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Published on: November 1, 2018
Enhanced detection of Theiler's virus RNA copy equivalents in the mouse central nervous system by real-time RT-PCR
Mark Trottier1, Brian P Schlitt, Howard L Lipton
1Department of Neurology, Evanston Hospital, 2650 Ridge Avenue, Evanston, IL 60201, USA.
Abstract:
Infection of mice by low-neurovirulence Theiler's murine encephalomyelitis virus (TMEV), such as BeAn and DA viruses, provides a relevant experimental animal model for multiple sclerosis (MS). As a step toward determining the kinetics of a persistent central nervous system (CNS) infection that leads to chronic demyelination, we adapted a rapid, accurate and highly specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection and quantitation of BeAn virus RNA copy equivalents in mouse tissues. The assay enabled detection of as few as 20-30 copies of BeAn virus RNA per microg of total RNA from infected mouse tissues and results for spinal cord revealed the same high levels of BeAn RNA as detected by Northern hybridization during the first 4 months of the persistent infection, but also was able to detect virus RNA copies as late as 1 year post-infection. Real-time RT-PCR analysis of BeAn virus RNA copy equivalents in different parts of the CNS, analyses not possible by Northern hybridization, revealed the following cline of virus persistence: spinal cord>brainstem/cerebellum>cerebrospinal fluid (CSF)>cerebral hemispheres. Systemic organs, including heart, intestine and mesenteric lymph nodes of infected mice, showed no evidence of viral persistence at 4 months post-infection.
Insights
A new real-time RT-PCR assay accurately detects Theiler's murine encephalomyelitis virus (TMEV) RNA in mouse tissues, revealing persistent central nervous system (CNS) infection for up to one year, crucial for multiple sclerosis (MS) research.
Area of Science:
- Neurovirology
- Infectious Diseases
- Immunology
Background:
- Theiler's murine encephalomyelitis virus (TMEV) infection in mice serves as a model for multiple sclerosis (MS).
- Understanding the kinetics of persistent TMEV central nervous system (CNS) infection is vital for studying chronic demyelination.
Purpose of the Study:
- To develop and validate a sensitive real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for TMEV RNA detection.
- To quantify BeAn virus RNA copy equivalents in mouse tissues to determine infection kinetics.
Main Methods:
- Adaptation and implementation of a rapid, accurate, and highly specific real-time RT-PCR assay.
- Quantification of BeAn virus RNA in various mouse tissues, including CNS and systemic organs.
- Comparison of real-time RT-PCR results with Northern hybridization for validation.
Main Results:
- The real-time RT-PCR assay detected as few as 20-30 copies of BeAn virus RNA per microgram of total RNA.
- High levels of BeAn RNA were detected in the spinal cord for up to 1 year post-infection, consistent with Northern hybridization for the first 4 months.
- A distinct gradient of virus persistence was observed in the CNS: spinal cord > brainstem/cerebellum > cerebrospinal fluid (CSF) > cerebral hemispheres.
- No viral persistence was detected in systemic organs (heart, intestine, mesenteric lymph nodes) at 4 months post-infection.
Conclusions:
- The developed real-time RT-PCR assay is a sensitive and specific tool for studying TMEV RNA kinetics in mouse models.
- The study elucidates the temporal and spatial dynamics of persistent TMEV infection in the CNS, providing insights into MS pathogenesis.
- The findings highlight the spinal cord as a primary site of long-term TMEV persistence in this experimental model.
