TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

Abstract

The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.

Characterization of the Localized Immune Response in the Respiratory Tract of Ferrets following Infection with Influenza A and B Viruses

“…Reverse transcription was performed as previously described (24). Primers and probes were previously described for all of the cytokine, chemokine, and housekeeping genes (24). Ferret granzyme B was amplified using previously described methods (24).…”

“…Primers and probes were previously described for all of the cytokine, chemokine, and housekeeping genes (24). Ferret granzyme B was amplified using previously described methods (24). Briefly, cDNA was prepared from ferret leukocytes stimulated in vitro for 72 h with concanavalin A (5 g/ml; Sigma) and recombinant human interleukin-2 (IL-2) (0.1 g/ml; Roche Diagnostics GmbH).…”

“…cDNAs were stored at Ϫ20°C. TaqMan real-time PCR assays were performed using cDNA samples, standards, and plasmid controls as previously described (24). All calculations were performed as previously described (24).…”

“…TaqMan real-time PCR assays were performed using cDNA samples, standards, and plasmid controls as previously described (24). All calculations were performed as previously described (24). The time zero control sample for all in vivo infection time courses was a pool of nasal washes from 50 uninfected ferrets (URT measures) or lung tissues from uninfected ferrets (LRT measures).…”

“…Comparisons of disease severities and immune responses induced by infection with A(H1N1)pdm09 virus and seasonal influenza A virus strains are limited in animal models (14,22,23), with no studies focusing on coexistent influenza A and B virus strains. We recently developed TaqMan real-time reverse transcription-PCR (RT-PCR) assays to detect 15 cytokines and chemokines associated with infection in the ferret (24). In the present study, these assays were used to characterize the immune responses elicited in ferrets following infection with two influenza A viruses [A(H1N1)(pdm09) and A(H3N2)] and an influenza B virus that cocirculated in the Australian population in 2009 and 2010.…”