GlaxoSmithKline, UK Virology Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
Correspondence
Rob Fenton
rob.j.fenton{at}gsk.com
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ABSTRACT |
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INTRODUCTION |
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Laboratory studies have shown that resistance can arise through mutation in either the thymidine kinase (TK) gene or the DNA polymerase gene, with most resistance involving alterations or deletions in the TK gene, leading to partial or complete failure to phosphorylate ACV (Coen & Schaffer, 1980; Collins & Darby, 1991
; Hill et al., 1991
).
There is considerable evidence from studies in animal models that TK-deficient variants of herpes simplex virus (HSV) are less virulent than wild-type viruses (Field & Wildy, 1978; Field & Darby, 1980
; Tenser et al., 1981
; Sibrack et al., 1982
). Furthermore, in murine models of HSV infection, TK-deficient viruses establish latency with lower efficiency and reactivate poorly (Tenser & Dunstan, 1979
; Efstathiou et al., 1989
; Coen et al., 1989
; Jacobson et al., 1993
). However, there have also been reports in the literature that some clinically derived TK-deficient viruses may retain a degree of virulence (Sakuma et al., 1988
; Erlich et al., 1989
; Kost et al., 1993
; Hwang et al., 1994
; Sasadeusz & Sacks, 1996
; Horsburgh et al., 1998
). Although these observations appear to conflict, it is often difficult to distinguish between partial and complete loss of TK activity and this may influence neurovirulence.
The experiments of Efstathiou et al. (1989) showing lack of neurovirulence were performed using a laboratory-generated TK deletion mutant that was unlikely to express any TK functionality. In clinical isolates, the most common mutation described to date is a single G insertion in the G-string motif, a run of seven guanosine nucleotides from position 433 to 439, which appears to be a hotspot for mutation (Sasadeusz et al., 1997
). This mutation results in a -1 frame-shift upstream of the critical nucleoside-binding residues (Darby et al., 1986
; Brown et al., 1995
) and the induction of a truncated TK polypeptide. However, viruses with a single G insertion have been shown to reactivate from latently infected ganglia (Hwang et al., 1994
; Sasadeusz et al., 1997
). The explanation proposed was that a low frequency compensatory +1 translational frame-shift in the G-string region, during protein synthesis, resulted in a small amount of wild-type protein being produced (Hwang et al., 1994
).
Certain other neurovirulent TK mutants isolated in the clinic appear to have single amino acid substitutions (Horsburgh et al., 1998), which would be consistent with the hypothesis that their TK polypeptides retain a level of TK functionality. Once again, the picture is not absolutely clear. Horsburgh et al. (1998)
described a mutant of this type that, even by the most sensitive techniques, appeared to be totally deficient in TK functionality and this variant retained neurovirulence. They suggested that there could be other gene functions in HSV that could compensate for the loss of TK, supporting their conclusion by demonstrating a TK deletion mutant constructed in the same genetic background retained neurovirulence.
It is clear that the function of TK in neurovirulence remains poorly understood. The aim of the present study was to investigate further the role of TK through characterization of a panel of HSV-1 and HSV-2 clinical isolates exhibiting resistance to ACV as a consequence of mutation in the TK gene, and to identify the mutations responsible for ACV resistance.
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METHODS |
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Cells.
Vero cells were grown in GME medium supplemented with 10 % foetal calf serum (FCS), 100 units penicillin ml-1, 100 µg streptomycin ml-1 and 0·01 mM glutamine.
BuBHK cells, derived from baby hamster kidney (BHK) cells, are devoid of cellular TK activity following passage in the presence of 5-bromo-2'-deoxyuridine. These cells were grown in the same medium as described above.
All cells were grown, and assays performed, at 37 °C in 5 % CO2.
TK assay.
Confluent monolayers of BuBHK cells were infected with 10 p.f.u. per cell of virus and incubated at 37 °C. Cells were harvested after 18 h and extracts were prepared as described previously (Larder & Darby, 1984). Sonicates were assayed for TK activity at 37 °C, as described by Klemperer et al. (1967)
, using [14C]thymidine as the substrate at 30 µM (1·6 µCi ml-1; 5·92x104 Bq).
Sequence analysis.
