Polyomavirus (BK Virus)

Polyomavirus BK (BKV) is small double stranded DNA virus that has been assigned to the Polyomaviridae family. The BK virus genome consists of a  non-coding control region National Coalition for Cancer Research (NCCR), the early coding region that transcribes the T antigen, and the late coding region, which codes for the viral capsid proteins (VP-1, VP-2, VP-3) and agnoprotein (123). The National Coalition for Cancer Research contains the origin of replication as well as enhancer elements that can modulate viral transcription. T antigen binds to tumor suppressor proteins Rb and p53, and initiates the cell cycle in host cells.

VP-1, VP-2, and VP-3 are structural proteins that make up the viral capsid. VP-1 interacts with cellular receptors and promotes viral entry.  This protein also displays considerable genetic heterogeneity, and this sequence variation is the basis of four major BK virus genotypes.

Minor viral coat proteins VP-2 and VP-3 share a common C-terminal with viroporin activity, a VP-1 interacting domain, a DNA binding domain that participates in virion assembly, and nuclear localization signals, which after exposure during viral disassembly, and target BK virus to the host nucleus (35, 54). The VP-2 protein is exposed in the activated viral particle, which integrates into and perforates the endoplasmic membrane during viral entry into the cell. This effect is mediated by hydrophobic transmembrane domains. VP-2 and VP-3 can induce apoptosis of host cells (69).  It has been shown that VP-3 activates a poly (ADP-ribose) polymerase, which results in ATP depletion and cell death. VP3, the longer protein has a N-terminal myristoylation sequence that also contributes to viral infectivity.

Agnoprotein is a dimeric protein that has been primarily studied in polyomavirus JCV and found to colocalize with cytoplasmic lipid droplets (158).  It has a Leu/Ile/Phe-rich domain and an amphipathic a -helix that promotes binding of Large T-antigen to the origin of replication, and regulates the splicing of late coding transcripts (33,132, 133). Agnoprotein  represses the action of T-antigen and VP-1 proteins, interferes with DNA repair and cell cycle regulation, assists viral capsid assembly in promyelocytic leukemia nuclear bodies (143, 153) and promotes virion release from the cell by acting as a viroporin (152). Agnogene deletion impairs JCV VP-1 expression, virion production, and produces non-functional BKV genomes (47, 108). Agnoprotein-negative mutants of polyomavirus JC and SV40 produce virions that are deficient in DNA content (134).

Serologic studies show widespread prevalence of BK virus infection in man (149). Evidence is accumulating that this is the result of multiple routes of infection (123, 129). Air borne transmission is supported by observations that BK virus DNA is found in 1% of nasopharyngeal aspirates obtained from infants with respiratory infection. The possibility of feco-oral transmission  is suggested by demonstration of BK virus DNA in metropolitan sewage, and in stool samples from hospitalized children and bone marrow transplant recipients (160).  Given that polyomavirus is widely latent in the kidney, renal transplantation is believed to be an important modality of infection in patients with end stage kidney disease (21). The presence of high titer anti-VP-1 DNA antibodies in donor serum is associated with increased risk of virus transmission and disease in the renal allograft recipient. Finally, there is evidence that transplacental transmission of polyomavirus can occur. The frequency of viral  reactivation in pregnancy has been variably estimated from rare to as high as 65% (73).

Risk factors may be donor, recipient, transplant, or virus related (19, 22, 25, 26, 27, 62, 75, 140, 147).

Reported donor-related factors include deceased-donor, living-donor transplant, ABO incompatibility, cytomegalovirus infection, donor seropositive status, and donor viruria (162).

 Recipient-related risk factors  that have been implicated in increased for nephropathy  include older age, male gender, Caucasian race, Asian ethnicity (78), African American race  (156), hemodialysis (compared to peritoneal dialysis) (105), diabetes mellitus, prior renal tubular injury, lower frequency of the activating natural killer cell receptor KIR3DS1 (157), and recipient serostatus (150).

Potential risk factors associated with the transplantation procedure are pre-transplant desensitization (51), ischemic/harvesting injury, delayed graft function, ureteric stenting, tacrolimus trough levels, calcineurin inhibitor based therapy, choice of mycophenolic acid over everolimus, mycophenolic acid area under the curve (AUC), intensity of immunosuppression, multiple episodes of acute rejection, and administration of lymphocyte depleting agents (22, 107). The effect of HLA matching is complex: while the risk of nephropathy is higher in mismatched patients (8), the incidence of graft loss is said to be lower in matched patients (38).  HLA-A2, HLA-B44,and HLA-DR15 appear to lower the risk of BK viremia (95).

Potential viral-related factors include sequence alterations in the VP-1 gene,rearrangements in the NCCR region, presence of viremia in pre-transplant or day 1-5 post-transplant samples, high viral load and rapid doubling of viral copy numbers. None of the listed risk factors consistently come out statistically significant in all studies. This may be due to the overriding effect of the type and intensity of immunosuppression, which is also the most modifiable factor in clinical practice (25, 104, 154, 155, 135).

