- Email: [email protected]
Significance of resistance of herpes simplex virus to acyclovir
Journal of the American Academy of Dermatology
Arndt
REFEREN~CES 1. Physicians' desk reference. 41st ed. Oradell, NJ: Medical "Economics, 1987. 2. Robinson GE, Weber/, Griffiths C, Underhill GS, Jeffries DJ, Goldmeir D. Cutaneous adverse reactions to acyclovir: case reports. Genitourin Med 1985;61:62-3. 3. Sylvester RK, Ogden WB, Draxler CA, Lewis B. Vesicular eruption: a local complication of concentrated acyclovir infusions. JAMA 1986;255:385-6. 4. Tucker WE Jr. Preelinical toxicology profile of acyclovir: an overview. Am J Med 1982;73:27-30. 5. Brigden D, Rosling AE, Woods NC. Renal function after acyclovir intravenous injection. Am J Med 1982;73(1A): 182-5.
I
6. Wade JC, Meyers JD. Neurologic symptoms associated with parenteral acyclovir treatment after marrow transplantation. Ann Intern Med 1983;98:921-5. 7. Cohen SMZ, Minkove JA, Zebley JW III, Mulholland JH. Severe but reversible neurotoxicity from acyclovir. Ann Intern Med 1984;100:920. 8. Krigel RL. Reversible neurotoxicity due to oral acyclovir in a patient with chronic lymphocytic leukemia. J Infect Dis 1986;154:189. 9. Abramson JS, Roach ES, Levy HB. Postinfectious encephalopathy after treatment of herpes simplex encephalitis with acyclovir. Pediatr Infect Dis 1984;3:146-7.
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Significance of resistance of herpes simplex virus to acyclovir Clyde S. Crumpacker, M . D . Boston, MA The genome of herpes simplex virus codes for several enzymes, including viral thymidine kinase and viral deoxyribonucleic acid (DNA) polymerase. When viral resistance develops, it does so by changes in these two enzymes. Three possible mechanisms of viral resistance to acyclovir include (1) selection of viral mutants that make little or no thymidine kinase and do not phosphorylate acyclovir adequately, (2) selection of mutants that can phosphorylate thymidine but cannot phosphorylate acyclovir (i.e., these viruses have thymidine kinases with altered substrate specificity), and (3) selection of viruses that have altered DNA polymerases that replicate viral DNA in the presence of acyclovir triphosphate. Thymidine kinase-deficient virus has been isolated from clinical isolates frequently, but few strains appear to be virulent for animals or humans and only a few seem to have caused clinical disease. Viruses with altered substrate specificity have been reported but viruses with an altered DNA polymerase have not occurred in clinical practice. Antiviral drugs should be used only when necessary to minimize the appearance of resistant strains o f virus. (J AM ACADDERMATOL1988; 18:190-5.)
We have had an antiviral program at Beth Israel Hospital for several years. This has been a very interactive program between infectious diseases,
From the Departmentof Medicine, Harvard Medical School,and the Division of InfectiousDiseases, BethIsrael Hospital. Reprint requests to: ClydeS. Crumpacker,M.D., Division of Infectious Diseases, BethIsraelHospital, 330BrooklineAve., No, 617, Boston, MA 02215.
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oncology, and dermatology, and it was initially put together by Dr. Irwin Freedberg when he was in Boston. I will divide m y discussion of viral resistance to acyclovir into two categories: (1) the mechanisms o f resistance and (2) the significance o f viral resistance in clinical use o f the drug. The fact that resistance can emerge is, in itself, an indication and measure of the specificity o f a given antimicrobial drug and this is the case with acy-
Volume 18 Number 1, Part 2 January 1988 clovir. ~ The genome of herpes simplex virus codes for two enzymes, viral thymidine kinase (TK) and viral deoxyribonucleic acid (DNA) polymerase, that differ from cellular enzymes and are the basis for the specificity of acyclovir. Viral thymidine kinase is required to phosphorylate and activate the drug and the activated triphosphate of acyclovir inhibits DNA polymerase so that viral replication is inhibited. When viral resistance develops, it does so by changes in these two enzymes, TK and DNA polymerase. There are three possible general mechanisms of viral resistance to acyclov~'4: (1) selection Of viral mutants that do not make TK (TK-) and therefore do not phosphorylate acycloVir.5"9 This is the main mechanism that has been proven to be important in the clinical use of acyclovir so far. (2) Mutants have been selected in tissue cultures that have TKs that can phosphorylate thymidine but cannot phosphorylate acyclovir. These viruses are said to have TKs with altered substrate specificity. A clinical isolate selected with acyclovir treatment that has an altered thymidine kinase has recently been described, l°,~ (3) Selection of viruses that have altered DNA polymerases that will replicate viral DNA in the presence of acyclovir triphosphateJ T M The last two are the most worrisome possibilities should they become widespread because we do not know the potential effects of these mutants. Selection is important in all three types of resistance. I believe that these mutants exist in any wild population of viruses and the use of the drug provides strong selective processes so that these resistant viruses emerge as the dominant members. 15Much of the discussion about altered pathogenicity of acyclovir-resistant virus is speculative and it is only the TK- mutants that appear to be clinically important at this time. It is important to note that strains of HSV producing intermediate amounts of TK may be confused with viral strains containing a TK with an altered substrate specificity unless appropriate tests are carried out to distinguish between these two mechanisms of resistance to acyclovir. Darby et a116 first described a herpes simplex mutant virus with altered substrate specificity. The viral TK can phosphorylate thymidine but it cannot phosphorylate acyclovir, and these viruses are
Resistance of herpes simplex to acyclovir
