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.
Equine herpesvirus type 1 (EHV-1) is one of the major horse diseases, causing considerable worldwide losses. A variety of techniques, including nested PCR, have been used to diagnose EHV-1 infections. In this paper, a real-time PCR assay that uses non-specific SYBR Green I® fluorochrome for the detection of EHV-1 DNA is described. 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 neuronal cell cultures and also different clinical samples. The technique is specific: it was not reactive with other herpesviruses or opportunistic bacterial pathogens such as Escherichia coli, Staphylococcus epidermidis and Enterococcus faecium. In comparison to virus isolation or the nested PCR used previously, the test was more sensitive and should be useful for the common diagnosis based on its specificity and rapidity.
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.
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