Primary infection, typically during childhood, is not associated with any well-defined clinical syndrome, although there are anecdotal reports of upper respiratory infection and acute cystitis.  After resolution of primary infection, BK virus enters a latent phase in the urogenital tract, and possibly peripheral blood mononuclear cells, tonsils, and other hematopoietic tissues (123).  Reactivation of latent virus occurs in old age, pregnancy, diabetes mellitus, congenital immunodeficiency, acquired immune deficiency syndrome (AIDS), and most importantly, organ transplantation. The first sign of reactivation is BK viruria, which has been reported in 15-60% of kidney transplant patients. This is followed by BK viremia and BK virus nephropathy (BKVN) seen respectively in 5-30% and 1-10% of kidney transplant patients. BK virus nephropathy presents with renal dysfunction, which may be confused with acute rejection.  The rate of graft loss was greater than 50% in the 1990’s, but has fallen to as low as 15% due to regular screening, and pre-emptive reduction of immunosuppression. The first reports of BK virus nephropathy were from adult recipients of kidney transplants, but it now clear that children can be affected (3, 57,59). Moreover, cases have now been recorded after kidney-pancreas, pancreas, liver, heart, lung, and bone marrow transplantation (10, 12, 56, 92, 139). In addition to renal parenchymal involvement, BK virus has been associated with ureteric stricture (53).

In bone marrow transplant recipients, hemorrhagic cystitis is an important clinical syndrome attributed to BK virus infection, particularly after radiation and chemotherapy associated urinary bladder injury have been excluded (11, 18, 46, 50, 144).  The latter occur very early after transplantation whereas viral infection has a later onset. Milder forms of BK virus hemorrhagic cystitis are self-limited, but 5-10% of cases may encounter severe bleeding that can require prolonged hospitalization.

Disease outside of the urogenital tract associated with BK virus has only rarely been seen in the form of myopathy in kidney transplantation or disseminated infection in AIDS patients (159, 114). A case of  BK virus associated colonic ulceration has been published (76). Diagnosis was based on immunohistochemistry and not confirmed by DNA sequencing. Unusual clinical presentations of BK virus infection that have not always been rigorously documented include meningo-encephalitis, salivary gland disease, oral ulcers, acute respiratory infection, pneumonia with viral cytopathic effect, hemophagocytic syndrome, bone marrow aplasia, neutropenia, bone marrow aplasia, systemic vasculopathy with capillary leakage syndrome, and pediatric skin eruptions (13, 40, 99, 110).

Several modalities have been used to make a diagnosis of BK virus infection in the laboratory, and are briefly summarized below.

Detection of BK virus during this latent phase of infection requires Southern blotting or highly sensitive PCR techniques, such as those that use nested primers or 45-60 cycles of amplification. With these methods, latent BK virus infection has been documented in the kidneys, urinary bladder, prostate, cervix, vulva, testes, prostate, semen, peripheral blood mononuclear cells, and tonsils (32, 58, 128).  Diagnosis of such low level infection has no known clinical significance, but has potential applications in tracing donor to recipient transmission of BKV.

Serologic testing has largely been performed in research laboratories with a special interest in BK virus infection. IgG antibodies to BK virus VP-1 are present in most healthy subjects. If serial samples are available for testing, a rise in IgG level > 0.577 optical density (OD) units and a rise in IgA or IgM level > 0.041 OD units have been used to detect active viral replication. IgA antibodies to VP-1 are present with greater frequency (81.4% vs 17.5%) and higher titer (mean optical density 0.11-0.15 vs 0.05-0.08) in patients who have BK viruria compared to those who do not. Transient episodes of reversion to a seronegative state can occur in immunosuppressed patients. Hence, quantitative PCR is a more sensitive and reliable method of monitoring BK virus reactivation than measurement of antibodies. However, serologic testing may have utility in risk assessment of virus transmission via organ transplantation: the rate of viruria in kidney recipients rises from 0% if the donor titer is <1:640 to 88% if the donor titer is >1:163840. The greatest risk of viral reactivation post-transplant (estimated to be 50%) is associated with a positive serostatus of both the donor and recipient, while a seronegative status for both the donor and recipient carries the lowest risk (11%) (20). There is evidence that antibody measurements may also have prognostic value. Thus, low titer anti-BKV IgG antibody measured pre-transplant, and absence of IgA antibodies at week 1 are associated with an increased risk of developing viremia (19, 20, 119).  Pseudovirion technology allows measurement of virus neutralizing antibodies specific to individual genotypes (111). It has been shown that patients seropositive for genotype I may be naive for genotype IV at the time of kidney transplantation and undergo seroconversion at a later time point. Neutralizing antibody studies suggest that genotypes I, II, III and IV are distinct serotypes with distinct cell tropism, and that genotype II is much more prevalent than has been hitherto appreciated (112, 137).

Urine cytology can be used to detect asymptomatic stages of viral activation by the presence of decoy cells, which are essentially urothelial or tubular epithelial cells with intranuclear viral inclusions. When decoy cells are associated with inflammation and present in numbers that exceed variably defined thresholds, such as 10 per cytospin (37), or more than 10 per high power field (146), a diagnosis of BK virus nephropathy should be strongly suspected. If decoy cell casts are present the diagnosis of nephropathy is virtually certain, although a biopsy is still desirable for staging of the disease and monitoring subsequent progression of interstitial fibrosis. Urine cytology cannot distinguish BK virus nephropathy from the rare cases of JC virus and SV40 virus nephropathy.

