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Seasonal influenza virus infections cause yearly epidemics which are the source of a significant public health burden worldwide. The ferret model for human influenza A virus (IAV) is widely used and has several advantages over other animal models such as comparable symptomology, similar receptor distribution in the respiratory tract to humans and the ability to be infected with human isolates without the need for adaptation. However, a major disadvantage of the model has been a paucity of reagents for the evaluation of the cellular immune response. Investigation of T-cell mediated immunity in ferrets is crucial to vaccine development and efficacy studies. In this study we have used commercially produced antibodies to ferret interferon gamma (IFN-γ) allowing us to reliably measure influenza-specific IFN-γ as a marker of the cellular immune response using both enzyme-linked immunospot (ELISpot) and enzyme-linked immunosorbent (ELISA) techniques. Here we demonstrate the application of these tools to evaluate cellular immunity in ferrets infected with clinically relevant seasonal H1N1 and H3N2 IAV subtypes at equivalent doses. Using small heparinised blood samples we were able to observe the longitudinal influenza-specific IFN-γ responses of ferrets infected with both seasonal subtypes of IAV and found a notable increase in influenza-specific IFN-γ responses in circulating peripheral blood within 8 days post-infection. Both seasonal strains caused a well-defined pattern of influenza-specific IFN-γ responses in infected ferrets when compared to naïve animals. Additionally, we found that while the influenza specific IFN-γ responses found in peripheral circulating blood were comparable between subtypes, the influenza specific IFN-γ responses found in lung lymphocytes significantly differed. Our results suggest that there is a distinct difference between the ability of the two seasonal influenza strains to establish an infection in the lung of ferrets associated with distinct signatures of acquired immunity.

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections.

Ferrets are widely used to study human influenza virus infection. Their airway physiology and cell receptor distribution makes them ideal for the analysis of pathogenesis and virus transmission, and for testing the efficacy of anti-influenza interventions and vaccines. The 2009 pandemic influenza virus (H1N1pdm09) induces mild to moderate respiratory disease in infected ferrets, following inoculation with 106 plaque-forming units (pfu) of virus. We have demonstrated that reducing the challenge dose to 102 pfu delays the onset of clinical signs by 1 day, and results in a modest reduction in clinical signs, and a less rapid nasal cavity innate immune response. There was also a delay in virus production in the upper respiratory tract, this was up to 9-fold greater and virus shedding was prolonged. Progression of infection to the lower respiratory tract was not noticeably delayed by the reduction in virus challenge. A dose of 104 pfu gave an infection that was intermediate between those of the 106 pfu and 102 pfu doses. To address the hypothesis that using a more authentic low challenge dose would facilitate a more sensitive model for antiviral efficacy, we used the well-known neuraminidase inhibitor, oseltamivir. Oseltamivir-treated and untreated ferrets were challenged with high (106 pfu) and low (102 pfu) doses of influenza H1N1pdm09 virus. The low dose treated ferrets showed significant delays in innate immune response and virus shedding, delayed onset of pathological changes in the nasal cavity, and reduced pathological changes and viral RNA load in the lung, relative to untreated ferrets. Importantly, these observations were not seen in treated animals when the high dose challenge was used. In summary, low dose challenge gives a disease that more closely parallels the disease parameters of human influenza infection, and provides an improved pre-clinical model for the assessment of influenza therapeutics, and potentially, influenza vaccines.

Animal models of influenza are important in preclinical research for the study of influenza infection and the assessment of vaccines, drugs and therapeutics. Here, we show that Golden Syrian hamsters (Mesocricetus auratus) inoculated via the intranasal route with high dose of influenza H1N1 display comparable disease kinetics and immune responses to the ‘gold standard’ ferret (Mustela furo) model. We demonstrate that both the hamster and ferret models have measurable disease endpoints of weight loss, temperature change, viral shedding from the upper respiratory tract and increased lung pathology. We also characterised both the humoral and cellular immune responses to infection in both models. The comparability of these data supports the Golden Syrian hamster model being useful in preclinical evaluation studies to explore the efficacy of countermeasures against influenza.

Abstract Live attenuated influenza vaccine (LAIV) is widely used to protect humans from seasonal influenza infection, particularly in children. In contrast to inactivated vaccines, the LAIV can induce both mucosal and cellular immune responses. Here we show that a single dose of monovalent H1N1pdm09-specific LAIV in the ferret model is fully protective against a subsequent wild-type H1N1pdm09 challenge, and furthermore reduces the severity of disease following challenge with a different influenza A subtype (H3N2). The reduced severity comprised reductions in weight loss and fever, as well as more rapid clearance of virus, compared to non-vaccinated H3N2-challenged ferrets. No H3N2-neutralizing antibodies were detected in vaccinated ferret sera. Rather, heterosubtypic protection correlated with interferon-gamma+ (IFN-γ+) T-cell responses measured in peripheral blood and in lung lymphocytes. The IFN-γ+ cells were cross-reactive to H3N2 virus even when obtained from vaccinated animals that had never been exposed to H3N2 virus. We believe this study provides compelling evidence that the LAIV can provide a significant reduction in infection and symptoms when challenged with heterosubtypic influenza strains not included in the LAIV, highlighting the importance of cross-reactive T-cells in the design of a universal influenza vaccine.

SARS-CoV-2 induces mild infection in ferret model. Here, Ryan et al. characterise optimal infection dosage inducing upper respiratory tract (UTR) viral shedding, progression time of viral shedding, and pathology in ferrets and finally provide evidence for protection after re-challenge.