Progressive multifocal leukoencephalopathy (PML), a rare but fatal demyelinating disease caused by JC virus (JCV), occurs mainly in immunocompromised patients. As PML develops in individuals with various underlying disorders sporadically and infrequently, a nationwide survey of PML is difficult. This study was conducted to elucidate the characteristics of PML in Japan through an internet-assisted laboratory surveillance program.

Background: Progressive multifocal leukoencephalopathy (PML) is a demyelinating disorder caused by JC virus (JCV). Although detecting JCV DNA in the cerebrospinal fluid (CSF) by real-time polymerase chain reaction (PCR) is useful, diagnosis is difficult when JCV concentrations are low. We therefore aimed to lower the detection limit of real-time PCR testing by enriching JCV in the CSF via ultrafiltration.Methods: Virus suspensions and CSF specimens from 20 untreated patients with suspected PML were collected and total DNAs were extracted. The JCV large T gene was detected by quantitative real-time PCR under condition with and without prior centrifugal ultrafiltration.Results: The JCV DNA was reliably detected to a lower limit of 10 copies/mL of virus suspension by real-time PCR with ultrafiltration. When using this method, the quantity of JCV DNA per PCR reaction increased 3.2- to 8.7-fold compared with the standard procedure. Seven patients were positive for JCV when using the standard procedure, and an additional patient was positive when using ultrafiltration. All JCV-positive patients had neurological features and magnetic resonance imaging findings compatible with PML.Conclusions: The detection limit of JCV DNA by real-time PCR can be lowered by viral enrichment using ultrafiltration. Our simple protocol offers a valuable tool for PML diagnosis when extremely low copy numbers of JCV are released into the CSF or when brain biopsy is not feasible.

Progressive multifocal leukoencephalopathy (PML) is a devastating demyelinating disease caused by JC virus (JCV), predominantly affecting patients with impaired cellular immunity. PML is a non-reportable disease with a few exceptions, making national surveillance difficult. In Japan, polymerase chain reaction (PCR) testing for JCV in the cerebrospinal fluid (CSF) is performed at the National Institute of Infectious Diseases to support PML diagnosis. To clarify the overall profile of PML in Japan, patient data provided at the time of CSF-JCV testing over 10 years (FY2011–2020) were analyzed. PCR testing for 1537 new suspected PML cases was conducted, and 288 (18.7%) patients tested positive for CSF-JCV. An analysis of the clinical information on all individuals tested revealed characteristics of PML cases, including the geographic distribution, age and sex patterns, and CSF-JCV-positivity rates among the study subjects for each type of underlying condition. During the last five years of the study period, a surveillance system utilizing ultrasensitive PCR testing and widespread clinical attention to PML led to the detection of CSF-JCV in the earlier stages of the disease. The results of this study will provide valuable information not only for PML diagnosis, but also for the treatment of PML-predisposing conditions.

During neurotropic virus infection, microglia act as a source of chemokines, thereby regulating the recruitment of peripheral leukocytes and the multicellular immune response within the CNS. Herein, we present a comprehensive study on the chemokine production by microglia in response to double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Transcriptional analyses of chemokine genes revealed that dsRNA strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. We also observed that the dsRNA stimulation triggered the activation of signaling pathways mediated by nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). The microglial CXCL10 response to dsRNA was induced via NF-kappaB, p38, and JNK pathways, whereas the dsRNA-induced CCL5 production was dependent on JNK, but not on the other signal-transducing molecules tested. In addition, the acidic environment of intracellular vesicles was required for the activation of cellular signaling in response to dsRNA. Taken together, these results suggest that the recognition of dsRNA structure selectively induces the CXCL10 and CCL5 responses in microglia through vacuolar pH-dependent activation of NF-kappaB and MAPK signaling pathways.