Original Article

Brief Report

Recurrent Acyclovir-Resistant Genital Herpes in an Immunocompetent Patient

Rhonda G. Kost, Edgar L. Hill, Michael Tigges, and Stephen E. Straus

N Engl J Med 1993; 329:1777-1782December 9, 1993

Article

Acyclovir has been widely used over the past decade as an effective and safe drug for the treatment of infections with herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)1. Resistance to acyclovir was initially noted only in studies of HSV infection in vitro and in animal models,2-4 then it was found in immunocompromised patients, and it has now become a well-documented clinical challenge to the care of patients infected with the human immunodeficiency virus (HIV)4-12. Acyclovir resistance has not been a problem to date in the treatment of immunocompetent people with HSV infection. Although we identified acyclovir-resistant isolates in a study of long-term suppressive treatment for genital herpes in normal hosts and later studies documented occasional shedding of acyclovir-resistant virus by immunocompetent patients before, during, or after therapy, no correlation has been established between such isolates and clinical outcomes13-17.

Acyclovir-resistant HSV is operationally defined by the need for a dose of more than 3 μg of drug per milliliter of culture medium to inhibit viral replication by 50 percent in vitro (ID₅₀)13. The antiviral activity of acyclovir requires that it first be converted to its monophosphate derivative by viral thymidine kinase. Cellular kinases then convert the drug to its active triphosphate form, which inhibits viral DNA polymerase and viral replication18-21. Mutations in either the viral thymidine kinase or polymerase genes can lead to acyclovir resistance. The normal viral thymidine kinase (the TK⁺ phenotype) phosphorylates acyclovir, thymidine, and other related substrates. Mutations in the viral thymidine kinase gene leading to the formation of premature stop codons result in thymidine kinase-deficient (TK^-) virus strains, whereas other point mutations alter the specificity of thymidine kinase for some of its potential substrates, yielding the thymidine kinase-altered (TK^A) viral phenotype2,22-24. Rare HSV strains maintain a TK⁺ phenotype but are rendered resistant to acyclovir by mutations in the viral DNA polymerase, the target of acyclovir triphosphate3,5,21,25,26.

We describe an immunologically normal man with frequently recurring, symptomatic, culture-positive outbreaks of genital herpes that were not suppressed despite treatment with 4.8 g of oral acyclovir a day and sustained, appropriate serum drug levels. Isolates recovered from six of his sequential outbreaks during and after therapy were resistant to acyclovir (ID₅₀, 6 to 16.3 μg per milliliter) and were identical according to multiple biochemical and molecular criteria. The isolates tested displayed a predominantly TK^A phenotype and, in comparison with an acyclovir-sensitive HSV strain, contained the same four point mutations, including one that resulted in an alteration in the putative acyclovir-binding domain of the viral thymidine kinase.

Methods

HSV was isolated from genital lesions by culture in human embryonic lung cells (WI-38, BioWhittaker, Walkersville, Md.), as described elsewhere13. HSV-2 virion DNA was purified from infected cells as described elsewhere27. Viral DNA was digested by the restriction endonucleases BamHI, BglII, EcoRI, HindIII, KpnI, and SalI (New England Biolabs, Beverly, Mass.). Virus type and genetic relatedness were determined by comparing the patterns of restriction-endonuclease digestion of the DNA from clinical isolates with those of a standard laboratory strain, HSV-2 33328,29.

HSV-2 isolates were tested for susceptibility to antiviral drugs with standard plaque-reduction assays6. The ID₅₀ was calculated by plotting the log₁₀ concentration against the percentage of suppression of plaques formed.

HSV strains were tested for their ability to express thymidine kinase activity by [¹²⁵I]iododeoxycytidine ([¹²⁵I]dC) plaque autoradiography, as recently described and modified30-32. This method enables one visually to distinguish virus with a TK^A phenotype, which results in faintly labeled plaques, from virus that is TK⁺ or TK^-; these respective phenotypes label plaques darkly or not all. Mixed populations of viruses that may include mutants or viruses that have reverted to the normal phenotype are easily identified by this method.

Extracts of HSV-infected 143B cells were assayed for thymidine kinase activity by methods described elsewhere33. These cells lack thymidine kinase and provide a negative background on which to characterize the HSV thymidine kinase biochemically.

The polymerase chain reaction (PCR) was used to amplify the thymidine kinase gene from HSV-2 333 and from the clinical isolates with modifications of methods described previously34. Two primers consisting of thymidine kinase-specific sequences were chosen from the published HSV-2 333 sequence35. The reactions were carried out in Taq polymerase reaction buffer (Boehringer-Mannheim, Indianapolis) at standard concentrations. Thermal cycling was performed with an automated thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.) for 35 cycles consisting of 30 seconds at 97 °C, 2 minutes at 55 °C, and 4 minutes at 72 °C. Two modifications, the use of the 7-deaza-2'-deoxyguanosine triphosphate nucleotide and the hot-start method, were required for optimal amplification of specific products34.

