ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1991, p. 2431-2433

Vol. 35, No. 11

0066-4804/91/112431-03$02.00/0 Copyright © 1991, American Society for Microbiology

Sulfated Polyanions Do Not Inhibit Duck Hepatitis B Virus Infection WOLF-BERNHARD OFFENSPERGER,l* SILKE OFFENSPERGER,l EIKE WALTER,' HUBERT E. BLUM,2 AND WOLFGANG GEROK' Department of Medicine, University of Freiburg, 7800 Freiburg, Germany,' and Gastrointestinal Unit and Molecular Hepatology, MGH Cancer Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 021292 Received 30 July 1991/Accepted 13 September 1991

On the basis of the antiviral action of sulfated polyanions in human immunodeficiency virus and other viral infections, we studied the effect of dextran sulfate and heparin on duck hepatitis B virus infection. These agents do not affect viral uptake and replication in liver cells in vitro or in vivo. Sulfated polyanions, therefore, appear to have no potential for the treatment of hepadnavirus infections.

ability of the in vitro and in vivo models to study DHBV infection, the data demonstrate that chloroquine, a lysosomotropic agent, blocks DHBV uptake into primary hepatocytes but not viral replication in already infected liver cells (lanes 2 and 4). By comparison, phosphonoformate, a known inhibitor of HBV and DHBV DNA polymerase and reverse transcriptase, efficiently blocks viral replication in hepatitis naive duck hepatocytes infected in vitro (Fig. 1B, lane 2), and in already infected hepatocytes (Fig. 1B, lane 4). These data demonstrate that primary duck hepatocyte cultures can serve as a useful in vitro system for the study of antiviral agents targeted to different stages of the life cycle of DHBV. The in vitro model of DHBV infection was extended to young ducklings, which can be efficiently infected by DHBV-positive serum. In this in vivo model, the application of suramin, a known inhibitor of viral uptake (20), results in an almost complete suppression of DHBV infection (data not illustrated), demonstrating the suitability of this system for the in vivo assessment of antiviral strategies. By using the model systems described above, the effect of sulfated polyanions on DHBV infection was first assessed in vitro. As shown in Fig. 2 for noninfected primary duck hepatocytes, dextran sulfate and heparin do not inhibit DHBV infection at concentrations up to 500 ,ug/ml. Similarly, in primary duck hepatocytes isolated from DHBVinfected ducks, viral replication was not affected by either drug. Also, polycations, such as poly-L-lysine (8 ,ug/ml) and DEAE-dextran (12 pLg/ml), were without effect on viral uptake (data not illustrated). For in vivo analyses, DHBV-negative ducklings were infected by DHBV in the presence of dextran sulfate or heparin. Dextran sulfate and heparin were given intravenously twice daily, starting 2 days after hatching at a dose of 50 p,g/g of body weight. One day later, the ducklings were infected by intravenous injection of 100 ,ul of DHBV DNApositive serum (approximately 1010 virions per ml), followed by the application of the polyanions until day 14, when the ducks were sacrificed. As shown in Fig. 3, dextran sulfate and heparin do not inhibit DHBV infection at a dose approximately corresponding to the maximal oral dosage given for treatment of AIDS patients (1). Sulfated polyanions are pivotal in many forms of cell recognition and adhesion (8). Their antiviral effect has been demonstrated for herpes simplex virus (18, 22, 26), cytomegalovirus, vesicular stomatitis virus, Sindbis virus, arenavi-

Dextran sulfate, heparin, and other sulfated polysaccharides have been shown to be potent antiviral agents (5, 11, 25). They primarily act via inhibition of viral absorption to target cells, such as the binding of the human immunodeficiency virus (HIV) to CD4-positive cells (4, 16). In addition, inhibitory effects on HIV replication, mediated through interference with RNase H, have been described (17). Sulfated polyanions are widely used as antilipemic agents and anticoagulants. Newer sulfated polysaccharides with marked antiviral and reduced antithrombin activity have been developed (3). Duck hepatitis B virus (DHBV) belongs to a group of hepatotropic DNA viruses (hepadnaviruses) which includes the human hepatitis B virus, the woodchuck hepatitis virus, the ground and tree squirrel hepatitis viruses, and the heron hepatitis B virus. Because of their close relatedness to retroviruses and because an effective therapy for hepadnavirus infections is not available to date, we studied the effect of sulfated polyanions on DHBV infection in vitro and in vivo.

