Equine Dermal Cells Research Articles

Equid herpesvirus type 3 infection produces membrane-associated and secreted forms of glycoprotein G that are not required for efficient cell-to-cell spread of the virus in vitro.

The ORF 70 gene of equid alphaherpesvirus type 3 (EHV-3) encodes glycoprotein G (gG), which is conserved in the majority of alphaherpesviruses. This glycoprotein is located in the viral envelope and has the characteristic of being secreted into the culture medium after proteolytic processing. It modulates the antiviral immune response of the host by interacting with chemokines. The aim of this study was to identify and characterize EHV-3 gG. By constructing viruses with HA-tagged gG, it was possible to detect gG in lysates of infected cells, their supernatants, and purified virions. A 100-, 60-, and 17-kDa form of the protein were detected in viral particles, while a 60-kDa form was identified in supernatants of infected cells. The role of EHV-3 gG in the viral infection cycle was assessed by the construction of a gG-minus EHV-3 mutant and its gG-positive revertant. When growth characteristics in an equine dermal fibroblast cell line were compared, the plaque size and the growth kinetics of the gG-minus mutant were similar to those of the revertant virus, suggesting that EHV-3 gG does not play a role in direct cell-to-cell transmission or virus proliferation of EHV-3 in tissue culture. The identification and characterization of EHV-3 gG described here provide a solid background for further studies to assess whether this glycoprotein has a function in modulating the host immune response.

The cannabinoid receptors system in horses: Tissue distribution and cellular identification in skin.

BackgroundThe endocannabinoid system (ECS) is composed of cannabinoid receptors type 1 (CBR1) and type 2 (CBR2), cannabinoid‐based ligands (endogenous chemically synthesized phytocannabinoids), and endogenous enzymes controlling their concentrations. Cannabinoid receptors (CBRs) have been identified in invertebrates and in almost all vertebrate species in the central and peripheral nervous system as well as in immune cells, where they control neuroimmune homeostasis. In humans, rodents, dogs, and cats, CBRs expression has been confirmed in the skin, and their expression and tissue distribution become disordered in pathological conditions. Cannabinoid receptors may be a possible therapeutic target in skin diseases.ObjectivesTo characterize the distribution and cellular expression of CBRs in the skin of horses under normal conditions.AnimalsFifteen healthy horses.MethodsUsing full‐thickness skin punch biopsy samples, skin‐derived primary epidermal keratinocytes and dermal‐derived cells, we performed analysis of Cnr1 and Cnr2 genes using real‐time PCR and CBR1 and CBR2 protein expression by confocal microscopy and Western blotting.ResultsNormal equine skin, including equine epidermal keratinocytes and dermal fibroblast‐like cells, all exhibited constant gene and protein expression of CBRs.Conclusions and Clinical ImportanceOur results represent a starting point for developing and translating new veterinary medicine‐based pharmacotherapies using ECS as a possible target.

Assessment of BPV-1 Mediated Matrix Metalloproteinase Genes Deregulation in the In Vivo and In Vitro Models Designed to Explore Molecular Nature of Equine Sarcoids.

Matrix metalloproteinases (MMPs) represent a family of enzymes capable of biocatalytically breaking down the structural and functional proteins responsible for extracellular matrix (ECM) integrity. This capability is widely used in physiological processes; however, imbalanced MMP activity can trigger the onset and progression of various pathological changes, including the neoplasmic transformation of different cell types. We sought to uncover molecular mechanisms underlying alterations in transcriptional profiles of genes coding for MMPs, which were comprehensively identified in equine adult dermal tissue bioptates, sarcoid-derived explants, and ex vivo expanded adult cutaneous fibroblast cell (ACFC) lines subjected to inducible oncogenic transformation into sarcoid-like cells. The results strongly support the hypothesis that the transcriptional activity of MMP genes correlates with molecular modifications arising in equine dermal cells during their conversion into sarcoid cells. The alterations in MMP transcription signatures occurs in both sarcoid tissues and experimentally transformed equine ACFC lines expressing BPV1-E4^E1 transgene, which were characterized by gene up- and down-regulation patterns.

