Further evidence from a murine infection model that famciclovir interferes with the establishment of HSV-1 latent infections

Alana M. Thackray and Hugh J. Field*

Centre for Veterinary Science, Cambridge University, Madingley Road, Cambridge CB3 0ES, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice were infected with herpes simplex virus type 1 (HSV-1) via the ear pinna. Famciclovir therapy was commenced on days 2–7 post infection (p.i.). The ipsilateral and contralateral trigeminal (TG) and third cervical ganglia (CIII) from individual mice were tested for latency 1 and 6 months after infection by explant culture or in situ hybridization for latency-associated transcripts (LAT). There were significantly fewer LAT-positive neurons in ipsilateral and contralateral TG (but not CIII) when therapy was delayed by up to 6 days. There was a low correlation between the number of LAT-positive neurons and reactivation by explant culture. Latency data for individual ganglia, compared with those from previous studies, allow us to rationalize differences between the effects of nucleosides on the establishment of latency in different anatomical sites and when tissues are evaluated using different techniques. The implications of the findings for the use of famciclovir to counter HSV latency in humans are addressed.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The central problem that confronts those developing chemotherapy for herpes simplex virus (HSV) is latency. During the acute infection that follows first exposure to HSV, the virus enters the peripheral nerves and establishes a lifelong latent infection in peripheral ganglionic neurons from which reactivations may occur from time to time. This provides a source of virus giving the familiar pattern of recurrent lesions, at or near, the site of the original infection. There is a consensus that chemotherapy using conventional nucleoside analogues, such as acyclovir, does not eliminate long-term latent HSV infection from the neural tissues of experimental animals.16 In one study,7 intensive acyclovir therapy was applied concurrently with experimental stimulation of reactivation in rabbits that had been previously inoculated via the cornea. Even this intensive treatment strategy failed to eliminate latent infection from the trigeminal ganglia. These results from experimental models are consistent with clinical data from several human trials. For example, in a multicentre study reported by Peacock et al.,8 45 patients with primary genital HSV-2 infections who had had lesions for <7 days were given 5 mg/kg acyclovir or placebo intravenously three times per day for 5 days. No differences were seen between the placebo- and acyclovir-treated groups for the proportion of patients with a recurrence, the time to recurrence or the mean monthly incidence of recurrences.

While there is general agreement that latent HSV is not affected by nucleoside analogue therapy, the effect of therapy on the acute infection during the period of establishment of latency is more controversial. Since acyclovir was first introduced there has been some evidence that treatment very early during the primary infection may interfere with the establishment of latency. It was reported in 19791 that acyclovir 50 mg/kg, given subcutaneously or intraperitoneally to mice starting 1 day before virus inoculation, reduced the proportion of mice in which virus could be reactivated when this was tested by explant cocultivation, although the protective effect depended on the inoculum dose. This effect on latency of acyclovir therapy starting within 24 h was confirmed in the same year in several different laboratories.911

More recently, we have reported on the effects of the oral prodrugs valaciclovir and famciclovir on the pathogenesis of HSV-1 and -2 in the murine ear pinna infection model.12,13 When therapy was initiated within 2–3 days of virus inoculation, both famciclovir and valaciclovir appeared to reduce the establishment of latency, tested for by explant cocultivation of the dorsal root ganglion or trigeminal ganglion (TG). We also showed that famciclovir was superior in this protective effect. The test for latency employed in these studies was explantation of the dorsal root ganglion or TG and maintenance in culture for several days followed by homogenization and titration of infectious virus. A positive result for one or more ganglia from an individual mouse proves that the animal carried latent HSV in the neural tissue, but a negative result was not definitive; indeed, more sensitive molecular techniques revealed the presence of cells containing HSV.14

This study explores further the effects on latency of famciclovir administered to HSV-1-infected mice during the acute virus infection. The results are compared with those reported from previous experiments and discussed in order to explain the apparently conflicting data produced by ourselves and others. The implication of these findings for the use of famciclovir in humans is addressed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal model

The virus used was HSV-1 SC16. This strain has been extensively characterized in mice15 and has been used previously for studying antiviral compounds.1,1618 Working stocks of virus were prepared at a low multiplicity of infection and titrated in baby hamster kidney (BHK-21) cells.