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markedly resistant to acyclovir. A single amino acid change in the viral enzyme produces a TK that is unable to phosphorylate acyclovir and other related antiviral drugs. These mutants are probably present in normal populations of herpes virus. A clinical isolate that is resistant to acyclovir and possesses an altered TK has also been reported. 10, 1~ Field 2 has isolated a similar mutant in an animal model. One of these mutants produced retinal changes, cataracts, and blindness in mice after intracerebral inoculation. 13 Virus strains that have altered D N A polymerase sequences have been produced by recombinant DNA techniques, x7These viruses are markedly resistant to acyclovir, BVDU (E-5-(2-bromovinyl)deoxyuridine), and adenine arabinoside .(vidarabine). This is an example of taking a piece of DNA that contains a mutation and codes for DNA polymerase and putting it into an otherwise normal herpes simplex virus type 2 genome in such a way as to alter the characteristics of the viral DNA polymerase. T h e s e viral polymerases are not inactivated by acyclovir triphosphate; in fact, their effect may be enhanced by the triphosphate. J8 So far, resistance from an altered DNA polymerase has not been seen in any clinical use of the drug. With continued use of acyclovir, mutants with an altered polymerase may be encountered. Schnipper and 119 published an article several years ago showing that wild herpes simplex virus type 1 isolates could be changed from being acyclovir sensitive to acyclovir resistant by several serial passages in tissue cultures containing acyclovir. This virus is resistant by virtue of no longer making TK. This resistance occurs readily in the test tube and it is the same mechanism that seems to be responsible for most of the clinical instances of resistance. In the presence of acyclovir, a virus is selected that is resistant because it is no !onger able to phosphorylate acyclovir. 20 I had a child in Boston with severe combined immunodeficiency syndrome with severe herpes of the face, herpes esophagitis, and herpes pneumonia. 21 There was dramatic healing after three doses of intravenous acyclovir at 250 mg/mL The pretreatment rising titer of virus in his saliva became completely negative. This treatment with acyclovir represents a real advance for immunosuppressed patients and patients with acquired ira-
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munodeficiency syndrome (AIDS). A second recurrence responded similarly to acyclovir. On the third recurrence treated with acyclovir the lesions did not heal as well and virus was continuously present. Acyclovir-resistant virus (100-fold less sensitive) with a marked decrease in TK activity was isolated. There are many ways to look for TK activity. 22-24 A test for TK activity was employed by Dr. Steve Marlowe in our laboratory in which radioactive t4C thymidine is added to primary rabbit kidney tissue cultures after herpes isolates have produced infected areas or plaques. If TK is present, radioactive areas at the herpes-infected plaques can be detected on x-ray film. The TKdeficient herpes virus isolated from the child was unable to phosphorylate acyclovir or another antiviral drug, bromovinyl deoxyuridine. Another test for TK involved radioimmunoprecipitation techniques and this test also showed that this acyclovir-resistant virus was deficient in making the TK protein (Hatis C. Personal communication). The potential to understand the regulation of viral processes is enormous with drugs like acyclovir. Acyclovir has selected out a mutant that has completely stopped making a viral protein, TK. There is an altered acyclovir that Burroughs Wellcome calls BW759, Syntex calls DHPG0 and Merck calls 2' NDG. (Names for this drug include ganciclovir, DHPG, 2'-NDG, BWB759U, BIOLF-62, 2'-nor-2'-deoxyguanosine, 9 - ( 1,3 -dihydroxy - 2 - propoxymethyl)guanine, and 9- [2-hydroxy- 1 - (hydroxymethyl)-ethoxymethyl]guanine. Ganciclovir is more readily taken up by cells and is more rapidly phosphorylated to the triphosphate form and its triphosphate form may be more highly selective for viral DNA polymerase. Its superior effect on human CMV compared with acyclovir is not fully understood. This drug is widely used in patients with AIDS to treat CMV infections. 25-35) There is another antiviral drug that we have been studying in Boston. 36.39 If you close two rings of acyclovir with a cyclic phosphate, the new drug no longer requires TK activity; it gets into cells and works Well against both T K - and TK d virus. The drug that was synthesized by Dr. Tolman at
Journal of the American Academy of Dermatology
Merck Sharp & Dohme is called 2'-nor-cGMP. This drug has not been used in clinical trials yet but it emphasizes that there will be other approaches to treating patients with TK- or deficient herpes simplex infections. In testing the 2'-NDG (BW759 or DHPG) and the 2'-nor-cyclic GMP drug against a resistant strain of virus, it took large amounts of the 2'-NDG, which works through TK, to inhibit the virus but the 2'-nor-cyclic GMP inhibited TK- herpes virus as well as it inhibited TK ÷ virus because inhibition did not depend on the TK enzyme being present. 2'-nor-cyclic GMP has a broad-spectrum antiviral effect against a variety of DNA viruses including both types of herpes simplex, varicella zoster virus, human CMV, vaccinia virus, simian virus 40, adenoviruses, and bovine papillomaviruses. It does not inhibit ribonucleic acid viruses. It is synthesized by phosphorylation of 2'-NDG. It is a cyclic phosphate of 2' NDG and an analog of cyclic guanosine monophosphate although it has no cyclic guanosine monophosphate activity. Its exact mechanism of action is not known. In more than 25 isolates studied at Beth Israel we could not find any isolates that had become resistant to acyelovir during topical treatment in patients with normal immune functions (Marlowe. Personal communication). So, development of resistance from topical use of the drug seems at this time to be a nonproblem in normal hosts. In a collaborative study by Nusinoff-Lehrman et al, 4° in which sensitivity levels of virus to acyclovir were available before, during, and after suppressive treatment of recurrent herpes, there was really no difference in sensitivity of viral isolates from patients who received placebo as opposed to those who received acyclovir. Of seven viral isolates taken during breakthrough recurrences with oral acyclovir suppressive therapy, six were available for testing and these six did not show decreased sensitivities to acyclovir. The use of acyclovir to suppress genital herpes simplex infections did not seem to produce a significant problem in terms of resistant virus. There is only one publication of resistance developing in virus taken from normal persons during acyclovir suppression of recurrent herpes. 1° The sensitivity of pretreatment viral isolates was
Volume 18 Number I, Part 2 January 1988
