune response of CD8+T cell from HLA-A2+ patients with Nasoparygeal cancer(NPC), induced by autologous dendritic cells(DC) loaded with HLA-A2-restricted epitope peptides from EBV latent membrane protein 2(LMP-2).Methods:Get DCs by culturing plastic-adherent monocytes isolated from NPC patients of HLA-A2+ peripheral blood with cytokine. Stimulating autologous CD8+T with DCs pulsed with the peptides CLGGLLTMV(CLG) and LTAGFIFL(LTA), the HLA-A2-restricted epitope peptides from LMP-2 protein. After 2 weeks for culture, frequencies of CD8+T cells to secret IFN-garmma in response to peptides were detected by Elispot assay and peptide-specific CD8+T cells were measured by tetramer staining.Results:The frequencies of effector cells to secrete IFN-gamma in response to CLG and LTA before DC presentation were (2.67±1.97)/well and (5.33 ±1.86)/well, respectively. After DC stimulation, the number was (42.67±33.79)/well and (25.67±18.25)/well respectively. They all were increased significantly after DC presentation(P<0.05). The median frequencies of CLG-specific CD8+T cell and LTA-specific CD8+T cell were 0.135% and 1.140% respectively before DC presentation; after presenting they were 1.045% and 1.945% respectively. The frequencies of the two specific CD8+T cells were enhanced significantly after DC stimulation(P<0.05).Conclusion:The DC loaded with the epitope peptides of LMP-2 from EBV can induce CD8+T cells to cytotoxic T lymphocytes(CTLs), with clinical potential for immunotherapy.展开更多
基金The paper was support by a grant from the Ministry Youth Research of China,No.98-1-269
文摘AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.
基金the Major State Basic Research Development Program of China(2010CB530105 and 2011CB504802)the National Natural Science Foundation of China(30900059,30870120 and 81000736)the Start-up Fund of the Hundred Talents Program of the Chinese Academy of Sciences(20071010-141)
文摘The function of the herpes simplex virus type 1 （HSV-1） UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study, fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein （EYFP）, the nuclear export signals （NES） of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition, the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattem of UL4. Furthermore, the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1 （CRM1）-dependent manner involving RanGTP hydrolysis
基金Wuhan Institute of Virology for financial support for the research(grant no.EISA020201).
文摘Herpes simplex virus type 1(HSV-1)causes lifelong infections worldwide,and currently there is no efficient cure or vaccine.HSV-1-derived tools,such as neuronal circuit tracers and oncolytic viruses,have been used exten-sively;however,further genetic engineering of HSV-1 is hindered by its complex genome structure.In the present study,we designed and constructed a synthetic platform for HSV-1 based on H129-G4.The complete genome was constructed from 10 fragments through 3 rounds of synthesis using transformation-associated recombination(TAR)in yeast,and was named H129-Syn-G2.The H129-Syn-G2 genome contained two copies of the gfp gene and was transfected into cells to rescue the virus.According to growth curve assay and electron microscopy results,the synthetic viruses exhibited more optimized growth properties and similar morphogenesis compared to the parental virus.This synthetic platform will facilitate further manipulation of the HSV-1 genome for the devel-opment of neuronal circuit tracers,oncolytic viruses,and vaccines.
文摘Objective:To study the immune response of CD8+T cell from HLA-A2+ patients with Nasoparygeal cancer(NPC), induced by autologous dendritic cells(DC) loaded with HLA-A2-restricted epitope peptides from EBV latent membrane protein 2(LMP-2).Methods:Get DCs by culturing plastic-adherent monocytes isolated from NPC patients of HLA-A2+ peripheral blood with cytokine. Stimulating autologous CD8+T with DCs pulsed with the peptides CLGGLLTMV(CLG) and LTAGFIFL(LTA), the HLA-A2-restricted epitope peptides from LMP-2 protein. After 2 weeks for culture, frequencies of CD8+T cells to secret IFN-garmma in response to peptides were detected by Elispot assay and peptide-specific CD8+T cells were measured by tetramer staining.Results:The frequencies of effector cells to secrete IFN-gamma in response to CLG and LTA before DC presentation were (2.67±1.97)/well and (5.33 ±1.86)/well, respectively. After DC stimulation, the number was (42.67±33.79)/well and (25.67±18.25)/well respectively. They all were increased significantly after DC presentation(P<0.05). The median frequencies of CLG-specific CD8+T cell and LTA-specific CD8+T cell were 0.135% and 1.140% respectively before DC presentation; after presenting they were 1.045% and 1.945% respectively. The frequencies of the two specific CD8+T cells were enhanced significantly after DC stimulation(P<0.05).Conclusion:The DC loaded with the epitope peptides of LMP-2 from EBV can induce CD8+T cells to cytotoxic T lymphocytes(CTLs), with clinical potential for immunotherapy.
