Hugh J. Field

Antiviral prodrugs – the development of successful prodrug strategies for antiviral chemotherapy

Following the discovery of the first effective antiviral compound (idoxuridine) in 1959, nucleoside analogues, especially acyclovir (ACV) for the treatment of herpesvirus infections, have dominated antiviral therapy for several decades. However, ACV and similar acyclic nucleosides suffer from low aqueous solubility and low bioavailability following oral administration. Derivatives of acyclic nucleosides, typically esters, were developed to overcome this problem and valaciclovir, the valine ester of ACV, was among the first of a new series of compounds that were readily metabolized upon oral administration to produce the antiviral nucleoside in vivo, thus increasing the bioavailility by several fold. Concurrently, famciclovir was developed as an oral formulation of penciclovir. These antiviral ‘prodrugs’ thus established a principle that has led to many successful drugs including both nucleoside and nucleotide analogues for the control of several virus infections, notably those caused by herpes‐, retro‐ and hepatitisviruses. This review will chart the origins and development of the most important of the antiviral prodrugs to date.

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.

Effect of acycloguanosine treatment of acute and latent herpes simplex infections in mice

Systemic treatment of mice with the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine [aciclovir]) was found to be highly effective against acute type 1 herpes simplex virus infection of the pinna. The drug ablated clinical signs and reduced virus replication both in tissue local to the inoculation site and within the nervous system. Provided that moderate-sized virus inocula were used, acycloguanosine treatment reduced or prevented the establishment of a latent infection in the dorsal root ganglia relating to the sensory nerve supply of the ear. However, although it aborted artificially produced infections in dorsal root ganglia, acycloguanosine was found not to be effective against the latent infection once established. This finding strongly indicated that latent herpes simplex virus in mice can exist in a nonreplicating form.

Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

The pathogenesis of equine herpesvirus type 1 in the mouse : a new model for studying host responses to the infection

An infection was established in adult BALB/c mice by means of intranasal inoculation of the AB4 strain of equine herpesvirus type 1 (EHV-1). The acute infection was confined to the respiratory tract and blood. Virus was shown to replicate in the nasal mucosa, trachea and lung for several days producing clinical signs of disease. Viraemia was also detected and a small proportion of peripheral blood cells contained virus at the peak of the infection. Histological and electron microscopic evidence were obtained which proved that productive virus replication occurred in the ciliated epithelial cells lining the bronchi and in pneumocytes in the lung, resulting in the destruction of these cells. Both humoral and cell-mediated responses to the infection were detected and monitored. By means of immunoprophylaxis or chemotherapy it was possible to modify the course of the infection. This infection model has many striking features in common with that observed in the natural host and the observations suggest that the mouse is a convenient and relevant model in which to study both host responses to EHV-1 infection and modification of the pathogenesis by means of immunoprophylaxis or therapy.

Herpes simplex virus antiviral drug resistance — current trends and future prospects

The various manifestations of herpes simplex virus (HSV) have been widely treated using antiviral agents for more than 40 years. Acyclovir (ACV) is the drug that has been most commonly used to date. When tested in cell culture, the majority of isolates of HSV are sensitive to ACV with ED50 values of approximately 0.1 microg/ml. ACV-resistant strains (defined as having ED50>2 microg/ml) are rarely encountered in clinical practice among normal patients (<1% isolates) and there is no firm evidence, to date, that this incidence is increasing. Resistant HSV occurs much more frequently, however, among immunocompromised patients during treatment (approximately 5% isolates) where this is recognised to be an important clinical problem leading to ineffective therapy. In this review it is argued that the rapid establishment of neuronal latency in the normal pathogenesis of HSV is the key to the low incidence of resistance development and leads to some optimism concerning future trends.

Cell-mediated immunity in herpes simplex virus-infected mice: induction, characterization and antiviral effects of delayed type hypersensitivity

Delayed type hypersensitivity (DTH) was induced ihe reaction was observed 4 to 5 days p.i. and could still be induced up to 18 months later. In contrast, the adoptive transfer of DTH using draining lymph node cells was only possible during the period 6 to 10 days p.i. The cells taken at these times also contained mediators of antiviral immunity, as determined by a marked reduction of virus titres in the ears of infected animals 1 to 3 days after transfer. Draining lymph node cells taken at later times contained mediators of virus immunity, but titres were not reduced until day 5 after the transfer. The cell type involved in both the DTH and antiviral activity was a T lymphocyte, although the particular T cell subsets involved have yet to be determined.

Pathogenesis of equine herpesvirus-1 in specific pathogen-free foals: primary and secondary infections and reactivation

SummarySix specific pathogen-free foals shown to be free of equine herpesvirus-1 and 4 (EHV-1 and -4) and lacking in maternally-derived antibodies were used to investigate the pathogenesis of EHV-1 in horses. Following primary intranasal inoculation with EHV-1 all foals showed signs of a mild, self-limiting upper respiratory tract infection. A leucopenia was observed, comprising both a lymphopenia and neutropenia. Virus was isolated from nasal mucus and buffy coat cells over several days during the clinical episode and after the animals became clinically normal. Notwithstanding the mildness of the clinical disease, virus was not eliminated completely and intravenous administration of dexamethasone resulted in reactivation of latent EHV-1 in animals which had received only a single dose of the virus. In a second infection given to four foals, 61 days after the primary inoculation, no clinical signs were observed, haematological changes were minimal and viraemia was absent.

Pathogenesis of Herpes Simplex Virus in Congenitally Athymic Mice: the Relative Roles of Cell-mediated and Humoral Immunity

Athymic nude (nu/nu) mice were inoculated in the ear pinna with 10(4) p.f.u. herpes simplex virus type 1 (strain SC 16). Initially, the virus was observed to replicate in the pinna, spreading via a neurological route to the dorsal root ganglia, spinal cord, brain and adrenal glands. Following the transfer of lymphoid cells from day 7 herpesvirus-infected hairy immunocompetent donors into infected nude mice, virus was not isolated from the pinna and nervous system of the majority of the mice. The passive transfer of neutralizing polyclonal anti-herpesvirus serum or neutralizing monoclonal anti-gp D serum did not reduce infectivity in the pinna, but markedly reduced the amount of virus in the ganglia and spinal cord. These data suggest that neutralizing antibodies play an important role in restricting the movement of virus to the nervous system, whereas cell-mediated immune (CMI) mechanisms are essential for eliminating virus from the pinna.

The distribution of herpes simplex type 1 antigen in mouse central nervous system after different routes of inoculation

Herpes simplex type 1 virus was inoculated into 3-week-old mice via four different routes; intracerebral, intravenous, intranasal and directly into the sciatic nerve. Virus antigen-containing cells in the central nervous system were identified by both an immunofluorescence and immunoperoxidase method. The portal of entry of virus into the CNS appeared to be the major determinant of distribution of virus antigen. Direct haematogenous seeding of virus into the CNS was not proven. It seems probable that infection was first established in sensory ganglia. Within the CNS, regions of high virus antigen concentration paralleled high cell density suggesting cell to cell spread. Consistent involvement of certain neuron groups may be due to their selective vulnerability. These animal experiments provide some explanation for the patterns of CNS herpetic infection observed in man.