compared with isolates obtained during recurrences. They found three isolates taken during treatment that were resistant to acyclovir. They postulated that breakthrough recurrences might be caused by selecting resistant virus. However, viruses from the next recurrence after therapy was stopped had come back to pretreatment sensitivity levels. Therefore I conclude that only 10 isolates obtained during suppressiye acyclovir treatment of otherwise normal persons had been studied by late 1985 and these have shown conflicting results. Many more isolates will have to be studied, but I think that resistant virus appearing during recurrences with oral suppressive therapy is not going to be a big problem. 41 In data accumulated by Burroughs Wellcome42 a study of more than 1400 viral isolates showed that about 10% were resistant to acyclovir, Ten percent resistant viral isolates seems like quite a small number and these few m a y also represent a skewed population because the isolates were obtained from patients receiving acyclovir who were already having problems with control of the disease. (Guest editor: Dr. Barry of Burroughs Wellcome [oral communication in June 1987] indicated that more than 2400 viral isolates have now been studied. About 7% of them have been resistant to acyclovir. Most of these isolates have been from problem cases in which patients are immunocompromised and resistant virus from otherwise normal patients treated with acyclovir is probably a much smaller percentage. A relatively high incidence of resistant virus has been obtained from patients undergoing bone marrow transplant and patients with AIDS.) My group in Boston 21 described a TKd-resistant strain of virus that developed in a child with agammaglobulinemia who had renal failure on renal dialysis. The resistant virus was noted after three courses of treatment with acyclovir. In this instance the viral lesions healed despite the presence of an acyclovir-resistant virus. In 1982 Burns et al43 reported on the emergence of TK d virus in two immunocompromised patients undergoing bone marrow transplant after treatment with acyclovir. The presence of the TK a virus did not interfere with recovery from the virus infection. In
Resistance of herpes simplex to acyclovir 193
1982 Sibraek et aP 4 reported a child with combined immunodeficiency disease with adenosine deaminase deficiency and a fetal liver transplant treated with acyclovir on three occasions for herpes simplex infections. Acyclovir-resistant TK a virus was isolated from tongue, lungs, and kidney at autopsy. In this case TK d vires may have produced clinical disease. In 1983 Wade et aP5 found a 6% incidence of resistant strains emerging during treatment of 52 patients undergoing bone marrow transPlant with intravenous acyclovir. The mechanism of resistance vias TKd or TK- activity. In 1984 Straus et al l° found three acyclovir-resistant viral isolates in otherwise normal patients receiving.oral acyclovir for suppression of recurrent genital herpes. Two of these were of the TK ~ variety and one strain was not studied at that time." After acyclovir was stopped, subsequent recurrences showed acyclovir-sensitive virus. In this same study some viral isolates obtained before administration of acyclovir were already resistent to acyclovir. In 1986 Schin azi et a146reported on herpetic infections in a man with AIDS. Of 10 viral isolates, three had no TK activity. These isolates appeared to cause clinical infections in the patient and they were virulent for mice. A 1985 report by Svennerholm et aP 7 of a similar TK- strain was clinically locally v i r u l e n t f None of the above studies have found evidence of dissemination of resistant virus to produce internal disease, hut it is still very early to say whether this will occur. Lesions caused by resistant virus may fail to heal unless another drug such as cyclic phosphate (2'-nor-cGMP) is used. Another concern is the possibility of transmission of resistant virus to other persons, especially if acyclovir is widely used, and the high likelihood that this virus would be transmitted to an infant at the time of delivery. We are all concerned about resistance of virus to antiviral drugs but, so far, this has not been documented to be a big problem. With the increasing interest and ability to measure sensitivity of virus to antiviral drugs we will find out more about viral resistance and its clinical significance. REFERENCES
1. Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, Schaeffer JH. Selectivity of action of an
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antihe~etic agent, 9-(2-hydroxyethoxymethyl)-guanine. Proc Nail Acad Sci USA 1977;74;5716-20. Field HJ. Resistance and latency. Br Med Bull 1985; 41:345-50. Larder BA, Darby G. Susceptibility to other antiherpes drugs of pathogenic variants of herpes simplex virus selected for resistance to acyclovir. Antimicrob Agents Chemother 1986;29:894-8. Larder BA, Darby G. Virus drug resistance: mechanisms and consequences. Antiviral Res 1984;4:1-42. Field HJ, Wildy P. The pathogenesis of thymidine kinase-deficient mutants of herpes simplex virus in mice. J Hyg (Lond) 1978;81:267-77. Tenser RB, Miller RL, Rapp F. Trigeminal ganglion infection by thy'midine kinase-negative mutants of herpes simplex virus. Science 1979;205:915-7. Tenser RB. Intracerebral inoculation of newborn and adult mice with thymidine kinase-deficient mutants of herpes simplex virus. I Infect Dis 1983;147:956. Tenser RB, Jones JC, Ressel SJ. Acute and latent infection by thymidine kinase mutants of herpes simplex type 2. J Infect Dis 1985;151:548-50. Field HJ. A perspective on resistance to acyclovir and herpes simplex virus. J Antimicrob Chemother 1983; 12(suppl): 129-35. Straus SE, Takiff HE, Seidlin M, et al. Suppression of frequently recurring genital herpes: a placebo-controlled double-blind trial of oral acyclovir. N Engl J Med 1984;310:1545-50. Ellis MN, Keller PM, Fyfe JA, et al. Clinical isolate of herpes simplex type 2 that induces a thymidine kinase with altered substrate specificity. Antimicrob Agents Chemother 1987;31:1117-25. Coen DM, Fleming HE Jr, Leslie LK, Retondo MJ. Sensitivity of arabinosyladenine-resistant mutants of herpes simplex virus to other antiviral drugs and mapping of drug hypersensitivity mutants to the DNA polymerase locus. J Virol 1985;53:477-88, Chiou HC, Kerns KM, Coen DM. Mutation within the herpes simplex virus DNA polymerase gene conferring resistance to (R)-9-(3,4-dihydroxybutyl)guanine(buciclovir). Antimicrob Agents Chemother 1986;30: 502-4. Field HJ', Coen DM. Pathogenicity of herpes simplex virus mutants containing drug resistance mutations in the viral DNA polymerase gene. J Virol 1986;60:286-9. Pards DS, Harrington JE. Herpes simplex virus variants resistant to high concentrations of acyclovir exist in clinical isolates. Antimicrob Agents Chemother 1982;22: 71-7. Darby G, Field HJ, Salisbury SA. Altered substrate specificity of herpes simplex virus thymidine kinase. Nature 1981;289:81-3. Crumpacker CS, Schnipper LE, Kowalsky P, Sherman D'. Resistance of herpes simplex virus to adenosine arabinoside (Ara-A) and E-5 (2-bromovinyl)-2'deoxyuridine (BVDU)--a physical analysis. J Infect Dis 1982;146:85-90. Knoff KW, Kauffman ER, Crumpacker CS. Physical mapping of drug resistance mutations defines an actiye center of the herpes simplex virus DNA polymerase enzyme. J Virol 1981;39:746-57.
