Antiviral drug resistance.

Recently the first report of zidovudine-resistant human immunodeficiency virus obtained from AIDS patients was published. Resistance to antiviral agents may result from single point mutations in the virus genome. Several mechanisms of resistance have already been elucidated at the biochemical level but the clinical significance of drug resistance is much more difficult to establish. Most attention is now focused on the immunocompromised host where clinically important resistance has been encountered most frequently. Hugh Field and Siân Goldthorpe describe the mechanisms of resistance in viruses that are currently targets for chemotherapy and discuss the likely future role of drug-resistant virus infections in man.

Sequence analysis of thymidine kinase-defective mutants of equine herpesvirus-1 (EHV-1).

We have amplified, cloned and sequenced the gene encoding the thymidine kinase (TK) of a wild-type strain (Ab4) of equine herpesvirus-1 (EHV-1) and two mutants with defective TK activity isolated for resistance to penciclovir (PCV). One of the mutants, PR1, has suffered a 879-bp deletion which reduces the size of TK to 180 bp. The other mutant, PR3, has an adenine to cytosine mutation resulting in a Lys38-->Thr change. This mutation modifies the amino acid sequence of a domain involved in binding ATP, leading to non-detectable enzymatic activity. Lys38 thus appears to be essential for the activity of the TK of EHV-1.

Isolation and characterization of acyclovir-resistant strains of herpes simplex virus.

A number of clinical studies have documented herpes simplex infections which appear to be resistant to nucleoside analogs; these include idoxuridine [1,2] and acyclovir [3]. Few, if any of the viruses isolated from such patients have yet been thoroughly characterized. We have isolated a number of acyclovir-resistant mutants by selection for resistance in tissue culture. The study of the biochemical and biological properties of these mutants has given some insight into the likely nature of resistant clinical strains. We have devised a number of simple tests to allow classification of laboratory mutants. We also draw attention to some of the difficulties the clinical virologist may encounter when analyzing putative resistant virus isolated from treated patients.

Molecular studies of the acute infection, latency and reactivation of equine herpesvirus-1 (EHV-1) in the mouse model.

The murine intranasal (i.n.) infection model was used to study the molecular distribution of equine herpesvirus-1 (EHV-1) during acute infection, latency and following a reactivation stimulus. After inoculation, infectious virus was detected in lungs, nasal turbinates, brains and olfactory bulbs during the acute phase. A nested PCR (nPCR) readily detected virus in these tissues and, in addition, virus was detected in spleens and (in the second round of nPCR) in peripheral blood mononuclear cells (PBMC). A digoxigenin-labelled in situ hybridization probe detected EHV-1 DNA in bronchiolar and vascular endothelium in the lungs and in and around germinal centres in the spleens. One month later, although infectious virus was absent from all tissues, the trigeminal ganglia, olfactory bulb and PBMC remained positive for virus DNA although this was detected only on the second round of nPCR. Furthermore, in situ hybridization, using either DNA or RNA probes, suggested that little or no transcription of virus occurred in neural tissues during the 'latent phase'. Following a reactivation stimulus, infectious virus was not isolated from any tissues, however, EHV-1 DNA was detected on the first round of nPCR in olfactory bulb, trigeminal ganglia and PBMC. This suggested a quantitative increase in EHV-1 DNA occurred following reactivation stimulus. The significance of these results is discussed in relation to the molecular state of EHV-1 in different tissues at various stages of infection and the validity of the murine model for studying latency and reactivation of EHV-1 in the horses.

Virological and molecular biological investigations into equine herpes virus type 2 (EHV-2) experimental infections.

Two 18-month-old naturally reared ponies were used to investigate the pathogenicity of EHV-2. After dexamethasone treatment, pony 1 was inoculated intranasally with EHV-2 strain T16, which has been isolated from a foal with keratoconjunctivitis superficialis and pony 2 was similarly inoculated with strain LK4 which was originally isolated from a horse with upper respiratory tract disease. Following virus inoculation, pyrexia was not detected in either pony but both developed conjunctivitis, lymphadenopathy, and coughing. EHV-2 was detected in nasal mucus samples up to day 12 post infection (p.i.), in eye swabs up to day 10 p.i., and in buffy coat cells throughout the investigation in both animals. EHV-2-specific antibody titres were raised significantly 18 days p.i. Following the administration of dexamethasone, 3 months p.i., infectious virus was again detected in nasal mucus and conjunctival swabs from both ponies for 7 days. The tissue distribution of EHV-2 genome was studied post mortem, by means of a nested PCR. EHV-2 was detected in lymphoid tissues, lung, conjunctiva, trigeminal ganglia and olfactory lobes of pony 2, whereas in pony 1 only the conjunctiva of the left eye was PCR positive.

