Antiviral effects of artesunate on JC polyomavirus replication in COS-7 cells.

The human JC polyomavirus (JCPyV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). A growing number of patients with induced or acquired immunosuppression are at risk for infection, and no effective antiviral therapy is presently available. The widely used antimalarial drug artesunate has shown broad antiviral activity in vitro but limited clinical success. The aim of this study was to investigate the effect of artesunate on JCPyV replication in vitro. The permissivity for JCPyV MAD-4 was first compared in four cell lines, and the monkey kidney cell line COS-7 was selected. Artesunate caused a concentration-dependent decrease in the extracellular JCPyV DNA load 96 h postinfection, with a 50% effective concentration (EC50) of 2.9 µM. This effect correlated with a decreased expression of capsid protein VP1 and a reduced release of infectious viral progeny. For concentrations of <20 µM, transient reductions in cellular DNA replication and proliferation were seen, while for higher concentrations, some cytotoxicity was detected. A selective index of 16.6 was found when cytotoxicity was calculated based on cellular DNA replication in the mock-infected cells, but interestingly, cellular DNA replication in the JCPyV-infected cells was more strongly affected. In conclusion, artesunate is efficacious in inhibiting JCPyV replication at micromolar concentrations, which are achievable in plasma. The inhibition at EC50 probably reflects an effect on cellular proteins and involves transient cytostatic effects. Our results, together with the favorable distribution of the active metabolite dihydroartemisinin to the central nervous system, suggest a potential use for artesunate in patients with PML.

The human fetal glial cell line SVG p12 contains infectious BK polyomavirus.

UNLABELLED: The human fetal glial cell line SVG was generated in 1985 by transfecting primary fetal brain cells with a plasmid containing an origin-defective mutant of simian virus 40 (SV40). The cells, which express SV40 large T-antigen, support the replication of human JC polyomavirus (JCPyV) and have been used for JCPyV studies but also for other studies in which cells of neural origin were desirable. We intended to use the SVG p12 cells from ATCC for antiviral drug studies with JCPyV. However, during initial experiments, immunofluorescence microscopy controls unexpectedly revealed cells expressing the late viral proteins VP1, VP2/VP3, and agno. This was confirmed by Western blotting. Since our agnoprotein antiserum is specific for BKPyV agnoprotein, infection with BKPyV was suspected. Indeed, specific BKPyV PCR of SVG p12 supernatants revealed a viral load of >1 × 10(10) genomic equivalents/ml. Negative-staining electron microscopy showed characteristic polyomavirus virions, and infectious BKPyV was transmitted from SVG p12 supernatant to other cells. Long-range PCR covering the viral genome, followed by DNA sequencing, identified BKPyV strain UT as well as deletion derivatives. This was confirmed by next-generation sequencing. JCPyV (MAD-4) was found to infect apparently uninfected and BKPyV-infected SVG p12 cells. In total, 4 vials from 2 different ATCC lots of SVG p12 cells dating back to 2006 contained BKPyV, whereas the subclone SVG-A was negative. In conclusion, SVG p12 cells from ATCC contain infectious BKPyV. This may have affected results and interpretations of previous studies, and caution should be taken in future experiments. IMPORTANCE: This work reveals that one of the most frequently used cell lines for JC polyomavirus (JCPyV) research, the SV40-immortalized human fetal glial cell line SVG p12 obtained directly from ATCC, contains infectious BK polyomavirus (BKPyV) of strain UT and a spectrum of defective mutants. Strain UT has been previously found in urine and in tumors of different patients but is also frequently used for research. It is therefore not clear if BKPyV was present in the brain tissue used to generate the cell line or if this is a contamination. Although productive JCPyV infection of SVG cells was not dependent on prior BKPyV infection, the unnoticed presence of BKPyV may have influenced the results of studies using these cells. The interpretation of past results should therefore be reconsidered and cells tested for BKPyV before new studies are initiated. The frequently used subclone SVG-A did not contain BKPyV and could be a useful substitute.

Antiviral effects of artesunate on polyomavirus BK replication in primary human kidney cells.

Polyomavirus BK (BKV) causes polyomavirus-associated nephropathy (PyVAN) and hemorrhagic cystitis (PyVHC) in renal and bone marrow transplant patients, respectively. Antiviral drugs with targeted activity against BKV are lacking. Since the antimalarial drug artesunate was recently demonstrated to have antiviral activity, the possible effects of artesunate on BKV replication in human primary renal proximal tubular epithelial cells (RPTECs), the host cells in PyVAN, were explored. At 2 h postinfection (hpi), RPTECs were treated with artesunate at concentrations ranging from 0.3 to 80 µM. After one viral replication cycle (approximately 72 hpi), the loads of extracellular BKV DNA, reflecting viral progeny production, were reduced in a concentration-dependent manner. Artesunate at 10 µM reduced the extracellular BKV load by 65%; early large T antigen mRNA and protein expression by 30% and 75%, respectively; DNA replication by 73%; and late VP1 mRNA and protein expression by 47% and 64%, respectively. Importantly, the proliferation of RPTECs was also inhibited in a concentration-dependent manner. At 72 hpi, artesunate at 10 µM reduced cellular DNA replication by 68% and total metabolic activity by 47%. Cell impedance and lactate dehydrogenase measurements indicated a cytostatic but not a cytotoxic mechanism. Flow cytometry and 5-ethynyl-2'-deoxyuridine incorporation revealed a decreased number of cells in S phase and suggested cell cycle arrest in G0 or G2 phase. Both the antiproliferative and antiviral effects of artesunate at 10 µM were reversible. Thus, artesunate inhibits BKV replication in RPTECs in a concentration-dependent manner by inhibiting BKV gene expression and genome replication. The antiviral mechanism appears to be closely connected to cytostatic effects on the host cell, underscoring the dependence of BKV on host cell proliferative functions.