Circulation and characterisation of Equid herpesvirus‐3 strains in France between 2010 and 2021

Background: Equine coital exanthema (ECE) is an acute viral infection of horses caused by equid herpesvirus-3 (EHV-3). The presence of the virus and its economic impact is well described in different countries such as Argentina or Japan. However, even though it is included in the HBLB code of practice (UK) and in the list of diseases monitored by the RESPE (FR), only a few studies have been performed in Europe. Objectives: To determine the circulation of EHV-3 in France and measure the efficacy of new antiviral compounds against EHV-3. Study design: Retrospective field study and in vitro experiments. Methods: 71 field samples from suspected ECE cases, and collected between 2010 and 2021, were analysed by qPCR (DNA polymerase gene) and phylogenetic analysis (gG gene) was performed on 10 positive samples. Antiviral compounds (aciclovir, ganciclovir, valganciclovir, decitabine, idoxuridine, pritelivir, cidofovir, aphidicolin, vidarabine) previously described with antiviral properties against EHV-1, were evaluated using Real-Time Cell Analysis (RTCA) on equine dermal cells with the reference strain EHV-3 VR3522, cytopathic effect monitoring (microscopy) and viral load quantification (qPCR). Results: 16/71 (22%) suspected ECE cases were confirmed positive by PCR with 0 to 3 cases per year. French strains were clustered into two of the three observed phylogenic groups, but there was no correlation between the phylogeny and the time of collection or location. Aphidicolin was the most effective compound with an EC50 value of 2.43±0.02 μM (RTCA) and 1.63±0.48 μM (qPCR). Pritelivir and cidofovir were less potent molecules against EHV-3 (EC50 >50 μM). Main limitation: Antiviral compounds were tested against the reference strain only. Conclusions: This study demonstrates for the first time the presence of EHV-3 over a twelve-year period in France. In vitro studies show the antiviral efficacy of aphidicolin.

Avaliação dos efeitos de acepromazina e xilazina na viabilidade de linhagem celular dérmica de equino

Xylazine and acepromazine are drugs used exclusively in veterinary medicine. Xylazineis used as a sedative, analgesic, and tranquilizer while acepromazine is used as a sedative, pre-anesthetic, and anesthetic adjuvant. In vitrodrug toxicity experimentation is essential to predict possible damage associated with treatment. This study was carried out to evaluate and compare the in vitroeffects of acepromazine and xylazine on cell viability. Equine Dermis cells lines were used to examine different drug concentrations (0.02 mg/mL, 0.01 mg/mL, 0.005 mg/mL and 0.0025 mg/mL). An MTT assay was carried out to reveal cell viability. Both tested drugs reduced the viability of ED cells at 0.02 and 0.01 mg/mL. At 0.005 mg/mL, only acepromazine presented an effect. These results corroborate previous studies with xylazine. On the other hand, this is thefirst report about acepromazine and cell viability. Previous studies suggest that the mechanisms involved in reducing cell viability are apoptosis for xylazine and the activation of the autophagic pathway for acepromazine. Both mechanisms have been seen in other drugs of the same classes. These findings reveal that both acepromazine and xylazine cause concentration-dependent cytotoxicity in vitro. Future experiments could further elucidate the mechanisms by which this effect happens and thus circumvent therisk of potential tissue damage in vivo.

Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs.

Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.

Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.

Equine herpesvirus type 1 affects mitochondrial network morphology and reactive oxygen species generation in equine dermal cell line.

In the present study, the influence of the infection with equine herpesvirus type 1 (non-neuro-pathogenic and neuropathogenic strains of EHV-1) on the morphology and distribution of mitochondrial network in equine dermal cell line was investigated. Our results indicate that EHV-1-infection caused changes in the mitochondrial morphology manifested mostly by fission and reactive oxygen species generation.

