Development and validation of puma (Felis concolor) cytokine and lentivirus real-time PCR detection systems Kerry Sondgeroth a , Christian Leutenegger b , Sue VandeWoude a, * a Department of Microbiology, Immunology and Pathology, 1619 Campus Delivery, Colorado State University, Fort Collins, CO 8052-1619, USA b Lucy Whittier Molecular Core Facility, Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA Accepted 30 November 2004 Abstract Studies of immune correlates of disease outcome associate humoral immune response mediated by T-helper 2 cytokines (IL- 4, IL-10) with more virulent disease relative to a cell-mediated response driven by T-helper 1 cytokines (IL-2, IFN-gamma), particularly in viral and other intra-cellular infections. Specifically, the kinetics of both human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV) infection are closely associated with Type 1 versus Type 2 cytokine profiles. Puma (Felis concolor) lentivirus (PLV) is closely related to FIV, but based on phylogenetic and clinical studies, is more ancient and less pathogenic. The aims of this study were to validate feline real-time PCR primer/probe systems for puma cytokines and PLVas sensitive, quantitative assays for use in investigations of PLV pathogenicity. We demonstrate that primer/probe systems for IL-4, IL-10, IFN-gamma, TNF-alpha, GAPDH, and the pol region of PLV-1695 amplify puma cytokines and PLV-1695 with high amplification efficiency and sensitivity. Detection of PLV-1695 provirus in experimentally inoculated domestic cats proved to be of equivalent sensitivity, specificity, and positive and negative predictivevalue to co-culture of one million peripheral blood mononuclear cells (PBMC). Evaluation of cytokine induction during naturally occurring PLV infection will allow insight into mechanisms of host control associated with apathogenic infection. In addition, determination of viral loads during different stages of PLV infection or in different tissues from domestic cats or pumas will further elucidate capacity of these viruses to replicate and establish infection. # 2004 Elsevier B.V. All rights reserved. Keywords: Lentivirus; Puma; Cytokine; Real-time PCR 1. Introduction Serologic surveys of 36 non-domestic feline species have revealed a minimum of 20 species that have antibodies that cross-react with feline immuno- deficiency virus (FIV) antigens (Olmsted et al., 1992; www.elsevier.com/locate/vetimm Veterinary Immunology and Immunopathology 104 (2005) 205–213 Abbreviations: FIV, feline immunodeficiency virus; GAPDH, GAP dehydrogenase; pol, polymerase; HIV, human immunodefi- ciency virus; IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cells; PCR, polymerase chain reaction; PLV, puma lentivirus * Corresponding author. Tel.: +1 970 491 7162; fax: +1 970 491 0523. E-mail address: suev@lamar.colostate.edu (S. VandeWoude). 0165-2427/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2004.11.009