The DNA from cloned viruses was analysed by cycle sequencing of PCR-generated products. A total of 12 overlapping sense and anti-sense internal primers that encompassed the entire HSV-1 and HSV-2 TK open reading frame was used (Table 2). Sequencing reactions were carried out using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit and electrophoresed on an ABI 377 sequencer, according to the manufacturer's instructions. The resulting sequence was analysed using Lasergene software (DNASTAR).
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In order to test the ability of viruses to reactivate from latency, cervical ganglia C2 and C3 were removed from mice at least 4 weeks after infection and cultured in medium 199 supplemented with 200 mM DMSO (Sasadeusz et al., 1997). Aliquots of medium (100 µl) were removed daily and assayed for infectious virus by plaque assay (Collins et al., 1982
; Collins & Oliver, 1986
). Aliquots were replaced with fresh medium (200 µl) on each occasion.
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RESULTS |
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Sequence analysis
HSV-1 isolates.
Paired clones from each of the six isolates had, in all cases but one, identical sequences (Table 3). The exceptions were the distinct clones from isolate 3. Candidate mutations in the TK gene that could explain the ACV-resistant phenotypes of these viruses (Fig. 1
) were identified initially by comparing sequences with those of the wild-type control, SC16. Of the 12 clones sequenced, eight had insertions or deletions in the G-string motif. The most common lesion, seen in five clones, was a single G insertion (clones C2b, C2c, C3b, C5a and C5c). Both clones from isolate 4 had a novel double G (GG) insertion and one clone from isolate 3 (clone C3c) had a single G deletion.
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The expected result of the single G deletion in clone C3c would be a +1 frame-shift and, in this case, the first stop encountered in the +1 reading frame would be that at residue 182, resulting in a predicted polypeptide of 181 aa. The GG insertion in clones C4a and C4b would cause a -2 frame-shift, resulting in the predicted polypeptide of 182 aa.
One other pair of clones would be expected to induce truncated polypeptides. Clones C1b and C1d both have nucleotide substitutions that result in the introduction of premature stop codons at residue 336. The predicted peptide would have the authentic TK sequence but lack the C-terminal 41 aa.
Only clones from isolate 6 would be expected to encode full-length polypeptides. Candidate lesions were identified by comparison of the sequence with that of SC16 and any ambiguities were resolved by extending the analysis to all available HSV-1 TK sequences. The lesion most likely to be responsible for ACV resistance was an amino acid substitution close to the C terminus at residue 364, LeuPro.
HSV-2 isolates.
Paired clones of the four HSV-2 isolates were analysed as described above (Fig. 1). Paired clones from each isolate had, in all but one case, identical sequences (Table 4
). Comparisons were made with the sequence of the wild-type HSV-2 strain 333 to detect candidate lesions that could be responsible for the ACV-resistant phenotypes and again ambiguities were removed by comparisons with all available HSV-2 TK sequences.
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The remaining four clones should induce full-length polypeptides with amino acid substitutions. In the case of clones from isolate 10, the sequences were identical, with an amino acid substitution at residue 201 (GlyAsp). The two clones from isolate 7, however, were different: clone C7a having a Glu
Lys substitution at residue 226 and clone C7c having a Leu
Pro substitution at residue 158.
In an attempt to confirm the size of the TK polypeptides expressed, Western blotting of the TK protein from a representative panel of clones was performed using a polyclonal rabbit antiserum produced against purified HSV-1 TK (data not shown).
Clones C2b, C3b, C4b, C5a and C6a all induced polypeptides of the expected sizes (clone C6a full-length and the remainder truncated). However, the predicted truncated polypeptide induced by clone C1b could not be detected (data not shown). This lack of recognition is probably a result of the absence of key epitopes in the truncated polypeptide.
Pathogenesis in the mouse zosteriform model
The pathogenesis of these drug-resistant clones was investigated in a mouse zosteriform model. The model distinguishes very clearly between the behaviour of the wild-type control virus, SC16, and the TK-deficient SC16-derived variant, DM21 (Table 5). Both viruses replicated efficiently at the primary inoculation site in the neck, achieving high titres at day 3. However, by day 6, the titre of the recovered DM21 was consistently lower than SC16. The wild-type virus was detected in the local cervical ganglia (C2 and C3) at high titres at day 3 and at low titres at the secondary peripheral site in the ear. By day 6, titres in the ganglia had waned but those in the ear had reached high levels. Consistent with previous observations, the deletion mutant, DM21, was not detected in the ganglia or at the peripheral site in the ear at either time.
