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0041-1337/04/7704-562/0 TRANSPLANTATION Copyright © 2004 by Lippincott Williams & Wilkins, Inc . Vol . 77, 562-567, No . 4, February 27, 2004 Printed in U.S.A . EFFECT OF ANTIMETABOLITE IMMUNOSUPPRESSANTS ON FLAVIVIRIDAE, INCLUDING HEPATITIS C VIRUS JASON R. STANGL, KATHLEEN Background. Recurrence of hepatitis C virus (HCV) after liver transplantation is almost universal and decreases both graft and patient survival . Medications that alter nucleic acid metabolism, including some common immunosuppressants used in HCV-infected patients, may affect viral replication . Methods . Bovine viral diarrhea virus (BVDV) is in the Flaviviridae family and is closely related to HCV . We measured the effect of two immunosuppressants, azathioprine (AZA) and mycophenolate acid (MPA), on both BVDV replication by plaque assay and host-cell replication by flow cytometry. We also compared the effect of ribavirin and AZA on the level of HCV replicon RNA by real-time reverse-transcriptase polymerase chain reaction. Results. At doses that achieved similar cytotoxicity, AZA decreased BVDV replication 10 times more than MPA. The inhibition of BVDV by AZA occurred at lower doses than the cellular cytotoxicity and did not depend on cytotoxicity. A two-log reduction in viral titers occurred despite blocking the cytotoxicity of AZA by inhibiting ribonucleotide reductase with high concentrations of thymidine . A metabolite of AZA, 6-mercaptopurine, still possessed this antiviral effect, but a metabolite further downstream, 6-thioguanine, did not, even though 6-thioguanine is the metabolite responsible for cellular toxicity . The effect of AZA on a HCV replicon was at least as large as that of ribavirin . Conclusions. This report suggests that AZA is a more potent antiviral than MPA for Flaviviridae and may exert a specific antiviral effect on HCV . Additional clinical studies to investigate this previously unanticipated antiviral effect of AZA on HCV in the posttransplant setting are indicated. Hepatitis C virus (HCV) is the most common indication for liver transplantation in developed countries . Reinfection of liver allografts by HCV is virtually universal . Moreover, the natural history of HCV is accelerated after liver transplantation, and there are reports that mortality after liver transplant for HCV is increasing recently (1) . The presence of active HCV infection decreases patient survival and liverallograft survival in recipients of orthotopic liver transplantation (2) . Hepatitis C viremia usually exceeds pretransplantation levels (3), and it is unknown whether new specific viral variants are selected after transplantation . In addition, the L. CARROLL, MITCHELL ILLICHMANN, AND ROBERT STRIKER amount each individual immunosuppressant drug contributes to the acceleration of the natural history of HCV is controversial (4) . Answering these questions is difficult given the lack of cell-culture models for HCV . This requires a study of HCV in the context of closely related viruses or with HCV replicons, which consist of RNA elements coding for nonstructural proteins of HCV that can autonomously replicate in Huh7 hepatoma cells (5) . Ribavirin has been shown to enhance the activity of interferon in the treatment of HCV and increase the percentage of sustained responders (6) . The exact mechanism of this enhancement is unknown, although at high concentrations, ribavirin can be incorporated into RNA viral genomes and decrease replication (7) . Antimetabolite immunosuppressants have some commonalties with ribavirin . Both ribavirin and mycophenolate acid (MPA) inhibit inosine monophosphate dehydrogenase (IMPDH) (8, 9) . This and other data have led some to quantify the antiviral effect of MPA and other IMPDH inhibitors (9) . At the same time, ribavirin and three metabolites of azathioprine (AZA) are all processed to monophosphate nucleotides by IMPDH intracellularly (8, 10), which then compete with endogenous nucleotide pools . Clinical data investigating potential antiviral effects of either AZA or MPA and whether