Differences in replication kinetics and cell tropism between neurovirulent and non-neurovirulent EHV1 strains during the acute phase of infection in horses.

Equine herpesvirus 1 (EHV1) replicates in the respiratory tract of horses, after which infected leukocytes transport virus throughout the body, resulting in abortion or nervous system disorders. Two EHV1 strains circulate in the field: neurovirulent and non-neurovirulent. To investigate differences in replication in the upper respiratory tract (URT), an experimental inoculation study in ponies was performed with both strains. Two groups of six ponies, were inoculated intranasally with 10(6.5) TCID(50) of either strain. Clinical signs, nasal shedding and viremia were evaluated. At early time points post-inoculation (pi), one pony of each group was euthanized. Tissues were collected for titration and immunostainings. Number and size of EHV1-induced plaques were calculated, and individual EHV1-infected cells were quantified and characterized. Inoculation with either strain resulted in nasal shedding and replication in several tissues of the URT. Both strains replicated in a plaquewise manner in epithelium of the nasal mucosa, but replication in epithelium of the nasopharynx was largely limited to non-neurovirulent EHV1. Plaques were never able to cross the basement membrane, but individual infected cells were noticed in the connective tissue of all examined tissues for both strains. The total number of these cells however, was 3-7 times lower with non-neurovirulent EHV1 compared to neurovirulent EHV1. CD172a(+) cells and CD5(+) lymphocytes were important target cells for both strains. Interestingly, in lymph nodes, B-lymphocytes were also important target cells for EHV1, irrespective of the strain. Viremia was detected very early pi and infected cells were mainly CD172a(+) for both strains. In summary, these results are valuable for understanding EHV1 pathogenesis at the port of entry, the URT.

Multiple oral dosing of valacyclovir in horses and ponies.

The aim of the current study was to investigate whether multiple oral dosing of valacyclovir could result in plasma concentrations exceeding the EC(50)-value of acyclovir against equine herpesvirus 1 (EHV1) during the majority of the treatment period. Additionally, we wanted to determine the concentration of acyclovir in nasal mucus and cerebrospinal fluid (CSF). Valacyclovir was administered to four horses and two ponies, three times daily, at a dosage of 40 mg/kg, for four consecutive days. Blood was collected prior to each administration and 1 h after dosing. Nasal mucus samples and CSF were collected once during treatment; 1 h after the last administration. This dosage regimen resulted in plasma concentrations that were higher than the EC(50)-value of 1.7 microg/mL, i.e. EC(50) of an isolate highly susceptible to acyclovir, for 80% of the treatment period; and higher than the EC(50)-value of 3.0 microg/mL, i.e. EC(50) of an isolate less susceptible to acyclovir, for 60% of the treatment period. Concentration in nasal mucus samples and CSF was 0.36-1.17 microg/mL and 0.11-0.23 microg/mL, respectively. This study illustrates that multiple dosing of valacyclovir may result in a therapeutic benefit as plasma concentrations could be maintained above the EC(50)-value of acyclovir against EHV1 for more than 50% of the treatment period. Acyclovir could be detected in both nasal mucus samples and CSF. However, these concentrations were lower than the EC(50).

Evaluation of orally administered valacyclovir in experimentally EHV1-infected ponies.

The purpose of the current study was to investigate the therapeutic efficacy of valacyclovir against EHV1 in a controlled study. Eight naïve Shetland ponies were inoculated with 10(6.5) TCID(50) of the neuropathogenic strain 03P37. Four ponies were treated with valacyclovir at a dosage of 40mg/kg bodyweight, 3 times daily, for 5 (n=2) or 7 (n=2) consecutive days, while the other four ponies served as untreated controls. The treatment regimen started 1h before inoculation. Ponies were monitored daily for clinical signs. At 0, 1, 2, 3, 4, 5, 7, 9, 11, 14, 17 and 21 days post inoculation (d pi), a nasopharyngeal mucus sample was taken to determine viral shedding. At the same time points, blood was collected and peripheral blood mononuclear cells (PBMC) were isolated to determine viremia. During the treatment, blood samples were collected 6 times daily, i.e. just before valacyclovir administration and 1h later, to determine the concentration of acyclovir in plasma. Also a nasopharyngeal swab was taken to measure the acyclovir concentration in nasal secretion. No differences could be noticed between valacyclovir-treated and untreated ponies. The clinical signs, the viral shedding and the viremia were similar in both the groups. Plasma acyclovir concentration could be maintained above the EC(50)-value of EHV1 during 50% of the entire treatment period in valacyclovir-treated ponies. Acyclovir could be detected in nasal swabs at concentrations varying from 50% to 100% of the corresponding plasma concentration. Although sufficiently high acyclovir levels could be reached in plasma and nasal mucus, no effect was seen of the treatment with valacyclovir on clinical signs, viral shedding and viremia of EHV1-infected ponies.