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The behaviour of clones C4a and C4b was quite distinct from that of the other clones. Virus was detected in the peripheral nervous system at days 3 and 6 and, by day 6, the infection had clearly spread to the secondary peripheral site in the ear.
In each case, attempts were made to sequence recovered virus, both directly from the recovered sample and after attempting to re-grow a stock to provide sufficient material for sequencing. However, with the exception of clones C4a and C4b, this proved to be unsuccessful.
Accumulating data indicated that clones C4a and C4b demonstrated properties that were distinct from those observed with the other viruses, which were included in these analyses. The behaviour of C4a and C4b was explored further and those analyses are the subject of the accompanying paper by Grey et al. (2003).
Reactivation from latency
Infected mice were housed for a minimum of 4 weeks, after which time they were sacrificed and the cervical ganglia C2 and C3 were excised and cultured in an attempt to reactivate latent virus (Table 5). Whilst there was evidence of limited/sporadic reactivation from ganglia infected with most clones, consistently higher levels of reactivation were observed from ganglia excised from mice infected with the control virus SC16 and clones derived from isolate 4. No reactivation was detected up to 10 days post-explantation from either the control TK-deficient virus, DM21, clones derived from isolates 1, 5 or 10 or from the unique clone C7c derived from isolate 7. Clone C7a and those derived from the remaining isolates (2, 3, 4, 6, 8 and 9) showed sporadic reactivation (sporadic reactivation being recovery of virus from 50 % or less of animals infected with clones from a specific isolate).
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DISCUSSION |
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Several insertions or deletions in the G-string motif were identified. These would be expected to result in translational frame-shifts, removing more than two-thirds of the wild-type polypeptide sequence [including regions important in nucleoside binding (Darby et al., 1986) and also the highly conserved aspartic acid residue, 162 (Brown et al., 1995
), which is believed to be responsible for Mg2+ co-ordination] and substitution of a random polypeptide sequence. It is extremely unlikely that these molecules would retain TK functionality and it has been assumed, therefore, that the observed insertions or deletions account for the TK-deficient phenotype.
Clones derived from isolate 1 had a nucleotide substitution that introduced a premature stop codon at residue 336 and would be expected to express a polypeptide with a 41 aa C-terminal deletion. Cys336 had itself been shown earlier to be functionally important in nucleoside binding (Darby et al., 1986) and so this may partially explain the TK-deficient phenotype of this variant.
The remaining clones predicted to express truncated polypeptides (isolates 2, 4, 5, 8 and 9 and the two distinct clones C3b and C3c) all had insertions or deletions in the G-string. The G-string is a homogeneous run of seven GC base pairs extending from nucleotide 433 to 439 in the TK gene. This is the longest homopolymer stretch in the TK gene and has been described previously (Sasadeusz et al., 1997) as a hotspot for mutation. Consistent with the work of Sasadeusz et al. (1997)
, the most common mutation was insertion of a single additional G residue, as seen in the clones from isolates 2, 5, 8 and 9 and clone C3b. Clone C3c had a single G deletion, as described previously in a laboratory isolate (Kit et al., 1983
). Most interesting were those clones derived from isolate 4 (clones 4a and 4b), where a novel double G insertion was identified.
Candidate mutations of those clones having only amino acid substitutions, isolates 6 and 10 and the two distinct clones from isolate 7, may explain the ACV resistance through TK deficiency. Although the substitution at Glu226 in clone C7a is in a residue known to contact the active site (Brown et al., 1995), the most perplexing substitution is the Leu364 substitution in clones from isolate 6, close to the C terminus (total polypeptide length 377 aa). This may be yet another indication that there is important, but as yet unidentified, functionality in the C terminus of the TK polypeptide (Darby et al., 1986
).
