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Real Time PCR-Based Infectivity Assay and Characterization of Cell Surface Receptors for Turkey Hemorrhagic Enteritis Virus
Mahsoub, Hassan Mostafa Mohammed
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Turkey hemorrhagic enteritis virus (THEV) is responsible for the hemorrhagic enteritis (HE) disease in commercial turkeys through infections by its virulent strains. HE is an acute condition characterized by depression, immunosuppression, bloody droppings, intestinal hemorrhage, and death. THEV (also known as turkey adenovirus 3) is an official member of the family Adenoviridae, genus Siadenovirus, species Turkey siadenovirus A. Two main types of live vaccines are currently used for the protection of turkeys against HE; a crude splenic vaccine propagated in live turkeys, and a cell culture-based vaccine generated in RP19 cells. The only laboratory-adapted tests for assessing the titers of these vaccines are agar gel immunodiffusion test and cell culture endpoint dilution, respectively. The assays suffer from low sensitivity, inaccuracy, and time consumption. A SYBR Green-based real time PCR assay for determining the genomic titer of THEV through the quantification of its hexon gene was developed. The assay was applied as a quality control for the titration of splenic vaccines and was found useful in distinguishing the differences in virus titer among many vaccine batches. Additionally, using the qPCR assay along with a cell culture system, a novel infectivity assay was developed for the titration of THEV, as an alternative for the endpoint dilution assay. Applying the assay on nine batches of commercial HE cell culture vaccines, high variations in infectious virus titers were detected. The new method is rapid, sensitive, and very accurate. A strong correlation was found between the genomic titer and qPCR infectious titer in HE cell culture vaccines. Moreover, the qPCR infectivity assay proved as an instrumental research tool. It was used to measure the effect of several treatments of RP19 cells on virus infection. The main target cell type for THEV infection and replication is B-lymphocytes, which are represented in vitro by the B lymphoblastoid, RP19 cells. The cellular surface components used by the virus to gain entry into cells are unknown. As an adenovirus, we hypothesized that THEV uses two different molecules on RP19 cells for the attachment and internalization. A recent study has shown that the synthesized THEV fiber knob domain binds to sialyllactose, based on a glycan array analysis. In our studies, the treatment of RP19 cells with neuraminidases and lectins resulted in high reduction of virus entry, which provide a strong evidence of the utilization of cell surface sialic acids as attachment receptor for THEV. Destruction of surface carbohydrates and proteins on RP19 cells also reduced virus entry, indicating that these components are part of the THEV receptor. Using virus overlay protein blot assay, THEV was found to specifically bind to two RP19 surface membrane proteins, most likely, representing primary and secondary receptors for virus entry. Further studies are required to identify these proteins and verify their role in THEV endocytosis in host cells.
- Doctoral Dissertations