Characterisation of a novel family of peptides with potent anti-viral activity against influenza viruses

Avian influenza viruses can cause devastating outbreaks in domesticated poultry, with rapid transmission of virus between birds and high mortality. Current measures for control of influenza involve surveillance, closure of live poultry markets and mass culling. However, despite implementing these measures, outbreaks continue. Considering the uncertainty over whether certain strains of avian influenza viruses will adapt to human transmission and limitations over how their spread may be controlled, this study considers the use of anti-viral peptides as protective agents against low pathogenicity avian influenza (LPAI) virus. This study investigated the activity and mode of action of two cell-penetrating anti-viral peptide families against influenza A virus infection: ‘FluPep’ (a family of short, hydrophobic peptides related to suppressor of cytokine signaling- 1 proteins) and ‘Entry Blocker’ (derived from the signal sequence of fibroblast growth factor-4). Plaque reduction assays demonstrated dose-dependent anti-viral activity of both peptide families against a panel of influenza viruses with diverse haemagglutinin subtypes. Determination of IC50 values showed strain-specific differences in sensitivity to FluPep but not Entry Blocker. The IC50 of FluPep 4 for A/PR/8/34 was reduced by reassorting in the HA and NA from a relatively sensitive avian strain using a reverse genetics approach, suggesting inhibitory effects on the viral glycoproteins. Accordingly, viral entry assays focusing on binding, internalisation, fusion and import were designed and optimised to dissect the mechanism(s) of action of the peptides. Results indicated that the peptides acted upstream of nuclear import of viral ribonucleoprotein complexes but did not reduce overall virus binding to cells. However, the peptides caused aggregation of the virus particles on the surface of the host cells and reduced their internalisation. Further work evaluated how the peptides may be delivered to the site(s) of viral replication in poultry. A screen of the current literature was completed to allow for the design of an expression cassette for poultry-derived Lactobacillus to express FluPep and Entry Blocker. Though the cassette has been reported to be suitable for expression of heterologous proteins in Lactobacilli, rescue of recombinants for expression of anti-viral peptides or a reporter protein proved challenging, possibly owing to toxicity. A stable construct for the expression of FluPep 4 in Lactobacillus was obtained but culture supernatant did not inhibit virus replication