Centre for Veterinary Science, Cambridge University, Madingley Road, Cambridge CB3 0ES, UK1
Author for correspondence: Hugh Field. Fax +44 1223 332998. e-mail hjf10{at}cam.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a series of experiments conducted over several years, we have compared the effects of ACV and PCV using a murine ear pinna infection model for HSV-1 or HSV-2, in order to determine whether the above biochemical differences are reflected in different effects in vivo. The compounds were administered orally in the form of famciclovir (FCV) or valaciclovir (VACV), which are rapidly metabolized in mice with similar kinetics to yield PCV and ACV, respectively (Field et al., 1995 ). Both compounds were effective in reducing clinical signs and infectious virus production during the acute infection. However, FCV was consistently more effective than VACV in limiting the establishment of latency, particularly when the onset of therapy was delayed for several days after virus inoculation (Thackray & Field, 1996a
, 1998
, 2000b
). However, the difference between the compounds was not easily reconciled with their relative effects on acute virus replication in the nervous system during the acute phase of the infection. The object of the present study was to assess the progress of infection in the relevant neural tissues by using a reporter gene to identify acutely infected cells.
Thus, the HSV-1 recombinant strain SC16 lacZ IE110, with the lacZ gene under the immediate-early gene promoter (Lachmann et al., 1999 ), was applied to the scarified skin of the neck and either FCV or VACV at 1 mg/ml was supplied ad libitum in the drinking water from days 1 to 9 post-inoculation (p.i.), this method for treating acute murine HSV infection having previously been shown by ourselves to be more effective than twice daily oral gavage at 50 mg/kg (Field & Thackray, 1995
). By this means, we could examine the effects of continuous therapy on the distribution and fate of infected neurons. Ganglia from other mice obtained from the same experimental groups were analysed 1·5 and 10 months later for evidence of latent infection and these data were matched with the results obtained from acutely infected ganglia.
The results emphasize how neither compound was able to prevent completely the relentless progression of HSV through the nervous system. However, they provide further evidence for a difference between the compounds in their interactions with HSV and neural tissues in vivo.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice and virus inoculation.
Female BALB/c mice were purchased (Harlan UK Ltd, Blackthorn, Bicester, UK) at 4 weeks of age and were inoculated at 5 weeks. Anaesthetized mice were shaved on the left side of the neck 3 days before inoculation, and 10 µl virus suspension containing 1x105 p.f.u. was placed onto a 1 cm2 scarified skin site, 0·5 cm lateral to the ventral mid-line. Mice were observed twice daily and clinical signs were recorded as described previously (Field et al., 1995 ).
Antiviral therapy.
FCV and VACV powders were supplied by SmithKline Beecham Pharmaceuticals. They were dissolved in drinking water at 1 mg/ml and were administered from day 1 to day 9 p.i. From day 10 p.i., mice were supplied with normal drinking water. Their total fluid consumption was used to calculate the dose, which was 160185 mg/kg/day for all groups. Consumption was not affected by the presence of the antiviral compounds or HSV infection.
Detection of
-galactosidase (
-gal)-positive neurons.
Ganglia were treated with X-Gal for 6 h prior to clarification in glycerol and enumeration of positive neurons by methods described previously (Lachmann & Efstathiou, 1997 ). After a preliminary enumeration of blue cells, the ganglia were wax-embedded and sectioned (5 or 10 µm). The number of positive neurons was then counted in each section with the aid of a microscope and results were recorded. Photographs of typical whole mounts and the corresponding sections are shown in Fig. 1
.
|
Detection of LATs by in situ hybridization.
Probes for the detection of LATs were made by T7 polymerase transcription of HindIII-linearized pSLAT 2 with a digoxigenin (DIG) detection system as described previously in detail (Arthur et al., 1993 ). The plasmid pSLAT 2 was a gift from Dr S. Efstathiou. In situ hybridization was carried out as described previously (Thackray & Field, 1998
), including the complementary strand to pSLAT 2 by way of a control. Positive neurons contained dark signal exclusively in the nucleus. A typical section is shown in Fig. 4(d)
.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of therapy on pathogenesis
Treatment with either VACV or FCV at 1 mg/ml in the drinking water commencing 24 h p.i. completely prevented death. Both drugs also reduced the intensity and duration of clinical signs as reflected in the zoster score, which was reduced significantly on several individual days (data not shown).
