Low Dose Influenza Virus Challenge in the Ferret Leads to Increased Virus Shedding and Greater Sensitivity to Oseltamivir

Abstract

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.

Correlation between Virus Replication and Antibody Responses in Macaques following Infection with Pandemic Influenza A Virus

“…Evaluation of different doses for viral inoculation has been described for mice, ferrets, and occasionally in humans, but not yet for nonhuman primates (5)(6)(7)(8)(9)(10)(17)(18)(19)(20)(21). These studies were either directed at finding the minimal infectious dose or minimal lethal dose or, in case of evaluation of attenuated viruses in humans, at finding the dose that resulted in adequate levels of infection while limiting development of clinical symptoms and fever.…”

“…A relation between inoculation dose and virus replication in nasal washes has been reported in several human studies (17,18,21), but other studies have shown no relation (19,20). Also, in ferrets conflicting results on the relation between virus inoculation dose and levels of virus replication have been reported (6,(8)(9)(10).…”

“…Novel candidate influenza vaccines are commonly tested for safety and efficacy in mice and ferrets and/or macaques before they are evaluated for immunogenicity in humans (2,3). However, whereas for mice and ferrets dose-finding studies have been described and implemented for testing of vaccines and antiviral agents (4)(5)(6)(7)(8)(9)(10)(11), for macaques usually a standard challenge dose is used, typically in the range between 10 6 and 10 7 50% tissue culture infective doses (TCID 50 ) (12)(13)(14)(15).…”

Complexities in Ferret Influenza Virus Pathogenesis and Transmission Models

“…However, this dose is unrealistically high compared with typical infectious doses in humans, which have been shown to be as low as 3 TCID 50 following exposure to small-particle influenza virus aerosols (34,35). Similarly, studies quantitating the 50% infectious dose of influenza viruses in the ferret (FID 50 ) have revealed that both avian and human influenza viruses are capable of high infectivity in this species, with doses under 10 PFU leading to productive ferret infection following inhalation of virus-containing aerosols (24,36). Low-dose infection has also been documented following inoculation of ferrets by the ocular route (28).…”

Evaluation of a dry powder delivery system for laninamivir in a ferret model of influenza infection

“…Ferrets are the preferred animal model to assess influenza virus infection, virulence and transmission (Govorkova et al, 2007;Itoh et al, 2009;Maines et al, 2009;Munster et al, 2009;Zhang et al, 2013), and have been widely used in antiviral studies to assess drug effectiveness (Govorkova et al, 2007(Govorkova et al, , 2011Marriott et al, 2014;Oh et al, 2015), different treatment strategies (Maines et al, 2009;Oh et al, 2014) and the selection of resistant viruses (Hurt et al, 2010). Because oseltamivir, the most widely used NAI globally, is an orally administered drug, delivery to ferrets (or other animals) is relatively straightforward.…”