Effects of Buprenorphine Treatment on Influenza Pathogenesis in the Ferret (Mustela putorius furo)

Ferrets are the gold-standard model for influenza A virus (IAV) research due to their natural susceptibility to human and zoonotic IAV, comparable respiratory anatomy and physiology to humans, and development of clinical signs similar to those seen in infected people. Because the presence and progression of clinical signs can be useful in infectious disease research, uncertainty in how analgesics alter research outcomes or compromise characteristics of disease progression have outweighed the concern regarding animal discomfort from these symptoms. Nonetheless, the principles of animal research require consideration of refinements for this important model for IAV research. Opioids offer a possible refinement option that would not directly affect the inflammatory cascade involved in IAV infection. Mirroring pathogenicity studies that use ferrets, 12 ferrets were inoculated intranasally with the A(H3N2) IAV A/Panama/2007/1999 and divided into 3 treatment groups (n = 4 each), of which 2 groups received buprenorphine treatments on different schedules and the third received a saline control. The duration and location of viral replication, lymphohematopoietic changes, and clinical signs were comparable across all groups at all time points. High quantities of infectious virus in nasal wash specimens were detected in ferrets from all groups through day 5 after inoculation, and peak viral titers from the upper respiratory tract did not differ between ferrets receiving buprenorphine treatments on either schedule. Compared with the saline group, ferrets receiving buprenorphine exhibited transient weight loss and pyrexia, but all groups ultimately achieved similar peaks in both of these measurements. Collectively, these findings support the continued evaluation of buprenorphine as a refinement for IAV-challenged ferrets.

Despite decades of international research and the availability of public health countermeasures, including vaccines and antivirals, influenza viruses remain a persistent threat to human and animal health.^26,35 Influenza A viruses (IAV) exhibit a diverse range of virulence, exist in several host reservoirs, and can show rapid rates of antigenic change.²⁶ As a result, IAV are associated with both seasonal epidemics and occasional pandemics in humans,³⁵ and animal infections with IAV have become key for understanding multifactorial traits that include pathogenicity, transmissibility, and vaccine efficacy. Due to their relatively small size, adaptability to the research setting, and similarities to human lung anatomy and physiology, ferrets provide an excellent model for respiratory diseases in humans and are a valuable small-animal model for such studies.^8,30 Data generated from ferrets are included in numerous risk-assessment rubrics evaluating the pandemic potential of novel and emerging influenza viruses, including those established by the Centers for Disease Control and Prevention and the World Health Organization.^14,51The study of influenza virus in ferrets dates back to the early 1930s, when this species was first found to be susceptible to influenza virus.⁴⁴ Ferrets are naturally susceptible to both human and zoonotic IAV.⁴⁷ After infection, ferrets present with clinical signs like those of humans; these signs are often not recapitulated in other species, such as mice and guinea pigs.^28,39,46 The severity and spectrum of clinical signs associated with influenza virus–inoculated ferrets can vary, depending on the virus strain, route and dose of inoculation, and various host parameters.⁵ Whereas influenza viruses with low virulence in ferrets may cause only acute pyrexia and mild to moderate weight loss, isolates with high virulence can cause severe, systemic illness with gastrointestinal and neurologic symptoms.⁴The 3Rs, replace, reduce, refine, encourage investigation of how research involving animals can be conducted in more humane ways.^2,13,37,41 Analgesia for symptoms of influenza in ferrets represents an opportunity for refinement, but this intervention could confound research assessing disease progression. NSAID and corticosteroids are often prescribed to treat the clinical signs associated with influenza in humans.⁴³ These interventions could alter the inflammatory cascade and subsequent pathophysiology of the disease, thus reducing the validity of studies designed to characterize and compare influenza viruses.^6,43 NSAID reportedly inhibit nuclear factor κB, a regulator of inflammatory processes that is involved in viral RNA synthesis.^25,27 In addition, NSAID have been found to increase survival rates in influenza virus-infected mice.⁵³ Therefore, the use of NSAID may be problematic in studies investigating the pathogenesis of influenza viruses.Buprenorphine, an opioid, is an established analgesic in ferrets that can be administered either intravascularly, intramuscularly, or subcutaneously at 0.01 to 0.05 mg/kg with an analgesic duration of 6 to 12 h.^{11,16,24,38,52} Historically buprenorphine has been described as a partial µ receptor agonist and κ and γ receptor antagonist,^22,29,40,48 but the drug recently was described to behave as a full µ agonist.³⁶ The ceiling effect of analgesia and the immunosuppressive effects reported with other opioids have not been documented to occur with buprenorphine.^15,36,42 However, the use of buprenorphine does have the possibility of adverse effects, including sedation, weight loss, constipation, and respiratory depression.^{10,15,16,22,23,34,42} Nonetheless, buprenorphine is a commonly prescribed analgesic for numerous small mammalian species used in research settings.^20,22,40Given that influenza is an ongoing threat to human and animal health and because no replacement is available for data gained with the ferret model, pain mitigation options for research conducted in this species must be addressed. To date, concerns about altering the course of the disease have precluded the evaluation of refinements options in IAV-infected ferrets. The goal of the current study was to assess the effects of buprenorphine treatments on the pathogenesis of a seasonal IAV in ferrets; this assessment was achieved by comparing virus-inoculated ferrets that were either sham-treated or that received buprenorphine according to 2 different dosing schedules. We hypothesized that buprenorphine treatments would not affect experimental readouts, including morbidity, viral shedding, lymphopenia, and seroconversion in convalescent serum; these parameters are commonly measured during IAV research. Study results indicate that buprenorphine did not uniformly or significantly modulate disease progression, peak viral titers in the upper respiratory tract, or clinical responses used to characterize viral pathogenicity in ferrets.