Determination of acyclovir in horse plasma and body fluids by high-performance liquid chromatography combined with fluorescence detection and heated electrospray ionization tandem mass spectrometry.

Two methods are presented for the determination of 'respectively' the plasma protein unbound and total concentration of acyclovir in horse plasma and body fluids: first, a liquid-liquid extraction was performed on plasma, combined with HPLC-fluorescence detection for the total plasma concentration; second a more sensitive method using high-performance liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) was described for plasma and for body fluids analysis. To obtain the unbound concentration of acyclovir in plasma, a simple deproteinization step using a Microcon filter was performed. Ganciclovir was used as an internal standard. Analysis was carried out on an Inertsil 5 ODS-3 column for the HPLC-fluorescence method. For the LC-HESI-MS/MS method a PLRP-S column was used. The limit of quantification (LOQ) for the total concentration was set at 50 and 2 ng mL(-1) for the HPLC-fluorescence method and the LC-HESI-MS/MS method, respectively. The limit of quantification for the unbound concentration was set at 5 ng mL(-1) and at 2 ng mL(-1) for body fluids. The methods were successfully used to perform pharmacokinetic and clinical studies in horses after intravenous and oral dosage of acyclovir and its prodrug valacyclovir.

Pharmacokinetics of acyclovir after intravenous infusion of acyclovir and after oral administration of acyclovir and its prodrug valacyclovir in healthy adult horses.

The purpose of this study was twofold. The first aim was to evaluate the oral bioavailability and pharmacokinetics (PKs) of acyclovir in horses after intravenous (i.v.) administration and after oral administration of acyclovir and its prodrug, valacyclovir. Second, we aimed to combine these PK data with pharmacodynamic (PD) information, i.e., 50% effective concentrations (EC(50) values) from in vitro studies, to design an optimal dosage schedule. Three treatments were administered to healthy adult horses: 10 mg of acyclovir/kg of body weight delivered as an i.v. infusion over 1 h, 20 mg of acyclovir/kg administered as tablets by nasogastric intubation, and 20 mg of valacyclovir/kg administered as tablets by nasogastric intubation. Total plasma concentrations were measured by a high-performance liquid chromatography method combined with fluorescence detection, while unbound plasma concentrations were determined by liquid chromatography-tandem mass spectrometry. The peak concentration of i.v. acyclovir was approximately 10 mug/ml for both the total and the unbound plasma concentrations. The mean half-life of elimination was between 5.05 h (total concentration) and 11.9 h (unbound concentration). Oral administration of acyclovir resulted in low maximum concentration in plasma (C(max)) and poor bioavailability. A 10-times-higher C(max) and an 8-times-higher bioavailability were achieved with oral administration of valacyclovir. The i.v. administration of 10 mg/kg acyclovir and the oral administration of 20 mg/kg valacyclovir achieved concentrations within the sensitivity range of equine herpesvirus type 1 (EHV-1). The higher bioavailability of valacyclovir makes it an attractive candidate for the prophylactic and/or therapeutic treatment of horses infected with EHV-1. The results from the PK/PD modeling showed that a dosage of 40 mg/kg valacyclovir, administered three times daily, would be sufficient to reach plasma concentrations above the EC(50) values.

In vitro susceptibility of six isolates of equine herpesvirus 1 to acyclovir, ganciclovir, cidofovir, adefovir, PMEDAP and foscarnet.

