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Amplification of JC virus regulatory DNA sequences from cerebrospinal fluid: diagnostic value for progressive
1Department of Viral Infection, University of Tokyo, Japan.
Abstract:
Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease in the central nervous system caused by a ubiquitous human polyomavirus designated as JC virus (JCV). PML affects individuals with decreased immune competence and is now one of the common opportunistic infections in patients with AIDS. JCV DNAs in the brain of PML patients contain various PML-type regulatory regions that were generated from the archetypal regulatory region during persistence. Recently, many studies have suggested that detection of JCV DNA from the cerebrospinal fluid (CSF) may offer a tool for diagnosing PML. However, in all of these studies, coding sequences within the T antigen or capsid protein gene have been targeted for amplification. To amplify the JCV regulatory region, we established a nested PCR that could efficiently amplify the regulatory region from most JCV subtypes prevalent in the world. Using this PCR, we amplified JCV regulatory regions from the CSF samples from 4 patients strongly suspected of PML, whereas amplification was negative from 80 CSF samples from patients without PML. Sequencing of the amplified fragments revealed that they had unique deletions and/or duplications. Furthermore, in 3 PML patients, we analyzed the structures of regulatory regions derived from the brain as well as CSF. In each of these cases, the major regulatory sequence of both origins were identical. This finding indicates that JCV DNA in brain lesions is excreted in the CSF. Since the structures of PML-type JCV regulatory regions are unique to individual patients, the current PCR, if the amplified fragments are sequenced, can eliminate false positives that may arise from contaminations.
Insights
Diagnosing progressive multifocal leukoencephalopathy (PML) can be improved by detecting JC virus (JCV) DNA regulatory regions in cerebrospinal fluid. This new nested PCR method accurately identifies unique JCV DNA sequences, aiding in PML diagnosis.
Area of Science:
- Neurovirology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system.
- PML is caused by the JC virus (JCV) and primarily affects immunocompromised individuals, including those with AIDS.
- Current diagnostic methods for PML often target JCV coding sequences, but amplifying the regulatory region may offer improved diagnostic accuracy.
Purpose of the Study:
- To develop and validate a nested PCR method for amplifying the JC virus (JCV) regulatory region from cerebrospinal fluid (CSF).
- To assess the utility of detecting JCV regulatory regions in CSF for diagnosing PML.
- To characterize the unique structures of PML-type JCV regulatory regions.
Main Methods:
- Development of a novel nested PCR assay to amplify the JCV regulatory region.
- Testing the assay on CSF samples from patients suspected of PML and control patients.
- Sequencing of amplified JCV regulatory regions to identify unique deletions and duplications.
- Comparison of JCV regulatory region structures from brain lesions and CSF in PML patients.
Main Results:
- The nested PCR successfully amplified JCV regulatory regions from CSF samples of 4 PML patients.
- Amplification was negative in 80 CSF samples from non-PML patients.
- Sequencing revealed unique deletions and/or duplications in the amplified JCV regulatory regions, specific to individual patients.
- JCV regulatory sequences from brain lesions and CSF were identical in 3 PML patients, indicating excretion of JCV DNA in CSF.
Conclusions:
- The developed nested PCR is an efficient method for amplifying diverse JCV regulatory regions.
- Detection of unique JCV regulatory regions in CSF can serve as a valuable tool for diagnosing PML.
- The method's ability to sequence unique regulatory regions can help eliminate false positives from contamination.