AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by i...AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect na＇ive cells. The presence of minus （antisense） RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques （flow cytometry and Western blot） respectively. RESULTS： The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies （anti-core, and anti-E1）. Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION： HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.展开更多
Because of limited viral replication and lack of cytopathic effect in cell culture,a new PCR-based rapid seroneutralization assay for detection of GII.4norovirus neutralized antibodies was developed with serum samples...Because of limited viral replication and lack of cytopathic effect in cell culture,a new PCR-based rapid seroneutralization assay for detection of GII.4norovirus neutralized antibodies was developed with serum samples from acute-phase patients,convalescent-phase patients and healthy controls.According to this study,neutralizing antibodies were detected in 100% ofconvalescent-phase sera, and in 2.5% of healthy controls sera. However, all of the acute-phase serum samples could not neutralize virus efficiently. Compared to the results from ELISA （96.2% at sensitivity and 80% at specificity）, the present in vitro neutralization assay is more specific and more sensitive.展开更多
We have previously reported that bovine papillomavirus type 1(BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae(budding yeast) cultures(Zhao an...We have previously reported that bovine papillomavirus type 1(BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae(budding yeast) cultures(Zhao and Frazer 2002,Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication(day 3–16), middle weak replication(day 23–34/45) and late stable replication(day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs(cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.展开更多
It was ever thought that genomic information is transmitted faithfully from generation to generation. But our current knowledge does not indicate that it is the case. For example, genomic variations can be generated f...It was ever thought that genomic information is transmitted faithfully from generation to generation. But our current knowledge does not indicate that it is the case. For example, genomic variations can be generated from DNA replication infidelity and unequal chromosome segregation. Natural decay of DNA molecules is also a fundamental source of changing genomic information. In addition, cellular and organismal exposure to exogenous genotoxic agents such as ultraviolet （UV） light, oxidative stress, chemical mutagens, and radiation can lead to a variety of modifications on DNA constituents, resulting in genome alterations. Fortunately, cells have evolved several response systems to tackle numerous DNA lesions in order to maintain their genome integrity. Among them, check- point control is probably the most well-known one. For exam- ple, checkpoint responds to replication stress, replication fork stalling, double-strand DNA breaks, and various other types of DNA lesions. Increasing experimental evidence indicates that genomic instability is probably the fundamental reason for carcinogenesis. Genomic instability is also found to be a main etiological factor of neurodegenerative diseases, aging, immunodeficiency, etc. Thus, to understand how cells regulate to maintain their genomic stability is of fundamental importance.展开更多
基金Supported by the Ministry of Scientific Research, Academy of Scientific Research and Technology, Medical Research Council Code: P5-MED-030-01 and US-Egypt joint project BIO7-002-011
文摘AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect na＇ive cells. The presence of minus （antisense） RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques （flow cytometry and Western blot） respectively. RESULTS： The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies （anti-core, and anti-E1）. Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION： HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.
文摘Because of limited viral replication and lack of cytopathic effect in cell culture,a new PCR-based rapid seroneutralization assay for detection of GII.4norovirus neutralized antibodies was developed with serum samples from acute-phase patients,convalescent-phase patients and healthy controls.According to this study,neutralizing antibodies were detected in 100% ofconvalescent-phase sera, and in 2.5% of healthy controls sera. However, all of the acute-phase serum samples could not neutralize virus efficiently. Compared to the results from ELISA （96.2% at sensitivity and 80% at specificity）, the present in vitro neutralization assay is more specific and more sensitive.
基金This work was funded in part by grants from the National Nature Science Foundation of China(81772791 and 81172463)。
文摘We have previously reported that bovine papillomavirus type 1(BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae(budding yeast) cultures(Zhao and Frazer 2002,Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication(day 3–16), middle weak replication(day 23–34/45) and late stable replication(day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs(cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.
文摘It was ever thought that genomic information is transmitted faithfully from generation to generation. But our current knowledge does not indicate that it is the case. For example, genomic variations can be generated from DNA replication infidelity and unequal chromosome segregation. Natural decay of DNA molecules is also a fundamental source of changing genomic information. In addition, cellular and organismal exposure to exogenous genotoxic agents such as ultraviolet （UV） light, oxidative stress, chemical mutagens, and radiation can lead to a variety of modifications on DNA constituents, resulting in genome alterations. Fortunately, cells have evolved several response systems to tackle numerous DNA lesions in order to maintain their genome integrity. Among them, check- point control is probably the most well-known one. For exam- ple, checkpoint responds to replication stress, replication fork stalling, double-strand DNA breaks, and various other types of DNA lesions. Increasing experimental evidence indicates that genomic instability is probably the fundamental reason for carcinogenesis. Genomic instability is also found to be a main etiological factor of neurodegenerative diseases, aging, immunodeficiency, etc. Thus, to understand how cells regulate to maintain their genomic stability is of fundamental importance.