The human polyomavirus BK (BKPyV): virological background and clinical implications.

Polyomavirus BK (BKPyV) infects most people subclinically during childhood and establishes a lifelong infection in the renourinary tract. In most immunocompetent individuals, the infection is completely asymptomatic, despite frequent episodes of viral reactivation with shedding into the urine. In immunocompromised patients, reactivation followed by high-level viral replication can lead to severe disease: 1-10% of kidney transplant patients develop polyomavirus-associated nephropathy (PyVAN) and 5-15% of allogenic hematopoietic stem cell transplant patients develop polyomavirus-associated haemorrhagic cystitis (PyVHC). Other conditions such as ureteric stenosis, encephalitis, meningoencephalitis, pneumonia and vasculopathy have also been associated with BKPyV infection in immunocompromised individuals. Although BKPyV has been associated with cancer development, especially in the bladder, definitive evidence of a role in human malignancy is lacking. Diagnosis of PyVAN and PyVHC is mainly achieved by quantitative PCR of urine and plasma, but also by cytology, immunohistology and electron microscopy. Despite more than 40 years of research on BKPyV, there is still no effective antiviral therapy. The current treatment strategy for PyVAN is to allow reconstitution of immune function by reducing or changing the immunosuppressive medication. For PyVHC, treatment is purely supportive. Here, we present a summary of the accrued knowledge regarding BKPyV.

Characteristics of polyomavirus BK (BKPyV) infection in primary human urothelial cells.

High-level polyomavirus BK (BKPyV) replication in urothelial cells is a hallmark of polyomavirus-associated hemorrhagic cystitis (PyVHC), a painful condition affecting bone marrow transplant recipients. In kidney transplant recipients, replication in tubular epithelial cells is associated with overt disease whereas high-level urothelial replication is clinically silent. We characterized BKPyV replication in primary human urothelial cells (HUCs) and compared it to replication in renal tubular epithelial cells (RPTECs). HUCs were easily infected, as shown by expression of T-antigens, VP1-3, and agnoprotein, and intranuclear virion production. Compared to RPTECs, progeny release was delayed by ≥24h and reduced. BKPyV-infected HUCs rounded up like "decoy cells" and detached without necrosis as shown by delayed cytokeratin-18 release, real-time viability monitoring and imaging. The data show that BKV infection of HUCs and RPTECs is significantly different and support the notion that PyVHC pathogenesis is not solely due to BKPyV replication, but likely requires urotoxic and immunological cofactors.

Fluoroquinolones inhibit human polyomavirus BK (BKV) replication in primary human kidney cells.

Reactivation of human polyomavirus BK (BKV) may cause polyomavirus-associated nephropathy or polyomavirus-associated hemorrhagic cystitis in renal- or bone marrow-transplant patients, respectively. Lack of treatment options has led to exploration of fluoroquinolones that inhibit topoisomerase II and IV in prokaryotes and possibly large T-antigen (LT-ag) helicase activity in polyomavirus. We characterized the effects of ofloxacin and levofloxacin on BKV replication in the natural host cells - primary human renal proximal tubular epithelial cells (RPTECs). Ofloxacin and levofloxacin inhibited BKV load in a dose-dependent manner yielding a ∼90% inhibition at 150 µg/ml. Ofloxacin at 150 µg/ml inhibited LT-ag mRNA and protein expression from 24h post infection (hpi). BKV genome replication was 77% reduced at 48 hpi and a similar reduction was found in VP1 and agnoprotein expression. At 72 hpi, the reduction in genome replication and protein expression was less pronounced. A dose-dependent cytostatic effect was noted. In infected cells, 150 µg/ml ofloxacin led to a 26% and 6% inhibition of cellular DNA replication and total metabolic activity, respectively while 150 µg/ml levofloxacin affected this slightly more, particularly in uninfected cells. Cell counting and xCELLigence results revealed that cell numbers were not reduced. In conclusion, ofloxacin and levofloxacin inhibit but do not eradicate BKV replication in RPTECs. At a concentration of ofloxacin giving ∼90% inhibition in BKV load, no significant cytotoxicity was observed. This concentration can be achieved in urine and possibly in the kidneys. Our results support a mechanism involving inhibition of LT-ag expression or functions but also suggest inhibition of cellular enzymes.