Female, 4-week-old BALB/c mice (Bantin and Kingman, Kingston, UK) were inoculated into the left ear pinna with HSV-1 at 4 x 105 pfu/mouse using previously published methods.18

Famciclovir was administered in the drinking water at 1 mg/mL commencing on day 2, 3, 4, 5, 6 or 7 post-infection (p.i.) and continued up to day 10. From day 11 p.i., the mice were supplied with normal drinking water. Their total intake was measured each day and the consumption used to calculate the average daily dose. Volumes consumed per day ranged from 2.2 to 3.3 mL/mouse, with a mean of 2.6 ± 0.4 mL/mouse. Consumption was not affected by the presence of famciclovir and corresponded to a mean dose of 140–200 mg/kg/day for all groups.

Mice were assessed subjectively once daily from day 0 to day 18 p.i. and all signs noted. Mean weights with s.d. were calculated from groups of eight mice. Mortality was assessed using separate groups of 20 mice.

Detection of latent virus in the ganglia by explant culture

One month and 6 months after infection, five mice per treatment regimen were tested for latency. Ipsilateral and contralateral TG and third cervical ganglia (CIII) were explanted and incubated independently for 5 days at 37°C in a 5% CO2 atmosphere. The ganglia were then homogenized and tested for infectious virus by plaque titration using BHK-21 cells.

Detection of LATs by in situ hybridization

Probes for the detection of latency-associated transcripts (LATs) were made by T7 polymerase transcription of HindIII-linearized pSLAT 2 with a digoxigenin (DIG) detection system as previously described in detail.19 The plasmid pSLAT 2 was a gift from Dr S. Efstathiou, Department of Pathology, Cambridge University, UK. After transcription, the reaction mixtures were precipitated with ethanol and the product was resuspended in 100 µL of 10 mM Tris–1 mM dithiothreitol with RNase inhibitor.

Single ganglia were fixed in periodate–lysine–paraformaldehyde at 4°C for 16 h, transferred to 50% ethanol and then embedded in paraffin. Sections (5 µm) were collected on glutaraldehyde-activated, 3-aminopropyltriethoxysilane-coated slides and dewaxed in xylene.

Sections were digested with proteinase K 100 mg/L at 37°C for 5 min for CIII and 6 min for TG. Overnight hybridization was carried out at 72°C (25°C below the theoretical melting temperature). One to three micrograms of DIG-labelled riboprobe was used in each 100 µL of hybridization solution. One stringent wash in 0.1 x SSC (1 x SSC contains 0.15 M NaCl and 0.015 M sodium citrate) with 30% formamide and 10 mM Tris–HCl (pH 7.5) was carried out at 75°C for 30 min. Bound probe was detected with alkaline phosphatase–conjugated anti-DIG Fab fragments according to the manufacturer's instructions (Boehringer Mannheim). Positive cells contained brown stain confined to the nucleus. The intensity of staining varied among individual neurons and from block to block. A characteristic nucleoplasmic signal was seen, although individual cells showed various signal distributions and intensities. Cell nuclei that clearly contained more than the background level of brown staining were scored as positive. The number of LAT-positive cells in each section was recorded by microscopic examination, and representative sections were photographed for future reference. Alternate sections from all tissues were counted and used to calculate the mean number of positive neurons per section. Five mice from each treatment group were tested and the aggregate mean and s.d. for the group was recorded. Every in situ hybridization test with the LAT probe included RNase- and DNase-treated sections to rule out spurious positives.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical signs

Mice given inoculations into the skin of the ear pinna developed clinical signs typical of HSV infection; without therapy, 50% died between days 5 and 9 p.i. (Figure 1Go). Untreated mice showed a marked cessation in weight gain which was most pronounced up to day 5 p.i. The surviving mice gained weight thereafter but remained significantly smaller than uninfected control mice up to 18 days p.i., the end of observation (Figure 2Go).