Journal of the American Academy of Dermatology
19. Schnipper LE, Crumpacker CS. Resistance of herpes simplex virus to acycloguanosine: role of viral thymidine kinase and DNA polymerase loci. Proc Natl Acad Sci USA 1980;77:2270-3. 20. McLaren C, Corey L, Dekker C, Barry DW. In vitro sensitivity to acyclovir in genital herpes simplex viruses from acyclovir-treated patients. J Infect Dis 1983; 148:868-75. 21. Crumpacker CS, Schnipper LE, Marlowe SI, Kowalsky PN, Hershey BJ, Levin MJ. Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with acyclovir. N Engl J Med 1982;306:343-6, 22. McLaren C, Ellis MN, Hunter GA. A colorimetric assay for the measurement of sensitivity of herpes simplex viruses to antiviral agents. Antiviral Res 1983;3:223-4. 23. Martin JC, Ellis MN, Keller PM, et al. A plaque autodiographic assay for the detection and quantitation of thymidine kinase mutants in clinical isolates. Antimicrob Agents Chemother 1985;28:181-7. 24. Harmenberg J, Sundquist V-A, Gadler H, Leven B, Brannstrom G, Wahren B. Comparative methods for detection of thymidine kinase-deficient herpes simplex virus type 1 strains. Antimicrob Agents Chemother 1986; 30:570-3. 25. Germershausen J, Bostedor R, Kirk-Field A, et al. A comparison of the antiviral agents 2'-nor-2'-deoxyguanosine and acyclovir: uptake and phosphorylation in tissue culture and kinetics of in vitro inhibition of viral and cellular DNA polymerase by the respective phosphates. Biochem Biophys Res Commun 1983;116:360-7. 26. Smee DF, Martin JC, Verheyden JPH, Matthews TR. Anti-herpesvirus activity of the acyclic nucleoside 9(1,3-dihydroxy-2-propoxymethyl)guanine. Antimicrob Agents Chemother 1983;23:676-82. 27. Cheng Y-C, Huang E-S, Liu J-C, et al. Unique spectrum of activity of 9-[(1,3-dihydroxy-2-propoxy)methyl] guanosine against herpesvirus in vitro and its mode of action against herpes simplex virus type 1. Proc Natl Acad Set USA 1983;80:2767-70. 28. St, Clair MH, Miller WH, Miller RL, Lambe CU, Furman PA. Inhibition of a cellular alpha DNA polymerase and herpes simplex virus-induced DNA polymerase by the triphosphate of BW759U. Antimicrob Agents Chemother 1984;25:191-4. 29. Felsenstein D, D'amico DJ, Hirsch MS, et al. Treatment of cytomegalovirus retinitis with 9-[2-hydroxy-1(hydroxymethyl)ethoxymethyl]guanlne. Ann Intern Med 1985;103:377-80. 30. Masur H, Lane HC, Palestine A, et al. Effect of 9-(1,3dihydroxy-2-propoxymethyl) guanine in serious cytomegalovirus disease in eight immunocompromised homosexual men. Ann Intern Med 1986;104"41-4. 31. Collaborative DHPG Treatment Study Group. Treatment of serious cytomegalovirus infections with 9-(1,3dihydroxy-2-propoxymethyl)guanine in patients with AIDS and other immunodeficiencies. N Engl J Med 1986;314:801-4. 32. Van der Horst CM, Lin J-C, Raab-Traub N, Smith MC, Pagano JS. Differential effects of acyclovir and 9-(1,3dihydroxy-2-propoxymethyl)guanine on herpes simplex virus and Epstein-Barr virus in a dually infected human lymphoblastoid cell line. J Virol 1987;61:607-10.
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33. Davies M-EM, Bondi JV, Grabowski L, Schofield TL, Field AK. 2'-nor-2'-deoxyguanosine is an effective therapeutic agent for treatment of experimental herpes kerajtitis. Antiviral Res 1987;7:119-25. 34. St Clair MH, Lambe CU, Furman PA. Inhibition of ganciclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information. Antimicrob Agents Chemother 1987;31:844-9. 35. Erice A, Jordan MC, Chace BA, Fletcher C, Chinnock B J, Balfour HH Jr. Ganciclovir treatment of cytomegalovirus disease in transplant recipients and other immunocompromised hosts. JAMA 1987;257:3082-7. 36. Oliver S, Bubley G, Crumpacker C. Inhibition of HSVtransformed murine cells by nucleoside analogs, 2~-NDG and 2'-nor-cGMP: mechanisms of inhibition and reversal by exogenous nucleosides. Virology 1985;145:84-93. 37. Tolman RL, Field AK, Karkas JD, et al. 2'-nor-cGMP: a seco-cyclic nucleotide with powerful anti-DNA-viral activity. Biochem Biophys Res Commun 1985;128: 1329-35. 38. Germershausen J, Bostedor R, Liou R, et al. Comparison of the modes of anfiviral action of 2'-nor-deoxyguanosine and its cyclic phosphate, 2'-nor-cyclic GMP. Antimicrob Agents Chemother 1986;29:1025-31. 39. Baba M, Mori S, Shigeta S, de Clercq E. Selective inhibitory effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine and 2'-nor-cyclic GMP in adenovirus replication in vitro. Antimicrob Agents Chemother 1987;31:337-9. 40. Nusinoff-Lehrman S, Douglas JM, Corey L, Barry DW,
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41. 42. 43. 44. 45.