different immunosuppressive regimens are associated with different rates of HCV recurrence are conflicting (11-15) . These trials are also difficult to compare given a number of variables, such as genotypically different viruses, different immunosuppressive regimens, and different patient populations . To address the limited question of whether MPA or AZA have specific antiviral effects because of their similarities to ribavirin, we turned to a virus closely related to HCV and in the same viral family, bovine viral diarrhea virus (BVDV), which can be grown in cell culture . Using BVDV as a surrogate for HCV, we directly measured the antiviral effect of antimetabolite immunosuppressants independent of their effects on the adaptive immune system . In this report, we demonstrate that AZA has significantly more specific antiviral activity than MPA . In addition, we show that the antiviral activity of AZA is comparable with that of ribavirin on an HCV replicon . RS received support from IRG-58-011-45-03 from the American Cancer Society and holds a basic science grant from the American Society of Transplantation, and JS is supported by an NIH fellowship T32 CA09614-15 . There are no conflicts of interest to report . Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin. Address correspondence to : Dr . Robert Striker, University of Wisconsin Medical School, 1300 University Avenue, Room 4638 MSC, Madison, WI 53706-1532 . E -mail : rtstriker@wisc .edu . Received 11 July 2003 . Accepted 16 September 2003 . 562 DOI : 10 .1097/01 .TP .0000114610 .40412 .C6 MATERIALS AND METHODS Madin-Darby bovine kidney (MDBK) cells (ATCC CCL22, Manassas, VA) were grown in Dulbecco's modified Eagle's medium-F12 (Cellgro, Herndon, VA) supplemented with 10% heat-inactivated bovine serum (Atlanta Biologicals lot #k0041, Norcross, GA) that was demonstrated to be free of cytopathic and noncytopathic BVDV by enzyme-linked immunoadsorbent assay and antibodies to BVDV type 1 strains by serum neutralization assay . The cells were also tested for BVDV contamination by reverse-transcriptase polymerase chain reaction (RT-PCR) (16) . Cytopathic pNADL BVDV viral stock that had been extensively passed in this media was kindly provided by Ron Schultz, University of Wisconsin . AZA, MPA, 6-methyl mercaptopurine, 6-thioguanine, and thymidine were purchased from February 27, 2004 STANGL ET AL . Sigma (St . Louis, MO) . 6-mercaptopurine and ribavirin were purchased from ICN (Costa Mesa, CA) . 563 al. (17) . Samples were analyzed with the ABI 7700 Sequencer (Langenfeld, Germany) and the OOCt calculated according to Stuyver et al. (18) . Plaque Assays Unless otherwise noted, freshly seeded MDBK cell monolayers (1x10 5 cells in a 100 mm dish) were seeded in the presence of varying concentrations of antimetabolite drugs and incubated for 3 hours at 37°C, 5% C02 . Then, a low multiplicity inoculum (approximately 0 .01 pfu/cell of cp BVDV) was added, and cells with virus were further incubated for 72 hours, after which the supernatant was collected . Mock-infected plates with the same drug exposure for the same amount of time were trypsinized and counted in triplicate with a flow cytometer . Serial dilutions of each supernatant were added to 4-hour old, newly seeded monolayers without any drug . One hour after infection, the inoculum was removed, and MDBK cell medium containing 1% methylcellulose was added to the monolayers . Plaques were counted 96 hours postinfection . Dilutions that gave approximately 25 to 75 plaques per plate were repeated in triplicate . Real-Time RT-PCR HCV 1bN replicon with no adaptive mutations transfected into Huh7 cells (clone 1) (5) was kindly supplied by Stanley Lemon, University of Texas, Galveston . Subconfluent cells were incubated for 72 hours in the presence of media containing 1 mM thymidine alone or thymidine with either 100 µM ribavirin or 100 µM AZA . RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) according to manufacturers instructions, and 50 ng of total RNA was used per replicate of the real-time PCR assay . Primers and probes for the HCV 