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Figure 1. Reduction in mortality resulting from famciclovir treatment commencing at various times after infection. Groups of 20 female BALB/c mice were inoculated via the left ear pinna with 4 x 105 pfu HSV-1 strain SC16. Famciclovir was administered in the drinking water at 1 mg/mL commencing on day 2 ({triangledown}), 3 (*), 4 ({blacktriangleup}), 5 ({square}), 6 ({diamondsuit}) or 7 ({diamond}) p.i. •, Control, untreated mice; {circ}, mock-infected mice. All therapy was stopped at the end of day 10 p.i.

 


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Figure 2. Effect of infection and famciclovir chemotherapy on weight gain. Groups of eight female BALB/c mice were inoculated and treated as described in the legend to Figure 1Go. Symbols as in Figure 1Go.

 
There was a graded response to famciclovir therapy according to the day on which treatment was started. When therapy began on day 2 or 3, there was 100% survival, but when the start of therapy was delayed to day 4 or later, some mice died from the infection (Figure 1Go); delay to day 5 or beyond afforded no significant protection. No further mice died in any group following cessation of therapy on day 10 p.i.

The effects of therapy were also reflected in the weight of the mice. Weight loss tended to be lower when therapy was started on day 2, 3 or 4, but only therapy starting on day 2 had a significant effect; in this group mice showed a marked increase in weight from day 6 p.i. (P < 0.01 for days 3, 5 and 7–10 p.i. for mice treated with famciclovir on days 2–10 in comparison with the infected, untreated controls) (Figure 2Go). Daily observations were continued until day 18, but no significant weight loss or other new clinical signs occurred after the end of therapy.

Assessment of latency

Explant culture.
All the ipsilateral ganglia (CIII and TG) from 10 untreated mice yielded infectious virus by explant culture. Of the contralateral ganglia, nine of 10 TG and six of 10 CIII were also positive (Table IGo).


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Table I. Assessment of latent infection by means of explant culture followed by detection of infectious virus in the trigeminal and third cervical ganglia 1 and 6 months after infection
 
Among the treatment groups, a significant reduction in latency was seen in mice that had been treated from day 2 p.i. and none of the TG were positive. For the days 2–10 treatment group there was a reduction of 75% for the left TG and CIII and a 90% reduction for the right TG and CIII compared with the infected, untreated controls (Table IGo) (P < 0.01). For left and right TG there was also a significant reduction in reactivation from latency in mice that had been treated from day 3 p.i. (P < 0.01). The days 3–10 treatment gave reductions of 20% and 45% for left and right TG and CIII in comparison with the infected, untreated controls. TG from mice treated from day 4 p.i. or later and CIII from mice treated from day 3 or later showed no significant reduction in their reactivation from latency by this test.

Detection of LAT-positive neurons by in situ hybridization.
Further groups of five mice were sampled 1 month after infection and sections were prepared from the same selection of left and right ganglia as above. The number of LAT-positive neurons was determined for each section (Figure 3Go) and mean values were obtained (Table IIGo). For untreated mice, the greatest number of LAT-positive cells was observed in the left TG. This number was reduced by therapy and the reductions were significant even when the start of therapy was delayed up to day 6 p.i. (P < 0.001). No reduction compared with controls was observed, however, in mice which had received therapy commencing on day 7 p.i.






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Figure 3. Representative sections of ganglia showing in situ hybridization for major LAT. Representative examples of ganglionic sections used to count LAT-positive cells yielding the data shown in Tables II and IVGoGo. Mice were sampled at 1 month after infection. Left and right ganglia were obtained from the same mouse in each case. The ganglia illustrated were from untreated mice. (a) Left TG. S, neuron containing a nucleus showing strong LAT-positive signal; n, LAT-positive nucleus; W, neuron containing a nucleus showing weak LAT-positive signal; N, LAT-negative neuron. (b) Right TG; (c) left CIII; (d) right CIII. The actual number of LAT-positive neurons scored under the microscope for the whole section in each case for panels a to d were: 90, 66, 27 and eight, respectively. Magnification: panels a and b, x100; panels c and d, x200.