Primary duck hepatocytes and ducklings were used as in vitro and in vivo models, respectively, of DHBV infection. Hepatocytes were isolated from DHBV-infected and uninfected ducks as described previously (19, 23). Hepatocytes from uninfected ducks were infected in vitro by the addition of 30 ,l of DHBV DNA-positive serum to a plate containing 2.5 ml of medium and 2 x 106 cells. After 2 h at 37°C, the cells were washed and incubated with fresh medium. Uninfected hepatocytes were routinely preincubated with the agent to be studied at day 2 after plating and infected with DHBV DNA-positive serum in the presence of the drug 24 h later. Cell viability was determined by trypan blue exclusion. As shown in Fig. 1A, primary duck hepatocytes can be efficiently infected in vitro (lane 1). Also, in vivo-infected hepatocytes can be kept in culture over prolonged periods of time (lane 3). The DHBV DNA hybridization pattern observed reflects replicative intermediates with the most prominent species in the 1.3-kbp position, representing full-length minus-strand molecules, and in the 3.0-kbp position, representing full-length virion DNA; the intermediate band at about 1.8 kbp represents covalently closed circular (supercoiled) DHBV DNA. In addition to demonstrating the suit*

Corresponding author. 2431

2432 NOTES 2432 NOTES AGENTS CHEMOTHER. ~~~~~~~~~~~~~~~ANTIMICROB. A 1 2

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FIG. 1. (A) Effect of chioroquine on DHBV infection and replication in vitro. Southern blot analysis of DNA isolated from primary duck hepatocytes after 2 weeks in culture was carried out as described earlier (19). Lanes 1 and 2, primary hepatocytes isolated from DHBV DNA-negative ducks, infected with DHBV DNApositive serum; lanes 3 and 4, primary hepatocytes isolated from DHBV DNA-positive ducks. Lanes 1 and 3, no chloroquine added; lanes 2 and 4, 50 p.M chloroquine added. (B) Effect of phosphonoformate on DHBV infection and replication in vitro. Lanes 1 and 2, primary hepatocytes isolated from DHBV DNA-negative ducks, infected with DHBV DNA-positive serum; lanes 3 and 4, primary hepatocytes isolated from DHBV DNA-positive ducks. Lanes 1 and 3, no phosphonoformate added; lanes 2 and 4, 500 p.g of phosphonoformate per ml added. Size markers are HindIII-digested lambda DNA and 10 pg of DHBV DNA. Autoradiographic exposure time was 2 days at -800C.

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FIG. 3. Effect of dextran sulfate and heparin on DHBV infection in vivo. Southern blot analysis of DNA isolated from duck liver was performed as described earlier (19). Sample sources: lanes 1 and 2, two control ducklings; lanes 3 and 4, two ducklings treated with dextran sulfate (50 p.g/g of body weight); lanes 5 and 6, two ducklings treated with heparin (50 p.g/g of body weight). For further experimental details, see the legend to Fig. 1. Molecular size markers are shown to the left in kilobase pairs.

ruses (2, 5), and HIV (11, 25). Their primary mode of action appears to be an inhibition of viral attachment and penetration (18). For HIV it has been shown that sulfated polysaccharides inhibit gpl2O binding by interacting with sites on the CD4 receptor (13). Inhibitory mechanisms of sulfated polyanions have further been shown to affect steps subsequent to viral uptake (17). In addition to these direct antiviral effects, sulfated polyanions are known to induce interferon, thereby stimulating the host's defense mechanisms (15). Ducklings and primary duck hepatocytes have been shown to be useful models for in vivo and in vitro testing of strategies aimed at termination of DHBV infection. Using these systems, nucleoside analogs (6, 7, 9, 14, 24, 27), phosphonoformate (9), the reverse transcriptase inhibitor HPA.23 (7), and supercoiled DNA-active and DNA-binding compounds (7) were shown to inhibit DHBV replication. Furthermore, suramin was shown to block DHBV uptake into primary hepatocytes but not viral replication (20). In vivo studies demonstrated a marked but transient inhibition of DHBV replication by nucleoside analogs (10, 12) and

phosphonoformate (21). On the basis of similarities between retroviruses and on DHBV infection was evaluated in vitro and in vivo. In contrast to the positive findings for the viruses mentioned above, most notably HIV, dextran sulfate and heparin do not affect DHBV uptake into liver cells or viral replication. This difference is possibly due to a different mode of entry of these viruses into target cells. Sulfated polyanions, there-

hepadnaviruses, the effect of sulfated polyanions

FIG. 2. Effect of dextran sulfate (A) and heparin (B) on DHBV infection in vitro. Primary hepatocytes were isolated from DHBV DNA-negative ducks, followed by infection with DHBV DNApositive serum. Lane 1, no drug added; lanes 2 to 4, 100 (lane 2), 250 (lane 3), and 500 (lane 4) p.g of the respective drug added per ml. For further experimental details, see the legend to Fig. 1.

