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Characterization and E protein expression of mutant strains during persistent infection of KN73 cells with Japanese
Guohe Feng1, Tsutomu Takegami, Guizhen Zhao
1Department of Infectious Diseases, Second Clinical College of China Medical University, Shenyang 110004, China.
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.
Insights
Mutants derived from Japanese encephalitis virus (JEV) persistent infections show reduced virulence. This is linked to significantly suppressed E protein expression in infected cells.
Area of Science:
- Virology
- Cell Biology
Background:
- Japanese encephalitis virus (JEV) causes persistent infections in host cells.
- Understanding JEV mutant characterization is crucial for antiviral strategies.
Purpose of the Study:
- To characterize JEV mutants from persistent infections.
- To investigate the relationship between mutant characteristics and E protein expression.
Main Methods:
- Established persistent JEV infection in human hepatoma KN73 cells.
- Collected and titrated cell-associated viruses.
- Utilized indirect immunofluorescence and Western blot assays for protein analysis.
Main Results:
- Persistent JEV infection led to significantly lower virus titers compared to acute infections.
- Viral antigen levels were reduced in persistently infected cells.
- E protein expression was markedly suppressed, while NS3 protein expression remained stable.
Conclusions:
- JEV mutants from persistent infections exhibit reduced virulence and replication.
- Decreased E protein expression is associated with these attenuated JEV mutants.
- These findings suggest a role for E protein suppression in JEV persistence and attenuation.
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