All predicted truncated polypeptides encoded by HSV-1 isolates with G-string modifications were detected by Western blotting, as were the full-length type 1 polypeptides induced by clones from isolate 6. However, the truncated polypeptide induced by the isolate 1 clones was not detected; this was unexpected, as the predicted polypeptide should have incorporated all the epitopes present in variants with G-string mutations. One possible explanation might be that removal of the C-terminal 41 aa introduces instability into the molecule so that the protein is rapidly degraded.
The mouse neck/ear model was used to investigate the biological phenotype of these cloned viruses. In this model, it was shown that the majority of TK-deficient viruses exhibit dramatically reduced neurovirulence, behaving similarly to the TK-deficient control virus DM21 (Table 4). They showed similar titres at the primary inoculation site, the neck, at days 3 and 6, with virtually no spread to the secondary site in the ear. However, it is notable that virus was recovered sporadically from cervical ganglia C2 and C3 at day 3 following infection with most clones.
Clones from isolate 4 showed a pattern of neurovirulence intermediate to that of DM21 and the wild-type type 1 strain SC16, in that virus was isolated from ganglia at day 6 and also from the secondary ear site at the same time. Furthermore, latent virus was recovered from ganglia 28 days post-infection from 21 of 22 animals tested.
Virus was also recovered sporadically from ganglia explanted from animals infected with many of the other clones. In fact, only paired clones from isolates 1, 5 and 10 and clone C7c, along with the control, DM21, showed no evidence of ganglionic virus. Previous reports have suggested that TK-deficient viruses with G-string insertions retain some neurovirulence (Hwang et al., 1994; Sasadeusz et al., 1997
), neurovirulence in these reports referring to a capability to reactivate from latent infection. The explanation put forward by Hwang et al. (1994)
was that +1 frame-shifting occurred at a lower frequency than translational frame-shifting during translation of the TK polypeptide, resulting in a small proportion of wild-type functional polypeptide. Our data are generally consistent with these observations, since ganglionic virus was isolated sporadically following infection with the paired clones from isolates 2, 7 and 8 and clone C3b (all with G-string insertions). The only clones having a G insertion that failed to show evidence of reactivation were from isolate 5. The explanation for this is unclear, but with a small sample size this may be due simply to chance. There does not appear to be anything in the acute mouse infections that distinguishes these clones from those of other similar isolates. A further possibility is that clones from isolate 5 contain additional unrecognized lesions.
Other clones that showed evidence of sporadic reactivation were clone C3c (a G-string deletion), those derived from isolate 6 (amino acid substitution) and clone C7a (another amino acid substitution). In the case of variants with amino acid substitutions, there may be low level TK activity facilitating reactivation. Two amino acid substitutions that appeared incompatible with reactivation were Leu158Pro, seen in clone C7c, and Gly201
Asp, seen in clones from isolate 10, possibly indicating a more profound effect of these substitutions on TK activity.
The explanation for the reactivation of clone C3c remains to be established, as does the high reactivation frequency of the paired clones from isolate 4. However, there is a parallel between the two. If it is assumed that a low level of functional TK is a pre-requisite for reactivation, then both clone C3c and the paired clones from isolate 4 must induce expression of functional TK activity in animals. In both cases, either translational frame-shifting (either +2 or -1 in the G-string) or mutation resulting in insertion of a single G into the G-string could result in translation in the correct frame and induction of a functional protein. Another possible explanation suggested by the work of Horsburgh et al. (1998) is that there may be functions elsewhere in the genomes of some isolates that can account for the retention of neurovirulence.
We believe that these studies have confirmed that the majority of TK-deficient viruses have low neurovirulence. Generally, true TK-negative viruses (such as TK-deletion mutants) are unable to replicate in the nervous system and, although they may reach local sensory ganglia and establish latent infections, they cannot be reactivated by simple co-cultivation. Viruses able to express a low level of TK functionality are also generally attenuated. They show no evidence of zosteriform spread during acute infections but they may reactivate somewhat inefficiently from latent infections. There are, however, some exceptions, such as isolate 4, which shows evidence of zosteriform spread and efficient reactivation.