The PCR-amplified products from clinical isolate HSV-2 4365-9 were cloned into pGEM2 vector (Promega Corp., Madison, Wis.) by standard techniques29. The resulting plasmid, pGTK9-2, contains the HSV-2 4365-9 thymidine kinase gene from bases -230 to +1275 relative to the start of transcription, as confirmed by sequencing. Sequencing primers were chosen to span the HSV-2 thymidine kinase gene at intervals of 200 to 250 bases and to provide serial overlapping sequences. The PCR-amplified products were sequenced according to a modified protocol using dimethylsulfoxide36. Cloned plasmids were sequenced with the Sequenase Version 2.0 kit (United States Biochemical, Cleveland)15. Band compressions were resolved with Deaza T7 Sequencing Mixes (Pharmacia LKB Biotechnology, Piscataway, N.J.) as described elsewhere.

The patient gave informed consent for HIV-antibody testing, phlebotomy, and apheresis. Testing for HIV antibody was performed with the Western blot assay (Dupont, Wilmington, Del.), and testing for p24 antigen with an enzyme-linked immunosorbent assay (ELISA) (Coulter Source, Marietta, Ga.). Two-pass lymphocyte apheresis was performed with citrate-dextrose type A anticoagulant and the Haemonetics VSO cell separator (Haemonetics, Braintree, Mass.). Lymphocytes were purified by Ficoll-Hypaque centrifugation (Pharmacia LKB Biotechnology), cryopreserved in a controlled-rate freezer, and stored in liquid nitrogen until needed. Antigen-driven lymphocyte proliferation was assayed in quadruplicate cultures37. The HSV antigens consisted of ultraviolet light-inactivated HSV-2 or HSV-1 (10⁵ plaque-forming-unit equivalents before irradiation) or recombinant HSV-2 glycoproteins D or B expressed in Chinese-hamster-ovary cells (1 μg per milliliter) (Chiron, Emeryville, Calif.). Tetanus toxoid (1 Lf unit per milliliter) and phytohemagglutinin A (1 μg per milliliter) were included as positive protein and mitogen controls, respectively.

Results

Case History

The patient was a 24-year-old man who presented in November 1990 with the onset of new, painful penile vesicles. Culture confirmed an HSV infection. Oral acyclovir was prescribed (200 mg five times daily) for five days, and the lesions resolved. The vesicles were first noted to recur in March 1991, and the patient began long-term therapy with acyclovir (200 mg twice daily). Despite this regimen, in June 1991 genital lesions began to recur every two to three weeks, yielding positive cultures for HSV. Lesions appeared only on the dorsum penis and consistently healed within seven days; over a period of months, however, escalating doses of oral acyclovir (up to 800 mg six times daily) failed to suppress recurrences (Figure 1Figure 1Clinical Course of a Man with Acyclovir-Resistant Genital Herpes.).

The patient reported no history of other recurrent infections or systemic illness. He was a sexually active homosexual who before November 1990 had only one partner. In the two weeks before his first outbreak he had three new sexual contacts, two of whom were positive for HIV antibody. One of these contacts acknowledged recurrent genital herpes that was suppressed with long-term acyclovir therapy; he had not noticed a recurrence in 18 months. The man declined further evaluation. Our patient reported his own antibody test for HIV as repeatedly negative over the past two years.

In May 1992, physical examination revealed only fresh, grouped vesicles on the dorsum penis. Acyclovir was withheld for six weeks, during which time three additional recurrences were documented, each healing within seven days and being no more severe or prolonged than those observed during drug treatment. Oral suppressive therapy was then resumed at a dose of 800 mg six times daily. In all, six positive viral cultures were obtained during and after acyclovir therapy over a six-month period (Figure 1).

Laboratory Evaluation

Laboratory studies of the patient's immune system were normal. The complete blood count, the differential count, and the results of a panel of 22 tests of blood chemistry and multiple serologic tests were normal. Immunoglobulin levels were normal. Serum antibodies to HSV-2 were detected by commercial enzyme immunoassay (BioWhittaker). The total CD4 lymphocyte count was 1001 cells per cubic millimeter. Enzyme immunoassay was negative for antibody to the human T-cell lymphotropic virus type I. ELISA and Western blotting for HIV antibody and ELISA for p24 antigen were all negative initially and again seven months later (in January 1993). Intradermal testing revealed normal delayed-type hypersensitivity responsiveness. Antigen-driven lymphocyte blastogenic responses to whole ultraviolet-inactivated HSV-2, ultraviolet-inactivated HSV-1, recombinant HSV-2 glycoproteins D and B, and tetanus toxoid, as well as the mitogenic response to phytohemagglutinin A, were normal as compared with the responses of 35 normal subjects with recurrent genital herpes. The serum level of acyclovir two hours after an oral dose of 800 mg (the peak level) was within the normally therapeutic range at 1.0 μg per milliliter, and the level immediately before a subsequent oral dose (the trough level) was 0.85 μg per milliliter.