5' untranslated region as well as cellular glyceraldehydes-3phosphate dehydrogenase were identical to that used by Cheney et RESULTS Comparison of Azathioprine and Mycophenolate on Bovine Viral Diarrhea Virus To begin to look for selective pressure that altered nucleotide pools may place on RNA viruses, we measured BVDV growth in MDBK cells exposed to MPA or AZA . When cells were actively replicating (subconfluent cells), both MPA, and to a much lesser extent AZA, had cytostatic and toxic effects in addition to any potential specific antiviral effects (data not shown) . The cytostatic and toxic effects of MPA, presumably attributable to decreased de novo purine synthesis from IMPDH inhibition, were such that even very low concentrations of MPA killed all the cells when the cells were rapidly dividing . By allowing cells to reach confluency and a slower growth rate before exposing cells to drug and virus, the effect of the drug on viral replication on an intact cell monolayer could be evaluated (Fig. 1) . Under these conditions, the antiviral effect of AZA was larger than that of MPA, with only 12% of the virus produced per living cell grown in AZA compared with the amount of virus produced per living cell grown in MPA when concentrations of both drugs caused an approximately similar (approximately 50%) decrease in cell growth . 1000 - "' a 100 L L O L d a E c - + 10 AZA cells -MPA cells --0--AZA virus - fl--MPA virus v w 0 R L e 0 0 2x 6x 18x Drug (uM) FIGURE 1 . Azathioprine (AZA) has a larger specific antiviral effect than mycophenolic acid (MPA) on bovine viral diarrhea virus (BVDV) in confluent cells . Confluent Madin-Darbin bovine kidney (MDBK) cells were grown in varying concentrations of AZA (circles) or MPA (squares), with (open symbols) and without (closed symbols) virus . After 72 hours, host cells were counted by flow cytometry, and virus was titered by plaque assay . Because of the cytotoxicity of MPA, confluent cells (closed symbols) were exposed to doses of AZA and MPA that allowed similar amounts of cellular growth (x=0 .1 for MPA and 1 for AZA) . The amount of BVDV virus (open symbols) generated was significantly less in cells exposed to AZA than MPA . 564 Vol . 77, No . 4 TRANSPLANTATION Effect of Azathioprine on Bovine Viral Diarrhea Virus does not Depend on Cytotoxic Effects an appreciable effect on cell growth, similar to the prodrug AZA itself. However, 6-mercaptopurine still caused a two-log The cytostatic and toxic effects of AZA are more modest decrease in BVDV replication, whereas 6-thioguanine had no than MPA and thus allowed the addition of AZA to rapidly effect . dividing cells, which in turn allowed more robust viral proHCV Replicon is More Sensitive to Azathioprine than duction . With increasing concentrations of AZA, viral producRibavirin at Equivalent Doses tion was significantly curtailed, even at concentrations where no detectable decrease in cell growth occurred (Fig . 2) . Cell confluency affects the amount of HCV replicon per cell The decrease in cell growth caused by AZA at higher concen(18, 20), and, because high concentrations of AZA without trations can be prevented by high concentrations of thymi- thymidine does affect Huh7 cell confluency (data not shown), dine (1 mM) . Under these conditions, DNA synthesis is lim- we also tested the HCV replicon in the presence of thymidine . ited, so there should be no production and incorporation of Huh7 cells bearing the 1bN replicon were grown in the presthe toxic metabolite of AZA, 6-thioguanosine triphosphate, ence of 1 mM thymidine with no other drug, AZA, or ribaviinto cellular DNA (10) (Fig . 3) and therefore no cytotoxicity. rin . After 72 hours, total RNA was isolated, and equivalent The viral suppression (Fig . 