Sequencing and Analysis of John Cunningham Polyomavirus DNA from Acquired Immunodeficiency Syndrome Patients with Progressive Multifocal Leukoencephalopathy
Progressive multifocal leukoencephalopathy (PML) is a rare but severe demyelinating disease of the central nervous system caused by the reactivation of the John Cunningham (JC) polyomavirus in immunocompromised individuals, particularly those with acquired immunodeficiency syndrome (AIDS). First identified in 1971 in a PML patient, JC polyomavirus has since been recognized as the etiological agent of this debilitating condition. The diagnosis of PML is categorized into two types: etiological diagnosis, which involves the detection of JC polyomavirus in cerebrospinal fluid (CSF) or brain tissue, and clinical diagnosis, which relies on typical clinical symptoms and magnetic resonance imaging (MRI) findings.
The JC polyomavirus genome is a closed circular double-stranded DNA molecule with a length of 5120 kilobases (kb). It is divided into three main regions: the early coding region, the late coding region, and the non-coding control region (NCCR). The early coding region encodes five proteins, including the large tumor (T) antigen, the small T antigen, and three T antigen-splicing variants (T’135, T’136, and T’165). These proteins play a crucial role in viral DNA replication and early gene transcription. The late coding region encodes the structural proteins VP1, VP2, VP3, and the agnoprotein. VP1 is the major structural protein, accounting for 70% of viral gene expression, and is essential for virus binding to host cell receptors and subsequent cellular entry. The NCCR is a highly variable region that can be divided into six blocks (A to F) and exists in two main forms: the archetype and the rearranged type. The archetype, represented by the CY strain, is commonly found in the kidney, urine, and lymphoid tissues but is rarely detected in the brain. In contrast, the rearranged type, also known as the PML type, is characterized by deletions, duplications, and rearrangements of specific sequence elements and is predominantly found in the CSF and blood of AIDS patients with PML.
JC polyomavirus typically remains latent in the urinary system after initial infection. However, in immunocompromised hosts, the virus reactivates and proliferates in the urinary system, leading to viruria. As the virus continues to replicate, it enters the peripheral blood, where it can be intermittently detected. The virus then spreads to lymphoid tissues, including the bone marrow, where NCCR rearrangements and VP1 amino acid (AA) substitutions may occur. When JC polyomavirus reaches the brain and infects glial cells, it results in positive detection in CSF and brain tissue, often leading to PML. Previous studies have shown that JC polyomavirus variants can be present in both CSF and blood, making these specimens valuable for diagnosis and analysis.
This study aimed to investigate the detection of JC polyomavirus in CSF and blood samples from AIDS patients clinically diagnosed with PML. The viral load and sequence variations of JC polyomavirus were analyzed in different types of specimens, including CSF, plasma, and peripheral blood mononuclear cells (PBMCs). The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board at the First Affiliated Hospital of Zhejiang University School of Medicine. Informed consent was obtained from all participants prior to data collection.
Six patients diagnosed with AIDS and clinically diagnosed with PML were included in the study. Clinical and laboratory data were obtained from electronic databases, and MRIs were reviewed by a neuroradiologist. A total of 17 samples were collected, comprising five CSF samples, six plasma samples, and six PBMC samples. Real-time polymerase chain reaction (PCR) and sequencing of JC polyomavirus were performed to analyze the nucleotide sequences of the VP1-coding region and the NCCR rearrangements. The sequences were aligned and analyzed using BioEdit and MEGA software.
The experimental results revealed that JC polyomavirus DNA was detected in four out of five CSF samples from clinically diagnosed PML patients, yielding a positive rate of 80%. In plasma samples, two out of six were positive for JC polyomavirus DNA, while only one out of six PBMC samples tested positive. Notably, patient number 9, who did not have a CSF specimen available, tested positive for JC polyomavirus DNA in both plasma and PBMC samples, with viral loads of 5.57 × 10^4 copies/mL and 3.28 × 10^4 copies/mL, respectively. Patient number 4 also had a positive plasma sample, with a viral load of 2.91 × 10^2 copies/mL. Based on these findings, four out of the six patients with clinically diagnosed PML could be etiologically diagnosed with PML due to the presence of JC polyomavirus DNA in CSF samples. The remaining two patients required further testing for confirmation.
Further sequencing analysis was conducted on the seven JC polyomavirus-positive samples, including four CSF samples, one PBMC sample, and two plasma samples. Four novel NCCR rearrangements were identified in samples CSF-15, PBMC-9, Plasma-9, and Plasma-4. In sample CSF-15, the NCCR rearrangement was characterized by the deletion of 34 nucleotides (nt28–nt61) in block D, resulting in the composition Ori-A-B-C-d-E-F. In sample Plasma-4, a 30-base pair (bp) duplicated sequence was inserted into the 23rd nucleotide of block F (nt23–nt52), forming the composition Ori-A-B-C-D-E-f-F. In samples PBMC-9 and Plasma-9, a 65-bp sequence was inserted into the F region between nucleotides 7 and 59, labeled as region V, resulting in the composition Ori-A-B-C-D-E-f-V-f. These rearrangements had not been previously reported and represent new variations identified in this study.
In addition to NCCR rearrangements, VP1 sequencing was performed on the seven JC polyomavirus-positive samples. Four nucleotide substitutions were identified at positions nt1622, nt1666, nt1843, and nt2253 on the CY sequence. These substitutions corresponded to AA positions 55, 69, 128, and 265 in the VP1 protein. Specifically, in sample CSF-15, leucine at AA position 55 was replaced by phenylalanine (L55F); in sample Plasma-4, glutamate at AA position 69 was replaced by aspartic acid (E69D); and in samples PBMC-9 and Plasma-9, asparagine at AA position 265 was replaced by threonine (N265T). These AA substitutions have been previously associated with PML pathogenesis and are believed to alter the virus’s affinity and specificity for host cell receptors, thereby influencing infectivity and pathogenicity.
The findings of this study highlight the importance of sequence variations and NCCR rearrangements in JC polyomavirus in the pathogenesis and tropism of PML. The NCCR contains transcription factor-binding sites, and rearrangements in this region can upregulate viral replication and transcription, leading to a more neurotropic virus. The detection of JC polyomavirus in peripheral blood, in addition to CSF, provides a valuable non-invasive biomarker for PML diagnosis, particularly when CSF or brain tissue samples are unavailable. Previous studies have suggested that a JC polyomavirus DNA load greater than 2.0 log copies/mL in the blood is associated with a worse prognosis in PML patients. Furthermore, the presence of plasma JC polyomavirus DNA can serve as a prognostic indicator of PML.
Despite the small sample size, this study provides significant insights into the detection and analysis of JC polyomavirus in AIDS patients with PML. The findings underscore the utility of blood samples for the etiological diagnosis of PML when CSF and brain tissue are inaccessible. Moreover, the identification of novel NCCR rearrangements and VP1 AA substitutions in JC polyomavirus sequences from CSF and blood samples highlights the role of viral variants in PML pathogenesis and tropism.
In conclusion, this study demonstrates that JC polyomavirus can be detected not only in CSF but also in blood samples from PML patients. The presence of NCCR rearrangements and VP1 AA substitutions in these samples underscores the importance of sequence variations in the pathogenicity and tropism of JC polyomavirus. These findings contribute to a better understanding of PML and provide valuable diagnostic and prognostic tools for managing this rare but devastating disease.
doi.org/10.1097/CM9.0000000000001225
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