Characterization of Virus Isolates

To confirm serial reactivations of the same strain of HSV-2, we performed restriction-endonuclease digestions with six enzymes28. These analyses demonstrated that the six clinical isolates were identical strains of HSV-2 and were distinct from reference strain HSV-2 333 (data not shown).

The antiviral susceptibilities of the clinical isolates were assessed in parallel with those of several wild-type and mutant strains of HSV-1 and HSV-2 previously characterized in one of our laboratories (Table 1Table 1In Vitro Drug-Sensitivity Testing of Viral Strains from the Patient and Reference Strains.)3,15,26. All six isolates obtained from the patient during or after treatment were moderately resistant to acyclovir (Table 1), with the ID₅₀ ranging from 6 to 16.3 μg per milliliter. The isolates were also resistant to ganciclovir, but they were sensitive to 1-beta-d-arabinofuranosyl thymine, which is phosphorylated by the viral thymidine kinase, and to two drugs that do not require activation by HSV thymidine kinase for viral inhibition, sodium phosphonoacetic acid and trisodium phosphonoformate (foscarnet) (Table 1)38,39. This pattern of in vitro drug sensitivity indicates a normal viral polymerase enzyme and implies a mutation in the viral thymidine kinase gene.

[¹²⁵I]dC autoradiography revealed that the clinical isolates contained a mixed population of viruses in which an abnormal thymidine kinase phenotype dominated. Within the four isolates studied, 92 to 98 percent of plaques were due to virus with the TK^A phenotype, whereas 2 to 8 percent had the wild-type TK⁺ phenotype (Figure 2Figure 2[¹²⁵I]dC Autoradiographs of Four Clinical Isolates Obtained from the Patient and Three Reference Strains.). To obtain pure stocks of TK^A virus from each of four clinical isolates, three cycles of plaque purification were performed and confirmed by [¹²⁵I]dC autoradiography.

To characterize further the thymidine kinase activity of the isolates from our patient, extracts were made of 143B cells infected with the clinical isolates, their derivative plaque-pure TK^A viruses, or two previously described reference viruses, TK⁺ HSV-2 12927 and TK^A HSV-2 1272023. The cell extracts were assayed for the ability to phosphorylate nucleosides. Phosphorylation of acyclovir by plaque-pure TK^A virus from the patient was depressed to 8 to 27 percent that of the TK⁺ HSV-2 12927 (data not shown). Although such phosphorylation by the patient's virus remained 54 times greater than that of the TK^A HSV 12720 control, the extent to which the patient's viruses phosphorylated acyclovir was not adequate to confer susceptibility to the drug on them. In addition the TK^A isolates from our patient demonstrated supranormal rates of thymidine phosphorylation (data not shown). Thus, the biochemical data support the theory that a thymidine kinase enzyme in the patient's virus was altered both in its ability to phosphorylate acyclovir and in its phosphorylation of its natural substrate thymidine.

We next defined the molecular basis for the thymidine kinase alterations in these clinical isolates. The cloned PCR-amplified product from isolate 4365-9 (plasmid pGTK9-2) was sequenced, and four discrete point mutations were identified in its thymidine kinase gene relative to the published sequence of HSV-2 strain 333 (Table 2Table 2Mutations Identified in the Thymidine Kinase Gene of Acyclovir-Resistant HSV-2 Clinical Isolates as Compared with Reference Strain HSV-2 333.). These mutations were confirmed by direct PCR sequencing, without intervening cloning, of the amplification products of the remaining clinical isolates, the plaque-purified viruses, and the amplified HSV-2 333 wild-type virus35. The mutation at nucleotide 529 was critical in altering the domain of the viral thymidine kinase purported to interact with acyclovir, the nucleoside-binding site; this mutation probably underlies our patient's clinical resistance to acyclovir22.

Discussion

Over the past decade, acyclovir-resistant HSV has been documented in vitro, in animal models, and in diverse populations of immunocompromised patients. Acyclovir-resistant TK⁺ and TK^A strains of HSV have been isolated from immunocompetent patients, but when evaluated carefully they proved to be transient and clinically unimportant23. We document here that an immunocompetent person can have repeated reactivations of drug-resistant HSV even when acyclovir is withdrawn. In the patient we describe, the resistant virus was clinically important because it was refractory to acyclovir treatment.

Acyclovir-resistant HSV is well documented in the HIV-infected community. It is possible that in the course of sexual contact our patient acquired a virus that was inherently resistant to acyclovir7,8. Alternatively, resistance may have evolved over the course of his long-term, slowly escalating therapy. Without knowing what viruses were harbored by his sexual partners, it is impossible to discern exactly how acyclovir resistance developed in this patient.