An exploration of antigen expression of hepatitis C entry receptors on equine cells in relation to equine hepacivirus A

Equine hepacivirus A (EqHV) belongs to the family Flaviviridae and has been identified as the hepacivirus phylogenetically most closely related to Hepatitis C virus (HCV). Like HCV, EqHV is a hepatotropic virus that has been reported in over fourteen countries from six continents. It has a high prevalence in some populations, in particular the Thoroughbred breed. However, much is still unknown about EqHV, such as its pathogenesis and mode of transmission. The main four receptors used by HCV for viral entry to human hepatocytes are Cluster of Differentiation-81 (CD-81), occludin (OCLN), claudin-1 (CLDN-1) and Scavenger Receptor Class B Member 1 (SCAR-B1). This study investigated the distribution and expression of these entry receptors on equine cells by flow cytometry, immunocytochemistry and immunohistochemistry. Using a human liver cell line (Huh-7) as a positive control, antibodies against HCV receptors appeared to cross-react with antigens on equine cells. The proportion of fetal horse kidney cells that expressed CD-81, OCLN, and CLDN-1 was 37.2 %, 16.0 %, and 7.0 %, respectively, whereas the equine dermal cells (E.Derms) expressed CD-81 (96.0 %), OCLN (1.2 %) and CLDN-1 (1.7 %). CD-81, OCLN and CLDN-1 were also expressed on E.Derms and Huh7 cells, as detected by immunocytochemistry and on equine liver cells and the allantochorionic region of Thoroughbred placenta by immunohistochemistry. These findings form the basis for further comparative investigation into the entry receptors used by EqHV to infect equine and human cells. Such information may inform future studies on EqHV pathogenesis and mode(s) of transmission.

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 integration of a macrophage-adapted live vaccine strain of equine infectious anaemia virus (EIAV) in the horse genome.

Integration is an important feature of retroviruses and retrovirus-based therapeutic transfection vectors. The non-primate lentivirus equine infectious anaemia virus (EIAV) primarily targets macrophages/monocytes in vivo. Investigation of the integration features of EIAVDLV121 strains, which are adapted to donkey monocyte-derived macrophages (MDMs), is of great interest. In this study, we analysed the integration features of EIAVDLV121 in equine MDMs during in vitro infection. Our previously published integration sites (IS) for EIAVFDDV13 in fetal equine dermal (FED) cells were also analysed in parallel as references. Sequencing of the host genomic regions flanking the viral IS showed that reference sequence (RefSeq) genes were preferentially targeted for integration by EIAVDLV121. Introns, AT-rich regions, long interspersed nuclear elements (LINEs) and DNA transposons were also predominantly biased toward viral insertion, which is consistent with EIAVFDDV13 integration into the horse genome in FED cells. In addition, the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, specifically gag junctions for EIAVDLV121 and tight junctions for EIAVFDDV13, are regulators of metabolic function, which is consistent with the common bioprocesses, specifically cell cycle and chromosome/DNA organization, identified by gene ontology (GO) analysis. Our results demonstrate that EIAV integration occurs in regions that harbour structural and topological features of local chromatin in both macrophages and fibroblasts. Our data on EIAV will facilitate further understanding of lentivirus infection and the development of safer and more effective gene therapy vectors.

Horse hair follicles: A novel dermal stem cell source for equine regenerative medicine.

The easily accessible niche represented by skin and its appendages may serve as a promising source to complement modern regenerative medicine for horses. In humans and in animal models for human medicine, the hair follicle and its stem cell niches are well characterized. Since literature in this field of equine research is scarce, we sought to analyze cells of the dermal stem cell niche of the equine hair follicle morphologically and for a subset of markers useful for cell characterization via immunolabeling. We cultured equine forelock skin explants to obtain cultures with cells migrating from the hair follicles. Isolation of cells revealed typical fibroblast morphology with a strong tendency to aggregate and form spheroids. For immunofluorescent characterization of primary isolations, we tested an antibody panel consisting of lineage makers for the dermal compartment of the hair follicle, markers associated with an undifferentiated cell status and markers for epithelial cell types as negative controls. All antibodies used were also tested on equine skin sections. The isolated cells displayed clear profiles of dermal and undifferentiated cells. To substantiate our findings, we tested our primary isolations for established equine multipotent mesenchymal stromal cell antigen expression markers in flow cytometry experiments yielding strong convergence. The data presented here provide insights to a stem cell source in horses almost unnoticed to date. The basic investigations of the equine dermal hair follicle stem cell niche confirm the expression of standard markers used in other species and lay the foundation for future studies on this easily available adult stem cell source. © 2017 International Society for Advancement of Cytometry.

Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo

Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Sensitivity and repeatability were established, and specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old fully differentiated (mature), newly seeded (immature) primary equine respiratory epithelial cell (ERECs), and equine dermal cell cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 14days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with equine herpesvirus 1 (EHV-1). The qPCR assay detected EHV-5 at stable concentrations throughout 14days in inoculated mature EREC and equine dermal cell cultures (peaking at 202 and 5861 viral genomes per 106 cellular β actin, respectively). EHV-5 copies detected in the immature EREC cultures increased over 14days and reached levels greater than 10,000 viral genomes per 106 cellular β actin. Moreover, EHV-5 was detected in the lymphocytes of 76% of horses and in the nasal swabs of 84% of horses experimentally infected with EHV-1 pre-inoculation with EHV-1. Post-inoculation with EHV-1, EHV-5 was detected in lymphocytes of 52% of horses while EHV-5 levels in nasal swabs were not significantly different from pre-inoculation levels. In conclusion, qPCR was a reliable technique to investigate viral load in in vivo and in vitro samples, and EHV-5 replication in equine epithelial cells may be influenced by cellular stages of differentiation.

Influence of long-term equine herpesvirus type 1 (EHV-1) infection on primary murine neurons\u2014the possible effects of the multiple passages of EHV-1 on its neurovirulence

Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first time that primary murine neurons are able to survive long-term EHV-1 infection. Positive results of real-time PCR test indicated a high level of virus DNA in cultured neurons, and during long-term infection, these neurons were still able to transmit the virus to the other cells. We also compared the neurovirulence of Rac-H and Jan-E EHV-1 strains after multiple passages of these strains in neuron cell culture. The results showed that multiple passages of EHV-1 in neurons lead to the inhibition of viral replication as early as in the third passage. Interestingly, the inhibition of the EHV-1 replication occurred exclusively in neurons, because the equine dermal (ED) cells co-cultivated with neuroculture medium from the third passage showed the presence of large amount of viral DNA. In conclusion, our results showed that certain balance between EHV-1 and neurons has been established during in vitro infection allowing neurons to survive long-term infection.

Evaluation of SmartFlare probe applicability for verification of RNAs in early equine conceptuses, equine dermal fibroblast cells and trophoblastic vesicles

Live cell RNA imaging has become an important tool for studying RNA localisation, dynamics and regulation in cultured cells. Limited information is available using these methods in more complex biological systems, such as conceptuses at different developmental stages. So far most of the approaches rely on microinjection of synthetic constructs into oocytes during or before fertilisation. Recently, a new generation of RNA-specific probes has been developed, the so named SmartFlare probes (Merck Millipore). These consist of a central 15-nm gold particle with target-specific DNAs immobilised on its surface. Because of their central gold particle, SmartFlare probes are detectable by transmission electron microscopy. The aim of the present study was to investigate the uptake and distribution of SmartFlare probes in equine conceptuses at developmental stages suitable for embryo transfer (Days 6-10), equine trophoblast vesicles and equine dermal fibroblast cell cultures, and to determine whether differences among these cell types and structures exist. Probe uptake was followed by transmission electron microscopy and fluorescence microscopy. Although the embryonic zona pellucida did not reduce uptake of the probe, the acellular capsule fully inhibited probe internalisation. Nanogold particles were taken up by endocytosis by all cell types examined in a similar manner with regard to time and intracellular migration. They were processed in endosomal compartments and accumulated within lysosomal structures after longer incubation times. In conclusion, the SmartFlare probe is applicable in equine conceptuses, but its use is limited to the developmental stages before the formation of the embryonic capsule.

Replication kinetics of neuropathogenic and non-neuropathogenic equine herpesvirus type 1 (EHV-1) strains in primary murine neurons and ED cell line.

