Related Experiment Videos
Detection of canine distemper virus in 100% of Paget's disease samples by in situ-reverse transcriptase-polymerase
A P Mee1, J A Dixon, J A Hoyland
1Bone Disease Research Centre, University Department of Medicine, Manchester Royal Infirmary, UK. andy.mee@man.ac.uk
Abstract:
Previous evidence implicating paramyxoviruses in the aetiopathology of Paget's disease of bone has been controversial. While several groups have demonstrated the presence of paramyxoviruses using electron microscopy, immunohistochemistry, and molecular biological techniques, others have found no evidence of viruses using reverse transcriptase-polymerase chain reaction (RT-PCR). We have previously provided evidence that canine distemper virus (CDV) is present in approximately 65% of samples of pagetic bone, using in situ hybridization and RT-PCR; however, these results have been criticized. To further investigate the possible Role of CDV, we have now developed the technique of in situ-RT-PCR (IS-RT-PCR) to examine for the presence of CDV-nucleocapsid (CDV-N) ribonucleic acid (RNA) in pagetic bone. Control samples consisted of uninvolved sites from patients with the disease, normal bone, and several active remodeling states. IS-RT-PCR was optimized to detect CDV-N using distemper-infected vero cells. The specificity of the technique was confirmed using vero cells infected with CDV, which showed amplified signal following IS-RT-PCR, and cells infected with measles virus (MV), in which no positive signal for CDV was detected by IS-RT-PCR. Following conventional in situ hybridization, CDV-N was detectable in 10 of 15 pagetic bone samples. However, after five, and particularly 10, cycles of IS-RT-PCR, CDV-N was found in all 15 samples. There was no evidence of CDV in four samples from uninvolved sites from pagetic patients, or in any of the other control samples. In this study, using the novel technique of IS-RT-PCR, CDV was found to be present in 100% of pagetic samples examined. There was no evidence of the virus in any of the control samples, including samples of bone from uninvolved sites from patients with Paget's disease. These results provide additional proof that CDV is present within pagetic bone and further support the hypothesis that paramyxoviruses are involved in the etiopathology of Paget's disease.
Insights
Canine distemper virus (CDV) was detected in 100% of Paget's disease bone samples using in situ-RT-PCR. This finding provides strong evidence for paramyxovirus involvement in Paget's disease aetiopathology.
Area of Science:
- Virology
- Bone Biology
- Pathology
Background:
- Paget's disease of bone aetiopathology remains unclear.
- Previous studies have controversially implicated paramyxoviruses.
- Canine distemper virus (CDV) has been suggested as a potential causative agent.
Purpose of the Study:
- To investigate the presence of CDV in Paget's disease bone samples.
- To further explore the role of CDV in the aetiopathology of Paget's disease.
- To develop and apply a novel in situ-RT-PCR (IS-RT-PCR) technique.
Main Methods:
- Development and optimization of IS-RT-PCR for detecting CDV-nucleocapsid (CDV-N) RNA.
- Testing IS-RT-PCR specificity using CDV and measles virus (MV) infected cells.
- Analysis of 15 Paget's disease bone samples and various control bone samples.
Main Results:
- Conventional in situ hybridization detected CDV-N in 10/15 Paget's samples.
- IS-RT-PCR detected CDV-N in all 15 (100%) Paget's samples after 10 cycles.
- No CDV evidence was found in control samples, including uninvolved bone from Paget's patients.
Conclusions:
- The novel IS-RT-PCR technique confirms 100% presence of CDV in Paget's disease bone.
- These findings strongly support the hypothesis linking paramyxoviruses to Paget's disease aetiopathology.
- CDV is a significant etiological factor in Paget's disease of bone.