2) still occurred, even though, amounts were assayed by real-time RT-PCR with probes to under these conditions, AZA caused no change in host cell the viral 5' untranslated region and a cellular housekeeping growth . Because only a small minority of hepatocytes in a gene to normalize the results . AZA reliably produced almost diseased liver (and even smaller minority in a normal liver) a one-cycle increase in the number of cycles required to reach are actively dividing (19), this cellular state of high thymi- the critical threshold (C t ) compared with no drug, which dine causing low cell turnover may mimic liver tissue better corresponds to only approximately 50% as much replicon in than rapidly dividing cells in a tissue culture dish . cells exposed to AZA (Fig . 5) . Although this change was small compared with the effect of AZA on BVDV, it was of similar 6-Mercaptopurine, but not the Downstream Metabolite 6magnitude or larger than the change on the HCV replicon Thioguanine, is the Likely Mediator of Azathioprine's because of equimolar amounts of ribavirin . Antiviral Effect To begin to determine the mechanism of the antiviral effect DISCUSSION of AZA, we examined whether any of the metabolites of AZA (Fig. 3) also had antiviral activity . 6-methyl mercaptopurine, AZA has been used in liver transplantation for more than 6-mercaptopurine, and 6-thioguanine were each individually 30 years . It is a prodrug that is converted to 6-mercaptopuadded to MDBK cells with or without BVDV . 6-methyl mer- rine and eventually into 6-thioinosine and 6-thioguanosine captopurine had no effect on cell growth or viral yield, triphosphate (Fig . 3) . The triphosphate of 6-thioguanosine is whereas both 6-mercaptopurine and 6-thioguanine decreased converted to deoxy6-thioguanosine and incorporated into celcell growth and viral yield (data not shown) . To isolate the lular DNA (21) . The thioribonucleotides are also available for antiviral effect, cells and virus were grown in the presence of inhibition or incorporation by viral enzymes including the thymidine with either 6-mercaptopurine or 6-thioguanine RNA polymerase . Therefore, AZA could share some proposed (Fig . 4) . In the presence of thymidine, neither metabolite had mechanisms with ribavirin that are dependent upon both 1000 --AZA inhibition of RNA viral replication is more profound than the inhibition of cell growth. MDBK cells were grown in varying concentrations of AZA with (dashed lines) and without (solid lines) virus and with (open symbols) and without (closed symbols) thymidine (T) . After 72 hours, host cells were counted by flow cytometry, and virus was titered by plaque assay . With increasing AZA, viral replication is inhibited 10- to 100-fold more than cell growth . In the presence of thymidine, no decrease in cell survival was seen, but the majority of the antiviral effect was maintained. This shows the antiviral effect of AZA occurs in the absence of cell death . FIGURE 2 . w ° L 100 ° ~~ v 10 - -k-cells - --virus cells with T o-- virus with T \~ - y ~~ V ~~ `o 0 0 10 20 30 AZATHIOPRINE (uM) 40 50 February 27, 2004 STANGL ET AL . H,C \ N~\\ Azathioprine NO, NH N S N ~ ~~ H H N_:'~ 6-mercaptopurine ~l 6-thioguanine 6-methyl mercaptopurine MeMPR ~- MPR time MetIMP MetlOP MetITP TGR tGMP ~, tIDP ttrP tGDP tGTP tdGTP_ . DNA Thiopurine metabolism . AZA is a prodrug that is metabolized to 6-mercaptopurine . 6-mercaptopurine is converted to 6-methyl mercaptopurine (MeMP) or 6-mercaptopurive riboside (MPR) . In turn, MPR is converted to mono-, di-, and triphosphate derivatives of thioinosine (tIM(D,T)P), methyl thioinosine (MetIM(D,T)P), and thioguanosine (tGM(D,T)P) . Any of these metabolites could be responsible for the antiviral effect, and any triphosphate could potentially be incorporated into the viral genome . Only 6-thioguanosine is processed by ribonucleotide reductase into a deoxyribonucleotide (tdGTP) and incorporated into cellular DNA, and this step is blocked by 1 mM thymidine. Incorporation of above thiopurine nucleotides into cellular RNA has not been observed (10) . Metabolites of AZA decrease purine synthesis through effects on glutamine-5-phosphoribosylpyrophosphate amidotransferase, whereas both mycophenolate acid (MPA) and ribavirin decrease GTP synthesis by inhibiting inosine monophosphate dehydrogenase . FIGURE 3. drugs being triphosphorylated and recognized by the viral replication machinery . The effect of ribavirin and many other antivirals as monotherapy is quite small and, at least in the case of HIV, the effect of nucleoside analogs is quickly obscured by the selection of viral resistance . In certain situations, the selection of less-fit HIV viruses with mutant polymerases can be clinically preferable to wild-type virulent HIV (22) . A similar detailed understanding regarding the selection of mutant HCV viruses is lacking even though the HCV polymerase is the likely target of ribavirin and possibly an indirect target of other antimetabolite drugs, including IMPDH inhibitors . 