Tel. +49 30 838 51833 Fax. +49 30 838 451847
The work presented here focused on the analysis of the sequence of the EHV-2-specific IL-10-gene of strain LK4 and its surrounding region. To prepare the development of vIL-10- mutants, different strategies to disrupt the open reading frame were investigated. Therefore a fragment of approximately 2000bp was amplified, cloned and sequenced. In contrast to published sequences, strain LK4 comprised of two uniques: an additional two repeats than previously described and the existence of a 15bp oligonucleotide located in front of the vIL-10-start codon. Investigation of the coding capacity revealed 14 open reading frames. Further analysis of the cloned sequence indicated that the transcription of the gene may be dependant on cellular transcription factors. The published amino acid sequence information for LK4-IL-10 could be confirmed. Although the sequences of the strains T400/3 and 86/67 were identical, strain LK4 showed seven point mutations, which were located in the N-terminal region of the gene. In addition, several binding sites for various cellular enzymes were identified, leading to the hypothesis that activation of the protein may possibly be mediated by cellular modification. For the inactivation of vIL-10, three expression cassettes used as visual markers, were selected and produced by PCR or REA. Experiments for the integration of these expression cassettes in the coding region of vIL-10 were performed with or without deletions. As no insert could be proven, possible causes were excluded extensively. In conclusion, the present results indicate a causal significance of the repetitive sequence upstream of the vIL-10. Based on the use of equine PBMC, a model system for characterization of wild-type virus in comparison with vIL-10 mutants was established. In-vitro-infected PBMC, as well as cells of an EHV-2-positive horse, were cultured over a period of 14 days. Infectious virus and viral DNA was frequently detected in experimentally infected cells up to 14 days p.i. by nPCR, cocultivation and plaque assay. An additional application of TPA allowed the verification of the latent state in naturally infected PBMC and also demonstrated EHV-2-reactivation by TPA. During a period of one month, the number of naturally infected PBMC was generally stable. To differ whether this phenomenon was the result of an apoptosis inhibition or a transformation potential, agar suspensions cultures were performed. Results confirmed earlier investigations: as no increase cell numbers was detectable, the survival of naturally infected PBMC seemed to be a result of mechanisms to inhibit apoptosis. For determination of viral antigens on the surface of naturally infected PBMC various monoclonal antibodies and a LK4-specific hyperimmune serum were utilised. The detection of antigen on the surface of both productively and latently infected cells was successful. In both populations the number of antigen expressing PBMC depended on the previous cultivation time. In order to determine a closer relationship between T-cells and EHV-2-infections as previously mentioned, in-vitro infected T-cells in addition to T-cells of a naturally infected horse were examined. While the former cell population was shown to be positive for EHV-2-DNA in any rate, the detection of viral DNA in naturally infected T-cells was successful only on one occassion after cell stimulation. The findings of this study in connection with published data concerning the potential of EBV to establish infections in human T-cells, indicate the need for further work to clarify this issue.