Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants

Abstract

Background JC Virus (JCV) is the etiologic agent for progressive multifocal leukoencephalopathy (PML), a demyelinating disease occurring in the brain of patients with underlying immune compromised states. All viable JCV genomes contain a conserved region in the T protein coding nucleotide sequence that when detected by PCR in CSF is a confirmatory diagnostic marker for PML along with clinical and neuroradiological evidence. The non-coding regulatory region (NCRR) is hypervariable, as evidenced by nucleotide sequence of the non-virulent variant, which is predominantly excreted in urine, versus that of virulent variants found in brain and CSF of PML patients. All variants can be found in blood. Objective A single assay that quantifies and identifies JCV DNA in clinical samples and discriminates between variants has significant value to physicians and patients at risk for PML. Study Design Separate primer pairs were tested together to quantitatively detect conserved viral DNA nucleotide sequence in patient samples, while simultaneously detecting the NCRR specific for the non-virulent variant. Results In testing using control plasmids and patients’ CSF, blood, and urine, PML patients predictably demonstrated the non-virulent, archetype NCRR in urine, but virulent NCRR variants in CSF and blood. Conclusion The JCV qPCR Multiplex assay targets two regions in JCV genomes to simultaneously identify and measure viral DNA, as well as distinguish between variants associated with PML and those that are not. The multiplex results could signal risk for PML if patients are viremic with JCV variants closely associated with PML pathogenesis.

Evidence linking HHV-6 with multiple sclerosis: an update

“…Or perhaps there are HHV-6 variants that differ between sites, for example the periphery and CNS? Studies of JC virus have identified sequences that lend to its classification as non-virulent (found in non-PML patients) or virulent (found in the brain and CSF of PML patients) [52]. As HHV-6 is at once ubiquitous and implicated in a non-ubiquitous pathology, perhaps there are genetic variants that are analogously associated with MS.…”

The JCPYV DNA load inversely correlates with the viral microrna expression in blood and cerebrospinal fluid of patients at risk of PML

“…The extracted DNA was tested in triplicate by a real-time PCR assay using primers and a probe targeting the Large T antigen coding region and NCCR; the sequences were specifically designed to identify the PML prototype (pathogenic virus) and archetype JCPyV (non-pathogenic virus) strains, as previously described [13]. Each reaction was carried out together with negative controls (no template) and plasmid DNA (diluted to contain 10 1 -10 6 copies per milliliter) containing the JCPyV genome.…”

Lymphocyte Gene Expression and JC Virus Noncoding Control Region Sequences Are Linked with the Risk of Progressive Multifocal Leukoencephalopathy

“…Deletion of the D DNA segment is a feature of almost every PML-type NCCR. A clinical test has been developed to identify JCV NCCR sequences with complete or partial deletions in the D segment (30). Almost all PML patients have changes in the NCCR, and almost every patient has unique changes (see Fig.…”