Acyclovir-resistant pools of HSV typically represent heterogeneous mixtures of virus, and the antiviral-susceptibility curves and thymidine kinase biochemical assays of a given strain reflect the net composition of that pool4. Thus, the mixture of TK^A and TK⁺ viruses in the isolates from our patient (Figure 2) is not surprising. Both viral phenotypes are probably derived from the same strain, persisting in ganglia and emerging episodically to become reactivated in this patient. The moderate reductions in phosphorylation of acyclovir by the virus mixture were not reduced further after plaque purification, indicating that the TK⁺ viruses did not contribute substantially to the total phosphorylating activity of the pool. Unlike TK^- mutants, which usually show diminished pathogenicity and capacity for reactivation in animal models, TK^A viruses are fully pathogenic and capable of reactivation2. It is precisely a TK^A strain that one would predict as most likely to underlie stable drug resistance in an immunocompetent person.

The conformation and active sites of the HSV-1 and HSV-2 thymidine kinase molecules have been proposed on the basis of amino acid homology with other known nucleoside-binding domains24,39-41. Most of the acyclovir-resistant thymidine kinase mutants studied to date contained single mutations or premature stop codons3,22,24. The virus shed by our patient was unusual in possessing four nonterminating mutations (Table 2). The amino acid substitutions at positions 78 and 220 are probably trivial, and the importance of the alteration in residue 140 is unknown. The mutation at nucleotide 529 lies in a location shown by Darby et al. to be sufficient when mutated to confer a TK^A phenotype in HSV-1,22 although in our patient's HSV-2 isolate the replacement of arginine by tryptophan presents a more drastic substitution than the glutamine substitution in Darby's strain Tr7. Thus, the substitution at base 529 in our patient's viral thymidine kinase may in itself be adequate to account for the decreased thymidine kinase phosphorylation of acyclovir.

Despite the occurrence of resistant HSV infections in severely immunocompromised patients, oral acyclovir has provided effective suppression of frequently recurring genital herpes in immunocompetent people for more than a decade14,42-44. Our patient presents a new limitation to this therapeutic approach. He carries and has repeated reactivations of an HSV-2 strain bearing thymidine kinase mutations that confer in vitro and clinically important resistance to acyclovir. He continues to have symptomatic recurrences, although his intact immune responses limit the duration and severity of each outbreak. Although high-dose intravenous acyclovir or intravenous foscarnet might suppress his recurrences, they are expensive and carry potential risk, and any benefit would probably be temporary.

The carriage and reactivation of acyclovir-resistant HSV may still be rare and unrecognized in immunocompetent people. Vigilance is needed, as are new therapeutic and preventive strategies for HSV disease.

We are indebted to the late Mr. Holly Smith for excellent technical assistance and invaluable contributions over four decades, to Dr. Paulo de Miranda for determining acyclovir serum levels, to Dr. Scott Fritz of PRI/CynCorp for purifying and cryopreserving the patient's lymphocytes, to Dr. Sharon Safrin for review of the manuscript, to Dr. James Fyfe for helpful discussion and review of the manuscript, and to William Barrick, R.N., for referring this patient for study.

Source Information

From the Medical Virology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. (R.G.K., S.E.S.); the Division of Virology, Burroughs Wellcome Company, Research Triangle Park, N.C. (E.L.H.); and Chiron Corporation, Emeryville, Calif. (M.T.).

Address reprint requests to Dr. Straus at the Laboratory of Clinical Investigation, Bldg. 10, Rm. 11N228, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892.

References

References

1. 1

   Whitley RJ, Gnann JW Jr. Acyclovir: a decade later. N Engl J Med 1992;327:782-789
   Full Text | Web of Science | Medline

2. 2

   Darby G, Field HJ, Salisbury SA. Altered substrate specificity of herpes simplex virus thymidine kinase confers acyclovir-resistance. Nature 1981;289:81-83
   CrossRef | Web of Science | Medline

3. 3

   Field HJ, Darby G, Wildy P. Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus. J Gen Virol 1980;49:115-124
   CrossRef | Web of Science | Medline

4. 4

   Field HJ. Development of clinical resistance to acyclovir in herpes simplex virus-infected mice receiving oral therapy. Antimicrob Agents Chemother 1982;21:744-752
   Web of Science | Medline

5. 5

   Sacks SL, Wanklin RJ, Reece DE, Hicks KA, Tyler KL, Coen DM. Progressive esophagitis from acyclovir-resistant herpes simplex: clinical roles for DNA polymerase mutants and viral heterogeneity? Ann Intern Med 1989;111:893-899
   Web of Science | Medline

6. 6

   Sibrack CD, Gutman LT, Wilfert CM, et al. Pathogenicity of acyclovir-resistant herpes simplex virus type 1 from an immunodeficient child. J Infect Dis 1982;146:673-682
   CrossRef | Web of Science | Medline

7. 7

   Chatis PA, Miller CH, Schrager LE, Crumpacker CS. Successful treatment with foscarnet of an acyclovir-resistant mucocutaneous infection with herpes simplex virus in a patient with acquired immunodeficiency syndrome. N Engl J Med 1989;320:297-300
   Full Text | Web of Science | Medline

8. 8

   Erlich KS, Mills J, Chatis P, et al. Acyclovir-resistant herpes simplex virus infections in patients with the acquired immunodeficiency syndrome. N Engl J Med 1989;320:293-296
   Full Text | Web of Science | Medline