Role of neutralizing antibodies and T-cells in pathogenesis of herpes simplex virus infection in congenitally athymic mice.

Congenitally athymic nude mice were infected with 10(4) p.f.u. herpes simplex type 1 (strain SC16). Following the passive transfer of neutralizing monoclonal antibodies (AP7, AP8 and AP12) it was observed that AP7 alone reduced the virus infectivity in the nervous system; AP8 and AP12 failed to protect mice probably due to poor in vivo binding to the neutralization site on the virus. Latent ganglionic infection could be established in nude mice following adoptive transfer of optimum number (2 x 10(7) cells/mouse) of immune lymph node cells from day 7 herpes virus-infected hairy immunocompetent donor mice. Moreover, in some of the immune lymph node cell protected nudes, latency could be maintained even in complete absence of neutralizing antibodies. Results of ear-ablation experiments revealed that removal of primary source of infection after day 5 of infection reduced the amount of virus in the ganglia and spinal cord. Acute neurological infection was not detected following transfer of protective anti-gp-D neutralizing antibody (LP2) in combination with removal of infected pinna. These data suggest that continuous seeding of virus occurs in related ganglia via the axonal route from infected ear pinna. It appears that local T-cell-mediated immune mechanisms are involved in maintenance of latency.

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.

Chemotherapy of Aujeszky's disease (pseudorabies) in the mouse by means of nucleoside analogues: bromovinyldeoxyuridine, acyclovir, and dihydroxypropoxymethylguanine.

Pseudorabies virus (PRV) infection was established in mice by means of inoculating the ear flap. The infection was universally fatal once clinical signs appeared. Bromovinyldeoxyuridine (BVDU) was a potent inhibitor of PRV in vitro, but this drug failed to protect mice and produced only marginal reductions in virus titre and slight prolongation of survival. Acyclovir (ACV) and dihydroxypropoxymethylguanine (DHPG) were both less active than BVDU when tested against the virus in BHK cells, yet DHPG therapy was extremely effective in mice; it reduced virus titres markedly and resulted in the long-term survival of mice given a potentially lethal infection. When ACV and DHPG were tested in vitro using murine rather than hamster cells, these compounds, especially DHPG, were shown to be much more active against PRV.

The inhibition of bovine herpesvirus-1 by methyl 2-pyridyl ketone thiosemicarbazone and its effects on bovine cells.

Methyl 2-pyridyl ketone thiosemicarbazone (MPKT) was found to inhibit bovine herpesvirus-1 (BHV-1) at an ED50 concentration of approximately 5-10 microM. Several virus strains were shown to have similar sensitivity to the drug and serial passage of virus in the presence of MPKT failed to yield resistant progeny. There was evidence for toxic effects on cells at drug concentrations similar to those required to inhibit virus and passage of cells in low concentrations of MPKT gave results suggesting cumulative toxicity. Pre-incubation of cells in the presence of MPKT produced a residual antiviral effect. Taken together, these observations cast doubt on the selectivity of the drug for BHV-1, at least in the bovine system under test.

The effect of (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC) on bovine herpesvirus-1 (BHV-1) infection and reactivation in cattle.

A reproducible pattern of respiratory disease was produced in calves inoculated intranasally with a pathogenic strain of bovine herpesvirus-1 (BHV-1). A latent infection was established which could be reactivated by means of corticosteroid administration. Groups of calves were given a single dose of 20 mg/kg of (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC) either the day before or the day following virus inoculation. The drug markedly reduced clinical signs and virus replication; the therapeutic dose appeared to be more effective than the dose given one day before virus inoculation. The establishment of latency was not prevented and a single dose of HPMPC, the day before a course of dexamethasone (6 weeks after the acute infection), did not prevent virus shedding.

Animal models for antiviral chemotherapy.

