Equid herpesvirus type 1 (EHV-1) disrupt actin cytoskeleton during productive infection in equine leukocytes

Equid herpesvirus type 1 (EHV-1) is a prevalent causative agent of equine diseases worldwide. After primary replication in the respiratory epithelium the virus disseminates systemically through a peripheral blood mononuclear cell (PBMC)-associated viraemia. EHV-1 is the only alphaherpes-virus known so far which is capable of establishing latent infection not only in neurons but also in immune system cells (mainly in lymphocytes and macrophages). Since leukocytes are not the target cells for viral replication but are used to transport EHV-1 to the internal organs, the question remains how the virus avoids the immune response and whether it could potentially be associated with virus-induced cytoskeletal rearrangements. Therefore, the aim of this study was to investigate the progress of EHV-1 replication in leukocytes stimulated by phytohemagglutinin and the impact of EHV-1 infection on the actin cytoskeleton. Using the real-time PCR method we evaluated the quantity of viral DNA from samples collected at indicated time points post infection. In order to examine possible changes in actin cytoskeleton organization due to EHV-1 infection, we performed immunofluorescent staining using TRITC-phalloidin conjugate. The results showed that EHV-1 replicates in leukocytes at a restricted level but with the accompaniment of chromatin degradation. Simultaneously, infection with EHV-1 caused disruption of the actin cytoskeleton; this was particularly apparent in further stages of infection. Disruption of the actin cytoskeleton may lead to the limited release of the virus from the cells, but may be also beneficial for the virus, since at the same time it potentially impairs the immune function of leukocytes

Influence of importin α/β and exportin 1 on equine herpesvirus type 1 (EHV-1) replication in primary murine neurons

Viruses replicating in the nucleus need to cross the nuclear membrane barrier during infection, therefore disruption of specific nuclear transport pathways is crucial for their replication cycle. In the present study we have investigated the influence of nucleo-cytoplasmic transport inhibitors – ivermectin and leptomycin B, on EHV-1 replication in primary murine neurons. Obtained results suggest that the examined proteins – exportin 1 and importin α/β may participate, but are not required, during EHV-1 infection. Based on these results, it can be assumed that EHV-1 is able to use other receptors for nucleo-cytoplasmic transport

HybProbes-based real-time PCR assay for rapid detection of equine herpesvirus type 2 DNA

Equid herpesvirus type 2 (EHV-2) together with equid herpesvirus type 5 are members of Gammaherpesvirinae subfamily, genus Rhadinovirus. EHV-2 is one of major agents causing diseases of horses common worldwide. A possible role of EHV-2 in reactivating latent equid herpesvirus type-1 has been suggested, because reactivation of latent EHV-1 was always accompanied by EHV-2 replication. Variety techniques, including cell culture, PCR and its modifications, have been used to diagnose EHV-2 infections. The aim of this study was to develop, optimize and determine specificity of real-time PCR (qPCR) for EHV-2 DNA detection using HybProbesR chemistry and to evaluate clinical samples with this method. The analytical sensitivity of assay was tested using serial dilutions of viral DNA in range between 70 and 7x105 copies/ml. The limit of detection (LOD) was calculated using probit analysis and was determined as 56 copies/ml. In further studies 20 different clinical samples were tested for the presence of EHV-2. Described in-house qPCR method detected viral DNA in 5 of 20 specimens used. The results of this work show that developed HybProbes-based real-time PCR assay is very reliable and valuable for detection and quantification of equid herpesvirus type 2 DNA in different clinical samples. The high level of sensitivity, accuracy and rapidity provided by the LightCycler 2.0 instrument are favorable for the use of this system in the detection of EHV-2 DNA in veterinary virology

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