9. 9

   Erlich KS, Jacobson MA, Koehler JE, et al. Foscarnet therapy for severe acyclovir-resistant herpes simplex virus type-2 infections in patients with the acquired immunodeficiency syndrome (AIDS): an uncontrolled trial. Ann Intern Med 1989;110:710-713
   Web of Science | Medline

10. 10

    Englund JA, Zimmerman ME, Swierkosz EM, Goodman JL, Scholl DR, Balfour HH Jr. Herpes simplex virus resistant to acyclovir: a study in a tertiary care center. Ann Intern Med 1990;112:416-422
    Web of Science | Medline

11. 11

    Hill EL, Hunter GA, Ellis MN. In vitro and in vivo characterization of herpes simplex virus clinical isolates recovered from patients infected with human immunodeficiency virus. Antimicrob Agents Chemother 1991;35:2322-2328
    Web of Science | Medline

12. 12

    Safrin S, Crumpacker C, Chatis P, et al. A controlled trial comparing foscarnet with vidarabine for acyclovir-resistant mucocutaneous herpes simplex in the acquired immunodeficiency syndrome. N Engl J Med 1991;325:551-555
    Full Text | Web of Science | Medline

13. 13

    Lehrman SN, Hill EL, Rooney JF, Ellis MN, Barry DW, Straus SE. Extended acyclovir therapy for herpes genitalis: changes in virus sensitivity and strain variation. J Antimicrob Chemother 1986;18:Suppl B:85-94
    Web of Science | Medline

14. 14

    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;310:1545-1550
    Full Text | Web of Science | Medline

15. 15

    McLaren C, Corey L, Dekket C, Barry DW. In vitro sensitivity to acyclovir in genital herpes simplex viruses from acyclovir-treated patients. J Infect Dis 1983;148:868-875
    CrossRef | Web of Science | Medline

16. 16

    Dekker C, Ellis MN, McLaren C, Hunter G, Rogers J, Barry DW. Virus resistance in clinical practice. J Antimicrob Chemother 1983;12:Suppl B:137-152
    Web of Science | Medline

17. 17

    Lehrman SN, Douglas JM, Corey L, Barry DW. Recurrent genital herpes and suppressive oral acyclovir therapy: relation between clinical outcome and in-vitro drug sensitivity. Ann Intern Med 1986;104:786-790
    Web of Science | Medline

18. 18

    Fyfe JA, Keller PM, Furman PA, Miller RL, Elion GB. Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine. J Biol Chem 1978;253:8721-8727
    Web of Science | Medline

19. 19

    Miller WH, Miller RL. Phosphorylation of acyclovir (acycloguanosine) monophosphate by GMP kinase. J Biol Chem 1980;255:7204-7207
    Web of Science | Medline

20. 20

    Miller WH, Miller RL. Phosphorylation of acyclovir diphosphate by cellular enzymes. Biochem Pharmacol 1982;31:3879-3884
    CrossRef | Web of Science | Medline

21. 21

    Furman PA, St Clair MH, Fyfe JA, Rideout JL, Keller PM, Elion GB. Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate. J Virol 1979;32:72-77
    Web of Science | Medline

22. 22

    Darby G, Larder BA, Inglis MM. Evidence that the `active centre' of the herpes simplex virus thymidine kinase involves an interaction between three distinct regions of the polypeptide. J Gen Virol 1986;67:753-758
    CrossRef | Web of Science | Medline

23. 23

    Ellis MN, Keller PM, Fyfe JA, et al. Clinical isolate of herpes simplex virus type 2 that includes a thymidine kinase with altered substrate specificity. Antimicrob Agents Chemother 1987;31:1117-1125
    Web of Science | Medline

24. 24

    Kit S, Sheppard M, Ichimura H, et al. Nucleotide sequence changes in thymidine kinase gene of herpes simplex virus type 2 clones from an isolate of a patient treated with acyclovir. Antimicrob Agents Chemother 1987;31:1483-1490
    Web of Science | Medline

25. 25

    Coen DM, Schaffer PA. Two distinct loci confer resistance to acycloguanosine in herpes simplex virus type 1. Proc Natl Acad Sci U S A 1980;77:2265-2269
    CrossRef | Web of Science | Medline

26. 26

    Collins P, Larder BA, Oliver NM, Kemp S, Smith IW, Darby G. Characterization of a DNA polymerase mutant of herpes simplex virus from a severely immunocompromised patient receiving acyclovir. J Gen Virol 1989;70:375-382
    CrossRef | Web of Science | Medline

27. 27

    Straus SE, Aulakh HS, Ruyechan WT, et al. Structure of varicella-zoster virus DNA. J Virol 1981;40:516-525
    Web of Science | Medline

28. 28

    Roizman B, Tognon M. Restriction enzyme analysis of herpesvirus DNA: stability of restriction endonuclease patterns. Lancet 1982;1:677-677
    Web of Science | Medline

29. 29

    Analysis and cloning of eukaryotic genomic DNA. In: Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Vol. 2. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989:9.31-9.62.
    

