Bacterial Toxins from Staphylococcus aureus and Bordetella bronchiseptica Predispose the Horse's Respiratory Tract to Equine Herpesvirus Type 1 Infection.

Respiratory disease in horses is caused by a multifactorial complex of infectious agents and environmental factors. An important pathogen in horses is equine herpesvirus type 1 (EHV-1). During co-evolution with this ancient alphaherpesvirus, the horse's respiratory tract has developed multiple antiviral barriers. However, these barriers can become compromised by environmental threats. Pollens and mycotoxins enhance mucosal susceptibility to EHV-1 by interrupting cell junctions, allowing the virus to reach its basolateral receptor. Whether bacterial toxins also play a role in this impairment has not been studied yet. Here, we evaluated the role of α-hemolysin (Hla) and adenylate cyclase (ACT), toxins derived from the facultative pathogenic bacterium Staphylococcus aureus (S. aureus) and the primary pathogen Bordetella bronchiseptica (B. bronchiseptica), respectively. Equine respiratory mucosal explants were cultured at an air-liquid interface and pretreated with these toxins, prior to EHV-1 inoculation. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed a decreased epithelial thickness upon treatment with both toxins. Additionally, the Hla toxin induced detachment of epithelial cells and a partial loss of cilia. These morphological changes were correlated with increased EHV-1 replication in the epithelium, as assessed by immunofluorescent stainings and confocal microscopy. In view of these results, we argue that the ACT and Hla toxins increase the susceptibility of the epithelium to EHV-1 by disrupting the epithelial barrier function. In conclusion, this study is the first to report that bacterial exotoxins increase the horse's sensitivity to EHV-1 infection. Therefore, we propose that horses suffering from infection by S. aureus or B. bronchiseptica may be more susceptible to EHV-1 infection.

Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: Efficacy of decitabine and valganciclovir alone or in combination.

Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.

Antiviral effect of sinefungin on in vitro growth of feline herpesvirus type 1.

Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

Intranasal treatment with CpG-B oligodeoxynucleotides protects CBA mice from lethal equine herpesvirus 1 challenge by an innate immune response.

Equine herpesvirus 1 (EHV-1) is the causative agent of a number of equine disease manifestations, including severe disease of the central nervous system, respiratory infections, and abortion storms. Our results showed that intranasal treatment with CpG-B oligodeoxynucleotides (ODN 1826) protected CBA mice from pathogenic EHV-1 RacL11 challenge. The IFN-γ gene and seven interferon-stimulated genes (ISGs) were upregulated 39.4- to 260.3-fold at 8 h postchallenge in the lungs of RacL11-challenged mice that had been treated with CpG-B ODN. Interestingly, IFN-γ gene expression was upregulated by 26-fold upon RacL11 challenge in CpG-B ODN-treated mice lungs as compared to that of CpG-A ODN (ODN 1585)-treated mice lungs; however, the seven ISGs were upregulated by 2.4-5.0-fold, suggesting that IFN-γ is a major factor in the protection of CBA mice from the lethal challenge. Pre-treatment with IFN-γ significantly reduced EHV-1 yield in murine alveolar macrophage MH-S cells, but not in mouse lung epithelial MLE12 cells. These results suggest that CpG-B ODN may be used as a prophylactic agent in horses and provide a basis for more effective treatment of EHV-1 infection.

Intranasal IgG4/7 antibody responses protect horses against equid herpesvirus-1 (EHV-1) infection including nasal virus shedding and cell-associated viremia.

Equid herpesvirus-1 (EHV-1) outbreaks continue despite widely used vaccination. We demonstrated previously that an ORF1/ORF71 gene deletion mutant of the EHV-1 strain Ab4 (Ab4ΔORF1/71) is less virulent than its parent Ab4 virus. Here, we describe the Ab4 challenge infection evaluating protection induced by the Ab4ΔORF1/71 vaccine candidate. Susceptible control horses developed respiratory disease, fever, nasal shedding, and viremia. Full protection after challenge infection was observed in 5/5 previously Ab4 infected horses and 3/5 Ab4ΔORF1/71 horses. Two Ab4ΔORF1/71 horses developed short-lasting viremia and/or virus shedding. Protective immunity in the respiratory tract was characterized by pre-existing EHV-1-specific IgG4/7 antibodies, the absence of IFN-α secretion and rapidly increasing IgG4/7 upon challenge infection. Pre-existing systemic EHV-1-specific IgG4/7 highly correlated with protection. T-cell immunity was overall low. In conclusion, protective immunity against EHV-1 infection including prevention of viremia was associated with robust systemic and intranasal IgG4/7 antibodies suggesting immediate virus neutralization at the local site.

Screening and evaluation of antiviral compounds against Equid alpha-herpesviruses using an impedance-based cellular assay.

