Dermatology Online Journal

Quinine sulfate and HSV replication
Ronni Wolf¹, Adone Baroni², Rita Greco³, Federica Corrado³, Eleonora Ruocco², Maria Antonietta Tufano³, and Vincenzo Ruocco²
Dermatology Online Journal 9(3): 3

1. Dermatology Unit, Kaplan Medical Center, Rechovot, 2. Department of Dermatology, Faculty of Medicine and Surgery, Second University of Naples 3. Department of Experimental Medicine, Microbiology Section, Faculty of Medicine and Surgery, Second University of Naples. MariaAn.tufano@unina2.it

---

Abstract

Although antimalarial drugs have been developed primarily to treat malaria, they are also beneficial for many dermatological, immunological, and rheumatological diseases, for which they are mostly used today in the Western world. The aim of the present study was to investigate the effect of quinine sulfate (QS) on the multiplication and adsorption of herpes virus type I (HSV-1). When Vero cells (African green monkey kidney) are infected with HSV-1 in the presence of QS, the viral adsorption is reduced, as demonstrated by a decrease of the number of microscopic plaques of the virus. When the virus-infected Vero cells are incubated in the presence of QS, the multiplication of HSV-1 is also reduced, and the diameter of the plaque are visibly smaller. The practical implications of the antiviral action of antimalarial drugs might be especially important to immunosuppressed patients who receive these drugs for autoimmune collagen-vascular diseases or as additional therapy for AIDS.

---

Introduction

Antimalarial drugs were first discovered in the seventeenth century, two and one-half centuries before the causative agent of malaria was identified.[1] Quinine, the first antimalarial agent, was derived from the cinchona tree. Quinine played a unique role in shaping today's world by enabling explorers and colonists from Europe to survive in tropical countries and to build their colonial empires despite the presence of lethal tropical malaria. When physicians were asked in 1945 to choose the ten most important drugs used in medicine, quinine and quinacrine were preceded in importance only by penicillin, sulfonamides, and blood derivatives.[2] Although antimalarial drugs have been developed primarily to treat malaria (and have never lost their place in treating this life-threatening disease that is still a major cause of infant death in the tropics), they are also beneficial for many dermatological, immunological, and rheumatological diseases, conditions for which they are used today mostly in the Western world.[3, 4]

In the beginning of the 1970s, researchers became interested in another property of antimalarial drugs, their antiviral activity. Thus ensued an evaluation of the affect of these substances on viruses of various structures and modes of replication. [5, 6, 7, 8, 9, 10, 11, 12, 13, 14]

The search for viruses that could be affected by antimalarials has continued over the years, and has resulted in an ever-growing list in which replication, activity, or infectivity are inhibited by these drugs. Antimalarials are found to affect vesicular-stomatitis virus[15, 16, 17], foot-and-mouth disease[18], yellow fever virus[19], varicella-zoster virus[20], Newcastle disease virus[21], African swine fever virus[22], Flavivirus[23], canine parvovirus[24], Epstein-Barr virus (EBV)[25], lymphocytic choriomeningitis virus[26, 27], Semliki Forest virus[28], bovine leukemia virus[29], feline calicivirus[30], infectious salmon anemia virus[31], rubella virus[32], hepatitis A virus[33], hepatitis B virus[34, 35, 36], rabies virus[37], Ebola virus[38], Borna disease virus[39], and porcine reproductive and respiratory syndrome virus.[40, 41] Some other viruses, such as poliovirus, are unaffected by antimalarials.[42, 43, 44]

The effect of antimalarials on herpes virus (HSV) is among the first to be studied. In 1971, Greenham and Poste show that lysosomal activation is a consistent feature of cell fusion induced by a range of viruses, including HSV.[10, 11] Lysosome-stabilizing agents (among them chloroquine) significantly reduce cell fusion without impairing virus replication. In contrast, Lancz et al. find that HSV replication is inhibited by chloroquine.[45] The higher concentrations of chloroquine in their experiment (using 60 µg/ml compared to 30 µg/ml in the earlier experiment) might be one explanation for the difference in the results. The findings of Lancz et al. are confirmed 2 years later by other authors, who also perform an in vivo experiment inoculating mice with HSV-1 and HSV-2 and find the course of infection to be unaltered by chloroquine treatment.[46]

Other investigators are able to demonstrate that 100 µM chloroquine inhibits multiplication of HSV-1 cultured in Vero cells in the late stage of infection, a result that indicates that the site of inhibition is in one of the steps within the maturation process of the virus.[47, 48] This inhibition is not attributed to any interference with the uncoating process. The drug also inhibits the syncytium formation induced by HSV-1 but does not affect the cell-to-cell spread of infection.