46.
47.
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Recurrent genital herpes and suppressive oral acyclovir therapy: relation between clinical outcome and in vitro drug sensitivity. Ann It~tem Med 1986;104;786-90. Ambinder RF, Bums WH, Lietman PS, Saral R. Prophylaxis: a strategy to minimize viral resistance. Lancet 1984;1:1154-5. Dekker C, Ellis MN, McLaren C, Hunter G, Rogers J, Barry DW. Virus resistance in clinical practice. J Antimicrob Chemother 1983;12(suppl):137-52. Burns WH, Santos GW, Saral R, et al. Isolation and characterization of resistant herpes simplex virus after acyclovir therapy. Lancet 1982;1:421-3. Sibrack CD, Gutman LT, Wilfert CM, et al. Pathogenicity of acyclovir-resistant herpes simplex virus type I from an immunodeficient child. J Infect Dis 1982;146:673-82. Wade JC, McLaren C, Myers JD. Frequency and significance of acyclovir-resistant herpes simplex virus isolated from marrow transplant patients receiving multiple courses of treatment with acyclovir. J Infect Dis 1983; 148:1077-82. Schinazi RF, del Bene V, Scott RT, Dudley-Thorpe J-B. Characterization of acyclovir-resistant and -sensitive herpes simplex viruses from a patient with an acquired immune deficiency. J Antimicrob Chemother 1986; 18(suppl): 127-34. Svennerholm B, Vahline A, Lowhagen GB, Widell A, Lycke E. Sensitivity of HSV strains isolated before and after treatment with acyclovir. Scand J Infect Dis 1985;47:149-54.
Arndt
REFEREN~CES 1. Physicians' desk reference. 41st ed. Oradell, NJ: Medical "Economics, 1987. 2. Robinson GE, Weber/, Griffiths C, Underhill GS, Jeffries DJ, Goldmeir D. Cutaneous adverse reactions to acyclovir: case reports. Genitourin Med 1985;61:62-3. 3. Sylvester RK, Ogden WB, Draxler CA, Lewis B. Vesicular eruption: a local complication of concentrated acyclovir infusions. JAMA 1986;255:385-6. 4. Tucker WE Jr. Preelinical toxicology profile of acyclovir: an overview. Am J Med 1982;73:27-30. 5. Brigden D, Rosling AE, Woods NC. Renal function after acyclovir intravenous injection. Am J Med 1982;73(1A): 182-5.
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6. Wade JC, Meyers JD. Neurologic symptoms associated with parenteral acyclovir treatment after marrow transplantation. Ann Intern Med 1983;98:921-5. 7. Cohen SMZ, Minkove JA, Zebley JW III, Mulholland JH. Severe but reversible neurotoxicity from acyclovir. Ann Intern Med 1984;100:920. 8. Krigel RL. Reversible neurotoxicity due to oral acyclovir in a patient with chronic lymphocytic leukemia. J Infect Dis 1986;154:189. 9. Abramson JS, Roach ES, Levy HB. Postinfectious encephalopathy after treatment of herpes simplex encephalitis with acyclovir. Pediatr Infect Dis 1984;3:146-7.
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Significance of resistance of herpes simplex virus to acyclovir Clyde S. Crumpacker, M . D . Boston, MA The genome of herpes simplex virus codes for several enzymes, including viral thymidine kinase and viral deoxyribonucleic acid (DNA) polymerase. When viral resistance develops, it does so by changes in these two enzymes. Three possible mechanisms of viral resistance to acyclovir include (1) selection of viral mutants that make little or no thymidine kinase and do not phosphorylate acyclovir adequately, (2) selection of mutants that can phosphorylate thymidine but cannot phosphorylate acyclovir (i.e., these viruses have thymidine kinases with altered substrate specificity), and (3) selection of viruses that have altered DNA polymerases that replicate viral DNA in the presence of acyclovir triphosphate. Thymidine kinase-deficient virus has been isolated from clinical isolates frequently, but few strains appear to be virulent for animals or humans and only a few seem to have caused clinical disease. Viruses with altered substrate specificity have been reported but viruses with an altered DNA polymerase have not occurred in clinical practice. Antiviral drugs should be used only when necessary to minimize the appearance of resistant strains o f virus. (J AM ACADDERMATOL1988; 18:190-5.)
We have had an antiviral program at Beth Israel Hospital for several years. This has been a very interactive program between infectious diseases,
From the Departmentof Medicine, Harvard Medical School,and the Division of InfectiousDiseases, BethIsrael Hospital. Reprint requests to: ClydeS. Crumpacker,M.D., Division of Infectious Diseases, BethIsraelHospital, 330BrooklineAve., No, 617, Boston, MA 02215.
