Agnogene Deletion in a Novel Pathogenic JC Virus Isolate Impairs VP1 Expression and Virion Production

Infection of glial cells by the human polyomavirus JC (JCV) causes progressive multifocal leukoencephalopathy (PML). JCV Encephalopathy (JCVE) is a newly identified disease characterized by JCV infection of cortical pyramidal neurons. The virus JCVCPN associated with JCVE contains a unique 143 base pair deletion in the agnogene. Contrary to most JCV brain isolates, JCVCPN has an archetype-like regulatory region (RR) usually found in kidney strains. This provided us with the unique opportunity to determine for the first time how each of these regions contributed to the phenotype of JCVCPN. We characterized the replication of JCVCPN compared to the prototype virus JCVMad-1 in kidney, glial and neuronal cell lines. We found that JCVCPN is capable of replicating viral DNA in all cell lines tested, but is unable to establish persistent infection seen with JCVMad-1. JCVCPN does not have an increased ability to replicate in the neuronal cell line tested. To determine whether this phenotype results from the archetype-like RR or the agnogene deletion, we generated chimeric viruses between JCVCPN of JCVMad-1. We found that the deletion in the agnogene is the predominant cause of the inability of the virus to maintain a persistent infection, with the introduction of a full length agnogene, either with or without agnoprotein expression, rescues the replication of JCVCPN. Studying this naturally occurring pathogenic variant of JCV provides a valuable tool for understanding the functions of the agnogene and RR form in JCV replication

JC Virus Encephalopathy Is Associated with a Novel Agnoprotein-Deletion JCV Variant

JC virus encephalopathy (JCVE) is a newly described gray matter disease of the brain caused by productive infection of cortical pyramidal neurons. We characterized the full length sequence of JCV isolated from the brain of a JCVE patient, analyzed its distribution in various compartments by PCR, and determined viral gene expression in the brain by immunohistochemistry(IHC). We identified a novel JCV variant, JCVCPN1, with a unique 143 bp deletion in the Agno gene encoding a truncated 10 amino acid peptide, and harboring an archetype-like regulatory region. This variant lacked one of three nuclear protein binding regions in the Agno gene. It was predominant in the brain, where it coexisted with an Agno-intact wild-type strain. Double immunostaining with anti-Agno and anti- VP1 antibodies demonstrated that the truncated JCVCPN1 Agno peptide was present in the majority of cortical cells productively infected with JCV. We then screened 68 DNA samples from 8 brain, 30 CSF and 30 PBMC samples of PML patients, HIV+ and HIV- control subjects. Another JCVCPN strain with a different pattern of Agno-deletion was found in the CSF of an HIV+/PML patient, where it also coexisted with wild-type, Agno-intact JCV. These findings suggest that the novel tropism for cortical pyramidal neurons of JCVCPN1, may be associated with the Agno deletion. Productive and lytic infection of these cells, resulting in fulminant JCV encephalopathy and death may have been facilitated by the co-infection with a wild- type strain of JCV

JCV GCN in a Natalizumab-Treated MS Patient is Associated With Mutations of the VP1 Capsid Gene

Objective: To describe the clinical, neuroimaging, immunologic, and virologic characteristics of JC virus-associated granule cell neuronopathy (JCV GCN) in a natalizumab-treated patient with multiple sclerosis (MS) who developed immune reconstitution inflammatory syndrome (IRIS) after natalizumab withdrawal. Methods: We obtained longitudinal clinical data as well as MRI and proton magnetic resonance spectroscopy from this patient with MS. We measured JCV-specific cellular immune response in his peripheral blood by intracellular cytokine staining and sequenced a fragment of JCV VP1 capsid gene detected in his CSF. We contrast our findings with the first recently reported case. Results: This patient presented with worsening cerebellar symptoms and progressive cerebellar atrophy without new MS lesions on MRI after 63 months of natalizumab monotherapy. JCV DNA was detected in his CSF by PCR and harbored novel GCN-type mutations in the VP1 gene. He developed IRIS upon discontinuation of natalizumab and plasma exchange, which manifested itself by a worsening of clinical symptoms and contrast enhancement in the cerebellum on MRI. Treatment with corticosteroids resulted in resolution of IRIS, as demonstrated by proton magnetic resonance spectroscopy. The patient had a strong JCV-specific T-cell response in his peripheral blood and remains alive after 15 months from onset of symptoms, although with significant disability. He did not have MS relapse on glatiramer acetate. Conclusions: JCV GCN should be considered in patients on natalizumab presenting with progressive cerebellar symptoms and cerebellar atrophy, and is associated with mutations in the JCV VP1 gene. Natalizumab withdrawal may be complicated by JCV GCN IRIS, and require treatment with corticosteroids