Distribution of -gal-expressing neurons
The temporal appearance of signal in the ganglionic neurons (in the absence of therapy) was assessed. Representative X-Gal-treated whole mounts and 10 µm sections of CIII and TG are shown in Fig. 1(ad). The first neurons that contained blue staining that was clearly above background were observed on day 2 p.i. in the left (ipsilateral) and right (contralateral) CIII (Table 1
; Fig. 2
). On day 3, while more positive neurons were detected in the right CIII, all TG neurons remained at or below background. However, on day 4, both left and right TG neurons gave positive signals, with more staining in the left than in the right.
|
|
|
Effects of therapy on distribution of -gal expression
In the cervical ganglia (CIII), there was relatively little reduction in the number of -gal-expressing neurons with either drug, and the reduction barely reached significance (Table 1
). However, in the TG with treatment, there was a marked reduction in the number of positive neurons; for FCV, this was highly significant (P
0·001) on days 6 and 7 p.i. for the left TG in comparison with the untreated controls and day 4 p.i. for VACV. For the contralateral (right) side, the reduction was highly significant on days 5 and 6 for FCV but only on day 6 for VACV (Table 1
; Fig. 2
). Scores for individual mice are also shown (Fig. 3
).
It was notable that therapy with either drug did not completely prevent the distribution of virus to any of the ganglia tested, and small numbers of positive neurons were observed in both left and right CIII with either therapy. In VACV-treated mice, the number of -gal-expressing cells in the TG exceeded 100 and 70 neurons per section for left and right TG, respectively. The comparable numbers for FCV were 35 and 25.
After therapy was discontinued (day 9) and all ganglia had become negative for 2 days, there was a single day (day 11) on which sections from VACV-treated mice only yielded positive signal in both left CIII and left TG neurons in 5/5 and 4/5 mice for CIII and TG, respectively (a representative whole mount and section are shown; Fig. 4a). The number of positive neurons ranged from 2 to 23 per section for CIII and 20 to 53 for TG (Figs 3
and 4a
; Table 1
).
A few ganglia were examined for -gal expression at 1·5 and 10 months p.i. and, in both cases, positive neurons (<20 positive cells per section) were detected in all ganglia (Figs 4bc
and 5a
). The mean numbers of positive neurons per section in the individual ganglia at 10 months for treated and untreated mice are shown (Table 2
).
|
|
When ganglia were tested by explant co-cultivation at 1·5 months p.i., infectious virus was reactivated from all the sampled CIII and right TG from untreated mice. However, only 4/5 of the left TG yielded infectious virus and the infectious virus titres for positive ganglia were low (geometric mean of 1·9 log10 p.f.u. per ganglion for the left TG compared with 2·1 for the right TG, 3·3 for the left CIII and 2·6 for the right CIII; data not shown). At 10 months p.i., additional mice were tested. In this case, all ganglia were positive except left TG (Fig. 5c). Since there may have been amplification during the 5 day culture period, the titration of infectious virus after 5 days provides only a semi-quantitative estimate of the reactivation. However, the highest titres of infectious virus were observed in the left CIII (Fig. 5c
). In contrast, no ganglia from either drug-treatment group reactivated infectious virus (Fig. 5c
). Thus, at 1·5 months p.i. and similarly at 10 months p.i., all ganglia from mice that had been treated with either FCV or VACV were negative for infectious virus after co-cultivation.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The TG showed the largest number of infected neurons during the acute phase, with or without treatment. The observed predominance of positive neurons in ganglia that are not directly innervated by the infection site is consistent with the previous publications of Ecob-Prince et al. (1993) and Simmons et al. (1992)
. They reported that the secondary sites for latency (i.e. those not directly innervated by the inoculation site) contained larger numbers of latently infected cells.
Our hypothesis to explain the failure of the antiviral compounds to prevent the spread of infection in the nervous system is that there were occasions during the treatment period when drug levels were sub-inhibitory and allowed replication to proceed, at least transiently, in some ganglionic neurons, such that axonal and trans-synaptic spread of infection was able to continue (Ecob-Prince et al., 1993 ). This may have resulted from uneven consumption of compound over the 24 h period (we have reported previously that mice consume most water during the night; Field & Thackray, 1995
). However, in our experience, providing compound ad lib is superior to twice-daily oral gavage on a strictly 12 h schedule (Field & Thackray, 1995
). We believe, therefore, that it is unlikely that constant exposure of the target cells at or above the inhibitory concentration for the compounds can be achieved by using conventional methods for supplying drug to the animal.