Traditionally animal models have formed a vital part of the preclinical evaluation of new forms of antiviral therapy. A variety of models used in the past or potentially useful in the future are considered in this short review. Several valuable and complex questions concerning virus-drug interactions in vivo have been successfully addressed by means of animal models. Better understanding of drug modes of action and virus pathogenesis in the models enable even more accurate predictions to be made for the outcome of antiviral therapy in man. The complexity of virus infections in man is such that animals are likely to remain an important part in drug evaluation for many years. To this end, new developments such as improved techniques in the production of transgenic animals are opening up a variety of completely novel methods for studying inhibitors of a wider group of viruses in vivo including the human immunodeficiency virus. However, the correct interpretation of animal data requires the critical evaluation of animal models. This review will identify several important difficulties which confront those working on antiviral chemotherapy in animals and which must continue to be addressed if confidence in animal data is to be maintained.

Characterization of latent infections in mice inoculated with herpes simplex virus which is clinically resistant to acyclovir.

Mice were inoculated into the ear pinna with herpes simplex virus (HSV) using a strain which is resistant to acyclovir (ACV) chemotherapy. The original inoculum was resistant to ACV because it contained a proportion of thymidine kinase-defective (TK-) virions. This had been obtained previously by passage of an HSV type 1 strain in mice undergoing suboptimal therapy. The cervical dorsal root ganglia were subsequently explanted from the infected mice and the presence of latent virus therein revealed by reactivation in vitro. These explant cultures yielded both TK+ and TK- viruses on reactivation. The establishment of latent infections was not affected by chemotherapy during the acute infection. One TK- ganglion isolate when studied in detail was found to be attenuated and thus resembled previously examined TK- strains which had been selected in vitro for ACV-resistance.

Isolation of bromovinyldeoxyuridine-resistant strains of herpes simplex virus and successful chemotherapy of mice infected with one such strain by using acyclovir.

Several strains of herpes simplex virus which were resistant to bromovinyldeoxyuridine were isolated by passaging the virus in the presence of the drug in tissue culture. The resistance of the majority of isolates was accounted for by their reduced ability to induce the enzyme thymidine kinase. These strains were co-resistant to acyclovir, but showed reduced pathogenicity in mice. However, another type of bromovinyldeoxyuridine-resistant virus was isolated which induced normal levels of thymidine kinase and retained virulence for mice. This resistant virus was sensitive to acyclovir and was successfully treated using oral acyclovir therapy.

The activity of (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC) against equine herpesvirus-1 (EHV-1) in cell cultures, mice and horses.

The activity of the nucleotide analogue, (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC), against equine herpesvirus-1 (EHV-1) was tested in cell culture, mice and foals. The ED50 for plaque reduction was found to be 0.07 and 0.03 microgram/ml in RK-13 and EEL cells respectively. In mice, a single administration of HPMPC (20 mg/kg, s.c.) was very effective at reducing clinical signs and virus replication if given on the day before intranasal inoculation with EHV-1. Treatment on the day of infection or day 1 p.i. was less effective, but still significantly reduced clinical signs and virus titres in the target organs (lungs and nasal tissue). Furthermore, HPMPC was found to protect mice partially from an intracerebral inoculation with EHV-1. Experiments in the horse suggested that HPMPC was also very active against EHV-1 in the natural host. Thus a single administration of HPMPC at 20 mg/kg, s.c., on the day of infection, markedly reduced clinical signs and nasal excretion of virus following intranasal inoculation with EHV-1. HPMPC given as a divided dose of 1 mg/kg on day 0 and day 3 p.i. had no effect on clinical signs but did reduce nasal excretion of virus. The significance of these results is discussed.

The acyclic nucleoside analogue penciclovir is a potent inhibitor of equine herpesvirus type 1 (EHV-1) in tissue culture and in a murine model.

Equine herpesvirus type 1 (EHV-1) was sensitive to the nucleoside analogue penciclovir (PCV) when tested in tissue culture; the ED50 was 1.6 micrograms/ml. Drug-resistant mutants were selected which were found to be TK-defective and approx. 45-fold less sensitive to PCV compared with the parental strain. PCV was compared with the phosphonyl derivative, HPMPA in mice infected with EHV-1. Both drugs were shown to be effective in vivo, limiting wild-type virus replication in respiratory tissues, and reducing viraemia. The treated mice also showed less clinical signs and reduced histopathology compared with placebo-treated controls. The establishment of latent EHV-1 in the mice, however, was not prevented. The results obtained with mice suggest that antiviral chemotherapy may be practical in the horse and that this possibility is worthy of further investigation in the natural host.

Herpes simplex virus zosteriform lesions with adoptive transfer of immune cells: a murine model which mimics human recurrent disease.

