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Updated: Aug 23, 2026
High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue
Published on: April 30, 2019
Preservation of rabies virus RNA from brain tissue using glycerine
Alvaro Aguilar-Setién1, Hector Aguila-Tecuatl, Emiliano Tesoro-Cruz
1Unidad de Investigación Médica en Inmunología, Coordinación de Investigación Médica, Instituto Mexicano del Seguro Social, México DF, México. aaguilas@data.net.mx
Abstract:
The challenge virus standard (CVS) strain and a wild isolate from a Mexican child who died of hematophagous bat (Desmodus rotundus)-transmitted rabies were injected intracerebrally into BALB/c mice. Brains obtained from infected mice were immersed in 80%, 50%, and 40% glycerine/phosphate-buffered saline (PBS). RNA was extracted from brains on days 1, 2, 3, 7, 21, and 60, and reverse transcriptase polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) tests were performed for rabies virus characterization. Storage temperature variation was recorded during the preservation period. The RT-PCR-RFLP tests were successfully performed on brain samples preserved in 50% glycerine/PBS, but not in those preserved in 80% or 40% glycerine/PBS. Temperatures ranged from 12 to 33 degrees C and were not harmful, provided that 50% glycerine/PBS was used. We concluded that brain samples obtained and stored under field conditions (i.e. without refrigeration) for up to 60 d can arrive at a reference laboratory in an adequate condition for viral RNA analysis.
Insights
Rabies virus RNA can be analyzed from brain samples stored without refrigeration for up to 60 days using a 50% glycerine/phosphate-buffered saline solution. This method enables reliable rabies virus characterization from field samples.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Public Health
Background:
- Rabies virus diagnosis is critical for public health, especially in regions with bat-transmitted rabies.
- Preservation of brain samples under field conditions is challenging due to limited access to refrigeration.
- Effective sample preservation is essential for accurate viral RNA analysis and molecular characterization.
Purpose of the Study:
- To evaluate the efficacy of different glycerine/phosphate-buffered saline (PBS) concentrations for preserving rabies virus RNA in mouse brain samples.
- To determine if rabies virus RNA can be successfully analyzed from samples stored under non-refrigerated field conditions for extended periods.
- To assess the impact of temperature fluctuations on the integrity of viral RNA during sample preservation.
Main Methods:
- BALB/c mice were intracerebrally inoculated with challenge virus standard (CVS) strain and a wild rabies virus isolate.
- Brain samples were preserved in 80%, 50%, and 40% glycerine/PBS solutions.
- Viral RNA was extracted at various time points (1, 2, 3, 7, 21, 60 days) post-preservation.
- Reverse transcriptase polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) was used for rabies virus characterization.
- Storage temperatures were monitored throughout the preservation period.
Main Results:
- RT-PCR-RFLP analysis was successful for brain samples preserved in 50% glycerine/PBS.
- Preservation in 80% or 40% glycerine/PBS did not yield successful RT-PCR-RFLP results.
- Temperatures ranging from 12 to 33 degrees C did not compromise viral RNA integrity when 50% glycerine/PBS was used.
- Successful viral RNA analysis was achieved from samples stored for up to 60 days under ambient temperatures.
Conclusions:
- Brain samples collected and preserved in 50% glycerine/PBS under field conditions are suitable for rabies virus RNA analysis.
- This preservation method facilitates molecular characterization of rabies virus, even without refrigeration.
- The findings support the use of non-refrigerated sample preservation for rabies surveillance and diagnosis in resource-limited settings.