Previously, we have reported a transient recurrence of infectious virus in ganglia and brain-stems of mice tested following the withdrawal of VACV (but not FCV) after a period of continuous therapy (Thackray & Field, 1996a , b
, 1997
, 2000a
). Here, we observed expression of
-gal in ipsilateral TG and CIII neurons of 8/10 mice tested on day 11, which was 2 days after therapy was discontinued and 3 days after reporter-gene expression had fallen to below the level of detection in the ganglia of surviving mice (with or without treatment). No observations were made immediately after day 11 in the present experiment, but extensive work with different models has shown that the recurrence of infectious HSV-1 (as evidenced by infectious virus) is transient. This occurred with higher doses of VACV and did not occur with sub-optimal doses of FCV (Thackray & Field, 2000b
). Previously, we followed the infection with daily observations for infectious virus for several weeks and saw no further evidence of virus activity in HSV-2-infected mice treated with either of the two drugs (Thackray & Field, 2000b
). Furthermore, we have observed in pilot experiments that there is little or no detectable
-gal expression during the period 23 weeks p.i. Fig. 4(a)
shows a representative CIII whole mount and section sampled on day 11 p.i. It was interesting that no blue cells were detected in the right TG, although this tissue contained a larger number of positive cells during the acute infection than the left CIII, which showed recurrence. We speculate that recurrence occurs in a sub-population of neurons that enter the lytic cycle on removal of the inhibitor. The fact that this does not occur on withdrawal of FCV may reflect the longer intracellular half-life of PCV triphosphate, although this seems unlikely, since in other extensive studies we have sampled ganglia from FCV-treated mice for up to 3 weeks after withdrawal of therapy, during which time no recurrences were observed (Thackray & Field, 2000a
). It seems more likely, therefore, that the sub-population of infected cells involved has already undergone lysis during the acute period of virus infection in the untreated mice or in the presence of PCV. This could be related to the ability of PCV (in contrast to ACV) to induce apoptosis (Thust et al., 1998
; Shaw et al., 1999
).
We showed in the present study that ganglia from surviving mice were latently infected in all cases. The ganglia contained LAT-positive neurons (Figs 4d and 5b
). We also observed some
-gal expression in a small number of neurons in mice from all three treatment groups (Fig. 4bc
and 5a
). Although the numbers were small, there was a trend towards the highest
-gal expression among the group of mice that had been treated with VACV, and this was consistent with the pattern of LAT expression (Fig. 5
). The continued sporadic expression of
-gal at late times after inoculation with this mutant has also been reported by Lachmann et al. (1999)
. The latent foci in the treated mice may have been established before the onset of therapy at 24 h p.i.; however, we have shown in a recent study (Field & Thackray, 2000
) that even commencing therapy 1 day before virus inoculation was unable to prevent the establishment of latently infected neurons as assessed by reporter gene or LAT expression.
Using the explant co-cultivation test for latency, all ganglia from treated mice were negative for infectious virus, and this confirms previous results: on numerous occasions, the ganglia from mice treated from 1 day p.i. or earlier have not yielded infectious virus by this test, despite the presence of LAT-positive neurons. The ganglia from control, untreated mice were positive for this test with the exception of the left TG at 10 months. This was an unexpected result and is not consistent with previous studies that used the ear pinna infection model. Furthermore, 4/5 left TG sampled at 5·5 months were positive for reactivation by means of co-cultivation, albeit with relatively low levels of infectious virus in the assay. However, we note that the left TG had the highest level of LAT-positive cells (both with and without treatment), and this may be important. We have previously observed no correlation between the number of LAT-positive neurons present in ganglia and the ability to reactivate by means of explant culture (Thackray & Field, 2000b ). Others have reported that there is no clear relationship between the presence of LAT expression and the ability to reactivate. For example, Ecob-Prince & Hassan (1994)
, using dual labelling techniques for LAT and a
-gal reporter gene, showed that at least a proportion of the cells that reactivate are neurons that appeared to be LAT-negative. We speculate that the neurons that are not directly innervated by the inoculation site, together with those from all ganglia from mice that received early antiviral therapy, contain fewer DNA copies per neuron, as shown for ACV therapy by Sawtell et al. (1998)
, and that this is a more important factor in determining the success of reactivation by means of explant culture.
We also observed that the right TG from mice that had received VACV therapy contained a significant excess of LAT-positive cells over controls. We have observed similar increased numbers of latently infected neurons in VACV-treated mice previously (e.g. Table 6 in Thackray & Field, 1998 ). Since this appears to be reproducible, it may reflect the sparing of infected neurons in the presence of ACV such that an increased number of latently infected neurons survive the primary infection. It is unlikely that the increase simply reflects the increased survival of mice that received therapy, since the excess was observed only for VACV and not with FCV therapy.
The general conclusion of this study is that, while neither VACV nor FCV can completely prevent the establishment of latency, both compounds do clearly affect the outcome of neural infection by HSV in vivo. Furthermore, the results of the present investigation confirm our earlier assertions that these two antiviral compounds are subtly different in how they interact with HSV-infected neurons. This is consistent with the known biochemical differences that exist between these two similar guanosine nucleoside analogues.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacon, T. H., Standring-Cox, R. & Howard, B. A. (1994). Comparative activity of penciclovir and acyclovir against herpes simplex virus type 2 in cell culture.Antiviral Research23, 25-36.