Existing murine models for cutaneous herpes simplex virus type 1 (HSV-1) infection have limited relevance to recurrent disease in humans, since the infection is usually primary rather than reactivated and infection occurs in the absence of an established immune response. To obtain a reproducible model to study the effects of topical antiviral therapy on recurrent disease we have adapted a mouse model which employs zosteriform spread of HSV-1 in the presence of adoptive transfer of immunity (ATI) which mimics human recrudescent lesions. Mice were infected with HSV-1 by scarification at the lateroventral line of the neck; 2 days later, the mice received adoptive transfer of immune cells from the cervical lymph nodes of syngeneic mice that had been infected in the ear pinna with the same strain of virus 7 days earlier. ATI resulted in a heightened inflammatory response in the target tissues for virus replication. Virus was cleared more quickly from the infected tissues in comparison with mice similarly inoculated without ATI, however, the intensity and duration of the inflammation was greater. The model was then used to test the effect of a topical formulation of foscarnet. The results presented demonstrate that the ATI model can provide useful data concerning the efficacy of topical antiviral chemotherapy in man.

Isolation of equine herpesvirus-1 mutants in the presence of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine: demonstration of resistance in vitro and in vivo.

The compound (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) had been previously shown to be highly effective in treatment of EHV-1 in a murine model for the equine disease. This paper describes the isolation of a series of mutants resistant to the drug. Resistance was demonstrated in cell culture and one mutant was tested in a murine model. The resistant mutant was pathogenic for mice; infectious virus was recovered from respiratory tissues and blood at levels similar to the parental virus. However, the mutant showed a significant degree of resistance in vivo, thus proving the virus-specific mode of action of the antiviral compound.

Critical determinants of antiherpes efficacy of buciclovir and related acyclic guanosine analogs.

Buciclovir is an example of an antiherpes, acyclic guanosine analog activated by the viral thymidine kinase and inhibiting viral DNA synthesis in infected cells. An investigation of closely related buciclovir-analogs with similar antiherpes activities in cell cultures and similar, or identical, modes of action but with disparate effects in vivo, revealed the following critical determinants of antiherpes efficacy. (1) The accumulation of guanosine analog-triphosphates in infected cells, which is cell-type-specific and analog-dependent. (2) The potencies of the triphosphates as inhibitors of the viral DNA polymerase. (3) The plasma kinetics of the analogs, which are widely different despite the similar structures. (4) The penetration into nervous tissue relative to penetration into non-nervous tissues, of importance in connection with the neurotropic behavior of the virus. (5) The concentration of the antagonist thymidine in certain tissues. (6) The difference in pathogenesis between primary infections and recurrent infections, exemplified in the different efficacies of topically applied drugs in cutaneous and genital HSV-2 infections in guinea pigs.

A reliable method for establishing viral infection in the rabbit by intranasal inoculation.

Bovine herpesvirus 1 (BHV-1) infection was established in the rabbit by a novel procedure which involved virus being injected through trephine openings on either side of the nose of anaesthetised animals. This method of intranasal inoculation produced more consistent reactions to BHV-1 inoculation, and both humoral and cell-mediated immune responses to the virus were of greater magnitude compared with those following per naris instillation of virus.

Combinations of antiviral and anti-inflammatory preparations for the topical treatment of herpes simplex virus assessed using a murine zosteriform infection model.

Recently we have reported a zosteriform murine infection model which employs the adoptive transfer of immune cells (ATI) to recipient infected mice to produce a disease that mimics human recurrent herpes simplex virus (HSV) disease. Mice were infected with HSV-1 by scarification at the lateroventral line of the neck; 2 days later, the mice received immune cells from HSV-1-infected syngeneic mice. Although virus was cleared more quickly from the target tissues of virus replication in recipient mice, ATI resulted in a heightened inflammatory response and delayed healing. This model was used to test the effects of topical formulations containing foscarnet and/or the anti-inflammatory agent, hydrocortisone. Virus clearance and clinical signs, including ear thickness and zosteriform spread of lesions, were studied. Treatment with 3% foscarnet accelerated virus clearance but had little effect on clinical parameters. By contrast, 0.5% hydrocortisone increased the titre and extended the presence of infectious virus for at least 6 days, although the reduction in clinical signs was greater than that obtained with topical foscarnet. Foscarnet in combination with hydrocortisone produced a marked reduction in clinical signs while virus replication was reduced. These results are discussed in relation to the inflammation and discomfort experienced by patients and a possible role for anti-inflammatory formulations in the treatment of HSV reactivation episodes in man.