Sequencing and Analysis of John Cunningham Polyomavirus DNA From Acquired Immunodeficiency Syndrome Patients With Progressive Multifocal Leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a rare, life-threatening brain disease caused by the John Cunningham (JC) polyomavirus—an infection carried by up to 80% of adults worldwide. For most people, JC virus remains dormant and harmless. But in those with weakened immune systems—like people living with AIDS—it can reactivate, infect brain cells, and trigger PML. Diagnosing PML typically requires finding JC virus in cerebrospinal fluid (CSF, the fluid surrounding the brain and spinal cord) or brain tissue. But what if those samples aren’t available? A 2020 study from researchers at Zhejiang University School of Medicine and Xixi Hospital of Hangzhou explored whether blood samples could fill that gap by analyzing JC virus DNA in AIDS patients with clinically diagnosed PML.

The Study: What They Did

Led by Cai-Qin Hu, Jun-Wei Su, and Biao Zhu (from the First Affiliated Hospital of Zhejiang University School of Medicine) and Meng-Yan Wang (from Xixi Hospital of Hangzhou), the study enrolled six AIDS patients with clinical PML—meaning their symptoms (like weakness, vision loss, or difficulty thinking) and MRI scans matched PML, but JC virus hadn’t been confirmed in CSF or brain tissue yet.

The team collected 17 samples:

5 CSF samples (one patient couldn’t provide CSF)
6 plasma samples (liquid part of blood)
6 PBMC samples (peripheral blood mononuclear cells, a type of immune cell)

Using real-time PCR (a sensitive test to detect viral DNA) and genetic sequencing, they measured:

Viral load: How much JC virus DNA was present.
Sequence changes: Mutations in two key parts of the virus genome:
- VP1: The gene that makes the virus’s outer “capsid” protein, which helps it attach to cells.
- NCCR: The non-coding control region, which regulates virus replication and transcription (how it makes more copies of itself).

Key Findings: JC Virus in CSF vs. Blood

The researchers found JC virus DNA most often in CSF—4 out of 5 available samples were positive. Plasma samples were next (2 out of 6 positive), followed by PBMCs (1 out of 6 positive). Two patients stood out:

Patient 9: High viral loads in both plasma (55,700 copies/mL) and PBMCs (32,800 copies/mL).
Patient 4: A lower viral load in plasma (291 copies/mL).

Four of the six patients could be definitely diagnosed with PML because their CSF tested positive for JC virus. For the other two, blood samples provided critical clues.

New Virus Variants Linked to PML

When the team sequenced the positive samples, they uncovered two important findings:

1. Never-Before-Seen NCCR Rearrangements

The NCCR has two forms:

Archetype: The “normal” form, found in kidneys, urine, or lymph tissue—rarely in the brain.
Rearranged: The PML-linked form, with deletions, duplications, or rearranged DNA that boosts virus replication and makes it more “neurotropic” (likely to target brain cells).

The study found four new NCCR rearrangements in CSF, plasma, and PBMC samples:

CSF-15: A 34-nucleotide chunk deleted from the “D block” of the NCCR.
Plasma-4: A 30-nucleotide sequence duplicated in the “F block.”
PBMC-9/Plasma-9: A 65-nucleotide “extra” sequence (labeled “V”) inserted into the F block.

These changes haven’t been reported before—and they’re thought to make the virus more harmful.

2. VP1 Mutations Tied to PML Pathogenesis

The VP1 protein helps JC virus attach to cells via sialic acid (a sugar on cell surfaces). Mutations in VP1 can change how the virus binds to receptors, increasing its ability to infect brain cells. The study found three such mutations:

L55F: Leucine replaced by phenylalanine at position 55 (in a CSF sample).
E69D: Glutamate replaced by aspartic acid at position 69 (in a plasma sample).
N265T: Asparagine replaced by threonine at position 265 (in plasma and PBMC samples from Patient 9).

All three mutations have been previously linked to PML in other studies—they alter the virus’s “key” to brain cells, making infection more likely.

Why This Matters for PML Diagnosis and Treatment

For patients with clinical PML, CSF is the gold standard for confirmation. But lumbar punctures (spinal taps to collect CSF) aren’t always feasible—especially in people with advanced AIDS. This study shows blood samples can be a valuable backup:

NCCR rearrangements in blood suggest the virus is active and neurotropic.
VP1 mutations in blood match those found in CSF, linking the virus to PML.
Viral load in blood (like Patient 9’s high levels) correlates with worse PML outcomes, per prior research.

The findings also add to our understanding of how JC virus becomes pathogenic: Rearranged NCCRs make the virus replicate faster, while VP1 mutations let it “switch” to targeting brain cells. Together, these changes turn a harmless infection into a deadly disease.

Limitations and Takeaways

The study’s biggest limitation is its small sample size—PML is rare, and enrolling more patients is challenging. But the results still offer a critical insight: When CSF or brain tissue isn’t available, blood tests can help diagnose PML by detecting harmful JC virus variants.

For doctors, this means blood samples could become a non-invasive tool to confirm PML in AIDS patients. For patients, it’s a reminder that even “hidden” virus in blood can reveal important clues about their condition.

This study was published in the Chinese Medical Journal in 2020 by Cai-Qin Hu, Jun-Wei Su, Meng-Yan Wang, Yong-Zheng Guo, Li-Jun Xu, Ran Tao, Yi-Rui Xie, Ying Huang, and Biao Zhu. It 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.

doi.org/10.1097/CM9.0000000000001225

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