Equid alpha-herpesviruses (EHV) are responsible for different diseases in equine population. EHV-1 causes respiratory diseases, abortions and nervous disorders, EHV-4 causes respiratory diseases and sporadic abortion, while EHV-3 is responsible of equine coital exanthema. In view of the lack of efficacy of vaccines against EHV-1 and EHV-4 and in the absence of vaccines against EHV-3, the use of antiviral treatment is of great interest. In this study, we documented the interest of the Real-Time Cell Analysis (RTCA) technology to monitor the cytopathic effects induced by these viruses on equine dermal cells, and established the efficacy of this method to evaluate the antiviral effect of aciclovir (ACV) and ganciclovir (GCV). In addition, the RTCA technology has also been found appropriate for the high-throughput screening of small molecules against EHV, allowing the identification of spironolactone as a novel antiviral against EHV.

The neuropathogenic T953 strain of equine herpesvirus-1 inhibits type-I IFN mediated antiviral activity in equine endothelial cells.

Equine herpesvirus-1 (EHV-1) infects equine endothelial cells (EECs) lining the small blood vessels in the central nervous system. However, the effect of type I IFN on EHV-1 replication in the EECs is not well studied. Thus, the primary objective of this study was to investigate the effect of type-I IFN on the replication of the neuropathogenic T953 strain of EHV-1 in vitro in EECs. The initial data showed that the EHV-1 was partly resistant to the biological effect of exogenously supplied recombinant equine IFN-α. Subsequent investigation into the mechanism of resistance showed that EHV-1 infection of EECs interfered with the STAT-1 phosphorylation through which type-I IFN exerts its antiviral effect. Immunofluorescence staining showed interference with the translocation of STAT-1 molecules from cytoplasm to nucleus confirming the virus mediated suppression of STAT-1 activation. Downstream of the JAK-STAT signaling, EHV-1 infection inhibited expression of cellular antiviral proteins including IFN-stimulated gene 56 (ISG56) and viperin. Taken together these findings suggest that the neuropathogenic T953 strain of EHV-1 evades the host innate immune response by inhibiting IFN and this may provide some insight into the pathogenesis of EHV-1 infection.

[DIFFERENTIAL SENSITIVITY OF MICROORGANISMS TO POLYHEXAMETHYLENEGUANIDINE].

Factors identified that affect the sensitivity of microorganisms to polyhexamethyleneguanidine (PHMG). Salts of PHMG chloride, valerate, maleate, succinate was to use. Test strains of Esherichia coli, Staphylococcus aureus, Bacillus cereus, Leptospira interrogans, Paenibacillus larvae, Mycobacterium bovis, M. avium, M. fortuitum, Aspergillus niger and some strains of viruses are taken as objects of research. We have determined that the cytoplasm membrane phospholipids is main "target" for the polycation molecules of PHMG. A differential sensitivity of the microorganisms to this drug is primarily determined by relative amount of lipids in membrane and their accessibility. Such trends exist: increase the relative contents of anionic lipids and more negative surface electric potential of membrane, and reduction of the sizes fat acid remainder of lipids bring to increase of microorganism sensitivity. Types of anion salt PHMG just have a certain value. Biocide activity of PHMG chloride is more, than its salts with organic acid. Feasibility of combining PHMG with other biocides in the multicomponent disinfectants studied and analyzed. This combination does not lead to a significant increase in the sensitivity of microorganisms tested in most cases. Most species of pathogenic bacteria can be quickly neutralized by aqueous solutions of PHMG in less than 1% concentrations.

Efficacy of five commercial disinfectants and one anionic surfactant against equine herpesvirus type 1.

We investigated the influences of various reaction conditions on equine herpesvirus type 1 (EHV-1) disinfection by 5 commercial disinfectants (3 quaternary ammonium compounds [QACs] and 2 chlorine-based disinfectants) and 1 anionic surfactant. QACs at their highest recommended concentrations had no virucidal effect on EHV-1 with a 10-min reaction time at 0°C or a 1-min reaction time at room temperature. Chlorine-based disinfectants achieved EHV-1 disinfection with a 10-min reaction time at -10°C or a 30-sec reaction time at room temperature. In the presence of 5% fetal bovine serum, QACs (except for benzalkonium chloride) showed more stable virucidal effects than did chlorine-based disinfectants. The virucidal effect of the anionic surfactant was almost equivalent to that of the QACs.

Evaluation of the antiviral activity of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine (A-5021) against equine herpesvirus type 1 in cell monolayers and equine nasal mucosal explants.

Equine herpesvirus 1 (EHV1) is a ubiquitous equine alphaherpesvirus that causes respiratory disease, neurological symptoms and abortions. Current vaccines are not fully protective and effective therapeutics are lacking. A-5021 [(1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine], previously shown to possess potent anti-herpetic activity against most human herpesviruses, was evaluated for its potential to inhibit EHV1 replication. In equine embryonic lung (EEL) cells, infected with either a non-neurovirulent (97P70) or a neurovirulent (03P37) EHV1 isolate, A-5021 proved to be about 15-fold more potent than acyclovir in inhibiting viral replication. Moreover, in equine nasal mucosal explants, A-5021 (at 8 and 32µM) was able to completely inhibit viral plaque formation whereas acyclovir did not exert an antiviral effect at these concentrations. Our data demonstrate that A-5021 is a potent inhibitor of EHV1 replication and may have potential for the treatment and/or prophylaxis of infections with this virus.

