Au moins 4 herpèsvirus majeurs peuvent infecter le cheval au niveau de ses voies respiratoires ; 2 alpha-herpèsvirus (les herpèsvirus équins 1 et 4) et 2 gamma-herpèsvirus (les herpèsvirus équins 2 et 5). Les deux gamma-herpèsvirus ont été identifiés partout dans le monde depuis très longtemps mais leur pouvoir pathogène demeure moins bien connu. Parallèlement, de nombreux travaux portant sur les maladies respiratoires sub-cliniques du cheval athlète ont pu mettre en évidence lintérêt de recherches approfondies sur le rôle des virus dans le déclenchement ou lentretien de ces pathologies souvent inflammatoires. . La prévalence de ces 4 virus a étéévaluée lors dune première étude portant sur plus de 700 liquides de lavages respiratoires à laide doutils de biologie moléculaire. Lherpèsvirus équin 2 sest avéré comme le plus présent dans un groupe de chevaux contre performants dans les liquides trachéaux. Une infection expérimentale conduite sur 6 chevaux a permis de reproduire la maladie, de détecter le virus dans divers compartiments biologiques dont la trachée pendant plus de 14 jours post infection. La présence du virus était contemporaine de ciliocytophtorie des cellules épithéliales et dune neutrophilie dans le liquide. Lherpèsvirus équin 2 peut donc induire une inflammation de la trachée et des anomalies morphologiques des cellules épithéliales ce qui en fait un co-facteur potentiel du syndrome dinflammation trachéale, préoccupant pour léconomie du cheval athlète / Equine gamma-herpesvirus 2 and 5 have been identified worldwide since many years. Their real pathogenic role has still to be determined. On the other hand, many important studies have been performed in order to better understand subclinical diseases of the athletic horse. Several publications pointed out the potential incidence of viruses in lower airway inflammation. Within the first step of this work, over 700 respiratory fluids were tested in order to detect and to evaluate the presence of equid herpesviruses in these biological samples. Using molecular tools, Equid herpesvirus 2 was found to significantly be the most important among the four herpesviruses detected in tracheal washes of a group of poorly-performing horses. An experimental infection with equid herpesvirus 2 was then conducted on 6 horses and succeeded in reproducing clinical and cytological signs of disease and virus shedding in most biological samples including tracheal washes. The virus shedding was concomitant with ciliocytophthoria of the exfoliated epithelial cells and liquid neutrophilia. Equid herpesvirus 2 can induce a tracheal inflammation and ciliocytophthoria. This virus could play a role in the new syndrome of tracheal inflammation, and could be an important feature for equine industry

All mammalian cells encode microRNAs (miRNAs), which are small non-coding RNAs (~ 22 nucleotides) that control numerous physiological processes via regulation of gene expression. A number of viruses, in particular herpesviruses, also encode miRNAs. Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi’s sarcoma associated herpesvirus (KSHV) are associated with lymphoproliferative disorders and some types of cancer in humans. Gammaherpesvirus-encoded miRNAs are predicted to contribute to pathogenesis and virus life cycle by suppressing host and viral target genes. However, the exact functions of these miRNAs during virus infection in the natural host are largely unknown. Strict species specificity has limited research on the human gammaherpesviruses mainly to in vitro studies. Murine gammaherpesvirus 68 (MHV-68) encodes at least 15 miRNAs and provides a unique tractable small animal model to investigate in vivo gammaherpesvirus pathogenic features that are difficult to assess in humans. Following intranasal infection of lab mice, the virus undergoes primary lytic infection in the lung epithelial cells and then spreads to the spleen establishing latent infection in splenic B lymphocytes, macrophages, and dendritic cells. The peak of the latent viral load occurs in the spleen at 14 dpi and then it decreases over time, but the virus is not completely eliminated and the latent viral genomes remain in the host cells for lifetime and can reactivate to produce infectious virus under certain conditions. The aims of my project were to: (1) establish and develop quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for quantification of the MHV-68 miRNAs, (2) determine the miRNAs expression profiles during the two stages of virus infection (lytic and latent infection), (3) investigate the kinetics of the miRNAs expression during latency in vivo, (4) construct an MHV-68 miRNA mutant virus lacking 9 miRNAs (designated MHV-68.ΔmiRNAs), and (5) carry out thorough phenotypic characterisation of this mutant virus in order to determine the possible functions MHV-68 miRNAs in the context of natural host infection. It was found that the MHV-68 miRNAs expression pattern varied during different stages of infection, suggesting a differential regulation of the expression of these miRNAs depending on the phase of infection. In order to investigate the kinetics of miRNAs expression during latency in vivo, BALB/c mice were infected intranasally with MHV- 68 virus and spleens were harvested at days 10, 14, 21, and 32 post infection. The levels of miRNAs expression were determined by qRT-PCR in the splenocytes from infected mice. Interestingly, in contrast to the lytic MHV-68 protein coding genes, the expression of the miRNAs increased over time after 21 dpi, suggesting that the MHV-68-encoded miRNAs may play more fundamental roles during later stages of latent infection. In order to determine the potential roles of the MHV-68 miRNAs in virus pathogenesis, a miRNA mutant virus lacking the expression of 9 miRNAs, named MHV- 68.ΔmiRNAs, was constructed. The miRNA mutant virus replicated with the same kinetics as wild type virus in vitro and in vivo demonstrating that the deleted MHV-68 miRNAs are dispensable for virus lytic replication. To examine the roles of the miRNAs during virus latency, the MHV-68.ΔmiRNAs virus was characterised throughout a 49- day course of infection. Although the level of ex vivo reactivation of the MHV-68.ΔmiRNAs virus was comparable to that of the WT virus during the establishment of latency and as late as 28 dpi, the reactivation of the MHV-68.ΔmiRNAs virus was approximately 18-times higher than that of the WT virus at 49 dpi despite the similar levels of the genomic viral DNA loads at the same time-point. This suggests that the MHV-68 miRNAs suppress virus reactivation and promote maintenance of long-term latency. Moreover, the lytic viral gene expression levels were higher in splenocytes from the MHV-68.ΔmiRNAs-infected mice than the basal expression levels in the splenocytes from WT MHV-68-infected mice, suggesting that the MHV-68 miRNAs may suppress viral lytic gene expression during long-term latency in vivo and thus help the virus lay low.

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