Publication Database

Molecular characterization and pathogenesis of equine and elephant herpesviruses (2019)

Art

Hochschulschrift

Autor

Selvaraj, Pavulraj (WE 5)

Quelle

Berlin, 2019 — XVIII, 165 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/26276

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Herpesviruses are important viral pathogens infecting human and animal populations worldwide. In the current study, we have investigated molecular mechanisms of equine herpesvirus type 1 (EHV-1) pathogenesis and characterized EHV-4 and elephant endotheliotropic herpesvirus type 1A (EEHV-1A) following disease outbreak in horses and elephants, respectively. To evaluate the role of viral proteins in cell-to-cell virus transfer between peripheral blood mononuclear cells (PBMC) and endothelium, we have constructed EHV-1 (Ab4 strain) mutants after deleting open reading frame 1 (ORF1), ORF2 and ORF17 genes, either as single gene deletions or in combinations. Ab4 deletion mutants were analysed for replication properties, PBMC infection, cell-to-cell virus transfer between epithelium-PBMC and PBMC-endothelium, virus induced changes in PBMC proteome and cytokine/chemokine expression profiles. All deletion mutants were successfully reconstituted. ORF1, ORF2 and ORF17 genes are not essential for virus replication, and ORF17 deletion containing mutants showed significant reduction in plaque size. ORF2 and ORF17 were implicated in virus spread to the endothelium as evaluated by contact and flow chamber models; however, virus transfer from epithelial cells to PBMCs was not affected. Interestingly, all PBMC subpopulations including B lymphocytes, T lymphocytes and monocytes were able to transfer EHV-1 to EC. Ab4 infection in PBMC upregulated host proteins associated with endocytosis, Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration, and downregulated chemokine signaling, herpesvirus infection, ribonucleic acid degradation and apoptotic pathways. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signalling, mitogen activated protein kinase (MAPK) and herpesvirus infection pathways in infected PBMC. We presume that reduction in virus transfer from PBMC to endothelium may be attributed to modulation of host signaling and immune pathways. MAPK pathway is implicated in signal transduction, cell adhesion, cell survival and virus replication; Herpesvirus pathway is mediating receptor signaling, pro-inflammatory cytokine release, inhibition of apoptosis, nuclear factor-kappa B and antigen processing and presentation; Chemokine signaling is essential for activation of various immune pathways. Proteomic results were further confirmed by chemokine assay where Ab4 infection completely reduced the cytokine/chemokine release in infected PBMC and deletion of ORF1, ORF2 and ORF17 genes restored expression. Collectively, ORF2 and ORF17 genes essentially involved in virus spread to endothelium and along with ORF1 could stimulate strong cytokine/chemokine response. Therefore, these gene deletion mutants could be the potential target for development of live attenuated-vaccine. EHV-4 is enzootic in equine population. We have investigated a large outbreak of respiratory infection occurred in a group of in-housed foals and mares, at a Standardbred horse breeding farm in northern Germany. Virological assay revealed the involvement of EHV-4 in all the cases of respiratory illness as confirmed by virus isolation, quantitative-polymerase chain reaction and serological follow-up in paired serum sample using virus neutralization test and peptide-specific enzyme linked immunosorbent assay. Different restriction fragment length polymorphism profiles of the four isolates suggested the involvement of more than one animal as an index case of infection either due to primary infection or reactivation from latency. Epidemiological investigation revealed multifactorial causation as stress caused by seasonal changes, management practices, routine equestrian activities and exercise contributed in EHV-4 outbreak. Our study necessitates the stress alleviating measures and management practices in stud farm in order to avoid immuosuppression and disease outbreak. EEHV causes fatal hemorrhagic disease in young Asian elephants. Our investigation on two young Asian elephants died due to acute hemorrhagic disease revealed the involvement of EEHV-1A as a cause of the death. Widespread distribution of EEHV-1A was observed in various organs and tissues of the infected elephants. In hepatic endothelial cells, enveloped viral particles were accumulated within and around cytoplasmic electron-dense bodies. Virus isolation study in different cell cultures showed only limited virus replication; however, late viral protein expression was detected in infected cells. We have further demonstrated that glycoprotein B of EEHV-1A possesses a conserved cleavage site Arginine-X-Lysine/Arginine-Arginine that is targeted by the cellular protease furin, as like other members of the Herpesviridae. Through next-generation sequencing, we have determined the complete 180 kilobase pair genome sequence of EEHV-1A isolated from the liver. Though our virus isolation was unsuccessful, only little information is available regarding virus culture and the function of viral proteins. We have attempted to take an initial step in the development of appropriate cell culture system and virus characterization. Further, the complete genome sequence of EEHV-1A from our investigation will facilitate future studies on the epidemiology and diagnosis of EEHV infection in elephants. Taken together, our findings provide new insights into EHV-1 pathogenesis, especially molecular mechanisms of virus spread between PBMC and endothelium. Further our study with EHV-4 and EEHV-1A makes a significant contribution to existing knowledge and offers an important resource on herpesvirology.