One of the difficulties in this field is the terminology used. Neurovirulence can be used to describe a range of interactions with the nervous system that have at one end of the spectrum neurovirulent viruses that replicate poorly, if at all, in sensory ganglia and show no evidence of zosteriform spread but which can be reactivated sporadically from latently infected ganglia. It appears that most of the so-called neurovirulent TK-deficient isolates are probably in this category. At the other end of the spectrum are viruses like those derived from isolate 4, described in this paper and the accompanying paper by Grey et al. (2003), viruses that are able to invade and replicate in sensory ganglia, spread to secondary zosteriform sites and reactivate efficiently from ganglia.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown, D. G., Visse, R., Sandhu, G., Davies, A., Rizkallah, P. J., Melitz, C., Summers, W. C. & Sanderson, M. R. (1995). Crystal structures of the thymidine kinase from herpes simplex virus type-1 in complex with deoxythymidine and ganciclovir. Nat Struct Biol 2, 876881.[Medline]
Christophers, J., Clayton, J., Craske, J., Ward, R., Collins, P., Trowbridge, M. & Darby, G. (1998). Survey of resistance of herpes simplex virus to acyclovir in northwest England. Antimicrob Agents Chemother 42, 868872.
Coen, D. M. & Schaffer, P. A. (1980). Two distinct loci confer resistance to acycloguanosine in herpes simplex virus type 1. Proc Natl Acad Sci U S A 77, 22652269.[Abstract]
Coen, D. M., Irmiere, A. F., Jacobson, J. G. & Kerns, K. M. (1989). Low levels of herpes simplex virus thymidine-thymidylate kinase are not limiting for sensitivity to certain antiviral drugs or for latency in a mouse model. Virology 168, 221231.[Medline]
Collins, P. & Oliver, N. M. (1986). Sensitivity monitoring of herpes simplex virus isolates from patients receiving acyclovir. J Antimicrob Chemother 18 (Suppl. B), 103112.[Abstract]
Collins, P. & Darby, G. (1991). Laboratory studies of herpes simplex virus strains resistant to acyclovir. Rev Med Virol 1, 1928.
Collins, P. & Ellis, M. N. (1993). Sensitivity monitoring of clinical isolates of herpes simplex virus to acyclovir. J Med Virol (Suppl. 1), 5866.
Collins, P., Appleyard, G. A. & Oliver, N. M. (1982). Sensitivity of herpes virus isolates from acyclovir clinical trials. Am J Med 73, 380382.[Medline]
Darby, G., Larder, B. A. & Inglis, M. M. (1986). 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 67, 753758.[Abstract]
Efstathiou, S., Kemp, S., Darby, G. & Minson, A. C. (1989). The role of herpes simplex virus type 1 thymidine kinase in pathogenesis. J Gen Virol 70, 869879.[Abstract]
Erlich, K. S., Mills, J., Chatis, P., Mertz, G. J., Busch, D. F., Follansbee, S. E., Grant, R. M. & Crumpacker, C. S. (1989). Acyclovir-resistant herpes simplex virus infections in patients with the acquired immunodeficiency syndrome. N Engl J Med 320, 293296.[Medline]
Field, H. J. & Wildy, P. (1978). The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice. J Hyg 81, 267277.
Field, H. J. & Darby, G. (1980). Pathogenicity in mice of strains of herpes simplex virus which are resistant to acyclovir in vitro and in vivo. Antimicrob Agents Chemother 17, 209216.[Medline]
Grey, F., Sowa, M., Collins, P., Fenton, R. J., Harris, W., Snowden, W., Efstathiou, S. & Darby, G. (2003). Characterization of a neurovirulent acyclovir-resistant variant of herpes simplex virus. J Gen Virol 84, 14031410.