Real time PCR is the method of choice for routine BK virus screening in organ transplant recipients. It is several times more sensitive than urine cytology, and can distinguish BK virus from polyomavirus JC. This distinction is important because JC virus can be shed in healthy individuals with significant frequency, and only rarely causes renal disease. High viral DNA copy numbers in the urine or plasma can act as a surrogate marker of BK virus nephropathy. The actual cuts off points for diagnosis vary in different institutions, as inter-laboratory standardization of PCR assays has not yet been achieved. Urine viral loads reported to correspond to a diagnosis of BK virus nephropathy in various studies include 5.2E+04 (90), 1E+05 (36), and 1E+07 copies/ml (62, 121).  Likewise, plasma or serum viral load cut offs mentioned by different investigators as an indication for clinical intervention include 1E+03 (36), 3E+03 (23) and 1E+04 copies/ml (62, 121).  Even low level viremia may be significant in case of infection with genotypes II and III because these variants may not be efficiently amplified by PCR primers derived from commonly used genotype I reference strains (68,122).

The gold standard for the diagnosis of BKVN is a biopsy of the allograft kidney. Renal tubular cells show intranuclear viral inclusions, associated with (a) an acute tubular necrosis like picture,  (b) interstitial nephritis mimicking acute rejection, or (c) chronic allograft nephropathy with advanced interstitial fibrosis and tubular atrophy (62). The inflammatory infiltrate in BK virus nephropathy may be B-cell rich with maturation to plasma cells. Although, BK virus is by far the most common cause of viral nephropathy, JC virus, SV40, cytomegalovirus and adenovirus may, rarely, result in viral inclusions. Hence, the diagnosis of BK virus nephropathy should be confirmed by ancillary techniques. For immunohistochemistry, a cross-reacting monoclonal antibody directed against the large T-antigen of the Simian virus 40 (clone PAb 416, Calbiochem) is commonly used. There is considerable inter-laboratory variation in staining intensity and assessment of percentage of infected cells, but the binary classification of biopsies into virus positive and negative is reliable (2, 100). Distinction between BKvirus, JC virus, and SV40 virus is currently best done by DNA sequencing.

In cases with low viral load, cytopathic effect may not be clearly demonstrable by light microscopy, and the diagnosis becomes apparent only after the biopsy is evaluated with the appropriate antibodies or DNA probes.  Another situation encountered from time to time is the presence of high copy numbers of BK virus DNA in the plasma or urine, but negative immunohistochemistry and in-situ hybridization studies. Even transient high level viremia in the absence of overt nephropathy is associated with higher serum creatinine and lower 6m estimated GFR worse graft function (45). These considerations have led to the concept of presumptive BK virus nephropathy reflecting the fact that a randomly taken renal allograft biopsy can miss a disease process, which is inherently focal in nature, and more pronounced in the medulla than in the cortex. The rate of sampling error is as high as 35%,  but can be minimized by taking two cores of biopsy tissue in cases where the clinical index of suspicion is high (38).  

Standardized assessment and reporting is suggested to ensure consistent biopsy readings across multiple institutions. Classification of PyVAN into categories PyVAN-A, -B and –C has been proposed based on the degree of inflammation and interstitial fibrosis (66, 67). The Banff Working Group formulated a working proposal wherein stage A and B were defined by the extent of BKPyV mediated cell injury. In this system, an identical stage can be assigned to biopsies which differ markedly in the degree of inflammation, and hence differ in prognosis (97). Inflammation and extent of fibrosis and tubular atrophy at diagnosis may be the most important predictors of a poor outcome.

Distinguishing between BK virus nephropathy in the inflammatory phase and acute rejection is an important clinical problem when viral infected cells are sparse. The occurrence of glomerulitis, intimal arteritis, and peritubular capillary C4d deposition in rejection is helpful in the differential diagnosis. Tubulitis is not a reliable discriminating parameter, even if present away from areas of viral cytopathic effect. MHC class II upregulation by the tubular epithelium is no longer considered a marker specific for rejection as it also occurs in the context of viral replication (102).

The incidence of acute T-cell mediated rejection after reduction of immunosuppression is approximately 10%. Kidney biopsies are particularly difficult to interpret during this phase. Morphologic criteria for resolving BK virus nephropathy and T-cell mediated rejection overlap, and the differential diagnosis is facilitated by careful clinical correlation and attention to serial trends in serum creatinine and viral loads in the urine and plasma (67, 102). Analyzing T-cell specificity by next generation sequencing of T-cell receptor usage might provide a future tool in the differential diagnosis (43). In general response to steroids is seen in only a third to half of biopsies that otherwise satisfy Banff criteria for acute rejection (14, 15). Clinical management in this setting will remain uncertain until better diagnostic criteria are developed and effective anti-BK virus drugs become available.

In biopsies with BK virus nephropathy it is usually possible to demonstrate clusters of viral particles measuring 40-50 nanometer in diameter in tubular epithelium by ultrastructural examination (85). This can suffice for a provisional diagnosis of polyomavirus nephropathy in laboratories that do not have a well standardized immunohistochemical or in-situ hybridization protocol available for more definitive identification. However, BK virus, JC, and SV40 viruses cannot be distinguished from each other by this method. Indeed, if measurements of viral capsid diameter are not made, even adenovirus and parvovirus are difficult to exclude on morphologic grounds. The presence of ultrastructural immune complex deposits in the tubular basement membranes tends to be associated with worse clinical outcome (24, 60). Electron microscopy of urinary sediments has been advocated as a screening tool for polyomavirus infection, but cannot reliably detect urinary loads less than 1E+06 to 1E+09 copies/ml (17, 146).  More recently, the presence of a three dimensional viral clusters (so called ‘haufen’) have been shown to be a good marker of BK virus nephropathy in viremic or persistently viruric patients with a negative biopsy (145).  A ‘haufen’ test should not be used as a substitute for a biopsy, which provides important information about concurrent rejection and degree of histologic chronicity. Moreover, therapeutic intervention is quite justified in a patient with persistent viremia whether or not ‘haufen’ can be documented in the urine. Indeed, the sensitivity and specificity of the haufen test for a diagnosis of presumptive nephropathy has not been  examined in a patient cohort of reasonable size.

