Centre for Veterinary Science, Cambridge University, Madingley Road, Cambridge CB3 0ES, UK
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Abstract |
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Introduction |
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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 23 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.
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Materials and methods |
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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 140200 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 Tris1 mM dithiothreitol with RNase inhibitor.
Single ganglia were fixed in periodatelysineparaformaldehyde 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 TrisHCl (pH 7.5) was carried out at 75°C for 30 min. Bound probe was detected with alkaline phosphataseconjugated 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.
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Results |
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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 1). 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 2
).
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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 710 p.i. for mice treated with famciclovir on days 210 in comparison with the infected, untreated controls) (Figure 2). 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 I).
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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 3) and mean values were obtained (Table II
). 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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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 II 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 II
).
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Discussion |
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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 II) 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 IV). 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.
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Acknowledgments |
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Notes |
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References |
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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, 4179.[Abstract]
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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, 246974.[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, 5949.[ISI][Medline]
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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, 46170.[ISI]
Received 14 October 1999; returned 23 November 1999; revised 1 December 1999; accepted 24 January 2000