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Abstract
Human polyomavirus JC (JCV), the etiological agent of the disease progressive multifocal leukoencephalopathy (PML) affects immunocompromised patients particularly patients with AIDS. In vitro studies of JCV infection are hampered by the lack of sensitive JCV quantitation tests. Although the hemagglutination (HA) assay has been routinely employed for in vitro quantitation of JCV, its sensitivity is severely limited. We have employed a real-time PCR assay which compares favorably with the HA assay for the in vitro quantitation of JCV. JCV(Mad1), propagated in primary human fetal glial (PHFG) cells in two independent laboratories, was purified and quantitated by the HA assay. Both batches of purified JCV(Mad1) were then serially diluted in Dulbecco's Modified Eagle's Medium to obtain HA titers ranging from 64 to 0.001 HA units (HAU) per 100 μL of virus suspension. DNA was extracted from 100 μL of virus suspension and eluted in 50 μL of buffer, and DNA amplification and quantitation were performed in the Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection System using T-antigen as the target gene. Real-time PCR for quantitation of JCV was sensitive and consistently detected 1.8 × 101 copies of JCV DNA, and as low as 0.001 HAU equivalent of JCV. Moreover, there was a strong linear correlation between the HA assay and the DNA copy number of JCV(Mad1). The intra-run and inter-run coefficients of variation for the JCV standard curve were 0.06% to 4.8% and 2.6% to 5.2%, respectively. Based on these data, real-time PCR can replace the less-sensitive HA assay for the reliable detection, quantitation and monitoring of in vitro JCV replication.
Highlights
Human polyomavirus JC (JCV), a small, non-enveloped virus with a closed circular double-stranded-DNA genome, is ubiquitous in nature with a seroprevalence of up to 80% among geographically isolated populations [13]
Most primary JCV infections occur during childhood [3], and are subclinical
JCV remains latent for life and may cause a fatal demyelinating disease, progressive mulifocal leukoencephalopathy (PML), among immunocompromised patients [5]
Summary
Human polyomavirus JC (JCV), a small, non-enveloped virus with a closed circular double-stranded-DNA genome, is ubiquitous in nature with a seroprevalence of up to 80% among geographically isolated populations [13]. The dynamic range of detection was determined by preparing 10-fold serial dilutions of JCV plasmid in the range of 10 pg to 1 fg, that represented 1.8 × 105 to 1.8 × 101 copies of JCV DNA, respectively. The reliability of real-time PCR was defined by calculating coefficients of variation of Ct values of replicates of standard curve dilutions [21,26].
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