Tissue damage in polyomavirus infection is believed to the result of lytic viral replication that results in cell death.  There is evidence that transition from latent to lytic viral infection is triggered by ischemic, drug induced, or rejection associated injury. The fact that the allograft kidney is prone to both ischemia and rejection may largely explain why most cases of BK virus nephropathy occur after renal transplantation, and only rarely following liver, heart, lung, and bone marrow transplantation (123).  Expression of proinflammatory cytokines and chemokines is mediated by activation of innate defense mechanism such as the Toll-like receptor 3 pathway (131). Using DNA microarray analysis of allograft kidney biopsies or cultured human endothelial cells, it has been shown that BK virus nephropathy is associated with up-regulation of several major groups of mRNA’s, including CD8, Interferon-gamma, CXCR3, perforin, and genes involved in cell cycle regulation, apoptosis, and anti-viral immunity (1, 55, 94).  It is notable that several of these molecules are also upregulated in acute cellular rejection, and this explains why the differential diagnosis between viral nephropathy and acute rejection is a vexing problem (125).  BK virus infection also results in the up-regulation of molecules associated with graft fibrosis, including matrix collagens, TGF-β, MMP2, MMP9, and markers of epithelial-mesenchymal transformation. Thus, persistent BK virus nephropathy can progress to chronic allograft nephropathy.

BKV is a strictly human pathogen and no suitable animal model exists. Therefore, drug discovery work is has been primarily done in vitro. Several compounds have been identified to be modestly active in cell culture systems as summarized below:

Cidofovir is Food & Drug Administration (FDA) approved for the therapy of cytomegalovirus retinitis. However, in vitro data indicates that it is a broad spectrum anti-viral agent with activity against Herpesviruses, adenovirus, pox virus, papillomavirus, and polyomavirus families.  For BK virus, in vitro 50% effective concentration of cidofovir (EC50) is 36.3 mg/ml (115.1 mM), and the 50% cytotoxic concentration (CC50) is 63.9 mg/ml (202.6 mM) (49). The selectivity index (SI) is 2.3, which is a reflection of its low therapeutic margin (49).  Esterification of cidofovir with hexadecyloxypropyl (HDP), octadecyloxyethyl (ODE), and oleyloxyethyl (OLE) group containing side chains results in up to a 3-log lowering of EC50 and a marked increase in selectivity index (120). These ester derivatives have the additional advantage of being orally bioavailable and less nephrotoxic than the parent compound. The principal concern is whether reduced renal uptake of these compounds will compromise their efficacy in BK virus nephropathy. It is  possible that this potential drawback would be compensated for by the 100 fold higher anti-viral efficacy.

Another nucleoside analogs with promising in vitro anti-BK virus effect is an adenine analog of Cidofovir called (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine or (S)-HPMPA (HDP-(S)-HPMPA). The EC50, CC50, and SI  of this compound are 0.015 mM, 0.8 mM, and 54.0 respectively (126). Favorable in vitro anti-BK virus activity, with SI has high as 232,  has been demonstrated for long fatty acid side chain analogs of 9-[2-(phosphonomethyoxy)ethyl]adenine (PMEA). PMEA is the derivative compound for a number of medications currently being used in the treatment of hepatitis B and HIV infection.  Finally, the nucleoside analog 2′,3′-Didehydro-3′-deoxythymidine has shown significant anti-BK virus effect with EC50, CC50, and SI of 5.1+/-1.9 mM, 404.3 mM, and 72.6, respectively. This compound is chemically related to 3’-fluoro-2’ deoxythymidine, which is a potent inhibitor of adenovirus (101), and to 3’-azido-thymidine (AZT), which is in clinical use for HIV infection. All of the aforementioned compounds are worthy of follow up for lead optimization, toxicology and safety studies.

Leflunomide belongs to a family of drugs called the malonitrilamides.  Using a real time PCR based drug sensitivity assay the EC50, CC50 and SI have reported as respectively 11.3 mg/ml, 39.7 mg/ml, and 3.8 respectively (49).

Quinolones inhibit gyrase and similar enzymes that are necessary for bacterial and viral DNA uncoiling prior to DNA replication. Nalidixic acid and oxolinic acid were the first compounds of this group shown to inhibit BK virus replication in vitro (116). More recently several fluoroquinolones in clinical use were shown to have EC50 varying from 13.6 to 266.6 mg/ml, CC50 varying from 10.6 – 283 mg/ml, and SI ranging from 0.7 (coumermycin) to 3.6 (ciprofloxacin) (127).

