Publikationsdatenbank

Development and molecular studies on Equine Herpesvirus type 1 (EHV-1) vaccine against foot and mouth disease virus (2018)

Art

Hochschulschrift

Autor

Kamel, Mohamed Salah Kamal (WE 5)

Quelle

Berlin: Mensch und Buch Verlag Berlin, 2018 — XVI, 169 Seiten

ISBN: 978-3-86387-934-1

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/23202

Kontakt

Institut für Virologie

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

Abstract / Zusammenfassung

Equine herpesvirus-1 is an alphaherpesvirus affecting equid species resulting in severe clinical outcomes manifested clinically as respiratory infection, abortion and nervous manifestations. EHV-1 establishes cell-associated viremia, following respiratory infection, reaching the target endothelial lining of blood vessel of reproductive tract and nervous system, resulting in vasculitis and thrombosis exhibited as clinical outcomes. Although PBMC-EC interface is very crucial in virus pathogenesis and clinical outcomes advent, the mechanisms underlying virus transfer from infected PBMC to EC are still unknown. To investigate these mechanisms, two-step en passant mutagenesis, functional assays (contact assays, transwell assays and flow chamber assay) together with confocal immunofluorescence data and laser scanning live cell imaging and electron microscope were executed. The single viral particle or viral cluster was found to colocalize with the extracellular matrix components. Viral particle or viral clusters embedding within these extracellular matrices are protected from the neutralizing antibodies and efficiently transmitted to endothelial cells. Destruction of this extracellular matrix from infected PBMC surface affects the infectious capability of PBMC. EHV-4, although found colocalized with ECM, is not transmitted from infected PBMC either in the static or dynamic conditions conjecturing their trapping and inactivation in these structures. Two genes, gB and gD have been implicated their role in virus transfer between PBMC and EC. Another two important mechanisms were assessed for the first time, membrane fusion between infected PBMC and endothelial cell as a mode of cell-to-cell transmission and the other one is the transcellular migration of PBMC into the endothelial cells transferring the virus to them. Two important events had been detected and caught by electron microscopy and require further and deep investigation. The first event is the first-time detection of microvesicle including equine herpesvirus-1 viral particle augmenting their role in viral life cycle and virus spread. EHV-1 was also found in a tight junction between infected and uninfected endothelial cells raising their role in viral cell-to-cell between endothelial cells. UL56 is a phosphorylated type II membrane early protein which has a function in downregulating MHC class I and other surface molecules. To assess their role in virus transfer between PBMC and endothelial cells in EHV-1 and EHV-4, different set of mutants were generated by two-step en passant mutagenesis. The deletion mutants and swapping mutants of UL56 in EHV-1/4 showed no significant difference in the in vitro growth properties compared to their parental viruses. The deletion mutants and swapping mutants of UL56 between the two viruses had no role in virus transfer in both static or dynamic conditions. The expression levels of adhesion molecules of the surface of mutant-infected PBMC very late antigen (VLA-4) and leucocyte function-associated antigen (LFA-1) showed no statistical significant difference compared to their parental viruses (EHV-1 or EHV-4 WT). The results manifested that UL56 alone or /swapping in or between EHV-1 and EHV-4 has no effect in virus transfer between PBMC and endothelial cells raising the questions of the other cellular and viral partner that could help UL56 to affect virus transfer as happens with MHC-1 downregulation. As FMD is one of the most contagious and highly economically viral disease affecting cloven-hoofed animals, it causes severe economic losses and social consequences. The most available used inactivated vaccines for FMD are short-lived humoral immunity, need cold chain containment, can`t protect against different serotypes and even sub serotypes and have difficulties in production. Construction of rH-P1 of FMD was performed and confirmed by PCR, RFLP, and sequencing for future using against FMD to differentiate between vaccinated and humoral immunity following natural infection (DIVA capability), not need for cold chain containment and finally, production of cellular and humoral immunity. Recombinant growth properties (plaque size and growth kinetics) were compared to their parental virus pRacH, P1 integration and expression were assessed by immunofluorescence and western blotting. The virus had been prepared and sent for future animal experiment challenge.