In situ hybridization of virulent canine distemper virus in brain tissue, using digoxigenin-labeled probes
A Zurbriggen1, C Müller, M Vandevelde
1Institute of Animal Neurology, University of Berne, Switzerland.
Abstract:
Only a few hybridization experiments have been performed for detection of canine distemper virus (CDV) nucleic acid sequences in tissue cultures and in various tissues. Those published studies used probes derived from tissue culture-adapted CDV, and hybridization signals were not obtained in the CNS tissue, although infective CDV and viral antigen were detectable in this tissue. We developed probes complementary to virulent CDV and were able to detect viral RNA not only in primary brain cell cultures, but also in brain tissues, by use of in situ hybridization. Sensitivity of the test at least equaled that of immunohistochemistry. We applied digoxigenin-labeled, strand-specific RNA probes complementary to the nucleoprotein-coding viral nucleic acid sequence. Our results indicate that to detect CDV nucleic acid sequences in brain tissues, it is essential to use probes derived from the virulent virus.
Insights
Developing new probes for canine distemper virus (CDV) detection enabled identifying viral RNA in brain tissues. This advancement is crucial for diagnosing CDV infections, especially in the central nervous system (CNS).
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Neuroscience
Background:
- Previous hybridization methods for canine distemper virus (CDV) nucleic acid detection showed limited success, particularly in central nervous system (CNS) tissues.
- Existing studies utilized probes from tissue culture-adapted CDV, failing to detect viral sequences in CNS despite the presence of infectious virus and antigen.
- This limitation hindered accurate diagnosis and understanding of CDV's neuropathogenesis.
Purpose of the Study:
- To develop and validate novel nucleic acid probes for sensitive detection of virulent canine distemper virus (CDV) in various tissues, including the CNS.
- To improve the diagnostic capability for CDV infections, especially in neurological tissues.
- To investigate the necessity of using virulent virus-derived probes for effective CDV RNA detection.
Main Methods:
- Development of digoxigenin-labeled, strand-specific RNA probes complementary to the nucleoprotein-coding sequence of virulent CDV.
- Application of in situ hybridization techniques to detect viral RNA in primary brain cell cultures and brain tissues.
- Comparison of the sensitivity of the developed method with immunohistochemistry.
Main Results:
- The newly developed probes successfully detected canine distemper virus (CDV) RNA in both primary brain cell cultures and brain tissues.
- In situ hybridization using probes derived from virulent CDV yielded positive signals in CNS tissues where previous methods failed.
- The sensitivity of this novel hybridization assay was found to be at least equivalent to that of immunohistochemistry.
Conclusions:
- Using probes derived from virulent canine distemper virus (CDV) is essential for accurate detection of viral nucleic acid sequences in brain tissues.
- The developed in situ hybridization method offers a sensitive and effective tool for diagnosing CDV infections, particularly in the CNS.
- This advancement improves the ability to study CDV's impact on the nervous system and aids in veterinary diagnostics.
