Early therapy with valaciclovir or famciclovir reduces but does not abrogate herpes simplex virus neuronal latency.

Mice were infected via the ear pinna using a recombinant strain of HSV-1 expressing the beta-gal gene under the LAT promoter. Mice were treated continuously with valaciclovir or famciclovir, from 1 day before or 1 day after virus inoculation for 10 days. Ipsilateral and contralateral trigeminal and cervical ganglia were later assessed by co-cultivation or for X-Gal-positive or LAT-positive neurons. Latency was markedly reduced by early therapy, however, a basal level of HSV-1-positive neurons was detected in all mice.

Evidence for co-infection of ovine prion strains in classical scrapie isolates.

The diversity of strains of ovine prions within classical scrapie isolates was investigated by transmission studies in wild type mice. To determine the maximum diversity of prion strains present in each ovine scrapie isolate examined, isolates from mice having the shortest and longest incubation times for terminal disease after primary inoculation were passaged serially. Serial passage of ARQ/ARQ scrapie isolates in RIII mice revealed the ME7 prion strain in mice with short incubation times for terminal prion disease and the 87A strain in those mice with long incubation times. Serial passage of VRQ/VRQ scrapie isolates in RIII mice led to emergence of the 221C prion strain in mice with short incubation times and a variant of the 221C strain in those mice with long incubation times. RIII mice with short incubation times had higher levels of total and proteinase K-resistant PrP(Sc) compared with those RIII mice with long incubation times, while mice with long incubation times had large aggregates and plaques of PrP(Sc). ME7 PrP(Sc) differed in stability compared with the 87A prion strain, while PrP(Sc) associated with 221C had similar stability to that of the 221C variant. Serial passage in VM mice led to identification of ME7 and 87V in the same scrapie isolate. The data show that different prion strains can emerge from the same ovine scrapie isolate following serial passage in wild type mice and that the transmission properties of these strains correlate with distinct patterns of PrP(Sc) deposition.

Famciclovir and valaciclovir differ in the prevention of herpes simplex virus type 1 latency in mice: a quantitative study.

Famciclovir (FCV) and valaciclovir (VACV) have previously been shown to be potent inhibitors of herpes simplex virus type 1 (HSV-1) in a murine cutaneous model. In the present study, mice were inoculated in the skin of the left ear pinna with herpes simplex virus (HSV) type 1. Antiviral therapy was started on different days postinoculation (p.i.), terminating at the end of day 10 p.i. The compounds were administered twice daily by oral gavage at 50 mg/kg of body weight/dose. Mice were sampled on day 5 p.i., during the acute phase of the infection, and the titers of infectious virus in the target tissues (ear, brain stem, and trigeminal ganglia) were determined. At 2 to 3 months p.i., the ipsilateral and contralateral trigeminal and cervical dorsal root ganglia were explanted, and four different methods were used to detect latent HSV. The methods were (i) conventional explant culture for 5 days followed by homogenization, (ii) long-term culture (up to 73 days) of whole ganglia, followed by homogenization, (iii) dissociation by enzymatic disaggregation and an infectious center assay, and (iv) in situ hybridization to detect latency-associated transcripts (LATs). The conventional explant culture method was the least sensitive method, while in situ staining for LAT was the most sensitive, and all mice, including those treated from early times with FCV, were shown to be latently infected. Significantly less latent virus was detected by all four methods, however, in ganglia obtained from mice that had been treated with FCV in comparison with the amount detected in ganglia from mice that had been treated with VACV. However, in no case was latency completely eliminated.

Persistence of infectious herpes simplex virus type 2 in the nervous system in mice after antiviral chemotherapy.

Young adult mice were inoculated with herpes simplex virus type 2 (HSV-2) in the ear pinna. A relatively severe infection resulted, and 45% of the mice died by 11 days postinfection. Therapy at 1 mg/ml by means of the drinking water with either famciclovir for periods of 5 or 10 days or valaciclovir for 5, 10, 15, or 20 days decreased clinical signs and reduced mortality to 15% or less. Throughout a period of 27 days, mice were tested daily for the presence of infectious virus in the ear pinna, brain stem, and ipsilateral trigeminal ganglia. Virus was cleared from these tissues in surviving, untreated animals by 12 days postinfection, and no infectious virus was detected subsequently in any tissue. Furthermore, no infectious virus was detected after day 9 in mice that had been treated with famciclovir. In mice that had received valaciclovir therapy, however, infectious virus was repeatedly detected in the trigeminal ganglia and brain stem tissue samples up to 7 days after treatment was discontinued. To date, no specific mechanism to account for these results has been discovered; however, possible mechanisms for the persistence of potentially infectious virus in neural tissue of treated mice are discussed.

