Japanese Encephalitis Virus Replication and Inhibitory Effect of shRNA in Mice

Japanese Encephalitis Virus (JEV) is responsible for over 30,000 annual cases of encephalitis worldwide, causing 30% mortality. JEV is thus a continuing threat to public health, so development of new antiviral drugs against JEV is desirable. Here, we examined JEV replication in mouse and used a short hairpin RNA JRi as the antiviral agent. The features of virus replication in neuron and survival rates of mice infected with JEV were different between virus strains. The mice infected with the virulent JEV strain (JaGAr01) were injected with pJRi (100 μg/mouse) which produced shRNAJRi. The survival rates of mice treated at 3 days before, the same day and 3 days after JEV infection were 22%, 78% and 44%, respectively. In addition, we demonstrated that the injection of pJRi induced interferon (IFN) production in cells and mice. These results suggest that the replication of JEV can be efficiently inhibited by RNAi and innate immunity including IFN. These data mean that pJRi has the inhibitory activity against JEV infection in vivo, and could be used as an antiviral drug to treat JEV infection.

Hepatitis C virus nonstructural protein NS_3 and telomerase activity

OBJECTIVE: To study the effect of hepatitis C virus nonstructural protein NS(3) (HCV NS3) on telomerase activity and carcinogenesis. METHODS: Streptavidin-peroxidase (SP) conjugated method was used to detect the expression of HCV NS(3) protein in NIH3T3 cells transfected with plasmid pRcHCNS(3)-5' and pRcHCNS(3)-3'. Telomerase activity was detected by an in situ telomerase activity labeling method, telomeric repeat amplification protocol polymerase chain reaction (TRAP-PCR) and telomerase PCR enzyme linked immunosorbent assay (ELISA) technology in the transfected and non-transfected NIH3T3 cells. RESULTS: HCV NS(3) protein was expressed in the NIH3T3 cells transfected with plasmid pRcHCNS(3)-5' expressing HCV NS(3) C-terminal deleted protein or with plasmid pRcHCNS(3)-3' expressing HCV NS(3) N-terminal deleted protein. The positive signal of HCV NS(3) protein was localized in the cytoplasm of NIH3T3 cells, and the signal intensity of the former was stronger. Telomerase activity in NIH3T3 cells transfected with plasmid pRcHCNS(3)-5' was stronger than that in NIH3T3 cells transfected with plasmid pRcHCNS(3)-3' (P

Characterization and E protein expression of mutant strains during persistent infection of KN73 cells with Japanese encephalitis virus

OBJECTIVE: To study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression. METHODS: Persistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins. RESULTS: In the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed. CONCLUSIONS: The virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.

肺腺癌HIF-1α／HIF-2α的表达与FDG摄取的关系

目的乏氧是肿瘤形成和进展的关键因素。乏氧诱导因子-1α（HIF-1α）可在短时间内诱导和调节对急性和重度乏氧的反应，而乏氧诱导因子-2α（HIF-2α）引起的反应持续时间更长，并能调控对慢性和中度乏氧的反应。乏氧可使细胞FDG摄取增加。因此，HIF可能在细胞脱氧葡萄糖（FDG）摄取的过程中起着重要的作用。