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Particle Agglutination Method for Poliovirus Identification
Published on: April 20, 2011
Isolation and Characterization of Poliovirus in Cell Culture Systems
Bruce R Thorley1, Jason A Roberts2
1National Enterovirus Reference Laboratory, Victorian Infectious Diseases Reference Laboratory, Doherty Institute, 792 Elizabeth Street, Melbourne, VIC, 3000, Australia. bruce.thorley@mh.org.au.
Abstract:
The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.
Insights
Cell culture remains crucial for poliovirus isolation, despite advances in genetic testing. This method is vital for the Global Polio Eradication Initiative, ensuring accurate identification of enteroviruses.
Area of Science:
- Clinical virology
- Molecular biology
- Public health virology
Background:
- Cell culture was the gold standard for enterovirus isolation.
- Reverse transcription polymerase chain reaction (RT-PCR) largely replaced cell culture for general enterovirus detection.
- The World Health Organization's Global Polio Eradication Initiative still requires cell culture for poliovirus isolation due to limitations in genetic testing.
Purpose of the Study:
- To describe established cell culture procedures for isolating and characterizing enteroviruses, particularly poliovirus.
- To provide protocols for establishing cell banks and routine cell line passaging.
- To highlight the continued importance of cell culture in polio eradication efforts.
Main Methods:
- Inoculation of mammalian cell lines (e.g., those expressing CD155 receptor) with clinical specimens.
- Passaging of infected cell cultures to increase virus titer.
- Establishing cell banks and performing routine cell line maintenance.
Main Results:
- Cell culture allows for the isolation and characterization of enteroviruses, including poliovirus.
- Virus replication in cell culture leads to a cytopathic effect, indicating infection.
- Cultured poliovirus isolates can be stored long-term at low temperatures for further analysis.
Conclusions:
- Cell culture remains indispensable for poliovirus isolation in the context of global eradication efforts.
- The described cell culture protocols are applicable to various enteroviruses.
- Despite advancements in molecular diagnostics, cell culture provides a reliable method for virus isolation and characterization.
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