In vitro assessment of the antiviral potential of trans-cinnamic acid, quercetin and morin against equid herpesvirus 1.

The antiviral activity of quercetin, morin and trans-cinnamic acid was evaluated in vitro against equid herpesvirus 1 (EHV-1) by determining the virucidal activity and using the time of addition assay to test inhibition of the viral replication cycle. The cytotoxicity of each substance was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. Quercetin showed virucidal action and inhibition of the viral replication cycle at 0 and 1h. Morin showed potential virucidal and viral replication cycle inhibition at 0 h. Trans-cinnamic acid did not show virucidal activity but inhibited the viral replication cycle at -1 and 0 h. This study demonstrates the potential of these compounds as future antiviral candidates in relation to viruses of importance in veterinary medicine.

Pharmacokinetics of penciclovir after oral administration of its prodrug famciclovir to horses.

We investigated the pharmacokinetics of penciclovir after oral administration of its prodrug famciclovir to horses. Following an oral dose of famciclovir at 20 mg/kg, maximum plasma concentrations of penciclovir occurred between 0.75 and 1.5 hr (mean 0.94 + or - 0.38 hr) after dosing and were in the range 2.22 to 3.56 microg/ml (mean 2.87 + or - 0.61 microg/ml). The concentrations of penciclovir declined in a biphasic manner after the peak concentration was attained. The mean half-life of the rapid elimination phase was 1.73 + or - 0.34 hr whereas that of the slow elimination phase was 34.34 + or - 13.93 hr. These pharmacokinetic profiles observed were similar to those of another antiherpesvirus drug, acyclovir, previously reported in horses following oral dosing of its prodrug valacyclovir.

Antiviral effect of recombinant equine interferon-gamma on several equine viruses.

Recombinant equine interferon-gamma (reIFN-gamma) was prepared using a baculovirus expression system and its antiviral activity was investigated using several equine viruses. The reIFN-gamma suppressed the replication of all equine viruses used in the present experiment in horse cell cultures, but did not affect the growth of host cells at concentrations of less than 1000 u/ml. A strong antiviral effect was observed, especially against RNA viruses. Equine picornavirus, equine rhinovirus and equine arteritis virus could not be propagated at all in 100 u/ml reIFN-gamma when 100 TCID(50) of infective viruses was inoculated to cultivated horse cells. DNA viruses, equine herpesvirus types 1, 2, 3 and 4 and equine adenovirus, were less sensitive to reIFN-gamma but their growth became less than 1/100 in the cells treated with 100 u/ml reIFN-gamma compared to untreated cells. The antiviral effects were decreased in the cells of heterologous species and more than 1000 u/ml reIFN-gamma was required to induce an antiviral effect.

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.

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.

Down-regulation of MHC class I expression by equine herpesvirus-1.

There is good evidence that cytotoxic T lymphocytes play an important role in the clearance of equine herpesvirus-1 (EHV1) in horses. We have demonstrated that, in common with other alphaherpesviruses, EHV1 infection can lead to dramatic down-regulation of MHC class I expression at the cell surface, a common strategy for pathogen evasion of the host immune response. This down-regulation is specific for MHC class I and does not reflect a general shut-off of host-cell protein synthesis. The use of monoclonal antibodies that recognize different MHC class I epitopes has demonstrated that the effect may be allele- or locus-specific. Use of the viral DNA synthesis inhibitor phosphonoacetic acid, which prevents late viral gene expression, showed that the effect is mediated by an immediate-early or early viral gene, and use of the protein translation inhibitor cycloheximide confirmed that an early gene is primarily responsible. The data indicate that EHV1 infection results in enhanced endocytosis of MHC class I from the cell surface; the only other herpesvirus reported to use this mechanism is human herpesvirus-8. Elucidation of the precise mechanisms used by EHV1 in this process and identification of the genes responsible may lead to improved vaccination strategies.

Adaptation of equine herpesvirus 1 to unnatural host led to mutation of the gC resulting in increased susceptibility of the virus to heparin.

Heparin extensively inhibited infection of MDBK cells by equine herpesvirus 1 (EHV-1) strains adapted to bovine cells or hamsters, while the reagent merely reduced infectivity of strains passaged only in equine cells. The gC of two strains adapted to non-equine cells seemed to have higher affinity for heparin, although the reagent bound to both the gC and gB of all strains tested. Amino acid substitutions of the gC of the EHV-1 strains adapted to non-equine cells converged on the hydrophilic regions, amino acid residues 92 to 175, resulting in the glycoprotein becoming more cationic. These results indicate that these hydrophilic regions of the gC may be responsible for binding to heparin.

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.