Human immunoglobulin preparations: Intravenous immunoglobulins from healthy subjects contain antibodies against variety common viral pathogens. This forms the basis for the administration of these preparations to patients with cytomegalovirus, varicella, hepatitis B, and other viral infections. We published in vitro data showing that a 5 gram vial of IVIG (Gammagard, Baxter Inc.) can neutralize approximately 5E+09 BK virus particles (124).

BK virus genome has a supercoiled double stranded DNA, which must be uncoiled by the action of topoisomerase enzymes prior to viral replication. Camptothecin is an FDA approved topoisomerase inhibitor that is used for the treatment of cancer. It can inhibit BK virus replication with an EC50 of 0.1 mM, CC50 of 0.9 mM, and an SI of 9.0 (unpublished data).  However, as an anti-cancer agent with multisystem toxic effects, this drug is not likely to be formally evaluated for treatment of BK virus infections in the clinical arena. Nevertheless, compounds belonging to the general class of topoisomerase inhibitors deserve further evaluation. We have found a compound designated NSC 270718, obtained from the National Cancer Institute, to be an order of magnitude less toxic.

BK virus uses primarily the host cell metabolic machinery to facilitate its own replication. Since a number of protein kinases are involved in host cell replication, this provides a rationale for exploring protein kinase inhibitors as potential therapeutic agents for BK virus infection. Targeting the host kinome offers prospects of developing broad spectrum anti-BK virus compounds encompassing multipronged mechanisms. Provided we can find relatively non-toxic agents, these compounds would also have the advantage of being less prone to development of drug resistance following continuous exposure.  In our search for anti-viral protein kinase inhibitors, we have found that ST0-8584, which is chemically 2,2-bis(4-chlorophenoxy)-N-[(6-bromo-2-oxochromen-3-yl)carbonylamino]acetamide, has an EC50 of 1.1 mM, CC50 of 21.1 mM, and SI of 19.2 (166). This observation indicates that screening of chemical libraries of protein kinase inhibitors may be a worthwhile avenue to pursue in the quest for anti-BKV drugs. Compounds containing benzaldehyde hydrazone and acetamide thiazole motifs will of particular interest.

BK virus receptor binding compounds: The primary receptor binding determinant on the BK virus capsid is the VP-1 protein. The corresponding host ligand is not completely characterized, but appears to contain a terminal α2-3-linked sialic acid associated with Type II gangliosides (41, 42, 49, 93).  In vitro studies show that BTB11968  or 2-(hydroxymethyl)-6-(nonyloxy)tetrahydro-2H-pyran-3,4,5-triol, has an EC50 = 6.4 mM, CC50 = 126.4,  and SI = 19.8. Thus, future work seeking to identify more potent receptor blocking agents appears to be justified.  The caveat is that receptor blockers tend to have low efficacy and are more suitable as prophylactic rather than therapeutic agents.

Treatment of BK virus infection is usually reserved for patients with viral nephropathy or persistent BK viremia.  There is evidence that therapy might also be considered for sustained viruria (9). Effectiveness of various antiviral regimens has been claimed in observational cohort studies, but not established by randomized controlled trials. Most studies performed to date suffer from an important caveat in that they are not controlled for simultaneous reduction of immunosuppression or addition of mTOR inhibitors (70, 91, 107,151).  Nevertheless, when adjunctive measures described below are not effective, it is reasonable to consider empiric use of drugs shown to have anti-BK virus activity in vitro. Off label use of cidofovir, leflunomide, quinolone antibiotics, and intravenous immunoglobulin (IVIG) are all potential options (Table 1). All of these compounds are FDA approved for other medical indications. There is not enough evidence to designate any of these agents as the drug of choice, or to support the use of combined therapy.  Indeed a meta-analysis did not show any graft survival benefit for any of the antiviral therapies in common use (71).  A review of the published literature allows us to offer the following clinical pharmacology perspective on the drugs currently used in the transplant clinic:

The mechanism by which cidofovir mediates anti-BKV activity is unclear. In the case of cytomegalovirus, cidofovir is believed to exert its effect by inhibiting viral DNA polymerase (109). The BK virus genome does not code for such an enzyme, but the viral large T-antigen does possess a functional domain with DNA polymerase activity. Since cidofovir is a nucleoside analog, its broad spectrum antiviral effect may also reflect direct inhibition of viral DNA synthesis by cellular DNA polymerases. 

For BK virus nephropathy or persistent viremia, cidofovir (Vistide, Gilead Sciences Inc.) should be used in a dose of 0.25-1.0 mg/Kg IV every 1-3 weeks.  The package insert of this drug recommends prehydration with 1 liter normal saline to minimize nephrotoxicity. However, the amount of fluid infused should take into account the degree of renal dysfunction, and cardiac function of the patient.  Treatment should continue at least till clearance of viremia, and preferably also clearance of viruria (118). In cases of  refractory viruria, a reasonable treatment end point could be achievement of  urinary viral load of 1E+05 copies per ml or less (61, 163).  The maximum number of permissible doses is not precisely defined, but up to 22 doses have been safely administered (84).

