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Wu, Ge (2020) Development of diagnostic assays for emergent RNA viruses. PhD thesis, University of Nottingham.

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Abstract

This study aims to develop novel diagnostic assays for zika virus (ZIKV) and equine influenza viruses (EIV) by

• Generating pseudotyped ZIKV as a surrogate for wild-type virus in the virus neutralisation (VN) test

• Producing plant-expressed ZIKV non-structural (NS) 1 protein as a surrogate for wild-type virus in the ELISA

• Identifying lineage-specific antibodies against EIV using Next Generation Phage Display (NGPD) to replace antibodies produced by immunizing animals for use in ELISA.

Co-circulation and cross-reaction among flaviviruses have become the major obstacle for an accurate diagnosis. Antigenic drift and shift of influenza viruses underline the importance of close monitoring and surveillance for precise diagnosis and vaccine production. This study aimed to develop novel diagnostic assays for emergent/re-emergent viruses, namely, Zika virus (ZIKV) and equine influenza virus (EIV) using various techniques including pseudotyping, plant expression and next-generation phage display (NGPD) to improve their current diagnostic assays.

Vesicular stomatitis virus (VSV) backboned pseudotype virus (PV) was generated by co-transfecting producer cells with commercially available VSV plasmids. ZIKV envelope protein (ZIKV E) was used to replace the VSV glycoproteins to generate VSV-backboned ZIKV PV, which showed no infectivity. However, VSV backboned Chikungunya virus (CHIKV) PV used as a control exhibited similar luciferase activity as the VSV PV (~1×107 RLU/ml), indicating successful production of VSV-backboned CHIKV PV. The virus budding site of ZIKV is different from CHIKV and VSV, which was identified as one of the possible reasons for the failure of infectivity of ZIKV PV. Future work would be to mutate the ER-retention signals in the transmembranes of ZIKV prME for its translocation to the plasma membrane and to incorporate furin proteases during pseudotyping for the ZIKV E to achieve a mature and infectious form.

Next, a ZIKV non-structural protein 1 (NS1) ELISA was developed using ZIKV NS1 proteins expressed in plants. Unlike envelope proteins, NS1 does not elicits flavivirus cross-reactive neutralisation antibodies but induces a non-neutralising yet virus-specific antibody response. NS1 gene was codon-optimised, synthesised and inserted into a plant expression vector pEAQ-HT to obtain expressed protein. Preliminary results using the resultant ELISA showed around 95% sensitivity and 95% specificity using 20 ZIKV positive human serum samples and 19 Dengue-only positive samples. For future work, a bigger sample size should be tested for the proper validation of this plant produced NS1 ELISA.

Finally, antibody peptide sequences that specifically recognise a strain of EIV from clade 2 and not the strain from clade 1 were identified using NGPD and data analysis. It is important to be able to distinguish different clades to keep current equine vaccine effective for the circulating strain. Future work would include the recovery of the peptide sequences using inverse PCRs and testing their specificities against other EIV strains in the two clades. Identified antibody peptides could be used to replace ferret antisera in the haemagglutination test for characterising circulating EIVs.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Daly, Janet Dunham, Stephen
Keywords:	Zika virus; Equine influenza viruses; Diagnostic assays; Flaviviruses; Pseudotyping; Plant expression; Next-generation phage display
Subjects:	Q Science > QR Microbiology > QR355 Virology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science
Item ID:	60143
Depositing User:	Wu, Ge
Date Deposited:	17 Jul 2020 04:40
Last Modified:	17 Jul 2020 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/60143

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