30. 30

    Martin JL, Ellis MN, Keller PM, et al. Plaque autoradiography assay for the detection and quantitation of thymidine kinase-deficient and thymidine kinase-altered mutants of herpes simplex virus in clinical isolates. Antimicrob Agents Chemother 1985;28:181-187
    Web of Science | Medline

31. 31

    Summers WC, Summers WP. [¹²⁵I]deoxycytidine used in a rapid, sensitive, and specific assay for herpes simplex virus type 1 thymidine kinase. J Virol 1977;24:314-318
    Web of Science | Medline

32. 32

    Tenser RB, Jones JC, Ressel SJ, Fralish FA. Thymidine plaque autoradiography of thymidine kinase-positive and thymidine kinase-negative herpesviruses. J Clin Microbiol 1983;17:122-127
    Web of Science | Medline

33. 33

    Fyfe JA, McKee SA, Keller PM. Altered thymidine-thymidylate kinases from strains of herpes simplex virus with modified drug sensitivities to acyclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine. Mol Pharmacol 1983;24:316-323
    Web of Science | Medline

34. 34

    Innis MA. PCR with 7-deaza-2'-deoxyguanosine triphosphate. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols. New York: Harcourt Brace Jovanovich, 1990:54-9.
    

35. 35

    Swain MA, Galloway DA. Nucleotide sequence of the herpes simplex virus type 2 thymidine kinase gene. J Virol 1983;46:1045-1050
    Web of Science | Medline

36. 36

    Winship PR. An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res 1989;17:1266-1266
    CrossRef | Web of Science | Medline

37. 37

    James SP. Immunologic studies in humans. In: Coligan JE, Kruisbeck AM, Margulis DH, Shevach EM, Strober W, eds. Current protocols in immunology. New York: Wiley Interscience, 1991:7.10.4-7.10.6.
    

38. 38

    Cheng Y-C, Dutschman G, Fox JJ, Watanabe KA, Machida H. Differential activity of potential antiviral nucleoside analogs on herpes simplex virus-induced and human cellular thymidine kinases. Antimicrob Agents Chemother 1981;20:420-423
    Web of Science | Medline

39. 39

    Field H, McMillan A, Darby G. The sensitivity of acyclovir-resistant mutants of herpes simplex virus to other antiviral drugs. J Infect Dis 1981;143:281-285
    CrossRef | Web of Science | Medline

40. 40

    Kit S. Thymidine kinase. Microbiol Sci 1985;2:369-375
    Medline

41. 41

    Davison AJ, Scott JE. The complete DNA sequence of varicella-zoster virus. J Gen Virol 1986;67:1759-1816
    CrossRef | Web of Science | Medline

42. 42

    Douglas JM, Critchlow C, Benedetti J, et al. A double-blind study of oral acyclovir for suppression of recurrences of genital herpes simplex virus infection. N Engl J Med 1984;310:1551-1556
    Full Text | Web of Science | Medline

43. 43

    Halsos AM, Salo OP, Lassus A, et al. Oral acyclovir suppression of recurrent genital herpes: a double-blind, placebo-controlled, crossover study. Acta Derm Venereol Suppl (Stockh) 1985;54:59-63
    

44. 44

    Kroon S, Petersen CS, Andersen LP, Rasmussen LP, Vestergaard BF. Oral acyclovir suppressive therapy in severe recurrent genital herpes. Dan Med Bull 1989;36:298-300
    Web of Science | Medline

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8. 8

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9. 9

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   CrossRef

10. 10

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    CrossRef

11. 11

    Lara B. Strick, Anna Wald, Connie Celum. (2006) HIV/AIDS: Management of Herpes Simplex Virus Type 2 Infection in HIV Type 1–Infected Persons. Clinical Infectious Diseases 43:3, 347-356
    CrossRef

12. 12

    G. A. Diaz, R. M. Rakita, D. M. Koelle. (2005) A Case of Ramsay Hunt-Like Syndrome Caused by Herpes Simplex Virus Type 2. Clinical Infectious Diseases 40:10, 1545-1547
    CrossRef

13. 13

    Adnan A. Bekhit, Ola A. El-Sayed, Hassan Y. Aboul-Enein, Yunus M. Siddiqui, Mohammed N. Al-Ahdal. (2005) Evaluation of Some Pyrazoloquinolines as Inhibitors of Herpes Simplex Virus Type 1 Replication. Archiv der Pharmazie 338:2-3, 74-77
    CrossRef

14. 14

    Stephen L Sacks, Fred Y Aoki. (2005) Famciclovir for the Management of??Genital Herpes Simplex in Patients??with Inadequate Response to??Aciclovir or Valaciclovir. Clinical Drug Investigation 25:12, 803-809
    CrossRef