It is to be noted that the doses recommended above for BK virus nephropathy or BK viremia are significantly less than the 5 mg/kg dose used for treatment of cytomegalovirus retinitis. There is understandable hesitation to administer cidofovir in full dose to kidney transplant recipients, who frequently have significant pre-existing renal parenchymal injury at the time antiviral therapy is initiated.  However, pharmacokinetic studies suggest that low dose cidofovir would not allow blood levels of cidofovir to reach EC50 values described in vitro (34, 106).  Unpublished data from my laboratory indicates that such a treatment regimen only produces a very transient reduction in plasma viral load with a quick return to pre-infusion levels. Nonetheless, limited anti-viral effect might still be achieved since this drug is concentrated within renal tubular cells. While probenecid, a drug that inhibits renal tubular excretion of Cidofovir and permits better plasma levels, is typically used in patients with cytomegalovirus retinitis, this is not done in the treatment of BK virus nephropathy. It is thought that probenecid induced lowering of intratubular excretion of cidofovir would potentially reduce its effective concentration in the urinary compartment which harbors most of the viral load in BK virus nephropathy.  Moreover, Probenecid does not seem to affect serum cidofovir concentrations when used in low doses in renal transplant patients (82).

Higher doses of cidofovir have been used for BK virus infection of the urinary bladder. Thus, in one study patients with hemorrhagic cystitis following bone marrow transplantation received a median of four doses of 3-5 mg/Kg IV with co-administration of probenecid (29). Instead of intravenous injection, 50-200 mg cidofovir can also be instilled directly into the urinary bladder.  Some clinicians have utilized a still higher intravesical dose of 350 mg or 5mg/Kg weekly diluted in 60 ml of 0.9% sodium chloride administered by a Foley’s catheter (130). In patients with indwelling catheters, it is recommended that the catheter be clamped for 1 hour following instillation of cidofovir.  Weekly instillations are continued till resolution of symptoms.

All patients on cidofovir should be carefully monitored for neutropenia, nephrotoxicity, intraocular hypotony, and uveitis, although these side effects are uncommon at the low doses employed in BK virus nephropathy. Nephrotoxicity typically manifests as rise in serum creatinine, and this may trigger an allograft kidney biopsy to distinguish between acute tubular necrosis, calcineurin inhibitor toxicity, acute rejection, and worsening of BK virus nephropathy. Proteinuria and glycosuria have been described as manifestations of cidofovir induced proximal tubular injury. Hence, concurrent administration of other nephrotoxic drugs (aminoglycosides, amphotericin B, foscarnet, vancomycin) should be avoided. Cidofovir given locally can precipitate bladder spasms, which can be managed with anti-spasmodics and analgesics. Finally, cidofovir is carcinogenic, teratogenic, and known to cause hypospermia in animal studies.

The mechanism of anti-BK virus action of leflunomide is not understood. Its activity against cytomegalovirus is said to result from defective tegumentation and viral assembly (77). In the case of BK virus, which is a non-enveloped virus, anti-viral effect may be related to inhibition of viral DNA synthesis (50).  This is accompanied by a significant host cytostatic effect with non-specific pyrimidine depletion (16).

Leflunomide (Arava, Aventis Corp.) treatment for BK virus nephropathy or persistent viremia consists of a 100 mg loading dose for 3-5 days, followed by a 20-60 mg/d maintenance dose (72).  A lower loading dose of 15-20 mg has been used in children aged 9-20 years (7). Individual pharmacokinetic variability makes it desirable to monitor plasma leflunomide levels. Recommended target trough levels are in the range 40-100 mg/ml. The immunosuppressive (in addition to antiviral) properties of leflunomide are said to useful in the management of patients where BK virus infection and acute rejection coexist.  Reported toxic effects include anemia, thrombocytopenia, CMV viremia, fungal pneumonia (48), hemolysis, and thrombotic microangiopathy (86).  Therefore, all patients on leflunomide therapy should be monitored with at least monthly viral load assessment, complete blood counts, serum creatinine, and liver function tests. Duration of therapy is determined by quantitative plasma and urine PCR for BK virus  DNA. Viral clearance in cases showing satisfactory response typically takes 2-3 months, and may be related more to recovery of cell mediated immunity than a true anti-viral effect.  Pharmacodynamic studies show no association between serum drug levels and reduction of viral load by PCR. In a multivariate analysis the administration of leflunomide was not an independent predictor of viral clearance (80).  A multicenter trial currently in progress seeks to evaluate if Leflunomide combined with Sirolimus may be clinically more efficacious (91).

The rationale behind the use of quinolones to treat BK viral infection is the ability of these compounds  to inhibit helicase activity of virus encoded large T-antigen (4).  Helicase enzyme is necessary for the unwinding and melting of double stranded viral DNA, which is the first step in DNA replication.

The in vitro selectivity index for inhibiting BK virus replication is low (142). Therefore, it is unlikely that these compounds will be useful in treating established disease.  On the other hand some but not all studies suggest some efficacy in an early stage of infection and as a prophylactic agent. Thus, in a series of bone marrow transplant recipients from Hong Kong, prophylactic administration of ciprofloxacin administration at a dose of 500 mg bid oral or 200 mg bid i/v, on days 1 thru 50 post-transplant, resulted in a significantly lower peak BK virus load, and a reduced incidence of hemorrhagic cystitis (88).  This effect was reproduced in another study from the University of South Carolina (103), but not in a third study from Singapore that was reported in the form of a letter to the editor  (115).