565 Ribavirin monophosphate is a competitive inhibitor of IMPDH and is further phosphorylated to a triphosphate nucleotide analog which, at least in vitro, is a substrate for the HCV genotype lb polymerase (23) . Metabolites of AZA also decrease purine synthesis through a different mechanism (inhibition of glutamine-5-phosphoribosylpyrophosphate aminotransferase) (21), whereas MPA and ribavirin both inhibit guanosine triphosphate (GTP) synthesis by inhibiting IMPDH . Because 6-mercaptopurine has an antiviral effect and 6-thioguanine does not, but both decrease purine synthesis, the indirect effects of AZA on purine synthesis do not seem to be sufficient for the antiviral activity . These results are consistent with two recent studies that show ribavirin has weak antiviral (hepatitis GB) (24) and anti-HCV replicon activity (25), but MPA has none . Furthermore, MPA has been shown to lack clinically significant antiviral activity as monotherapy in nontransplant HCV patients (26) . Ours is the first study to demonstrate antiviral activity of AZA and compare its magnitude with that of MPA and ribavirin . Mycophenolate mofetil, the prodrug of MPA, clearly decreases the risk of rejection relative to AZA in clinical trials (11, 12) . Yet, even in the face of more rejection, AZA has been associated with a variable amount of HCV recurrence . One study showed less recurrence with an AZA-containing regimen versus a non-AZA-containing regimen (13), whereas another found more HCV recurrence in patients with higher doses of AZA and corticosteroids (14) . Meanwhile, mycophenolate mofetil has also been associated with less HCV recurrence than AZA (11), no benefit compared with a regimen without mycophenolate mofetil or AZA (12), or an increased risk of graft failure (15) . Given the molecular effect demonstrated in this report, several issues need to be considered that might explain the disparities in clinical trials . First, the degree of sensitivity to AZA may depend on the specific strain of HCV . Second, immunosuppressive regimens in transplantation are rapidly evolving multicomponent cocktails that are difficult to compare between, and sometimes within, one study population . Third, thiopurine metabolism differs between patients because of polymorphisms in thiopurine methyltransferase and possibly other loci, which may lead to variability in thiopuring levels after drug administration . Bergan et al . (27) showed significant variability in 6-mercaptopurine plasma levels after oral AZA dosing in renal-transplant patients, with plasma concentrations up to approximately 1 µM . Although this represents the lower range of 6-mercaptopurineused in our study, previous data indicates that thiopurine efficacy correlates more with intracellular nucleotide concentrations as opposed to plasma levels (28) . Furthermore, studies in mice suggest that hepatic tissue levels of thiopurine metabolites are approximately fourfold greater than plasma levels (29) . In conclusion, we present evidence that the antiviral effect of AZA is mediated by a thioinosine metabolite, perhaps by being incorporated into the viral genome, as has been suggested to occur with ribavirin . If it is incorporated, the antiviral effect could be mediated either through the induction of mutations (5) or by altering RNA structure (18), which, in turn, may affect enzyme processivity, ribosome translation, or other properties of an RNA genome . Whether this will prove to be clinically useful depends in part upon how large the antiviral effect is relative to its immunosuppressive effect 566 Vol. 77, No . 