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Leverett, H.E. (2025) New approaches to study canine and equine influenza A virus. PhD thesis, University of Nottingham.

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Abstract

Equine and canine influenza A viruses (IAV) have been isolated for the past 70 and 25 years, respectively. Currently, a single subtype of equine influenza A virus (H3N8) circulates. This subtype jumped species to canines in the late 1990s in the US and circulated until 2016. In 2004, an avian H3N2 subtype virus was transmitted to dogs and spread into the US where it continues to circulate. Limited research is ongoing to further understand these two neglected viruses although within the research community, new approaches are more often applied to emerging and human influenza viruses. In this research, how novel approaches can be used to generate reagents and further develop the understanding of these two viruses was investigated.

Next generation phage display was used to generate variable heavy chain (VHH) antibodies that bound to recombinant equine IAVs (eIAV) with haemagglutinin (HA) surface glycoproteins from isolates representing the two current circulating clades of equine H3N8 subtype (Florida clade 1 and clade 2). The analysis highlighted 70 potential binders and a trial number of 10 were expressed using bacterial and mammalian expression systems to produce maltose binding protein (MBP) and fragment crystallizable (Fc) fused VHH antibodies. Three of each Fc and MBP fused VHH were successfully generated. One Fc fused antibody selectively bound influenza viruses in ELISA but did not differentiate between the two clades. Nonetheless, the VHH could have other applications in research, diagnosis or treatment of equine influenza.

Three cell lines (equine E. derm and extEqFL and canine DH82α) were explored for suitability for researching equine and canine IAVs by titrating viruses by 50% tissue culture infectious dose and viral replication kinetics by M gene RT-qPCR. Equine and canine IAVs primarily bind to α2,3 sialic acids (SA) so receptor staining was performed to confirm their presence on the cell lines. Generation of recombinant reverse genetics virus was undertaken to obtain the canine H3N2 subtype. Investigation into the innate immune response was completed using RNA sequencing and RT-qPCR for species specific cytokines. The extEqFL and E. derm cell lines were rejected as potential candidates due to poor lectin staining, viral replication and lack of cytokine expression. The DH82α cell line demonstrated efficient viral replication and appropriate lectin staining. The RNA sequencing data highlighted 21 genes from the IAV pathway indicating expression with variations seen between the 6- and 24-hour samples. Cytokine responses were lower than anticipated and not all expected genes were upregulated post-infection with virus.

Receptor specificity assays were applied to determine whether changes in the HA of eIAVs aided transmission into dogs. This was investigated using solid phase binding assays and biolayer interferometry. Equine IAV pseudotype viruses (PV) with mutations in the HA were generated however receptor binding could not be investigated with either method because of low viral titres and non-specific binding. Attempts to rescue viruses with mutated HA proteins by reverse genetics that could produce higher titres were not successful. Pseudotyped viruses were used to confirm that MDCK, DH82α and E. derm cells did not permit virus entry with equine or canine cells.

This thesis outlines how new approaches were developed and successfully utilised to study canine and equine influenza A viruses. The generation of antibodies using next generation phage display is a promising concept, and DH82α cells are identified as a potentially useful cell line for IAV research.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Daly, J. Dunham, S. Foster, T.
Keywords:	Influenza virus, Phage display, Canine, Equine
Subjects:	S Agriculture > SF Animal culture
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science
Item ID:	81169
Depositing User:	Leverett, Hope
Date Deposited:	24 Jul 2025 04:40
Last Modified:	24 Jul 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/81169

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