Submitted to: Animal Influenza Virus
Publication Type: Book / Chapter
Publication Acceptance Date: 8/13/2014
Publication Date: 10/13/2014
Citation: Kapczynski, D.R., Jiang, H., Kogut, M.H. 2014. Characterization of cytokine expression induced by avian influenza virus infection with real-time RT-PCR. In: Spackman, E., editor. Animal Influenza Virus. 2nd edition. New York, NY: Springer. p. 217-233.
Interpretive Summary: Protective immunity against viruses is mediated by the early reactions of innate immunity and the later responses of adaptive immunity. The innate immune response of birds to avian influenza infection includes the stimulation of protein molecules that defend the animal against disease. These molecules are secreted by cells of the immune system and direct the development of protective immunity. Detection and quantitation of these proteins is useful in determining how different birds respond to a particular virus. Although reagents available to measure these response molecules are expanding, molecular techniques utilizing the polymerase chain reaction (PCR) have provided excellent tools to measure these important mediators of immunity. The PCR methods provided in this chapter detail the steps and techniques necessary to detect the avian immune response against avian influenza viruses.
Technical Abstract: Knowledge of how birds react to infection from avian influenza virus is critical to understanding disease pathogenesis and host response. The use of real-time (R), reverse-transcriptase (RT), PCR to measure innate immunity, including cytokine and interferon gene expression, has become a standard technique employed by avian immunologists interested in examining these responses. This technique utilizes nucleotide primers and fluorescent reporter molecules to measure amplification of the gene of interest. The use of RRT-PCR negates the need for Northern blot analysis or DNA sequencing. It is simple, specific and sensitive for the gene of interest. However, it is dependent on knowing the target sequence prior to testing so that the optimal primers can be designed. The recent publication of genomic sequences of Gallus gallus, Meleagris gallopavo and Anas platyrhynchos species makes it possible to measure cytokine expression in chicken, turkey and duck species, respectively. Although these tests do not measure functionally expressed protein, the lack of antibodies to identify and quantify avian cytokines from different avian species makes this technique critical to any characterization of innate immune responses through cytokine and interferon activation or repression.