 

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Table II. Assessment of latency by means of in situ hybridization for LAT in third cervical (CIII) and trigeminal ganglia (TG) 1 month after infectiona
 
The right TG from untreated mice contained approximately half as many LAT-positive neurons as the left TG, but similar reductions were observed following therapy in both ipsilateral and contralateral TG. The cervical ganglia from untreated mice contained fewer LAT-positive cells. There was comparatively little reduction in the number of cells scoring positive for LAT despite early onset of famciclovir therapy (Table IIGo); significant reductions in LAT-positive neurons in CIII ganglia were observed only when treatment was started on day 3 or sooner.

All ganglia from treated mice contained some positive cells with the single exception of the right TG obtained from the group of mice that had been treated from the earliest time (day 2 p.i.); these sections yielded negative results for all five mice tested.

It is apparent from Tables I and IIGoGo that there was a very poor correlation between the mean number of LAT-positive neurons and the proportion of ganglia that scored positive or negative for latency following explant culture. Thus, treatment from day 2 p.i. yielded LAT counts of 7, 11, 0 and 12 positive neurons/section for left TG, left CIII, right TG and right CIII, respectively, which corresponded to reactivation rates of 0, 40, 0 and 0%, respectively, for these ganglia from groups of mice sampled on the same occasion. The equivalent counts for treatment starting on day 3 were 12, 16, 8 and 11 LAT-positive neurons/section compared with 60, 100, 40 and 100% reactivation rate, respectively, for the groups of explanted ganglia tested by culture on the same occasion. Thus, a count of 11 or 12 LAT-positive neurons/section yielded reactivation rates that ranged from 0 to 100% for ganglia tested from the equivalent groups of mice that had received the same period of treatment (Tables I and IIGoGo).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Before drawing conclusions from this experiment we compared the results with those reported in previous studies of ours, to see how consistent the results were. The proportion of mice (expressed as per cent) from which infectious virus was recovered from one or more ganglia following explant culture in the present study is shown in Table IIIGo in comparison with two studies where the same ganglia were tested.6,14 There was good concordance between the three studies for the left ganglia except that in one of the earlier studies6 a greater degree of protection was recorded when the start of famciclovir therapy was delayed to >=3 days p.i. (Table IIIGo). The mean number of latency positive cells in each ganglia was then expressed as the percentage of that in ganglia from untreated mice. Again, there is very good concordance between the two methods and between the two studies for individual ganglia (Table IVGo).


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Table III. The effect of famciclovir therapy on the establishment of latency assessed by explant culture in this study (A) in comparison with two previously published studies, references 14 (B) and 6 (C)
 

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Table IV. Effects of famciclovir on the establishment of latency assessed by in situ hybridization or infectious centre assay 1–2 months after infection in comparison with a previously published study14
 
The main findings from the present study confirm and extend previous data, proving that famciclovir therapy afforded protection against the establishment of latency as judged by explant culture or by counting cells expressing LATs. When the start of therapy was delayed for >=3 days, the protective effect was smaller, but significant reductions in LAT-positive cells were recorded in certain ganglia when therapy was delayed for up to 6 days p.i. (Table IIGo). Therapy appeared to reduce latency more, as judged by both methods, for TG compared with CIII, irrespective of whether they were from the ipsilateral or contralateral side.

The prolonged effect on latency suggests that famciclovir interacts with HSV in neurons during the establishment of latency and that the protective effect is not simply a consequence of reduced virus replication in peripheral tissues before entry of HSV into axons and colonization of neurons.

Early publications concerning the effects of acyclovir on the prevention of latency suggested that the protection afforded by therapy was dependent on inoculum dose.1 Although the dose of virus used in each experiment in the present series remained constant, and much effort was made to minimize the biological variables, marked fluctuations in mortality have been observed between experiments and subtle differences in conditions may alter the intensity of the primary infection which in turn would vary the burden of latent virus. The method of inoculation is also likely to be important. In our case virus was introduced by discrete intradermal inoculation (in 10 µL) rather than by means of scarification, an alternative method favoured for some HSV animal infection models.