In the present study we investigate the effect of quinine sulfate (QS) on HSV-1 multiplication and adsorption.

Materials and methods

Cells and culture conditions

Vero cells (African green monkey kidney) were grown in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10-percent heat-inactivated fetal calf serum (FCS), in flask 25 cm² at 37° C in a 5-percent CO₂ incubator. Cells were grown to confluence for 3 days.

Chemicals

QS (molecular weight 783 kDa) was dissolved in a 1:1 mixture of ethanol and water.

Toxicity of QS on cultured cells

The cells that had propagated in tissue-culture clusters were incubated with different concentrations of QS (0, 1, 10, 25, 50, and 100 µM) at 37° C for 24, 48, and 72 hours in RPMI medium. The cell monolayers were then washed with phosphate-buffered saline (PBS) and stained with Giemsa diluted 1:20.

Virus preparation and infections

HSV-1 was propagated in vitro by infecting Vero cells at a multiplicity of infection (MOI) of 10.

Cells were incubated in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 percent FCS. At about 3 days post-infection, the virus was harvested by sonication and clarified by centrifugation.

A virus stock solution containing approximately 10⁹ plaque-forming units (PFU)/ml was used in all experiments.

Monolayer Vero cells were cultured in a multiwell plate containing 12 wells and washed with Dulbecco's PBS. Then 0.2 ml virus dilutions with or without QS were added at concentrations of 1 µM and 10 µM, at a MOI of 0.1 and 0.01. After incubating the multiwell plate for 1 hour at 37° C, the inocula were removed, and the infected monolayers were incubated for additional 72 hours in RPMI medium with carboxymethyl cellulose (CMC) (ratio1:1) with or without QS.

The cells were fixed and stained with 1 percent crystal violet dissolved in a 10 percent formalin solution, and the number of microscopic plaques was counted under a microscope.

Results

QS is cytocidal at concentrations of 25, 50, and 100 µM, but not at concentrations of 1 µM and 10 µM. The cells that are incubated for 24, 48, and 72 hours at these lower concentrations are viable. Moreover, QS at concentrations of 1 µM and 10 µM does not show any virucidal activity.

When the cells are infected with HSV-1 in the presence of QS, the viral adsorption is reduced as demonstrated by a decrease of the number of microscopic plaques of the virus, expressed as PFUs (Table I).

Table 1

Effect of quinine sulfate (QS) on adsorption and replication of herpes simplex virus type 1 (HSV-1) in Vero cells
Condition of incubation with QS	MOI	HSV-1	HSV-1 with QS 1mM	% of decrease of PFU	HSV-1 with QS 10 mM	% of decrease of PFU
Preadsorption	0.1	780	432	45	108	86
Pre- and postadsorption	0.1	700	320	54	192	72.5
Preadsorption	0.01	476	128	73	90	81
Pre- and postadsorption	0.01	432	52	88	40	90
Results are expressed as plaques forming units (PFU)/ml. MOI = multiplicity of infection.

When the virus-infected Vero cells are incubated in the presence of QS the multiplication of HSV-1 is also reduced and the diameters of the plaques are visibly smaller (Fig. 1).

Figure 1

Discussion

We are able to show that QS reduces adsorption and multiplication of HSV-1 at very low concentrations (1 µM and 10 µM), levels more than ten-fold lower than the 50-percent inhibitory concentration of the drug (IC₅₀), as well as lower than concentrations of chloroquine used on viruses in previously reported experiments by others.[49, 50, 51]

Apart from the theoretical considerations of our findings, they might also have practical implications in illuminating the antiviral action of antimalarial drugs, especially in regard to immunosupressed patients who receive these drugs for autoimmune-collage-vascular diseases, or as additional therapy for AIDS.

References

1. Isaacson D, Elgart M, Turner M. Antimalarials in dermatology. Int J Dermatol. 1982;21:379-395.

2. Wallace D. The history of antimalarials. Lupus. 1996;5 (Suppl. 1):S2-S3.

3. Greenwood B, Greenwood A, Bradley A, Tulloch S, Hayes R, Oldfield F. Deaths in infancy and early childhood in a well-vaccinated, rural, West African population. Ann Trop Pediatr. 1987;7:91-99.

