Cancer is one of the most frequent causes of death in Poland and in the world. The low efficacy of conventional treatment, as well as the high toxicity of the usual therapies, have stimulated the search for alternative methods. One of them is the deployment of oncolytic viruses. Oncolytic viruses have a natural ability to lyse tumor cells or can obtain this ability through certain modifications. The aim of virotherapy is to discover a virus that will lyse only tumor cells, and will not be dangerous to healthy cells, and moreover will not cause an undesirable response from the host’s immune system. Animal viruses with oncolytic abilities are very promising, because they are not pathogenic for humans and often show a high specificity for human cancerous cells.
The aim of the study was to investigate the influence of equine herpesvirus type 1 (EHV-1) infection on actin cytoskeleton in ED (equine dermal). Cells in vitro.ED cells were infected with a strain of Jan-E of EHV-1, fixed and stained for the presence of actin and virus antigen. Results were evaluated by confocal microscopy. The assembly of the actin cytoskeleton was heavily disturbed. In order to affirm whether changes in cytoskeleton of EHV-1 infected cells depend on the type of cells, we infected Vero cell culture with 2 different standard strains of EHV-1 - Rac-H, AIV - and Jan-E isolated from an aborted fetus. Unfortunately, the infection of Vero cells with the strain Jan-E of EHV-1 failed because this strain was not adapted to the heterogeneous cell line. Only strain Rac-H and AIV can replicate in Vero cells, which was determined through the application of PCR.
In previous experiments, we have demonstrated that the presence of equine herpesvirus 2 (EHV-2) enhanced plaque formation in cell cultures infected with equine herpesvirus type 1. To determine whether a specific region of the EHV-2 genome is responsible for this effect, we have constructed a library of Bam HI fragments of the EHV-2 genome ligated into pcDNA plasmid. Equine dermal (ED) cell cultures were subsequently transfected with the constructs, passaged 5 times, tested for the presence of the plasmids and infected with EHV-1 at MOI=0.01. Only in cultures transfected with the pcDNA/Bam HI[G]construct, designated Δ2/4, the mean number of plaques at 24 hrs p.i. was approximately 10 times higher than in non-transfected controls. Virus titers in culture supernatants as well as in freeze-thawed cells were 4- and 5-fold higher, respectively, than in non-transfected cultures. These differences were observed only at 24 hrs p.i. At 48 hrs p.i. cultures were completely destroyed and, surprisingly, the virus titer was slightly lower in the supernatant of transfected cells. However, the titer of EHV-1 in freeze-thawed culture was exactly the same as in the control. These results suggest that the presence of Bam HI[G] fragment of the EHV-2 genome stimulates (accelerates?) plaque formation only at earlier stages of infection but does not influence the total yield of EHV-1 at 48 hrs p.i. The exact mechanism of this stimulation remains unclear and further experiments are necessary to determine the role of putative EHV-2 proteins encoded by Bam HI [G] fragment of the EHV-genome.
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.
Equine herpesvirus type 1 (EHV-1) is one of the major viral agents causing diseases in horses common worldwide. A variety of techniques, including PCR, have been used to diagnose EHV-1 infections. In this paper, an attempt of real-time PCR has been described, which uses specific fluorochrome-labeled TaqMan probes for detection of viral DNA. This method does not require post-amplification manipulations, thereby reducing the risk of cross-contamination. The assay was sensitive enough to detect EHV-1 sequences in different clinical samples, as well in mice neuronal cell cultures. The technique was also very specific – there was no cross reaction with other human and equine herpesviruses. Compared to previously used nested PCR technique, the test was more sensitive and should be useful for the common diagnosis based on its specificity and rapidity.
Herpesviridae is a large family of DNA viruses, capable of infecting higher as well as lower vertebrates. To date, more than one hundred species have been isolated and identified, and new species are still being discovered. For many years mammalian herpesviruses have been of interest to researchers because of their prevalence and pathogenicity, as well as significant economic losses associated with herpesviral infections, especially in livestock. In the course of their evolution, herpesviruses have perfectly adapted to their hosts and have developed the ability to establish latency. For years, many diverse efforts have been made to eliminate herpesviral infections, but the vaccines produced are generally ineffective and do not provide protection against the establishment of latency. Therefore, further research on their pathogenesis and continuous monitoring are needed to prevent the occurrence and spread of herpesvirus infections, particularly in farm and companion animals.
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.
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.
Real-time cell electronic sensing (RT-CES) based on impedance measurements is an emerging technology for analyzing the status of cells in vitro. It allows label-free, real time monitoring of the biological status of cells. The present study was designed to assess dynamic data on the cell processes during equine herpesvirus type 1 (EHV-1) infection of ED (equine dermal) cells and primary murine neuronal cell culture. We have demonstrated that the xCELLigence system with dynamic monitoring can be used as a rapid diagnostic tool both to analyze cellular behavior and to investigate the effect of viral infection.
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.
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