190
oncology, and dermatology, and it was initially put together by Dr. Irwin Freedberg when he was in Boston. I will divide m y discussion of viral resistance to acyclovir into two categories: (1) the mechanisms o f resistance and (2) the significance o f viral resistance in clinical use o f the drug. The fact that resistance can emerge is, in itself, an indication and measure of the specificity o f a given antimicrobial drug and this is the case with acy-
Volume 18 Number 1, Part 2 January 1988 clovir. ~ The genome of herpes simplex virus codes for two enzymes, viral thymidine kinase (TK) and viral deoxyribonucleic acid (DNA) polymerase, that differ from cellular enzymes and are the basis for the specificity of acyclovir. Viral thymidine kinase is required to phosphorylate and activate the drug and the activated triphosphate of acyclovir inhibits DNA polymerase so that viral replication is inhibited. When viral resistance develops, it does so by changes in these two enzymes, TK and DNA polymerase. There are three possible general mechanisms of viral resistance to acyclov~'4: (1) selection Of viral mutants that do not make TK (TK-) and therefore do not phosphorylate acycloVir.5"9 This is the main mechanism that has been proven to be important in the clinical use of acyclovir so far. (2) Mutants have been selected in tissue cultures that have TKs that can phosphorylate thymidine but cannot phosphorylate acyclovir. These viruses are said to have TKs with altered substrate specificity. A clinical isolate selected with acyclovir treatment that has an altered thymidine kinase has recently been described, l°,~ (3) Selection of viruses that have altered DNA polymerases that will replicate viral DNA in the presence of acyclovir triphosphateJ T M The last two are the most worrisome possibilities should they become widespread because we do not know the potential effects of these mutants. Selection is important in all three types of resistance. I believe that these mutants exist in any wild population of viruses and the use of the drug provides strong selective processes so that these resistant viruses emerge as the dominant members. 15Much of the discussion about altered pathogenicity of acyclovir-resistant virus is speculative and it is only the TK- mutants that appear to be clinically important at this time. It is important to note that strains of HSV producing intermediate amounts of TK may be confused with viral strains containing a TK with an altered substrate specificity unless appropriate tests are carried out to distinguish between these two mechanisms of resistance to acyclovir. Darby et a116 first described a herpes simplex mutant virus with altered substrate specificity. The viral TK can phosphorylate thymidine but it cannot phosphorylate acyclovir, and these viruses are
Resistance of herpes simplex to acyclovir
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markedly resistant to acyclovir. A single amino acid change in the viral enzyme produces a TK that is unable to phosphorylate acyclovir and other related antiviral drugs. These mutants are probably present in normal populations of herpes virus. A clinical isolate that is resistant to acyclovir and possesses an altered TK has also been reported. 10, 1~ Field 2 has isolated a similar mutant in an animal model. One of these mutants produced retinal changes, cataracts, and blindness in mice after intracerebral inoculation. 13 Virus strains that have altered D N A polymerase sequences have been produced by recombinant DNA techniques, x7These viruses are markedly resistant to acyclovir, BVDU (E-5-(2-bromovinyl)deoxyuridine), and adenine arabinoside .(vidarabine). This is an example of taking a piece of DNA that contains a mutation and codes for DNA polymerase and putting it into an otherwise normal herpes simplex virus type 2 genome in such a way as to alter the characteristics of the viral DNA polymerase. T h e s e viral polymerases are not inactivated by acyclovir triphosphate; in fact, their effect may be enhanced by the triphosphate. J8 So far, resistance from an altered DNA polymerase has not been seen in any clinical use of the drug. With continued use of acyclovir, mutants with an altered polymerase may be encountered. Schnipper and 119 published an article several years ago showing that wild herpes simplex virus type 1 isolates could be changed from being acyclovir sensitive to acyclovir resistant by several serial passages in tissue cultures containing acyclovir. This virus is resistant by virtue of no longer making TK. This resistance occurs readily in the test tube and it is the same mechanism that seems to be responsible for most of the clinical instances of resistance. In the presence of acyclovir, a virus is selected that is resistant because it is no !onger able to phosphorylate acyclovir. 20 I had a child in Boston with severe combined immunodeficiency syndrome with severe herpes of the face, herpes esophagitis, and herpes pneumonia. 21 There was dramatic healing after three doses of intravenous acyclovir at 250 mg/mL The pretreatment rising titer of virus in his saliva became completely negative. This treatment with acyclovir represents a real advance for immunosuppressed patients and patients with acquired ira-
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munodeficiency syndrome (AIDS). A second recurrence responded similarly to acyclovir. On the third recurrence treated with acyclovir the lesions did not heal as well and virus was continuously present. Acyclovir-resistant virus (100-fold less sensitive) with a marked decrease in TK activity was isolated. There are many ways to look for TK activity. 22-24 A test for TK activity was employed by Dr. Steve Marlowe in our laboratory in which radioactive t4C thymidine is added to primary rabbit kidney tissue cultures after herpes isolates have produced infected areas or plaques. If TK is present, radioactive areas at the herpes-infected plaques can be detected on x-ray film. The TKdeficient herpes virus isolated from the child was unable to phosphorylate acyclovir or another antiviral drug, bromovinyl deoxyuridine. Another test for TK involved radioimmunoprecipitation techniques and this test also showed that this acyclovir-resistant virus was deficient in making the TK protein (Hatis C. Personal communication). The potential to understand the regulation of viral processes is enormous with drugs like acyclovir. Acyclovir has selected out a mutant that has completely stopped making a viral protein, TK. There is an altered acyclovir that Burroughs Wellcome calls BW759, Syntex calls DHPG0 and Merck calls 2' NDG. (Names for this drug include ganciclovir, DHPG, 2'-NDG, BWB759U, BIOLF-62, 2'-nor-2'-deoxyguanosine, 9 - ( 1,3 -dihydroxy - 2 - propoxymethyl)guanine, and 9- [2-hydroxy- 1 - (hydroxymethyl)-ethoxymethyl]guanine. Ganciclovir is more readily taken up by cells and is more rapidly phosphorylated to the triphosphate form and its triphosphate form may be more highly selective for viral DNA polymerase. Its superior effect on human CMV compared with acyclovir is not fully understood. This drug is widely used in patients with AIDS to treat CMV infections. 25-35) There is another antiviral drug that we have been studying in Boston. 36.39 If you close two rings of acyclovir with a cyclic phosphate, the new drug no longer requires TK activity; it gets into cells and works Well against both T K - and TK d virus. The drug that was synthesized by Dr. Tolman at