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

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.

Comparison of effects of famciclovir and valaciclovir on pathogenesis of herpes simplex virus type 2 in a murine infection model.

The effects of famciclovir (FCV) and valaciclovir (VACV) were compared in a cutaneous infection model for herpes simplex virus type 2 (HSV-2). The compounds were administered orally from day 1 to day 5 postinfection. Both compounds reduced local inflammation and virus replication in the skin. FCV markedly reduced mortality and virus replication in the nervous system. On the cessation of therapy after 5 days, when the levels of infectious virus in the tissues were reduced to below the level of detection, there followed a rebound of virus replication in the ganglia and brain stems of mice that had been treated with VACV. The recurrence of infection in the brain stem occurred on three separate occasions. No such recurrences were observed following FCV treatment. When ganglia were explanted from survivors 6 weeks later, latent virus was shown to be reactivated in all 10 of 10 control, untreated mice. The number of mice whose ganglia yielded virus was reduced to 60% in mice that had been treated with VACV, whereas no mice that had been treated with FCV had evidence of latent infection by this test.

Differential effects of famciclovir and valaciclovir on the pathogenesis of herpes simplex virus in a murine infection model including reactivation from latency.

The ability of famciclovir and valaciclovir to affect the establishment and maintenance of latency in mice with a cutaneous herpes simplex type 1 (HSV-1) infection was examined. Mice were treated via drinking water starting at various times between days 1 and 5 and terminating on day 10 after inoculation. Clinical signs and viral replication in the target tissues were monitored. Three to four months later, trigeminal and dorsal root ganglia were explanted from groups of 16 mice and examined for latent virus by cocultivation. The two compounds differed in their effects on the acute neural infection, and ganglia explanted from famciclovir-treated mice were markedly reduced in their ability to reactivate virus, although neither drug affected latency if treatment was delayed for several months. The difference between the compounds is likely to reflect differences in the metabolism of their respective products, penciclovir and acyclovir, in infected neurons.

Reduced herpes simplex virus type 1 latency in Flt-3 ligand-treated mice is associated with enhanced numbers of natural killer and dendritic cells.

We have investigated the effect of Flt-3 ligand (Flt-3L) on the resistance to herpes simplex virus type-1 (HSV-1) infection in BALB/c mice which are normally highly susceptible to challenge with this virus. We have confirmed data by others that in vivo treatment with Flt-3L causes an increase in dendritic cells (DC) and natural killer (NK) cells in lymphoid tissue. Increasing doses of Flt-3L caused a corresponding increase in liver and spleen CD11c+ DC which were increased up to 20-fold compared with control levels. A significant expansion of NK cells was seen in the spleen of Flt-3L-treated mice where the number of DX5+ cells was increased by up to fivefold. We subsequently tested the hypothesis that Flt-3L treatment, at the time of viral infection, might lead to enhanced immunity and protection against viral pathogenesis. Two murine models of HSV-1 (SC16) infection were used. In the first model, mice were injected with Flt-3L daily for 9 days. Control mice received mouse serum albumin (MSA). On day 7 of the Flt-3L treatment 106 plaque-forming units (PFU) of SC16 was inoculated into the ear pinna. Flt-3L treatment significantly reduced mortality following virus inoculation, with 80% survivors in this group compared with 20% survivors in the MSA-treated group. In the second model, Flt-3L-treated mice were scarified with 104 PFU of SC16. In this case there was 60% survival in the Flt-3L-treated group of mice compared with 10% survival in the MSA-treated group. Assessment by in situ hybridization for latency-associated transcripts showed that Flt-3L treatment reduced the amount of latent virus within infected neurons. These studies show that in vivo treatment with Flt-3L results in protection against challenge with live HSV-1, which may be a consequence of enhanced numbers of DC and/or NK.

Comparison of efficacies of famciclovir and valaciclovir against herpes simplex virus type 1 in a murine immunosuppression model.