15. 15

    Myron J. Levin, Teresa H. Bacon, Jeffry J. Leary. (2004) Resistance of Herpes Simplex Virus Infections to Nucleoside Analogues in HIV‐Infected Patients. Clinical Infectious Diseases 39:s5, S248-S257
    CrossRef

16. 16

    Lawrence Corey, Anna Wald, Connie L. Celum, Thomas C. Quinn. (2004) The Effects of Herpes Simplex Virus-2 on HIV-1 Acquisition and Transmission: A Review of Two Overlapping Epidemics. JAIDS Journal of Acquired Immune Deficiency Syndromes 35:5, 435-445
    CrossRef

17. 17

    Crumpacker, Clyde S., . (2004) Use of Antiviral Drugs to Prevent Herpesvirus Transmission. New England Journal of Medicine 350:1, 67-68
    Full Text

18. 18

    Florence Morfin, Danielle Thouvenot. (2003) Herpes simplex virus resistance to antiviral drugs. Journal of Clinical Virology 26:1, 29-37
    CrossRef

19. 19

    Daniele Esquenazi, Marcia D. Wigg, Mônica M.F.S. Miranda, Hugo M. Rodrigues, João B.F. Tostes, Sonia Rozental, Antonio J.R. da Silva, Celuta S. Alviano. (2002) Antimicrobial and antiviral activities of polyphenolics from Cocos nucifera Linn. (Palmae) husk fiber extract. Research in Microbiology 153:10, 647-652
    CrossRef

20. 20

    Yu-Chun Wang, Chuan-Liang Kao, Wu-Tse Liu, Jun-Ren Sun, Yi-Er Tai, Szu-Hao Kung. (2002) A cell line that secretes inducibly a reporter protein for monitoring herpes simplex virus infection and drug susceptibility. Journal of Medical Virology 68:4, 599-605
    CrossRef

21. 21

    Stephen K. Tyring, David Baker, Wendy Snowden. (2002) Valacyclovir for Herpes Simplex Virus Infection: Long‐Term Safety and Sustained Efficacy after 20 Years’ Experience with Acyclovir. The Journal of Infectious Diseases 186:s1, S40-S46
    CrossRef

22. 22

    Christelle Danve, Florence Morfin, Danielle Thouvenot, Michèle Aymard. (2002) A screening dye-uptake assay to evaluate in vitro susceptibility of herpes simplex virus isolates to acyclovir. Journal of Virological Methods 105:2, 207-217
    CrossRef

23. 23

    Christian Gilbert, Julie Bestman-Smith, Guy Boivin. (2002) Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms. Drug Resistance Updates 5:2, 88-114
    CrossRef

24. 24

    Kimberly A. Yeung-Yue, Mathijs H. Brentjens, Patricia C. Lee, Stephen K. Tyring. (2002) The management of herpes simplex virus infections. Current Opinion in Infectious Diseases 15:2, 115-122
    CrossRef

25. 25

    Anil Mendiratta, James E. Peacock. (2002) EMPIRIC THERAPY WITH ACYCLOVIR IN PATIENTS WITH PRESUMED VIRAL ENCEPHALITIS: CLINICAL AND ECONOMIC IMPLICATIONS. Infectious Diseases in Clinical Practice 11:2, 58-65
    CrossRef

26. 26

    Daniel T. Leung, Stephen L. Sacks. (2000) Current Recommendations for the Treatment of Genital Herpes. Drugs 60:6, 1329-1352
    CrossRef

27. 27

    Florence Morfin, Danielle Thouvenot, Michle Aymard, Grard Souillet. (2000) Reactivation of acyclovir-resistant thymidine kinase-deficient herpes simplex virus harbouring single base insertion within a 7 Gs homopolymer repeat of the thymidine kinase gene. Journal of Medical Virology 62:2, 247-250
    CrossRef

28. 28

    W Lawrence Drew. (2000) Ganciclovir resistance: a matter of time and titre. The Lancet 356:9230, 609-610
    CrossRef

29. 29

    Astrid Meerbach, Renate Klöcking, Chris Meier, Andreas Lomp, Björn Helbig, Peter Wutzler. (2000) Inhibitory effect of cycloSaligenyl-nucleoside monophosphates (cycloSal-NMP) of acyclic nucleoside analogues on HSV-1 and EBV. Antiviral Research 45:1, 69-77
    CrossRef

30. 30

    Richard L. Miller, Linda M. Imbertson, Michael J. Reiter, John F. Gerster. (1999) Treatment of primary herpes simplex virus infection in guinea pigs by imiquimod. Antiviral Research 44:1, 31-42
    CrossRef

31. 31

    Gertrude B. Elion, Phillip A. Furman, James A. Fyfe, Paulo de Miranda, Lilia Beauchamp, Howard J. Schaeffer. (1999) The selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine. Reviews in Medical Virology 9:3, 147-153
    CrossRef

32. 32

    G. Boivin. (1998) Drug-resistant herpesviruses: Should we look for them?. European Journal of Clinical Microbiology & Infectious Diseases 17:8, 539-541
    CrossRef

