Detection of reactivation and size variation in the regulatory region of JC virus in brain tissue.

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Abstract
AIMS--To develop a sensitive and specific polymerase chain reaction (PCR) based system for detecting genomic variation in JC virus. To apply this system to formalin fixed, paraffin wax embedded brain tissue from patients with and without progressive multifocal leucoencephalopathy (PML). METHODS--A pair of primers (JC1 and JC2) were designed to be complementary to the early and late regions of JC and BK polyomaviruses, respectively. A third primer (JC3), internal to JC1 and JC2, was designed to be specific for JC virus. The specificity of JC3 was investigated by amplifying plasmids with BK or JC virus genomes. Sensitivity was estimated by titration of a plasmid containing JC virus genome. Seven brains from patients with PML (PMLB) and 30 from patients without PML (non-PMLB) were amplified using JC1 and JC2, followed by JC1 and JC3. Amplification of the beta globin gene was used as an amplification control. RESULTS--Amplification with JC1 and JC2 was common for JC and BK viruses, but with JC1 and JC3 it was specific for JC virus. The sensitivity of the system was 25 copies of JC plasmid per 10 microliters of digested tissue. Five out of seven PMLB and 28 of the 30 non-PMLB amplified for beta globin, but only the PMLB gave a signal with polyoma primers. Hypervariation of the length of the regulatory region of the JC isolates in the PML tissues was consistent with the presence of multiple strains of JC. CONCLUSIONS--Variation in the regulatory region of JC virus can be specifically and sensitively detected from routinely processed, paraffin wax embedded brain tissue. Variation in the regulatory region is common in PML derived JC strains, but JC virus was not detectable in non-PMLB tissue.