Using a murine flank inoculation model with scarification, Simmons et al.20 and Slobedman et al.21 reported that the number of HSV-1 genomes per LAT-positive neuron was dependent on the anatomical relationship between the infection site in the skin and particular ganglia. These workers reported that, on recovery from acute infection, latent virus DNA was most abundant in sites directly innervated by the skin site, with lower levels in ganglia corresponding to secondary sites. Conversely, the secondary sites contained more LAT-positive neurons. These authors concluded that input (i.e. unamplified) and progeny (i.e. amplified) DNA sequences persist in the peripheral nervous systems of mice infected with HSV-1 strain SC16, with possibly different biological consequences. In our case, using the same virus strain in the ear pinna model, we observed that CIII is more directly innervated than TG based on the time of first detection of infectious virus in the various ganglia (day 3 for CIII and day 4 for TG; data not shown). Our results (Table IIGo) indicated that there were more LAT-positive cells in TG than in CIII following ear pinna inoculation; this is consistent with the data of Simmons et al.20 who showed an inverse relationship between the number of DNA copies per neuron and the number of LAT-positive neurons per ganglion following flank inoculation. We speculate that early therapy is likely to have less influence on the earliest sites of neural infection.

It is interesting, therefore, to compare our data on the protective effects of therapy with those recently published by LeBlanc et al.22 These workers observed no difference between the effects of famciclovir and valaciclovir on the quantity of HSV DNA in latently infected TG. However, there were no survivors in the untreated groups, so no ganglia were available to compare with those obtained from treated mice. The experiment involved high-titre virus (106 pfu/mouse) applied bilaterally direct to the cornea; this resulted in rapid and high (100%) mortality by day 5 p.i. in untreated mice, and some mortality in treated mice. Furthermore, therapy was discontinued on day 7 p.i., when virus reached a peak titre in the brains of treated animals. This method of inoculation would favour direct uptake of virus into the axons and transfer to the ganglionic neurons before initiation of therapy (which commenced from day 1 p.i.) and the establishment of unamplified latency as described by Simmons et al.20 Therefore, the effects of therapy in this ocular model would be expected to be smaller, and potential differences between the two drugs may have been obscured. This may be comparable to the situation for left CIII following ear pinna inoculation in the experiment reported in the present study, in which mortality in untreated mice was relatively high (50%). Further work is required to establish whether this high-lethality model reflects naturally acquired disease in humans more accurately than the more moderate pathogenesis such as described in the present study.

As reported previously,14 famciclovir therapy reduced the number of LAT-positive cells, although their absolute number did not correlate closely with the ability to reactivate virus by explant culture. The likely explanation for this is that the nucleoside restricts the number of HSV genome copies that accumulate during the establishment of latency and which may vary from between one and 1000 DNA copies/cell;23 this may account, at least in part, for the reduced ability for ganglia to reactivate infectious virus. The in situ hybridization method, which is undoubtedly a more sensitive indicator of latent infection, detects transcription products that occur in multiple copies in the order of 50000/cell24 in the cells that contain HSV (strain SC16) genomes. It is not known, however, which method is more likely to reflect the pattern of recurrent disease that follows a period of latency in humans.

We found no evidence, either in the present study or from previous experiments, for an increase in the number of LAT-positive cells following famciclovir therapy. It has been suggested that acyclovir may protect infected neurons from lysis during the acute phase of infection and, theoretically, this could result in an increased number of neurons that carry latent infection. We have observed increases in LAT-positive cells to 109% and 118% compared with untreated controls in two experiments in which animals were treated with valaciclovir from day 2 to day 10 p.i.14 No such effect was observed in famciclovir-treated animals, either in the earlier experiments or in the present study where sections from treated mice contained as many LAT-positive cells as, or fewer LAT-positive cells than, untreated control mice. This could be related to the fact that penciclovir but not acyclovir induces apoptosis.25