Journal of the American Academy of Dermatology
Merck Sharp & Dohme is called 2'-nor-cGMP. This drug has not been used in clinical trials yet but it emphasizes that there will be other approaches to treating patients with TK- or deficient herpes simplex infections. In testing the 2'-NDG (BW759 or DHPG) and the 2'-nor-cyclic GMP drug against a resistant strain of virus, it took large amounts of the 2'-NDG, which works through TK, to inhibit the virus but the 2'-nor-cyclic GMP inhibited TK- herpes virus as well as it inhibited TK ÷ virus because inhibition did not depend on the TK enzyme being present. 2'-nor-cyclic GMP has a broad-spectrum antiviral effect against a variety of DNA viruses including both types of herpes simplex, varicella zoster virus, human CMV, vaccinia virus, simian virus 40, adenoviruses, and bovine papillomaviruses. It does not inhibit ribonucleic acid viruses. It is synthesized by phosphorylation of 2'-NDG. It is a cyclic phosphate of 2' NDG and an analog of cyclic guanosine monophosphate although it has no cyclic guanosine monophosphate activity. Its exact mechanism of action is not known. In more than 25 isolates studied at Beth Israel we could not find any isolates that had become resistant to acyelovir during topical treatment in patients with normal immune functions (Marlowe. Personal communication). So, development of resistance from topical use of the drug seems at this time to be a nonproblem in normal hosts. In a collaborative study by Nusinoff-Lehrman et al, 4° in which sensitivity levels of virus to acyclovir were available before, during, and after suppressive treatment of recurrent herpes, there was really no difference in sensitivity of viral isolates from patients who received placebo as opposed to those who received acyclovir. Of seven viral isolates taken during breakthrough recurrences with oral acyclovir suppressive therapy, six were available for testing and these six did not show decreased sensitivities to acyclovir. The use of acyclovir to suppress genital herpes simplex infections did not seem to produce a significant problem in terms of resistant virus. There is only one publication of resistance developing in virus taken from normal persons during acyclovir suppression of recurrent herpes. 1° The sensitivity of pretreatment viral isolates was
Volume 18 Number I, Part 2 January 1988
compared with isolates obtained during recurrences. They found three isolates taken during treatment that were resistant to acyclovir. They postulated that breakthrough recurrences might be caused by selecting resistant virus. However, viruses from the next recurrence after therapy was stopped had come back to pretreatment sensitivity levels. Therefore I conclude that only 10 isolates obtained during suppressiye acyclovir treatment of otherwise normal persons had been studied by late 1985 and these have shown conflicting results. Many more isolates will have to be studied, but I think that resistant virus appearing during recurrences with oral suppressive therapy is not going to be a big problem. 41 In data accumulated by Burroughs Wellcome42 a study of more than 1400 viral isolates showed that about 10% were resistant to acyclovir, Ten percent resistant viral isolates seems like quite a small number and these few m a y also represent a skewed population because the isolates were obtained from patients receiving acyclovir who were already having problems with control of the disease. (Guest editor: Dr. Barry of Burroughs Wellcome [oral communication in June 1987] indicated that more than 2400 viral isolates have now been studied. About 7% of them have been resistant to acyclovir. Most of these isolates have been from problem cases in which patients are immunocompromised and resistant virus from otherwise normal patients treated with acyclovir is probably a much smaller percentage. A relatively high incidence of resistant virus has been obtained from patients undergoing bone marrow transplant and patients with AIDS.) My group in Boston 21 described a TKd-resistant strain of virus that developed in a child with agammaglobulinemia who had renal failure on renal dialysis. The resistant virus was noted after three courses of treatment with acyclovir. In this instance the viral lesions healed despite the presence of an acyclovir-resistant virus. In 1982 Burns et al43 reported on the emergence of TK d virus in two immunocompromised patients undergoing bone marrow transplant after treatment with acyclovir. The presence of the TK a virus did not interfere with recovery from the virus infection. In
Resistance of herpes simplex to acyclovir 193
1982 Sibraek et aP 4 reported a child with combined immunodeficiency disease with adenosine deaminase deficiency and a fetal liver transplant treated with acyclovir on three occasions for herpes simplex infections. Acyclovir-resistant TK a virus was isolated from tongue, lungs, and kidney at autopsy. In this case TK d vires may have produced clinical disease. In 1983 Wade et aP5 found a 6% incidence of resistant strains emerging during treatment of 52 patients undergoing bone marrow transPlant with intravenous acyclovir. The mechanism of resistance vias TKd or TK- activity. In 1984 Straus et al l° found three acyclovir-resistant viral isolates in otherwise normal patients receiving.oral acyclovir for suppression of recurrent genital herpes. Two of these were of the TK ~ variety and one strain was not studied at that time." After acyclovir was stopped, subsequent recurrences showed acyclovir-sensitive virus. In this same study some viral isolates obtained before administration of acyclovir were already resistent to acyclovir. In 1986 Schin azi et a146reported on herpetic infections in a man with AIDS. Of 10 viral isolates, three had no TK activity. These isolates appeared to cause clinical infections in the patient and they were virulent for mice. A 1985 report by Svennerholm et aP 7 of a similar TK- strain was clinically locally v i r u l e n t f None of the above studies have found evidence of dissemination of resistant virus to produce internal disease, hut it is still very early to say whether this will occur. Lesions caused by resistant virus may fail to heal unless another drug such as cyclic phosphate (2'-nor-cGMP) is used. Another concern is the possibility of transmission of resistant virus to other persons, especially if acyclovir is widely used, and the high likelihood that this virus would be transmitted to an infant at the time of delivery. We are all concerned about resistance of virus to antiviral drugs but, so far, this has not been documented to be a big problem. With the increasing interest and ability to measure sensitivity of virus to antiviral drugs we will find out more about viral resistance and its clinical significance. REFERENCES