Hill, T. J., Field, H. J. & Blyth, W. A. (1975). Acute and recurrent infection with herpes simplex virus in the mouse: a model for studying latency and recurrent disease. J Gen Virol 28, 341353.[Abstract]
Hill, E. L., Hunter, G. A. & Ellis, M. N. (1991). In vitro and in vivo characterization of herpes simplex virus clinical isolates recovered from patients infected with human immunodeficiency virus. Antimicrob Agents Chemother 35, 23222328.[Medline]
Horsburgh, B. C., Chen, S. H., Hu, A., Mulamba, G. B., Burns, W. H. & Coen, D. M. (1998). Recurrent acyclovir-resistant herpes simplex in an immunocompromised patient: can strain differences compensate for loss of thymidine kinase in pathogenesis? J Infect Dis 178, 618625.[Medline]
Hwang, C. B., Horsburgh, B. C., Pelosi, E., Roberts, S., Digard, P. & Coen, D. M. (1994). A net +1 frameshift permits synthesis of thymidine kinase from a drug-resistant herpes simplex virus mutant. Proc Natl Acad Sci U S A 91, 54615465.[Abstract]
Jacobson, J. G., Ruffner, K. L., Kosz-Vnenchak, M., Hwang, C. B., Wobbe, K. K., Knipe, D. M. & Coen, D. M. (1993). Herpes simplex virus thymidine kinase and specific stages of latency in murine trigeminal ganglia. J Virol 67, 69036908.[Abstract]
Kit, S., Kit, M., Qavi, H., Trkula, D. & Otsuka, H. (1983). Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene. Biochim Biophys Acta 741, 158170.[Medline]
Klemperer, H. G., Haynes, G. R., Shedden, W. I. & Watson, D. H. (1967). A virus-specific thymidine kinase in BHK-21 cells infected with herpes simplex virus. Virology 31, 120128.[Medline]
Kost, R. G., Hill, E. L., Tigges, M. & Straus, S. E. (1993). Brief report: recurrent acyclovir-resistant genital herpes in an immunocompetent patient. N Engl J Med 329, 17771782.
Larder, B. A. & Darby, G. (1984). Virus drug-resistance: mechanisms and consequences. Antiviral Res 4, 142.[Medline]
Larder, B. A. & Darby, G. (1985). Selection and characterisation of acyclovir-resistant herpes simplex virus type 1 mutants inducing altered DNA polymerase activities. Virology 146, 262271.[Medline]
Lefkovits, I. & Waldemann, H. (1979). Limiting Dilution Analysis of Cells in the Immune System. Cambridge: Cambridge University Press.
Nugier, F., Colin, J. N., Aymard, M. & Langlois, M. (1992). Occurrence and characterization of acyclovir-resistant herpes simplex virus isolates: report on a two-year sensitivity screening survey. J Med Virol 36, 112.[CrossRef][Medline]
O'Brian, J. J. & Campoli-Richards, D. M. (1989). Acyclovir. An updated review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy. Drugs 37, 233309.[Medline]
Parris, D. S. & Harrington, J. E. (1982). Herpes simplex virus variants resistant to high concentrations of acyclovir exist in clinical isolates. Antimicrob Agents Chemother 22, 7177.[Medline]
Sakuma, S., Yamamoto, M., Kumano, Y. & Mori, R. (1988). An acyclovir-resistant strain of herpes simplex virus type 2 which is highly virulent for mice. Arch Virol 101, 169182.[Medline]
Sasadeusz, J. J. & Sacks, S. L. (1996). Spontaneous reactivation of thymidine kinase-deficient, acyclovir-resistant type-2 herpes simplex virus: masked heterogeneity or reversion? J Infect Dis 174, 476482.[Medline]
Sasadeusz, J. J., Tufaro, F., Safrin, S., Schubert, K., Hubinette, M. M., Cheung, P. K. & Sacks, S. L. (1997). Homopolymer mutational hot spots mediate herpes simplex virus resistance to acyclovir. J Virol 71, 38723878.[Abstract]
Sibrack, C. D., McLaren, C. & Barry, D. W. (1982). Disease and latency characteristics of clinical herpes virus isolated after acyclovir therapy. Am J Med 73, 372375.[CrossRef]
Tenser, R. B. & Dunstan, M. E. (1979). Herpes simplex virus thymidine kinase expression in infection of the trigeminal ganglion. Virology 99, 417422.[Medline]
Tenser, R. B., Ressel, S. & Dunstan, M. E. (1981). Herpes simplex virus thymidine kinase expression in trigeminal ganglion infection: correlation of enzyme activity with ganglion virus titer and evidence of in vivo complementation. Virology 112, 328341.[Medline]
Wade, J. C., Newton, B., McLaren, C., Flournoy, N., Keeney, R. E. & Meyers, J. D. (1983). Intravenous acyclovir to treat mucocutaneous herpes simplex virus infection after marrow transplantation: a double-blind trial. Ann Intern Med 96, 265269.
Received 7 October 2002;
accepted 21 February 2003.