Finally, our results suggest that a significant number of neurons become latently infected despite continuous therapy starting from 2 days p.i. (Tables II and IVGoGo). In another study26 we showed that, even when continuous therapy with famciclovir or valaciclovir (by means of the drinking water) was initiated the day before virus inoculation, this did not completely prevent the establishment of latency as judged by the detection of LATs. Although all the ganglia from these mice were negative for latency when tested by explant culture, a basal level of LAT-positive cells was detected in both ipsilateral and contralateral TG and CIII.26

A crucial question that underlies all these studies is, to what extent can data obtained from the murine infection model be extrapolated to humans? Little is known about the typical inoculum dose in a human exposure to HSV and the extent and duration of local replication required before entry of the virus into the axons. It seems likely to us that murine models exaggerate the quantity of latent HSV DNA established; particularly that which occurs from early times and this, together with the greater distance between the injection site and the sensory neurons, would imply that virus takes five to 10 times longer to reach the neurons in humans than it does in mice. This suggests that therapy during the first few days after exposure could influence the balance of latency in the neurons and its subsequent ability to reactivate to produce disease. Although the prospects for early therapy reducing the incidence or intensity of subsequent reactivations may be somewhat poor, our animal data provide a glimmer of hope that it may be possible to influence the pathogenesis of recurrent disease and that this should, therefore, remain a subject of interest and for further investigation in humans.


    Acknowledgments
 
We would like to thank Dr Stacey Efstathiou, Department of Pathology, Cambridge University, for his helpful discussion of our data.


    Notes
 
* Corresponding author. Tel: +44-1223-330810; Fax: +44-1223-332998; E-mail: hjf10{at}cam.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Field, H. J., Bell, S. E., Elion, G. B., Nash, A. A. & Wildy, P. (1979). Effect of acycloguanosine treatment of acute and latent herpes simplex infections in mice. Antimicrobial Agents and Chemotherapy 15, 554–61.[ISI][Medline]

2 . Blyth, W. A., Harbour, D. A. & Hill, T. J. (1980). Effect of acyclovir on recurrence of herpes simplex skin lesions in mice. Journal of General Virology 48, 417–9.[Abstract]

3 . Field, H. J. & De Clercq, E. (1981). Effects of oral treatment with acyclovir and bromovinyldeoxyuridine on the establishment and maintenance of latent herpes simplex virus infection in mice. Journal of General Virology 56, 259–65.[Abstract]

4 . Klein, R. J., DeStefano, E., Friedman-Kien, A. E. & Brady, E. (1981). Effect of acyclovir on latent herpes simplex virus infections in trigeminal ganglia of mice. Antimicrobial Agents and Chemotherapy 19, 937–9.[ISI][Medline]

5 . Darby, G. & Field, H. J. (1983). Latency and acquired resistance—problems in chemotherapy of herpes infections. Pharmacology and Therapeutics 23, 217–51.[Medline]

6 . Thackray, A. M. & Field, H. J. (1996). Differential effects of famciclovir and valaciclovir on the pathogenesis of herpes simplex virus in a murine infection model including reactivation from latency. Journal of Infectious Diseases 173, 291–9.[ISI][Medline]

7 . Nesburn, A. B., Willey, D. E. & Trousdale, M. D. (1983). Effect of intensive acyclovir therapy during artificial reactivation of latent herpes simplex virus. Proceedings of the Society for Experimental Biology and Medicine 172, 316–23.[Abstract]

8 . Peacock, J. E., Kaplowitz, L. G., Sparling, P. F., Durack, D. T., Gnann, J. W., Whitley, R. J. et al. (1988). Intravenous acyclovir therapy of first episodes of genital herpes: a multicenter double-blind, placebo-controlled trial. American Journal of Medicine 85, 301–6.[ISI][Medline]

9 . Klein, R. J., Friedman-Kien, A. E. & DeStefano, E. (1979). Latent herpes simplex virus infections in sensory ganglia of hairless mice prevented by acycloguanosine. Antimicrobial Agents and Chemotherapy 15, 723–9.[ISI][Medline]

10 . Park, N.-H., Pavan-Langston, D. & McLean, S. L. (1979). Acyclovir in oral and ganglionic herpes simplex virus infections. Journal of Infectious Diseases 140, 802–6.[ISI][Medline]

11 . Pavan-Langston, D., Park, N.-H. & Hettinger, M. (1981). Ganglionic herpes simplex and systemic acyclovir. Archives of Ophthalmology 99, 1417–9.[Abstract]

12 . Field, H. J. & Thackray, A. M. (1996). Valaciclovir and famciclovir—differences between two similar guanosine analogue prodrugs emerge from laboratory models. International Antiviral News 4, 23–7.