Detection of JC Virus-Specific Immune Responses in a Novel Humanized Mouse Model

Progressive Multifocal Leukoencephalopathy (PML) is an often fatal disease caused by the reactivation of the JC virus (JCV). Better understanding of viral-host interactions has been hampered by the lack of an animal model. Engrafting NOD/SCID/IL-2-Rg (null) mice with human lymphocytes and thymus, we generated a novel animal model for JCV infection. Mice were inoculated with either a PML isolate, JCV Mad-4, or with JCV CY, found in the kidney and urine of healthy individuals. While mice remained asymptomatic following inoculation, JCV DNA was occasionally detected in both the blood and the urine compartments. Mice generated both humoral and cellular immune responses against JCV. Expressions of immune exhaustion marker, PD-1, on lymphocytes were consistent with response to infection. Using this model we present the first in vivo demonstration of virological and immunological differences between JCV Mad-4 and CY. This model may prove valuable for studying JCV host immune responses

Frequent Infection of Cerebellar Granule Cell Neurons by Polyomavirus JC in Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) occurs most often in immunosuppressed individuals. The lesions of PML result from astrocyte and oligodendrocyte infection by the polyomavirus JC (JCV); JCV has also been shown to infect and destroy cerebellar granule cell neurons (GCNs) in 2 HIV-positive patients. To determine the prevalence and pattern of JCV infection in GCNs we immunostained formalin-fixed, paraffin-embedded cerebellar samples from 40 HIV-positive and 3 HIV-negative PML patients for JCV, glial and neuronal markers. JCV infection was detected in 30 patients (70%); 28 (93%) of these had JCV-infected cells in the granule cell layer (GCL); JCV-infected GCNs were demonstrated in 15/19 (79%) tested cases. JCV regulatory T antigen (T Ag) was expressed more frequently and abundantly in GCNs than JCV VP1 capsid protein. None of 37 HIV-negative controls but 1/35 (3%) HIV-positive subjects without PML had distinct foci of JCV-infected GCNs. Thus, JCV infection of GCNs is frequent in PML patients and may occur in the absence of cerebellar white matter demyelinating lesions. The predominance of T Ag over VP1 expression in GCNs suggests that they may be the site of early or latent central nervous system JCV infection. These results indicate that infection of GCNs is an important, previously overlooked aspect of JCV pathogenesis in immunosuppressed individuals

Frequent Infection of Neurons by SV40 Virus in SIV-Infected Macaque Monkeys with Progressive Multifocal Leukoencephalopathy and Meningoencephalitis

Simian virus 40 (SV40), family Polyomaviridae, in immunocompromised macaques can cause fatal demyelinating central nervous system disease analogous to progressive multifocal leukoencephalopathy caused by John Cunningham (JC) virus in immunocompromised humans. Recently, we have demonstrated that JC virus can infect cerebellar granule cell neurons and cortical pyramidal neurons in immunosuppressed people. To examine whether SV40 neuronal infection occurs spontaneously in immunosuppressed macaques, we analyzed archival brain specimens from 20 simian immunodeficiency virus–infected rhesus with AIDS and 1 cynomolgus post-transplant selected with SV40 brain infection from archival records from 1991 to 2012. In addition to white matter SV40 distribution in classic demyelinating progressive multifocal leukoencephalopathy, some of the 21 monkeys exhibited meningeal, subpial neocortical, and periventricular virus. This distribution pattern corresponded to broader viral tropism with neuronal infection in 14 (66.7%) of 21 cases. In all 14 cases, identified neurons were positive for early SV40 transcript large T antigen, but only 4 of the 14 cases exhibited late viral transcript viral protein 1–positive neurons. SV40-infected neurons were detected in frontal, parietal, occipital, and temporal cortices, hippocampus, thalamus, and brain stem. These observations confirm that spontaneous SV40 neuronal infection occurs in immunosuppressed macaques, which parallels JC virus–neuronal infection in immunosuppressed patients. Neuronal infection may be an important aspect of both SV40 and JC virus neuropathogenesis in their respective hosts

Progressive multifocal leukoencephalopathy in a patient without apparent immunosuppression

An 80-year-old man with no history of an immune-compromising disorder was diagnosed with progressive multifocal leukoencephalopathy (PML). He presented with dysphagia and left-sided weakness; magnetic resonance imaging demonstrated marked signal abnormality in the subcortical white matter of the left frontal lobe and in the posterior limb of the right internal capsule. Polymerase chain reaction (PCR) analysis of the cerebrospinal fluid (CSF) was negative for John Cunningham (JC) virus. On brain biopsy, foamy macrophages infiltrating the white matter were identified, staining positive for anti-simian virus 40 antibodies. Postoperatively, PCR for JC viral DNA in the CSF was positive, establishing the diagnosis of PML. Extensive investigation for an occult immunocompromising disorder was negative. The patient's neurologic deficits rapidly increased throughout his hospital stay, and he died 3.5 months after his diagnosis

JC Virus Antibody and Viremia as Predictors of Progressive Multifocal Leukoencephalopathy in Human Immunodeficiency Virus-1-Infected Individuals

We examined whether prediagnostic John Cunningham virus (JCV) antibodies and viremia are predictors of progressive multifocal leukoencephalopathy (PML) in 83 PML cases and 240 human immunodeficiency virus (HIV) disease-matched controls. JCV viremia was not predictive of PML, but some patients showed higher anti-JCV immunoglobulin G (IgG) responses 6 months prior to diagnosi