Boyd, M. R., Bacon, T. H., Sutton, D. & Cole, M. (1987). Antiherpesvirus activity of 9-(4-hydroxy-3-hydroxy-methylbut-1-yl)guanine (BRL 39123) in cell culture.Antimicrobial Agents and Chemotherapy31, 1238-1242.[Medline]
Earnshaw, D. L., Bacon, T. H., Darlison, S. J., Edmonds, K., Perkins, R. M. & Vere Hodge, R. A. (1992). Mode of antiviral action of penciclovir in MRC-5 cells infected with herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus .Antimicrobial Agents and Chemotherapy36, 2747-2757.[Abstract]
Ecob-Prince, M. & Hassan, K. (1994). Reactivation of latent herpes simplex virus from explanted dorsal root ganglia.Journal of General Virology75, 2017-2028.[Abstract]
Ecob-Prince, M. S., Preston, C. M., Rixon, F. J., Hassan, K. & Kennedy, P. G. E. (1993). Neurons containing latency-associated transcripts are numerous and widespread in dorsal root ganglia following footpad inoculation of mice with herpes simplex virus type 1 mutant in1814.Journal of General Virology74, 985-994.[Abstract]
Field, H. J. (1996). Famciclovir origins, progress and prospects.Expert Opinion on Investigational Drugs5, 925-938.
Field, H. J. & Thackray, A. M. (1995). The effects of delayed-onset chemotherapy using famciclovir or valaciclovir in a murine immunosuppression model for HSV-1.Antiviral Chemistry & Chemotherapy6, 210-216.
Field, H. J. & Thackray, A. M. (2000). Early therapy with valaciclovir or famciclovir reduces but does not abrogate herpes simplex virus neuronal latency.Nucleosides, Nucleotides and Nucleic Acids19, 461-470.[Medline]
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 Chemotherapy39, 1114-1119.[Abstract]
Ilsley, D. D., Lee, S.-H., Miller, W. H. & Kuchta, R. D. (1995). Acyclic guanosine analogs inhibit DNA polymerases alpha, delta, and epsilon with very different potencies and have unique mechanisms of action.Biochemistry34, 2504-2510.[Medline]
Lachmann, R. H. & Efstathiou, S. (1997). Utilization of the herpes simplex virus type 1 latency-associated regulatory region to drive stable reporter gene expression in the nervous system.Journal of Virology71, 3197-3207.[Abstract]
Lachmann, R. H., Sadarangani, M., Atkinson, H. R. & Efstathiou, S. (1999). An analysis of herpes simplex virus gene expression during latency establishment and reactivation.Journal of General Virology80, 1271-1282.[Abstract]
Sawtell, N. M., Poon, D. K., Tansky, C. S. & Thompson, R. L. (1998). The latent herpes simplex virus type 1 genome copy number in individual neurons is virus strain specific and correlates with reactivation.Journal of Virology72, 5343-5350.
Shaw, M. M., Watts, P. A. & Field, H. J. (1999). Effects of ganciclovir, penciclovir and acyclovir on apoptosis.Antiviral Research41, A66.
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 Virology73, 1287-1291.[Abstract]
Thackray, A. M. & Field, H. J. (1996a). 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 Diseases173, 291-299.[Medline]
Thackray, A. M. & Field, H. J. (1996b). Comparison of effects of famciclovir and valaciclovir on pathogenesis of herpes simplex virus type 2 in a murine infection model.Antimicrobial Agents and Chemotherapy40, 846-851.[Abstract]
Thackray, A. M. & Field, H. J. (1997). The influence of cyclosporin immunosuppression on the efficacy of famciclovir or valaciclovir chemotherapy studied in a murine herpes simplex virus type 1 infection model.Antiviral Chemistry & Chemotherapy8, 317-326.
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 Chemotherapy42, 1555-1562.
Thackray, A. M. & Field, H. J. (2000a). Persistence of infectious herpes simplex virus type 2 in the nervous system in mice after antiviral chemotherapy.Antimicrobial Agents and Chemotherapy44, 97-102.
Thackray, A. M. & Field, H. J. (2000b). Further evidence from a murine infection model that famciclovir interferes with the establishment of HSV-1 latent infections.Journal of Antimicrobial Chemotherapy45, 825-833.
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 Research37, A81.
Vere Hodge, R. A. & Perkins, R. M. (1989). Mode of action of 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (BRL 39123) against herpes simplex virus in MRC-5 cells.Antimicrobial Agents and Chemotherapy33, 223-229.[Medline]
Received 15 April 2000;
accepted 20 June 2000.