Equine herpesvirus type 1 (EHV-1) causes respiratory infections, abortion and neurological disorders in horses. Molecular epidemiology studies have demonstrated that a single-point mutation in DNA polymerase gene, resulting in an amino acid variation (N752/D752), is significantly associated with the neuropathogenic potential of EHV-1 strains. The aim of the study was to elucidate if there are any differences between neuropathogenic (EHV-1 26) and non-neuropathogenic (Jan-E and Rac-H) EHV-1 strains in their ability to infect neuronal cells. For the tested EHV-1 strains, cytopathic effect (CPE) was manifested by changed morphology of cells, destruction of actin cytoskeleton and nuclei degeneration, which led to focal degeneration. Moreover, EHV-1 26 strain caused fusion of the infected cells to form syncytia in culture. Real-time PCR analysis demonstrated that both neuropathogenic and non-neuropathogenic EHV-1 strains replicated in neurons and ED cells (equine dermal cell line) at a similar level. We can assume that a point mutation in the EHV-1 polymerase does not affect viral replication in this cell type.

Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3)

Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterized by pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomic sequence of EHV-3 has been recently made available, its genomic content remains poorly characterized and the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitate genetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterial artificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenic region between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologous recombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporated into E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of the EHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from those of the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinant viruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli and in vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74) coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene, and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensable for EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells; (iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic and transmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning of EHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis and host immune responses.

Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. This study shows that VEGF-E and ovIL-10 are active on equine dermalcells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses.

Real-time monitoring of Equid herpesviruses infectivity in equine dermal cell based on impedance measurements

In virology, different conventional methods are commonly used for virus titration and infectivity studies. Most of them are based on direct examination of viral cytopathic effect, which is time consuming, tedious and requires an endpoint reading. The RTCA xCELLigence system (ACEA Biosciences) has been developed to follow cellular events and their dynamics in real time using a micro-electrical biosensor technology. The aim of this study was to evaluate the feasibility of using this biosensor technology to measure the effect of different equid herpesviruses on equine dermal cell cultures, with or without antiviral acyclovir treatment. Cells were infected with EHV-1 (VR700), EHV-4 (VR2230), EHV-2 (VR701) or EHV-3 (VR352). The viral effects were monitored by 3 different methods: impedance measurements (RTCA), cytopathic effects recording (microscopy) and viral load quantification (qPCR). The RTCA technology showed a dose dependent drop of the cellular index induced by specific impedance variations measured during growth of each virus tested. These results correlated with the cytopathic effect induced by the different herpesvirus species, as well as viral loads measured by qPCR. For example, after 3 days of incubation with EHV-1, RTCA results showed an increase of the cellular index from 0% (infection without antiviral) to 45% and 100% in presence of Acyclovir (10μl/mL and 100 μg/mL, respectively). These results are correlated with the decreased of viral load measured in the presence of Acyclovir: 1010 genome copies/mL with EHV-1 only, 106 copies/mL with EHV-1 + Acyclovir 10 μg/ mL and 105 copies/mL with EHV-1 + Acyclovir 100 μg/ mL. In conclusion, the cellular impedance monitoring system is a new real-time method to monitor cell growth and viral proliferation in cell cultures. This technology is well adapted for high-throughput screening of antiviral molecules. In fact, the xCELLigence system enables continuous impedance measurement and quantification of cell adhesion, proliferation, cell death and detachment during viral infection.

The first virus isolation and partial characterization of equine herpesvirus-4 in a horse, South Korea

An equine herpesvirus-4 (EHV-4) was isolated in nasal swabs collected in a horse showing respiratory clinical signs. Equine dermis cells inoculated with the sample were observed with characteristic viral cytopathic effects after 3 days of postinoculation and the infected cells exhibited bright intracelluar fluorescence by indirect immunofluorescence assay. At the nucleotide level, the partial glycoprotein B gene of the Korean EHV-4 isolate (K001) had 99.9% identity to 1942 strain (GenBank No. M26171). To author's knowledge, the report describes the first isolation and partial characterization of EHV-4 in Korea. The virus can be used for further study of EHV-4.