13 . Field, H. J. & Thackray, A. M. (1997). Can herpes simplex virus latency be prevented using conventional nucleoside analogue chemotherapy? Antiviral Chemistry and Chemotherapy 8, 59–66.[ISI]

14 . Thackray, A. M. & Field, H. J. (1998). Famciclovir and valaciclovir differ in the prevention of herpes simplex virus type 1 latency in mice: a quantitative study. Antimicrobial Agents and Chemotherapy 42, 1555–62.[Abstract/Free Full Text]

15 . 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. Journal of General Virology 28, 341–53.[Abstract]

16 . Boyd, M. R., Bacon, T. H. & Sutton, D. (1988). Antiherpes virus activity of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) in animals. Antimicrobial Agents and Chemotherapy 32, 358–63.[ISI][Medline]

17 . Sutton, D. & Boyd, M. R. (1993). Comparative activity of penciclovir and acyclovir in mice infected intraperitoneally with herpes simplex virus type 1 SC16. Antimicrobial Agents and Chemotherapy 37, 642–5.[Abstract]

18 . Field, H. J., Tewari, D., Sutton, D. & Thackray, A. M. (1995). Comparison of efficacies of famciclovir and valaciclovir against herpes simplex virus type 1 in a murine immunosuppression model. Antimicrobial Agents and Chemotherapy 39, 1114–9.[Abstract]

19 . Arthur, J., Efstathiou, S. & Simmons, A. (1993). Intranuclear foci containing low abundance herpes simplex virus latency-associated transcripts visualized by non-isotopic in situ hybridization. Journal of General Virology 74, 1363–70.[Abstract]

20 . Simmons, A., Slobedman, B., Speck, P., Arthur, J. & Efstathiou, S. (1992). Two patterns of persistence of herpes simplex virus DNA sequences in the nervous systems of latently infected mice. Journal of General Virology 73, 1287–91.[Abstract]

21 . Slobedman, B., Efstathiou, S. & Simmons, A. (1994). Quantitative analysis of herpes simplex virus DNA and transcriptional activity in ganglia of mice latently infected with wild-type and thymidine kinase-deficient viral strains. Journal of General Virology 75, 2469–74.[Abstract]

22 . LeBlanc, R. A., Pesnicak, L., Godleski, M. & Straus, S. E. (1999). The comparative effects of famciclovir and valacyclovir on herpes simplex virus type 1 infection, latency, and reactivation in mice. Journal of Infectious Diseases 180, 594–9.[ISI][Medline]

23 . Sawtell, N. M. (1997). Comprehensive quantification of herpes simplex virus latency at the single-cell level. Journal of Virology 71, 5423–31.[Abstract]

24 . Speck, P. G. (1992). Analysis of establishment of latent infection with a virulent strain of herpes simplex type 1. Ph.D. thesis, University of Adelaide.

25 . Thust, R., Klöcking, R., Voutilainen, N., Wutzler, P. & Kaina, B. (1998). Similarities and differences in the genotoxic and apoptosis-inducing capacity of ganciclovir and penciclovir, respectively, in HSVtk+ transfectants of Chinese hamster ovary cells. Antiviral Research 37, A81.

26 . Field, H. J. & Thackray, A. M. (2000). Early therapy with valaciclovir or famciclovir reduces but does not abrogate herpes simplex virus neuronal latency. Nucleosides and Nucleotides 19, 461–70.[ISI]

Received 14 October 1999; returned 23 November 1999; revised 1 December 1999; accepted 24 January 2000