| Summary: | 碩士 === 國立中興大學 === 微生物暨公共衛生學研究所 === 106 === Orf virus （ORFV）, the prototype of the Parapoxvirus, is a zoonotic etiologic agent that infects small ruminants and causes contagious ecthyma. The resulting lesion presenting extensive vascular changes has been linked to a homolog of vascular endothelial growth factor （VEGF） encoded by ORFV. Viral genome consists of a large linear double-stranded DNA encoding many non-essential proteins. Among those, VEGF, vIL10, and OV20.0 have been identified as virulent factors. Several studies indicate live attenuated ORFV could serve as an ideal vector for expressing exogenous antigens. However, the narrow host range（i.e. sheep or goats） would be cumbrous for the evaluation of antibody response elicited by ORFV. The current study aimed to modify ORFV to a safer and more efficient viral vector platform by several approaches, including: deletion of the virulent factor （VEGF）, insertion of screening marker （eGFP）, as well as a screening marker（vvTK）. Initially, a transfer vector that harbors the expression cassette of eGFP and vvtk flanked with upstream and downstream sequences of vegf gene was constructed. By homologous recombination, the vvTK-eGFP expression cassette was incorporated into the vegf locus that leads to deletion of vegf coding region from viral genome. With the aid of eGFP expression, plaques of the recombinant ORFV （namely, VEGFΔ-vvTK-eGFP ORFV） was distinguishable under a fluorescent microscope and able to be isolated. Subsequently, genotype of this virus was verified by PCR using gene specific primer sets. Addition of compound bromodeoxyuridine（BrdU）resulted in decreased virus replication, indicating vvTK indeed could serve as a suicide protein for the subsequent run of recombinant ORFV generation. Compared with WT ORFV, VEGFΔ-vvTK-eGFP ORFV produced smaller plaques and lower yield. Collectively, we have successfully attenuated ORFV and with the vvTK insertion it would be employed as an ideal vector for development of multivalent vaccine or for oncolytic purpose.
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