Background:  JC virus (JCV) causes a lytic infection of oligodendrocytes in the central nervous system (CNS) white matter, leading to progressive multifocal leukoencephalopathy (PML). We have described a productive JCV infection of cerebellar granule cell neurons in 1 HIV⁺/PML case and in 1 HIV⁺ case with cerebellar atrophy but no PML. We called this novel clinical syndrome distinct from PML JC virus granule cell neuronopathy (JCV_GCN). We sought to determine the prevalence of granule cell neurons infection by JCV in PML patients and controls.

Methods: We collected formalin-fixed, paraffin-embedded archival cerebellum samples from 46 patients with histologically confirmed PML (44 HIV⁺, 2 HIV^–), as well as 44 control samples (3 HIV⁺, 41 HIV^–) of patients without PML. Antibodies were subjected to single and multiple immunohistochemistry and immunofluorescence staining, revealing in JCV-infected cells:  SV40 VP1 and SV40 T antigen cross-reacting with JCV, granule cell neurons NeuN and MAP-2; and in oligodendrocytes and myelin:  -myelin oligodendrocyte specific protein (MOSP) and '3'-cyclic nucleotide-3'-phosphodiesterase (CNPase); in astrocytes, glial fibrillary acidic protein (GFAP).

Results:  Of 44 HIV⁺/PML cases, 32 (73%) had JCV⁺ cells in the granule cell layer of the cerebellum compared with 0 of 2 HIV^–/PML and 0 of 44 controls without PML. Of the 32 patients with JCV⁺ cells in GCL, 24 (75%) had PML lesions in the nearby white matter, while 6 (19%) had JCV⁺ cells mainly in the GCL, and 2 (6%) had rare JCV⁺ cells in the granule cell layer and nearby white matter. The phenotype of the JCV⁺ cells in the granule cell layer was determined in 13 cases by double immunohistochemistry or immunofluorescence assay. Granule cell neuron infection was confirmed in 9 (69%), showing expression of T antigen only in 5 of 9 (22 %), VP1 only in 2 of 9 (22%), and VP1 and T antigen in 2 of 9 (22%). When both VP1 and T antigen were present, T antigen⁺ cells were 10 times more numerous than VP1⁺ cells. Numerous JCV⁺ oligodendrocytes and astrocytes were also present within the granule cell layer. The ratio of JCV⁺ granule cell neurons/JCV⁺ glial cells was between 1 of 10 and 1/ of 1000 in cases with PML lesions in nearby white matter, and ranged from 1 of 1 to 1 of 10 when JCV⁺ cells were primarily in the GCL.

Conclusions:  JCV⁺ cells are present in the granule cell layer of 73% HIV⁺/PML patients. Among these, granule cell neuron infection by JCV can be demonstrated by double immunostaining in 69% cases (50% of total number of HIV⁺/PML cases). JCV T antigen was found more frequently than VP1 protein, perhaps indicating an abortive JCV infection in granule cell neurons. JCV infection may spread to granule cell neurons from oligodendrocytes and astrocytes in nearby PML lesions, or arise primarily in the granule cell layer. Granule cell neuron infection is quite frequent in HIV⁺/PML, and may be an important component of JCV pathogenesis in these patients.