4 TRANSPLANTATION 1000 - ------------- 100 g a o \ \ -~ 6MP cells _ -a-6TG cells \ u o o ° o \ 10- \ \ --0--6MP virus \ --0--6TG virus \ \ O --------------------- 0 1 1 0 10 20 30 40 50 Drug (uM) 4. Antiviral effect of AZA is mediated through 6-mercaptopurine but not 6-thioguanine . Viral plaque-forming units and cell numbers were determined as a function of drug equivalent thiopurines . The addition of 6-mercaptopurine (open circles, dashed line) significantly reduced the amount of virus produced in a dose-dependent manner, but 6-thioguanine (open squares, dashed line) did not . Their effects on the cells, however, are similar . FIGURE and obviously requires validation in more clinical trials . Case AZA causes the overall effect to be proviral, the antiviral reports exist of patients with both autoimmune hepatitis and effect may still select for specific viral mutants that are hepatitis C treated with AZA . However, the effect of AZA on synergistic (or antagonistic) to other antivirals . With this in HCV viral load in this setting is uncertain and is potentially mind, AZA's role in liver transplantation should be reevaluconfounded by the concomitant use of corticosteroids (30) . ated to determine whether it affects viral load or selects for Theoretically, even if the immunosuppressive property of AZA-resistant HCV . In a broader sense, the studies described here indicate an important line of future inquiry into the effects of HCV and other viruses on allograft integrity . They .4 1 show that with appropriate models, it should be possible to parse the conflicting actions of immunosuppressants on viro7 .2 logic behavior through their direct actions, without the confounding effects of altered adaptive immunity . Acknowledgments. The authors thank Ron Schultz for providing 0 .8 guidance on BVDV free serum and viral stocks and Michael Lucey AACt for helpful discussions . 0 .6 REFERENCES 0 .4 0 .2 . 0 -AZA RIB 5. Antiviral effect of AZA is equivalent or larger than equimolar amounts of ribavirin on hepatitis C virus (HCV) 1bN replicon RNA level . Cells were grown in the presence of thymidine alone, thymidine+100 .sM ribavirin, or thymidine + 100 µM AZA . The amount of HCV replicon as well as cellular glyceraldehyde-3-phosphate dehydrogeFIGURE nase (GADPH) mRNA was quantitated by reverse-transcriptase polymerise chain reaction (PCR) . OCt is the difference in critical PCR threshold (HCVCt-GADPHCt ) . DOCt is the difference of ACt with drug minus ACt without drug . Therefore, larger AACt represents more inhibition of the HCV replicon. 1 . Berenguer M. Natural history of recurrent hepatitis C . Liver Transpl 2002 ; 8(10 Suppl 1) : S14 . 2 . Forman LM, Lewis JD, Berlin JA, et al . The association between hepatitis C infection and survival after orthotopic liver transplantation. Gastroenterology 2002 ; 122(4) : 889 . 3 . Gane EJ, Naoumov NV, Qian KP, et al . A longitudinal analysis of hepatitis C virus replication following liver transplantation . Gastroenterology 1996 ; 110(1) : 167 . 4 . Everson GT . Impact of immunosuppressive therapy on recurrence of hepatitis C . Liver Transpl 2002 ; 8(10 Suppl 1) : S19 . 5. Ikeda M, Yi M, Li K, et al . Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. J Virol 2002 ; 76(6) : 2997 . 6. McHutchison JG, Gordon SC, Schiff, ER et al . Interferon alfa-2b alone or Cn combination with rib avirin a lG treatment roup . N for chronic Med 1 998 ; C. Interventional Therapy EngI J 1998 ; 339(21) :1485 . 7 . Crotty S, Maag D, Arnold JJ, et al . The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen . Nat Med 2000 ; 6(12): 1375 . February 27, 2004 STANGL ET AL . 8 . Willis RC, Carson DA, Seegmiller JE . Adenosine kinase initiates the major route of ribavirin activation in a cultured human cell line . Proc Natl Acad Sci U S A 1978 ; 75(7) : 3042 . 9 . Markland W, McQuaid TJ, Jain J, et al . Broad-spectrum antiviral activity of the IMP dehydrogenase inhibitor VX-497 : a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon . Antimicrob Agents Chemother 2000 ; 44(4) : 859 . 