Equine herpesvirus 1 (EHV-1) is an important equine pathogen that causes respiratory disease, abortion, neonatal death and paralysis. Although vaccines are available, they are not fully protective and outbreaks of disease may occur in vaccinated herds. Therefore, there is an urgent need for effective antiviral treatment. For three abortigenic (94P247, 97P70 and 99P96) and three neuropathogenic isolates (97P82, 99P136 and 03P37), the effect of acyclovir, ganciclovir, cidofovir, adefovir, 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) and foscarnet on plaque number was studied. Additionally, for isolate 97P70, the effect on plaque size was investigated. Ganciclovir was most potent in reducing plaque number, followed by PMEDAP and acyclovir. Adefovir and cidofovir were less effective and foscarnet was the least effective compound. There were no differences detected for acyclovir, ganciclovir, adefovir and PMEDAP between the abortigenic and neuropathogenic isolates. One abortigenic isolate (99P96) was more susceptible to cidofovir and two neuropathogenic isolates (99P136 and 03P37) were less susceptible to foscarnet. For isolate 97P70, all compounds resulted in a significant reduction of plaque size. The most remarkable effect was observed for cidofovir. It was 40-fold more effective in reducing plaque size than in reducing plaque number. In conclusion, ganciclovir was the most potent compound and therefore, may be a valuable candidate for the treatment of EHV-1 infections in horses. The antiviral effect of foscarnet on plaque number was highly dependent on the viral isolate tested. Therefore, it is no valuable antiviral for the treatment of herpesvirus-infections. Cidofovir, although less effective in reducing plaque number, had a strong effect on plaque size.

In vitro comparison of antiviral drugs against feline herpesvirus 1.

BACKGROUND: Feline herpesvirus 1 (FHV-1) is a common cause of respiratory and ocular disease in cats. Especially in young kittens that have not yet reached the age of vaccination, but already lost maternal immunity, severe disease may occur. Therefore, there is a need for an effective antiviral treatment. In the present study, the efficacy of six antiviral drugs, i.e. acyclovir, ganciclovir, cidofovir, foscarnet, adefovir and 9-(2-phosphonylmethoxyethyl)-2, 6-diaminopurine (PMEDAP), against FHV-1 was compared in Crandell-Rees feline kidney (CRFK) cells using reduction in plaque number and plaque size as parameters. RESULTS: The capacity to reduce the number of plaques was most pronounced for ganciclovir, PMEDAP and cidofovir. IC50 (NUMBER) values were 3.2 microg/ml (12.5 microM), 4.8 microg/ml (14.3 microM) and 6 microg/ml (21.5 microM), respectively. Adefovir and foscarnet were intermediately efficient with an IC50 (NUMBER) of 20 microg/ml (73.2 microM) and 27 microg/ml (140.6 microM), respectively. Acyclovir was least efficient (IC50 (NUMBER) of 56 microg/ml or 248.7 microM). All antiviral drugs were able to significantly reduce plaque size when compared with the untreated control. As observed for the reduction in plaque number, ganciclovir, PMEDAP and cidofovir were most potent in reducing plaque size. IC50 (SIZE) values were 0.4 microg/ml (1.7 microM), 0.9 microg/ml (2.7 microM) and 0.2 microg/ml (0.7 microM), respectively. Adefovir and foscarnet were intermediately potent, with an IC50 (SIZE) of 4 microg/ml (14.6 microM) and 7 microg/ml (36.4 microM), respectively. Acyclovir was least potent (IC50 (SIZE) of 15 microg/ml or 66.6 microM). The results demonstrate that the IC50 (SIZE) values were notably lower than the IC50 (NUMBER) values. The most remarkable effect was observed for cidofovir and ganciclovir. None of the products were toxic for CRFK cells at antiviral concentrations. CONCLUSION: In conclusion, measuring reduction in plaque number and plaque size are two valuable and complementary means of assessing the efficacy of an antiviral drug. By using these parameters for six selected antiviral drugs, we found that ganciclovir, PMEDAP, and cidofovir are the most potent inhibitors of FHV-1 replication in CRFK cells. Therefore, they may be valuable candidates for the treatment of FHV-1 infection in cats.