4. Wolf R, Wolf D , Ruocco V. Antimalarials: Unapproved uses or indications. Clin Dermatol. 2000;18:17-35.

5. Mallucci L. Effect of chloroquine on lysosomes and on growth of mouse hepatitis virus (MHV-3). Virology. 1966;28:355-362.

6. Inglot A. Comparison of the antiviral activity in vitro of some non-steroidal anti-inflammatory drugs. J Gen Virol. 1969;4:203-214.

7. La Placa M, Brodano G, Portolani M, Palenzona A, Bernardini A. On the mechanism of polykaryocyte formation in virus-infected cell cultures. A histochemical study on lysosome activation during virus-induced oplykaryocytogenesis in cell cultures. Giorn Microbiol. 1969;17:67-77.

8. Durand D, Chalgren S, Franke V. Effect of chloroquine on myxovirus replication. Antimicrob Agents Chemother. 1970;10:105-108.

9. Waring M. Variation of the supercoils in closed circular DNA by binding of antibiotics and drugs: Evidence for molecular models involving intercalation. J Mol Biol. 1970;54:247-279.

10. Poste G. The role of lysosomes in virus-induced cell fusion. 2. Modification of the cell surface. Microbios. 1971;3:105-112.

11. Greenham L, Poste G. The role of lysosomes in virus-induced cell fusion. 1. Cytochemical studies. Microbios. 1971;3:97-104.

12. Wilson D. Chloroquine: protection against virus-induced cell damage without inhibition of virus growth. J Gen Virol. 1972;14:107-109.

13. Shimizu Y, Yamamoto S, Homma M, Ishida N. Effect of chloroquine on the growth of animal viruses. Arch Gesamte, Virusforsch. 1972;36:93-104.

14. Pazmino N, Yuhas J, Tennant R. Inhibition of murine RNA tumor virus replication and oncogenesis by chloroquine. Int J Cancer. 1974;14:379-385.

15. Miller D, Lenard J. Inhibition of vesicular stomatitis virus infection by spike glycoprotein. Evidence for an intracellular, G protein-requiring step. J Cell Biol. 1980;84:430-436.

16. Dille B, Johnson T. Inhibition of vesicular stomatitis virus glycoprotein expression by chloroquine. J Gen Virol. 1982;62:91-103.

17. Fredericksen B, Whitt M. Attenuation of recombinant vesicular stomatitis viruses encoding mutant glycoproteins demonstrate a critical role for maintaining a high pH threshold for membrane fusion in viral fitness. Virology. 1998;240:349-358.

18. Baxt B. Effect of lysosomotropic compounds on early events in foot-and-mouth disease virus replication. Virus Res. 1987;7:257-271.

19. Miller B, Tsai T, Mitchell C. Aedes aegypti and yellow fever virus: the effect of chloroquine on infection and transmission rates. Trans R Soc Trop Med Hyg. 1987;81:111-112.

20. Pontesilli O, Carotenuto P, Levin M, Suez D, Hayward A. Processing and presentation of cell-associated varicella-zoster virus antigens by human monocytes. Clin Exp Immunol. 1987;70:127-135.

21. Yoshida T, Takao S, Kiyotani K, Sakaguchi T. Endoproteolytic activation of Newcastle disease virus fusion proteins requires an intracellular acidic environment. Virology. 1989;170:571-574.

22. Alcami A, Carrascosa A, Vinuela E. The entry of African swine fever virus into Vero cells. Virology. 1989;171:68-75.

23. Randolph V, Winkler G, Stollar V. Acidotropic amines inhibit proteolytic processing of flavivirus PrM protein. Virology. 1990;174:450-458.

24. Basak S, Turner H. Infectious entry pathway for caninc parvovirus. Virology. 1992;186:368-376.

25. Miller N, Hutt-Fletcher L. Epstein-Barr vurus enters B cells and epithelial cells by different routes. J Virol. 1992;66:3409-3414.

26. Di Simone C, Zandonatti M, Buchmeier M. Acidic pH triggers LCMV membrane fusion activity and conformational change in the glycoprotein spike. Virology. 1994;198:455-465.

