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Molecular and immunological methods to detect rotavirus in formalin-fixed tissue
Kathleen M Tatti1, Jon Gentsch, Wun-Ju Shieh
1Division of Viral and Rickettsial Disease, Centers for Disease Control and Prevention, National Center for Infectious Diseases, Infectious Disease Pathology Activity, 1600 Clifton Road, NE, Mailstop G30, Atlanta, GA 30333, USA. ket2@cdc.gov
Journal of Virological Methods
|September 25, 2002
Summary
New methods detect rotavirus in tissue samples. These techniques, including RT-PCR, IHC, and ISH, aid in studying rotavirus vaccine safety and natural infections.
Area of Science:
- Virology
- Pathology
- Immunology
Background:
- A rhesus-based rotavirus vaccine was withdrawn in 1999 due to intussusception reports.
- Detecting rotavirus in fixed tissues is crucial for vaccine safety studies and understanding disease.
- Current diagnostic methods may be insufficient for formalin-fixed, paraffin-embedded samples.
Purpose of the Study:
- To develop and validate methods for detecting rotavirus in formalin-fixed pathology specimens.
- To assess the utility of RT-PCR, IHC, and ISH for rotavirus detection in tissues.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) targeting the VP4 gene.
- Immunohistochemistry (IHC) using polyclonal and monoclonal antibodies.
- In situ hybridization (ISH) with riboprobes for VP4 and NSP4 genes.
Main Results:
- RT-PCR successfully detected simian rotavirus strains (RRV, YK-1) in infected animal tissues.
- IHC identified rotavirus antigens in infected mouse liver.
- ISH confirmed the presence of rotavirus genes (VP4, NSP4) in infected liver tissues.
Conclusions:
- RT-PCR, IHC, and ISH are effective for detecting rotavirus in formalin-fixed tissues.
- These methods can aid in investigating rotavirus vaccine-associated intussusception.
- The techniques will advance the study of rotavirus pathogenesis and natural infections.