In kidney transplants ciprofloxacin administered in the first 30 days of transplantation lowered the risk of BK viremia at 3 months but not at later time points (164).  Investigators at the Brigham and Womens Hospital in Boston have had a long-standing interest in this issue. Their initial studies described patients with persistent urinary decoy cells who were treated with 400 mg per day of gatifloxacin for ten days without alteration of the immunosuppressive regimen. Seven of ten treated patients showed >80% reduction of viremia or disappearance of decoy cells (30).  Subsequently, this group published a larger retrospective study confirming a prophylactic effect in kidney transplant patients receiving either ciprofloxacin or levofloxacin (52).  However, they could not subsequently confirm this effect in a prospective randomized placebo controlled trial of patients who received a 30 day course of Levofloxacin (87).

At the time of this writing there are two ongoing trials (NCT 01789203 and NCT 01353339) which seek to determine if extending the period of prophylaxis to 3 months will result in greater anti-viral efficacy than has been achieved to date. However, there also is concern that prolonged use of  quinolone antibiotics may result in an increased incidence of drug resistant urinary tract infections and Clostridium difficile infections (113).

The principal mechanism of anti-BK virus action of IVIG is direct neutralization of virus by antibodies directed against the viral capsid protein VP-1. Potential non-neutralizing mechanisms  include steric hindrance, conformational change in the bound protein, complement dependent cytotoxicity or cell lysis, viral agglutination, and phagocytosis (74, 79,83).  The extent to which IVIG administered parenterally can access the site of viral replication in renal tubules is unknown. However, even if intra-cellular penetration does not occur, the introduction of neutralizing antibody in the peritubular capillary bed would reduce circulating load of viable viral particles, limit cell to cell spread of virus, and allow the immune system to better cope with viral infection. It is also pertinent to recall that IVIG has a variety of immunomodulatory properties, which form the basis of its use in patients with refractory T-cell mediated rejection or antibody mediated rejection. Indeed, it has been argued that IVIG therapy deserves particular consideration in those patients with BK virus nephropathy, in whom acute rejection is thought to co-exist.

Treatment of BK virus nephropathy with IVIG is not standardized. One published regimen calls for a dose of 2 g/Kg divided over 2-5 days along with reduced immunosuppression (138). A second study suggests using either 1-2 g/Kg IV X 1-2 doses or 150 mg/Kg IV biweekly for 8 weeks (163). Children have been treated with 60 mg/Kg for 5 doses given every 4-6 weeks (148) or as 600 mg/Kg every 4-6 weeks, with the first dose divided over three days (141).  There are several studies reporting that   reports that IVIG administration does not result in prompt resolution of responses in BK virus nephropathy (138, 141, 163). However, treatment in these studies was (a) initiated at a late stage of disease, (b) not based on lot-specific stoichiometric measurements of actual antibody titer or half-life, and (c) typically monitored by PCR-based viral DNA assays that do not distinguish between infectious and neutralized virions. The doses used were empirical, limited by cost, and varied widely (124).  More rational clinical trials based on sound pharmacokinetic principles are needed to better understand potential and limitations of IVIG therapy for BK virus infections.

The mainstay of therapy for BK virus nephropathy or persistent viremia is to reduce or discontinue immunosuppressive drugs, thereby allowing the host immune system to mount a successful anti-viral response. Most patients with definitive BK virus nephropathy are on triple therapies consisting of a calcineurin inhibitor (tacrolimus, cyclosporine A), an anti-proliferative drug (mycophenolic acid, azathioprine, sirolimus), and steroids. Two principal strategies have been reported : 

(a) Dose reduction of calcineurin inhibitors by 25% - 50%; followed by reducing the antiproliferative drug by 50%; followed by discontinuing the latter.

(b) Reducing the antiproliferative drug by 50%, followed by reducing calcineurin inhibitors by 25% -50%, followed by discontinuation of antiproliferative drugs such as mycophenolate. While mycophenolate has a paradoxic BKV inhibitory effect in vitro this does not seem to translate to tangible clinical benefit (26).

In both strategies, oral prednisone is typically maintained at 10 mg or less daily dose.  Alternate treatment approaches reported in the literature include switching from tacrolimus to low-dose cyclosporine, from mycophenolic acid to low-dose sirolimus, from calcineurin inhibitor to low-dose sirolimus, or from mycophenolic acid to leflunomide. These interventions are supported to some extent by epidemiologic as well as laboratory studies. Thus, registry data show a lower incidence of BK virus-treatment episodes in patients maintained on mTOR inhibitors.  Sirolimus, unlike calcineurin inhibitors, does not inhibit BK virus-specific T-cells (44). Tacrolimus activates BK virus replication in human tubular epithelial cells while sirolimus has an inhibitory effect mediated by protein kinase pathways (91, 64).

Reduction of immunosuppression should be titrated to the blood levels of individual therapeutic agents. Target tacrolimus trough levels of <6 ng/mL, cyclosporine trough levels of <150 ng/mL, and sirolimus trough levels of <6 ng/mL are desirable.  It is also important to follow serum or plasma BK virus load every 2 weeks, as it is responds earlier than the urine viral load. Given the known variability of PCR assays, a decline in viral load of at least 5-10 fold should be regarded as significant. In responsive cases, clearance of BK viremia may take several months. Once plasma BK virus DNA has become negative, serial quantitation of urine BK virus load can be initiated.  The ideal goal is to obtain complete urinary clearance of virus, but this is not always possible. As a compromise, a urine viral load of 1E+05 copies/mL is suggested as the end-point for stopping anti-viral therapy. Monitoring of serum creatinine is also critical, and a significant rise above baseline should trigger a renal biopsy.  Biopsy findings can help distinguish whether worsening graft function is a manifestation of acute rejection (secondary to reduced immunosuppression), progression of BKV nephropathy, or an unrelated incidental insult.