27. Borrow P, Oldstone M. Mechanism of lymphocytic choriomeningitis virus entry into cells. Virology. 1994;198:1-9.

28. Perez L, Irurzun A, Carrasco L. Activation of phospholipase activity during Semliki Forest virus infection. Virology. 1993;194:28-36.

29. Mager A, Masengo R, Mammerickx M, Letesson J. T cell proliferative response to bovine leukaemia virus (BLV): identification of T cell epitopes on the major core protein (p24) in BLV-infected cattle with normal haematological values. J Gen Virol. 1994;75:2223-2231.

30. Kreutz L, Seal B. The pathway of feline calcivirus entry. Virus Res. 1995;35:63-70.

31. Dannevig B, Falk K, Press C. Propagation of infectious salmon anaemia (ISA) virus in cell culture. Vet Res. 1995;26:438-442.

32. Petruzziello R, Orsi N, Macchia S, Tieti S, Frey T, Mastromarino P. Pathway of rubella virus infectious entry into Vero cells. J Gen Virol. 1996;77:303-308.

33. Bishop N. Examination of potential inhibitors of hepatitis A virus uncoating. Intervirology. 1998;41:261-271.

34. Offensperger W, Offensperger S, Walter E, Blum H, Gerok W. Inhibition of duck hepatitis B virus infection by lysosomotropic agents. Virology. 1991;183:415-418.

35. Schirmbeck R, Melber K, Reimann J. Hepatitis B virus small surfact antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I-restricted epitope presentation. Eur J Immunol. 1995;25:1063-1070.

36. Helbling B, Reichen J. Reactivation of hepatitis B following withdrawal of chloroquine. Schweiz Med Wochenschr. 1994;124:759-762.

37. Lewis P, Lentz T. Rabies virus entry into cultured rat hippocampal neurons. J Neurocytol. 1998;27:559-573.

38. Rouven J, Lewis W, Bates P. Characterization of Ebola entry by using pseudotyped viruses: Identification of receptor-deficient cell lines. J Virol. 1998;72:3155-3160.

39. Gonzalez-Dunia D, Cubitt B, de la Torre J. Mechanism of Borna disease virus entry into cells. J Virol. 1998;72:783-788.

40. nauwynck H, Duan X, Favoreel H, Van Oostveldt P, Pensaert M. Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytocic. J Gen Virol. 1999;80:297-305.

41. Kreutz L, Ackermann M. Porcine reproductive and respiratory syndrome virus enters cells through a low pH-dependent endocytic pathway. Virus Res. 1996;42:137-147.

42. Kronenberger P, Vrijsen R, Boeye A. Chloroquine induces empty capsid formation during poliovirus eclipse. J Virol. 1991;65:7008-7011.

43. Gromeier M, Wetz K. Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment. J Virol. 1990;64:3590-3597.

44. Perez L, Carrasco L. Entry of poliovirus into cells does not require low-pH step. J Virol. 1993;67:4543-4548.

45. Lancz G, McLaren L, James C, Scaletti J. Chloroquine mediated alterations in mammalian cell metabolism and viral replication. Proc Soc Exp Biol Med. 1971;136:1289-1293.

46. Banfield W, Kirsch A. The effect of chloroquine on herpesvirus infection in vitro and in vivo. Proc Soc Exp Biol Med. 1973;142:1018-1022.

47. Uchida T, Monura M. Aliphatic amines inhibit the formation of syncytia induced by herpes simplex virus type 1. Microbiol Immunol. 1984;28:243-249.

48. Koyama A, Uchida T. Inhibition of multiplication of herpes simplex virus type 1 by ammonium chloride and chloroquine. Virology. 1984;138:332-335.

49. Duraisingh M, Roper C, Walliker D, Warhurst D. Increased sensitivity to antimalarials mefloquine and artemisinin in conferred by mutations in the pfmdr1 gene of Plasmodium falciparum. Molecular Microbiology. 2000;36:955-961.

50. Pradines B, Tall A, Rogier C, Spiegel A, et al. In vitro activities of ferrochloroquine against 55 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs. Trop Med Int Health. 2002;7:265-270.

51. Ndong J, Atteke C, Aubouy A, Bakary M, Lebibi J, Deloron P. in vitro activity of chloroquine, quinine, mefloquine and halofantrine against Gabonese isolates of Plasmodium falciparum. Trop Med Int Health. 2003;8:25-29.

© 2003 Dermatology Online Journal