VOL. 35, 1991

fore, hold little promise for the treatment of hepadnavirus infections. The study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 154 TP-A2 and TP-A5) and a Hermann-andLilly-Schilling professorship from the Stifterverband dur die deutsche Wissenschaft to H.E.B. The expert technical assistance of Birgit Hockenjos is gratefully acknowledged. REFERENCES 1. Abrams, D. I., S. Kuno, R. Wong, K. Jeffords, M. Nash, J. B. Molaghan, R. Gorter, and R. Ueno. 1989. Oral dextran sulfate (UA 001) in the treatment of the acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. Ann. Intern. Med. 110:183-188. 2. Andrei, G., and E. De Clercq. 1990. Inhibitory effect of selected antiviral compounds on arenavirus replication in vitro. Antiviral Res. 14:287-300. 3. Baba, M., E. De Clercq, D. Schols, R. Pauwels, R. Snoeck, C. van Boeckel, G. van Dedem, N. KraaUeveld, P. Hobbelen, H. Ottenheim, and F. Den Holiander. 1990. Novel sulfated polysaccharides: dissociation of anti-human immunodeficiency

virus activity from antithrombin activity. J Infect. Dis. 161:208213. 4. Baba, M., R. Pauwels, J. Balzarini, J. Arnout, J. Desmyter, and E. De Clercq. 1988. Mechanism of inhibitory effect of dextran sulfate and heparin on replication of human immunodeficiency

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11. Ito, M., M. Baba, A. Sato, R. Pauwels, E. De Clercq, and S. Shigeta. 1987. Inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus (HIV) in vitro. Antiviral Res. 7:361-367. 12. Kassianides, C., J. H. Hoofnagle, R. H. Miller, E. Doo, H. Ford, S. Broder, and H. Mitsuya. 1989. Inhibition of duck hepatitis B virus replication by 2',3'-dideoxycytidine. Gastroenterology 97: 1275-1280. 13. Lederman, S., R. Gulick, and L. Chess. 1989. Dextran sulfate and heparin interact with CD4 molecules to inhibit the binding of coat protein (gpl20) of HIV. J. Immunol. 143:1149-1154. 14. Lee, B., W. Luo, S. Suzuki, M. J. Robins, and D. L. J. Tyrrell. 1989. In vitro and in vivo comparison of the abilities of purine and pyrimidine 2',3'-dideoxynucleosides to inhibit duck hepadnavirus. Antimicrob. Agents Chemother. 33:336-339. 15. Merigan, C., and M. S. Finkelstein. 1968. Interferon-stimulating and in vivo antiviral effects of various synthetic anionic polymers. Virology 35:363-374. 16. Mitsuya, H., D. J. Looney, S. Kuno, R. Ueno, F. Wong-Staal, and S. Broder. 1988. Dextran sulfate suppression of viruses in the HIV family: inhibition of virion binding to CD4+ cells. Science 240:646-649. 17. Moelling, K., T. Schulze, and H. Diringer. 1989. Inhibition of human immunodeficiency virus type 1 RNase H by sulfated polyanions. J. Virol. 63:5489-5491. 18. Nahmias, A. J., and S. Kibrick. 1964. Inhibitory effect of heparin on herpes simplex virus. J. Bacteriol. 87:1060-1066. 19. Offensperger, W.-B., S. Offensperger, E. Walter, H. E. Blum, and W. Gerok. 1991. Inhibition of duck hepatitis B virus infection by lysosomotropic agents. Virology 183:415-418. 20. Petcu, D. J., C. E. Aldrich, L. Coates, J. M. Taylor, and W. S. Mason. 1988. Suramin inhibits in vitro infection by duck hepatitis B virus, Rous sarcoma virus, and hepatitis delta virus. Virology 167:385-392. 21. Sherker, A. H., K. Hirota, M. Omata, and K. Okuda. 1986. Foscarnet decreases serum and liver duck hepatitis B virus DNA in chronically infected ducks. Gastroenterology 91:818824. 22. Takemoto, K. K., and P. Fabisch. 1964. Inhibition of herpes virus by natural and synthetic acid polysaccharides. Proc. Natl. Acad. Sci. USA 116:140-144. 23. Tuttleman, J. S., J. C. Pugh, and J. W. Summers. 1986. In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus. J. Virol. 58:17-25. 24. Tyrrell, D. L. J. 1988. Inhibition of duck hepatitis B virus replication by purine 2',3'-dideoxynucleosides. Biochem. Biophys. Res. Commun. 156:1144-1151. 25. Ueno, R., and S. Kuno. 1987. Dextran sulfate, a potent anti-HIV agent in vitro having synergism with zidovudine. Lancet ii:1379. 26. Vaheri, A., and C. Cantell. 1963. The effect of heparin on herpes simplex virus. Virology 21:661-662. 27. Yokoto, T., K. Konno, E. Chonan, S. Mochizuku, K. Kojima, S. Shigeta, and E. De Clercq. 1990. Comparative activities of several nucleoside analogs against duck hepatitis B virus in vitro. Antimicrob. Agents Chemother. 34:326-330.

Sulfated polyanions do not inhibit duck hepatitis B virus infection.

On the basis of the antiviral action of sulfated polyanions in human immunodeficiency virus and other viral infections, we studied the effect of dextr...
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