A mouse model of herpes simplex virus type 1 infection in an immunocompromised host was established by using cyclosporin-A to impair T-cell function. Following inoculation of herpes simplex virus type 1 into the skin of the ear pinna, cyclosporin-A prolonged virus replication in the skin and neural tissues compared with that in immunocompetent mice. This model was used to investigate the activity of famciclovir (FCV) and valaciclovir (VACV), which are oral products of the antiherpesvirus agents penciclovir and acyclovir, respectively. Both prodrugs gave similar blood profiles of the antiherpesvirus agents in normal and cyclosporin-treated mice. The compounds were administered by the oral route at 50 mg/kg per dose twice daily for 5 days. Both compounds were very effective at clearing infectious virus from the tissues despite the immunosuppression; FCV-treated animals cleared virus from the ear pinna more rapidly than VACV-treated animals. The areas under the concentration-time curve (AUC) for virus replication with time were reduced to 50 and 30% of control values for ear pinna and brain stem, respectively, with VACV therapy and to < 5% in both tissues by FCV. When treatment was continued to day 10, the reductions in AUC for ear and brain stem, respectively, were to 33 and 26% of control values with VACV and to < 3 and < 5% with FCV. However, on cessation of the antiviral treatment, there was a reproducible recurrence of infectious virus in the tissues obtained from VACV-treated mice. The recurrence of infectious virus was also evident after 10 days of treatment with VACV. In mice which had received FCV for 10 or 5 days, these was no resumption of virus replication in the ear pinna or brain stem. When dosing was reduced to once per day, both compounds were less effective at controlling the infection. Nevertheless, no recurrence of infectious virus was observed on cessation of FCV therapy.

The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in the horse.

Four specific pathogen-free ponies were infected intranasally with equine herpesvirus 1 (EHV-1) and two were similarly infected with an EHV-1 thymidine kinase deletion mutant. The primary infections were characterized by a transient fever accompanied by virus shedding into nasal mucus and viraemia. No virus was detected in clinical specimens after 15 days post-infection. Two months later a reactivation stimulus was administered to all six ponies and only the four that had been previously inoculated with wild-type EHV-1 shed virus into nasal mucus (for 10 days), proving the presence of a latent infection. No recurrence of viraemia was observed. The animals were monitored for a further 6 weeks and were consistently shown to be free from infectious virus. Tissues were then obtained postmortem. Co-cultivation of explanted trigeminal ganglia from two out of the four ponies that carried the wild-type virus yielded cultures positive for infectious virus. Apart from nasal epithelium, no infectious virus was recovered from any other tissue. PCR confirmed the presence of virus DNA in the ganglia from all six ponies. Lymphoid tissues also yielded positive signals using this technique. The relevance of virus detection by PCR in lymphoid and neural tissues is discussed in relation to the potential for reactivation of latent virus in the host. However, evidence is presented to show that EHV-1 is neurotropic and, in common with other members of the alpha-herpesvirus subfamily, establishes latency in sensory ganglia from which virus can be reactivated.

The pathogenesis of wild type and drug resistant mutant strains of bovine herpesvirus-1 (BHV-1) in the natural host.

An experimental infection with bovine herpesvirus-1 was established in calves by means of intranasal inoculation. Three calves were infected with the parental strain BHV-1 w/t, three with the TK-defective strain, B 1 and four with the HPMPA-resistant strain, 3 A. Inoculation with w/t virus resulted in a reproducible clinical disease characterised by respiratory distress, fever and the presence of virus in nasal mucus. Following the acute infection, w/t-inoculated animals became seropositive for BHV-1 specific antibody. The TK-defective mutant (BHV-1 B 1) produced an acute infection similar to the parental virus in all three calves inoculated. The HPMPA-resistant mutant (BHV-1 3 A), however, showed a reduced pattern of infection and virus of lower titre was isolated from three of four calves; the antibody responses were generally lower, and one calf remained seronegative until reactivation. Following stimulation with dexamethasone 72 days after the primary inoculation, virus was re-isolated from all wild type-inoculated calves. In contrast, no evidence of reactivation was obtained from the three B 1-inoculated animals. However, all four animals inoculated with the mutant 3 A showed virus reactivation including the calf which had remained seronegative following primary virus inoculation. Previous studies have suggested that drug-resistance mutations in herpesviruses frequently are associated with reduced pathogenicity on the basis of experiments in laboratory models. The importance of the present study is the demonstration that two different drug-resistant variants of an alpha herpesvirus both have altered pathogenicity in the natural host for that infection. These results also have implications for the design and use of attenuated vaccine strains.