Annick Gryspeerdt

Pharmacokinetics of Acyclovir after Intravenous Infusion of Acyclovir and after Oral Administration of Acyclovir and Its Prodrug Valacyclovir in Healthy Adult Horses

ABSTRACT 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 (EC50 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 μg/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 (Cmax) and poor bioavailability. A 10-times-higher Cmax 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 EC50 values.

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.

Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants

Equine herpesvirus type 1 (EHV-1) is the causative agent of equine herpes myeloencephalopathy, of which outbreaks are reported with increasing frequency throughout North America and Europe. This has resulted in its classification as a potentially emerging disease by the US Department of Agriculture. Recently, it was found that a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30) at aa 752 (N-->D) is associated with the neurovirulent potential of EHV-1. In the present study, equine respiratory mucosal explants were inoculated with several Belgian isolates typed in their ORF30 as D(752) or N(752), to evaluate a possible difference in replication in the upper respiratory tract. In addition, to evaluate whether any observed differences could be attributed to the SNP associated with neurovirulence, the experiments were repeated with parental Ab4 (reference neurovirulent strain), parental NY03 (reference non-neurovirulent strain) and their N/D revertant recombinant viruses. The salient findings were that EHV-1 spreads plaquewise in the epithelium, but plaques never cross the basement membrane (BM). However, single EHV-1-infected cells could be observed below the BM at 36 h post-inoculation (p.i.) for all N(752) isolates and at 24 h p.i. for all D(752) isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D(752) isolates compared with N(752) isolates at every time point analysed. Finally, this study showed that equine respiratory explants are a valuable and reproducible model to study EHV-1 neurovirulence in vitro, thereby reducing the need for horses as experimental animals.

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.

Equine alphaherpesviruses (EHV-1 and EHV-4) differ in their efficiency to infect mononuclear cells during early steps of infection in nasal mucosal explants

Equine herpesvirus type 1 (EHV-1) replicates extensively in the epithelium of the upper respiratory tract, after which it can spread throughout the body via a cell-associated viremia in mononuclear leukocytes reaching the pregnant uterus and central nervous system. In a previous study, we were able to mimic the in vivo situation in an in vitro respiratory mucosal explant system. A plaquewise spread of EHV-1 was observed in the epithelial cells, whereas in the connective tissue below the basement membrane (BM), EHV-1-infected mononuclear leukocytes were noticed. Equine herpesvirus type 4 (EHV-4), a close relative of EHV-1, can also cause mild respiratory disease, but a cell-associated viremia in leukocytes is scarce and secondary symptoms are rarely observed. Based on this striking difference in pathogenicity, we aimed to evaluate how EHV-4 behaves in equine mucosal explants. Upon inoculation of equine mucosal explants with the EHV-4 strains VLS 829, EQ(1) 012 and V01-3-13, replication of EHV-4 in epithelial cells was evidenced by the presence of viral plaques in the epithelium. Interestingly, EHV-4-infected mononuclear leukocytes in the connective tissue below the BM were extremely rare and were only present for one of the three strains. The inefficient capacity of EHV-4 to infect mononuclear cells explains in part the rarity of EHV-4-induced viremia, and subsequently, the rarity of EHV-4-induced abortion or EHM.

In vitro culture of equine respiratory mucosa explants.

An in vitro model of the upper respiratory tract of the horse was developed to investigate mechanisms of respiratory diseases. Four tissues of the upper respiratory tract of three horses were collected. Explants were maintained in culture at an air-liquid interface for 96h. At 0, 24, 48, 72 and 96h of cultivation, a morphometric analysis was performed using light microscopy, scanning electron microscopy and transmission electron microscopy. The explants were judged on morphometric changes of epithelium, basement membrane and connective tissue. Viability was evaluated using a fluorescent Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labelling (TUNEL) staining. No significant changes in morphometry and viability of any of the explants were observed during cultivation. Hence, the in vitro model may be useful to study infectious and non-infectious diseases at the level of the equine respiratory tract, with potential application to the development of vaccines and treatments for diseases of the respiratory tract.

Expression of late viral proteins is restricted in nasal mucosal leucocytes but not in epithelial cells during early-stage equine herpes virus-1 infection

Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.

Molecular characterization of equine rotaviruses isolated in Europe in 2013: Implications for vaccination

Abstract Equine group A rotavirus (RVAs) mainly cause disease in foals under the age of 3 months. Only sporadic data are available on the circulation of RVAs in equine populations in Europe. In this study, 65 diarrheic samples from foals under 4 months of age were collected in Belgium (n =32), Germany (n =17), Slovenia (n =5), Sweden (n =4), Hungary (n =3), Italy (n =2), France (n =1) and The Netherlands (n =1). Forty percent of these samples (n =26) were found to be RVA positive by a quantitative RT-PCR assay. The viral load in 11 of these samples was sufficiently high to be (partially) genotyped. G3, G14 and P[12] were the main genotypes detected, and phylogenetic analyses revealed that they were closely related to contemporary equine RVA strains detected in Europe as well as in Brazil and South Africa. Regional variation was observed with only G14 and P[12] being detected in Germany, whereas mainly G3P[12] was encountered in Belgium. Surprisingly the only G14P[12] RVA strain detected in Belgium was also found to possess the very rare P[18] genotype, which has been described only once from equine RVA strain L338 detected in the UK in 1991. Despite the identification of this uncommon P[18] genotype, G3P[12] and G14P[12] RVA strains remained the most important genotypes in Europe during the study period. Based on this finding and the knowledge that G3P[12] and G14P[12] serotypes are partially cross-reactive it can be assumed that a vaccine based on an inactivated virus of the G3P[12] genotype is still relevant in the current European epidemiological situation, although the addition of a G14 strain would most likely be beneficial.