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19. Schnipper LE, Crumpacker CS. Resistance of herpes simplex virus to acycloguanosine: role of viral thymidine kinase and DNA polymerase loci. Proc Natl Acad Sci USA 1980;77:2270-3. 20. McLaren C, Corey L, Dekker C, Barry DW. In vitro sensitivity to acyclovir in genital herpes simplex viruses from acyclovir-treated patients. J Infect Dis 1983; 148:868-75. 21. Crumpacker CS, Schnipper LE, Marlowe SI, Kowalsky PN, Hershey BJ, Levin MJ. Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with acyclovir. N Engl J Med 1982;306:343-6, 22. McLaren C, Ellis MN, Hunter GA. A colorimetric assay for the measurement of sensitivity of herpes simplex viruses to antiviral agents. Antiviral Res 1983;3:223-4. 23. Martin JC, Ellis MN, Keller PM, et al. A plaque autodiographic assay for the detection and quantitation of thymidine kinase mutants in clinical isolates. Antimicrob Agents Chemother 1985;28:181-7. 24. Harmenberg J, Sundquist V-A, Gadler H, Leven B, Brannstrom G, Wahren B. Comparative methods for detection of thymidine kinase-deficient herpes simplex virus type 1 strains. Antimicrob Agents Chemother 1986; 30:570-3. 25. Germershausen J, Bostedor R, Kirk-Field A, et al. A comparison of the antiviral agents 2'-nor-2'-deoxyguanosine and acyclovir: uptake and phosphorylation in tissue culture and kinetics of in vitro inhibition of viral and cellular DNA polymerase by the respective phosphates. Biochem Biophys Res Commun 1983;116:360-7. 26. Smee DF, Martin JC, Verheyden JPH, Matthews TR. Anti-herpesvirus activity of the acyclic nucleoside 9(1,3-dihydroxy-2-propoxymethyl)guanine. Antimicrob Agents Chemother 1983;23:676-82. 27. Cheng Y-C, Huang E-S, Liu J-C, et al. Unique spectrum of activity of 9-[(1,3-dihydroxy-2-propoxy)methyl] guanosine against herpesvirus in vitro and its mode of action against herpes simplex virus type 1. Proc Natl Acad Set USA 1983;80:2767-70. 28. St, Clair MH, Miller WH, Miller RL, Lambe CU, Furman PA. Inhibition of a cellular alpha DNA polymerase and herpes simplex virus-induced DNA polymerase by the triphosphate of BW759U. Antimicrob Agents Chemother 1984;25:191-4. 29. Felsenstein D, D'amico DJ, Hirsch MS, et al. Treatment of cytomegalovirus retinitis with 9-[2-hydroxy-1(hydroxymethyl)ethoxymethyl]guanlne. Ann Intern Med 1985;103:377-80. 30. Masur H, Lane HC, Palestine A, et al. Effect of 9-(1,3dihydroxy-2-propoxymethyl) guanine in serious cytomegalovirus disease in eight immunocompromised homosexual men. Ann Intern Med 1986;104"41-4. 31. Collaborative DHPG Treatment Study Group. Treatment of serious cytomegalovirus infections with 9-(1,3dihydroxy-2-propoxymethyl)guanine in patients with AIDS and other immunodeficiencies. N Engl J Med 1986;314:801-4. 32. Van der Horst CM, Lin J-C, Raab-Traub N, Smith MC, Pagano JS. Differential effects of acyclovir and 9-(1,3dihydroxy-2-propoxymethyl)guanine on herpes simplex virus and Epstein-Barr virus in a dually infected human lymphoblastoid cell line. J Virol 1987;61:607-10.
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33. Davies M-EM, Bondi JV, Grabowski L, Schofield TL, Field AK. 2'-nor-2'-deoxyguanosine is an effective therapeutic agent for treatment of experimental herpes kerajtitis. Antiviral Res 1987;7:119-25. 34. St Clair MH, Lambe CU, Furman PA. Inhibition of ganciclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information. Antimicrob Agents Chemother 1987;31:844-9. 35. Erice A, Jordan MC, Chace BA, Fletcher C, Chinnock B J, Balfour HH Jr. Ganciclovir treatment of cytomegalovirus disease in transplant recipients and other immunocompromised hosts. JAMA 1987;257:3082-7. 36. Oliver S, Bubley G, Crumpacker C. Inhibition of HSVtransformed murine cells by nucleoside analogs, 2~-NDG and 2'-nor-cGMP: mechanisms of inhibition and reversal by exogenous nucleosides. Virology 1985;145:84-93. 37. Tolman RL, Field AK, Karkas JD, et al. 2'-nor-cGMP: a seco-cyclic nucleotide with powerful anti-DNA-viral activity. Biochem Biophys Res Commun 1985;128: 1329-35. 38. Germershausen J, Bostedor R, Liou R, et al. Comparison of the modes of anfiviral action of 2'-nor-deoxyguanosine and its cyclic phosphate, 2'-nor-cyclic GMP. Antimicrob Agents Chemother 1986;29:1025-31. 39. Baba M, Mori S, Shigeta S, de Clercq E. Selective inhibitory effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine and 2'-nor-cyclic GMP in adenovirus replication in vitro. Antimicrob Agents Chemother 1987;31:337-9. 40. Nusinoff-Lehrman S, Douglas JM, Corey L, Barry DW,
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Recurrent genital herpes and suppressive oral acyclovir therapy: relation between clinical outcome and in vitro drug sensitivity. Ann It~tem Med 1986;104;786-90. Ambinder RF, Bums WH, Lietman PS, Saral R. Prophylaxis: a strategy to minimize viral resistance. Lancet 1984;1:1154-5. Dekker C, Ellis MN, McLaren C, Hunter G, Rogers J, Barry DW. Virus resistance in clinical practice. J Antimicrob Chemother 1983;12(suppl):137-52. Burns WH, Santos GW, Saral R, et al. Isolation and characterization of resistant herpes simplex virus after acyclovir therapy. Lancet 1982;1:421-3. Sibrack CD, Gutman LT, Wilfert CM, et al. Pathogenicity of acyclovir-resistant herpes simplex virus type I from an immunodeficient child. J Infect Dis 1982;146:673-82. Wade JC, McLaren C, Myers JD. Frequency and significance of acyclovir-resistant herpes simplex virus isolated from marrow transplant patients receiving multiple courses of treatment with acyclovir. J Infect Dis 1983; 148:1077-82. Schinazi RF, del Bene V, Scott RT, Dudley-Thorpe J-B. Characterization of acyclovir-resistant and -sensitive herpes simplex viruses from a patient with an acquired immune deficiency. J Antimicrob Chemother 1986; 18(suppl): 127-34. Svennerholm B, Vahline A, Lowhagen GB, Widell A, Lycke E. Sensitivity of HSV strains isolated before and after treatment with acyclovir. Scand J Infect Dis 1985;47:149-54.