33. 33

    S.M. Blower, T.C. Porco, G. Darby. (1998) Predicting and preventing the emergence of antiviral drug resistance in HSV-2. Nature Medicine 4:6, 673-678
    CrossRef

34. 34

    Emanuela Pelosi, Gilbert B Mulamba, Donald M Coen. (1998) Penciclovir and pathogenesis phenotypes of drug-resistant Herpes simplex virus mutants. Antiviral Research 37:1, 17-28
    CrossRef

35. 35

    M.M.F.S. Miranda, A.P. Almeida, S.S. Costa, M.G.M. Santos, M.H.C. Lagrota, M.D. Wigg. (1997) In vitro activity of extracts of Persea americana leaves on acyclovir-resistant and phosphonoacetic resistant Herpes simplex virus. Phytomedicine 4:4, 347-352
    CrossRef

36. 36

    Richard L. Hodinka. (1997) WHAT CLINICIANS NEED TO KNOW ABOUT ANTIVIRAL DRUGS AND VIRAL RESISTANCE. Infectious Disease Clinics of North America 11:4, 945-967
    CrossRef

37. 37

    Emily Erbelding, Thomas C. Quinn. (1997) THE IMPACT OF ANTIMICROBIAL RESISTANCE ON THE TREATMENT OF SEXUALLY TRANSMITTED DISEASES. Infectious Disease Clinics of North America 11:4, 889-903
    CrossRef

38. 38

    Stephen H. Gillespie, Timothy D. McHugh. (1997) The biological cost of antimicrobial resistance. Trends in Microbiology 5:9, 337-339
    CrossRef

39. 39

    Barbara Vonau, Naomi Low-Beer, Simon E. Barton, J. Richard Smith. (1997) Antenatal serum screening for genital herpes: a study of knowledge and attitudes of women at a central London hospital. BJOG: An International Journal of Obstetrics and Gynaecology 104:3, 347-349
    CrossRef

40. 40

    P. Reusser. (1996) Herpesvirus resistance to antiviral drugs: a review of the mechanisms, clinical importance and therapeutic options. Journal of Hospital Infection 33:4, 235-248
    CrossRef

41. 41

    Teresa H. Bacon. (1996) Famciclovir, from the bench to the patient — a comprehensive review of preclinical data. International Journal of Antimicrobial Agents 7:2, 119-134
    CrossRef

42. 42

    Bernard Weber, Jindrich Cinatl. (1996) Antiviral therapy of herpes simplex virus infection: recent developments 1. Journal of the European Academy of Dermatology and Venereology 6:2, 112-126
    CrossRef

43. 43

    H. HUNTER HANDSFIELD. (1996) Acyclovir Should Not Be Approved for Marketing Without Prescription. Sexually Transmitted Diseases 23:3, 171-173
    CrossRef

44. 44

    KENNETH H. FIFE. (1996) Over-the-Counter Acyclovir. Sexually Transmitted Diseases 23:3, 174-176
    CrossRef

45. 45

    Karl Broman, Terry Speed, Michael Tigges. (1996) Estimation of antigen-responsive T cell frequencies in PBMC from human subjects. Journal of Immunological Methods 198:2, 119-132
    CrossRef

46. 46

    OMEED M. MEMAR, STEPHEN K. TYRING. (1995) ANTIVIRAL AGENTS IN DERMATOLOGY: CURRENT STATUS AND FUTURE PROSPECTS. International Journal of Dermatology 34:9, 597-606
    CrossRef

47. 47

    David W. Kimberlin, Clayde S. Crumpacker, Stephen E. Straus, Karen K. Biron, W.Lawrence Drew, Frederick G. Hayden, Mark McKinlay, Douglas D. Richman, Richard J. Whitley. (1995) Antiviral resistance in clinical practice. Antiviral Research 26:4, 423-438
    CrossRef

48. 48

    David W. Kimberlin, Donald M. Coen, Karen K. Biron, Jeffrey I. Cohen, Robert A. Lamb, Mark McKinlay, Emilio A. Emini, Richard J. Whitley. (1995) Molecular mechanisms of antiviral resistance. Antiviral Research 26:4, 369-401
    CrossRef

49. 49

    Silvio Masci, Massimo Gravante, Maria Andreassi, Pierluigi Amerio, Gino Santini, Claudio de Simone, Giuseppe Famularo, Marina Ciancarelli. (1995) Intravenous Immunoglobulins Suppress the Recurrences of Genital Herpes Simplex Virus: A Clinical and Immunological Study. Immunopharmacology and Immunotoxicology 17:1, 33-47
    CrossRef

50. 50

    Donald M. Coen. (1994) Acyclovir-resistant, pathogenic herpesviruses. Trends in Microbiology 2:12, 481-485
    CrossRef

51. 51

    G. Carrega, E. Castagnola, A. Canessa, P. Argenta, R. Haupt, G. Dini, A. Garaventa. (1994) Herpes simplex virus and oral mucositis in children with cancer. Supportive Care in Cancer 2:4, 266-269
    CrossRef