10 . Nelson JA, Carpenter JW, Rose LM, et al . Mechanisms of action of 6-thioguanine, 6-mercaptopurine, and 8-azaguanine . Cancer Res 1975 ; 35(10) : 2872 . 11 . Wiesner R, Rabkin J, Klintmalm G, et al . A randomized double-blind comparative study of mycophenolate mofetil and azathioprine in combination with cyclosporine and corticosteroids in primary liver transplant recipients . Liver Transpl 2001; 7(5) : 442 . 12 . Jain A, Kashyap R, Demetris AJ, et al . A prospective randomized trial of mycophenolate mofetil in liver transplant recipients with hepatitis C . Liver Transpl 2002 ; 8(1): 40 . 13 . Hunt J, Gordon FD, Lewis WD, et al . Histological recurrence and progression of hepatitis C after orthotopic liver transplantation : influence of immunosuppressive regimens . Liver Transpl 2001 ; 7(12) : 1056 . 14 . Berenguer M, Prieto M, Cordoba J, et al . Early development of chronic active hepatitis in recurrent hepatitis C virus infection after liver transplantation : association with treatment of rejection . J Hepatol 1998 ; 28(5) : 756 . 15 . Burak KW, Kremers WK, Batts KP, et al . Impact of cytomegalovirus infection, year of transplantation, and donor age on outcomes after liver transplantation for hepatitis C . Liver Transpl 2002 ; 8(4) : 362 . 16 . Becher P, Orlich M, Konig M, et al . Nonhomologous RNA recombination in bovine viral diarrhea virus : molecular characterization of a variety of subgenomic RNAs isolated during an outbreak of fatal mucosal disease . J Virol 1999 ; 73(7) : 5646 . 17 . Cheney IW, Naim S, Lai VC, et al . Mutations in NS5B polymerase of hepatitis C Virus : Impacts on in vitro enzymatic activity and viral rna replication in the subgenomic replicon cell culture . Virology 2002 ; 297(2) : 298 . 18 . Stuyver LJ, Whitaker T, McBrayer TR, et al . Ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis C viruses in 567 culture . Antimicrob Agents Chemother 2003 ; 47(1) : 244. 19 . Negro F, Papotti M, Taraglio S, et al . Relationship between hepatocyte proliferation and hepatitis delta virus replication in neoplastic and non-neoplastic liver tissues . J Viral Hepat 1997 ; 4(2): 93. 20 . Gun JT, Bichko VV, Seeger C . Effect of alpha interferon on the hepatitis C virus replicon . J Virol 2001 ; 75(18) : 8516 . 21 . Van Scoik KG, Johnson CA, Porter WR . The pharmacology and metabolism of the thiopurine drugs 6-mercaptopurine and azathioprine . Drug Metab Rev 1985 ; 16(1-2) : 157 . 22 . Loveday C . International perspectives on antiretroviral resistance . Nucleoside reverse transcriptase inhibitor resistance . J Acquir Immune Defic Syndr 2001; 26(Suppl 1): S10 . 23 . Maag D, Castro C, Hong Z, et al . Hepatitis C virus RNA-dependent RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin . J Biol Chem 2001; 276(49) : 46094 . 24 . Lanford RE, Chavez D, Guerra B, et al . Ribavirin induces error-prone replication of GB virus B in primary tamarin hepatocytes . J Virol 2001 ; 75(17) : 8074. 25 . Zhou S, Liu R, Baroudy BM, et al . The effect of ribavirin and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA. Virology 2003 ; 310(2): 333 . 26 . Firpi RJ, Nelson DR, Davis GL . Lack of antiviral effect of a short course of mycophenolate mofetil in patients with chronic hepatitis C virus infection . Liver Transpl 2003 ; 9(1) : 57 . 27 . Bergan S, Rugstad HE, Bentdal 0, et al . Kinetics of mercaptopurine and thioguanine nucleotides in renal transplant recipients during azathioprine treatment. Ther Drug Monit 1994; 16(1) : 13 . 28 . Rundles RW, Elion GB . Mercaptopurine "bioavailability". N Engl J Med 1984 ; 310(14) : 929 . 29 . Gunnarsdottir S, and Elfarra AA. Distinct tissue distribution of metabolites of the novel glutathione-activated thiopurine prodrugs cis-6-(2acetylvinylthio)purine and trans-6-(2-acetylvinylthio)guanine and 6-thioguanine in the mouse . Drug Metab Dispos 2003 ; 31(6): 718 . 30 . Bellary S, Schiano T, Hartman G, et al . Chronic hepatitis with combined features of autoimmune chronic hepatitis and chronic hepatitis C : favorable response to prednisone and azathioprine . Ann Intern Med 1995 ; 123(1): 32.