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Isolation, Transfection, and Culture of Primary Human Monocytes
Published on: December 16, 2019
Theiler's virus infection of primary cultures of bone marrow-derived monocytes/macrophages
Cécile Martinat1, Ignacio Mena, Michel Brahic
1Unité des Virus Lents, CNRS URA 1930, Département de Virologie, 28 rue du Docteur Roux, Institut Pasteur, 75724 Paris Cedex 15, France.
Abstract:
Theiler's virus, a murine picornavirus, causes a persistent infection of macrophage/microglial cells in the central nervous systems of SJL/J mice. Viral replication is restricted in the majority of infected cells, whereas a minority of them contain large amounts of viral RNA and antigens. For the present work, we infected primary cultures of bone marrow monocytes/macrophages from SJL/J mice with Theiler's virus. During the first 10 h postinfection (p.i.), infected monocytes/macrophages were round and covered with filopodia and contained large amounts of viral antigens throughout their cytoplasm. Later on, they were large, flat, and devoid of filopodia and they contained only small amounts of viral antigens distributed in discrete inclusions. These two types of infected cells were very reminiscent of the two types of infected macrophages found in the spinal cords of SJL/J mice. At the peak of virus production, the viral yield per cell was approximately 200 times lower than that for BHK-21 cells. Cell death occurred in the culture during the first 24 h p.i. but not thereafter. No infected cells could be detected after 4 days p.i., and the infection never spread to 100% of the cells. This restriction was unchanged by treating the medium at pH 2 but was abolished by treating it with a neutralizing alpha/beta interferon antiserum, indicating a role for this cytokine in limiting virus expression in monocyte/macrophage cultures. The role of alpha/beta interferon was confirmed by the observation that monocytes/macrophages from IFNA/BR(-/-) mice were fully permissive.
Insights
Theiler's virus infection in mice macrophages shows restricted replication, with alpha/beta interferon limiting viral spread. This cytokine plays a key role in controlling Theiler's virus persistence in the central nervous system.
Area of Science:
- Neurovirology
- Immunology
- Cell Biology
Background:
- Theiler's virus establishes persistent infections in macrophage/microglial cells within the mouse central nervous system.
- Infected cells display heterogeneous viral RNA and antigen levels, suggesting differential replication.
- SJL/J mice are a key model for studying Theiler's virus-induced persistent infections.
Purpose of the Study:
- To investigate the in vitro behavior of Theiler's virus in primary bone marrow monocyte/macrophage cultures from SJL/J mice.
- To characterize the morphological changes and viral antigen distribution in infected macrophages.
- To elucidate the role of cytokines, specifically alpha/beta interferon, in restricting Theiler's virus replication in these cells.
Main Methods:
- Primary bone marrow monocyte/macrophage cultures from SJL/J mice were infected with Theiler's virus.
- Infected cells were analyzed for morphology and viral antigen presence at various time points postinfection.
- Viral yield was compared to that of BHK-21 cells.
- Intervention with pH 2 treatment and alpha/beta interferon antiserum was performed.
- Experiments were repeated using macrophages from IFNA/BR(-/-) mice.
Main Results:
- Infected macrophages exhibited distinct morphological changes and viral antigen distribution patterns over time, mirroring in vivo observations.
- Viral yield per cell was significantly lower compared to permissive cell lines like BHK-21.
- Cell death occurred within the first 24 hours postinfection, with no spread to 100% of cells.
- Interferon (IFN) alpha/beta antiserum treatment abolished viral replication restriction, while pH 2 treatment had no effect.
- Macrophages from IFNA/BR(-/-) mice showed fully permissive viral replication.
Conclusions:
- Alpha/beta interferon plays a critical role in restricting Theiler's virus replication and spread in primary macrophage cultures.
- The observed in vitro characteristics of infected macrophages closely resemble the in vivo pathology of Theiler's virus infection in SJL/J mice.
- Understanding interferon's role is crucial for developing strategies against persistent Theiler's virus infections in the central nervous system.