Factors associated with successful clearance of virus include female sex, transient or persistent plasma viral load <10,000 genomic equivalents, and non-doubling of viral copy numbers (45).  Once viral clearance occurs constant vigil is needed for the detection and early treatment of acute rejection since these patients are on lower than usual immunosuppression (165).

No vaccines against BK virus are available at this time, but laboratory studies in pursuit of this goal are ongoing. It has been shown that the VP-1 protein in the BK virus capsid can elicit virus neutralizing antibodies (111,112). This raises hope that a multivalent prophylactic vaccine based on recombinant virus like particles can be developed, as has been successfully accomplished for human papillomavirus. Whether such a vaccine will protect individuals already exposed to BKV earlier in life is uncertain. Effort is also underway to define antigenic epitopes that are recognized by the cellular immune system (31, 81, 89, 117, 167). Such peptides may become components of T-cell therapeutic vaccines for patients who have already developed BK virus nephropathy.

Most patients with end stage kidney disease are already exposed to BK virus at the time of transplantation.  The virus is latent primarily in the kidney, and organs selected for renal transplantation typically also have latent BK virus, which may be potentially transmitted to the recipient (136). It has been shown that donors with active BK virus replication, as evidenced by high titer anti-VP-1 antibodies, have greater likelihood of transmitting infection to recipients (19, 20).  Currently, it is difficult to identify such high risk organs during routine clinical practice, since serologic assays to measure anti-BKV antibody titers are not widely available.  Theoretically, transplantation of kidneys from BK virus seronegative subjects  to seronegative recipients could markedly reduce the risk of BKV transmission by organ transplantation. However, this would sharply reduce the pool of organs available for transplantation, since BK virus seropositivity is almost universal.

Once kidney transplantation has been performed the risk of active BK virus infection can be minimized by avoiding aggressive immunosuppression. Major transplant centers in theUS now have screening programs in place to diagnose BK virus replication at an early stage before it progresses to irreversible nephropathy and graft loss. Screening for BK virus using either urine cytology or quantitative PCR should be performed at least 3 monthly during the first 2 years post-transplant, and then annually (62).  Pre-emptive reduction of immunosuppression in patients with persistent viremia prevented the occurrence of subsequent nephropathy in one large study at the University of Saint Louis (25). It has also been shown that early diagnosis of nephropathy prior to rise of serum creatinine results in better clinical outcome than cases where diagnosis is delayed (28).  Unfortunately, no screening program can provide an early diagnosis for all cases. In some kidney transplant recipients viruria and viremia have been documented as early as 12 hours after transplantation (6).

The best screening sample for outpatient monitoring is controversial. The Kidney Disease Initiative for Global Outcomes (KDIGO) expressly recommends plasma (5). However, the American Society of Transplantation Guidelines notes that urine allows earlier diagnosis of viral reactivation  before the disease becomes clinically significant, and has a better sensitivity in diagnosis of hemorrhagic cystitis after hematopoietic stem cell transplantation (67). Moreover, evidence is accumulating that sustained viruria in the absence of viremia is also detrimental to graft function (9, 96, 97). 

Once BK virus nephropathy or persistent BK viremia has been documented, different regimens for reducing overall immunosuppression are followed. The most optimal strategy has not been defined by prospective randomized control trials.  Likewise, a role of anti-viral treatment in favorably altering the course of infection is not universally accepted. Another controversial area is the interpretation of lymphocytic infiltrates and tubulitis in biopsies with BK virus nephropathy. One school of thought believes that this reflects a component of graft rejection and calls for a brief course of steroid therapy (63, 98). An alternate view point is that the virus itself can induce an active inflammatory response with tubulitis, and further augmentation of immunosuppression is not indicated (161).

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Tables

Table 1: Antiviral Agents for BK Virus Infection

Drug	Trade Name	Manufacturer	Dose	Duration
Cidofovir	Vistide	Gilead Sciences	(a) For BK viremia or nephropathy: 0.25-1.0 mg/kg IV every 1-3 weeks (b) For hemorrhagic cystitis: 3-5 mg/kg IV weekly with probenecid or an intravesical dose of 50-350 mg in 60 ml normal saline	Response monitored by viral load* & serum creatinine   Till resolution of cystitis
Leflunomide	Arava	Aventis Corporation	100 mg oral loading dose x 3-5 days followed by 20-60 mg/day maintenance dose	Response monitored by viral load* and serum creatinine
Ciprofloxacin or other quinolone	Ciflox**	Bayer Corporation**	500 mg oral twice a day	Response monitored by viral load* and serum creatinine
Intravenous Immunoglobulin	Gammagard**	Baxter Corporation**	2 g/kg IV divided over 2-5 days or 150 mg/kg IV every 2 weeks	2-5 days   8 weeks

*Drug therapy should be considered at least till clearance of viremia. It is not always possible to eradicate low level viruria. However, an attempt should be made to reduce viral load to less than 100.00 genomic equivalents per ml of urine. A rise in serum creatinine after initial response may require a biopsy to rule out acute rejection.

**Several other pharmaceutical preparations are also commercially available.

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