Virus Research 127 (2007) 9–16 Cellular segregation of feline leukemia provirus and viral RNA in leukocyte subsets of long-term experimentally infected cats Andrea C. Pepin a , Ravi Tandon a , Valentino Cattori a , Eva Niederer b , Barbara Riond a , Barbara Willi a , Hans Lutz a , Regina Hofmann-Lehmann a,∗ a Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Switzerland b Institute of Biomedical Engineering, ETH and University of Zurich, Switzerland Received 4 October 2006; received in revised form 2 March 2007; accepted 13 March 2007 Available online 16 April 2007 Abstract Cats exposed to feline leukemia virus (FeLV) may develop different outcomes of the infection. However, during acute infection blood proviral and viral RNA loads of cats with progressive and regressive infection are not significantly different. Thus, not the overall loads but rather those of specific leukocyte subsets may influence the infection outcome. By combining fluorescence activated cell sorting (FACS) with sensitive real-time TaqMan ® PCR and reverse transcriptase (RT) PCR, we established in the present study the methods to determine FeLV proviral and viral RNA loads in specific leukocyte subsets. In addition, they were applied to analyze long-term persistently FeLV-infected (p27-positive) and FeLV exposed but nonantigenemic (p27-negative), nonviremic cats. In the latter animals, CD4 + and B lymphocytes exhibited the highest proviral loads, whereas in p27-positive cats, all leukocyte subsets showed similar high loads. In p27-positive cats, monocytes and granulocytes bore the highest viral RNA loads, whereas only one p27-negative cat was positive for viral RNA in T lymphocytes. To our knowledge, this is the first study to investigate FeLV proviral and viral RNA loads in leukocyte subsets of FeLV exposed cats. The herein described methods are important prerequisites to gain a deeper insight into the pathogenesis of FeLV infection. © 2007 Elsevier B.V. All rights reserved. Keywords: Feline leukemia virus; Quantitative real-time PCR; FACS; Leukocytes 1. Introduction Feline leukemia virus (FeLV) is a naturally occurring gam- maretrovirus in cats. It can cause both cytoproliferative (e.g. lymphomas, myeloproliferative disorders) and cytosuppressive (e.g. immunodeficiency, anemia) diseases. Altogether, four dif- ferent courses of infection have been described (Hoover and Mullins, 1991; Lutz et al., 1983b): (i) progressive infection with persistent viremia, regressive infection with (ii) transient or (iii) undetectable viremia, and (iv) localized FeLV-infection in tis- sues, such as spleen, lymph nodes, small intestine and mammary glands, without concurrent viremia (Hayes et al., 1989; Pacitti et al., 1986). Latent nonproductive infection characterized by the absence of viremia and persistence of the virus in the bone ∗ Corresponding author at: Clinical Laboratory, Vetsuisse-Faculty, University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland. Tel.: +41 44 635 83 22; fax: +41 44 635 89 23. E-mail address: rhofmann@vetclinics.uzh.ch (R. Hofmann-Lehmann). marrow has been described in cats that ostensibly recovered from a regressive infection (Madewell and Jarrett, 1983; Rojko et al., 1982). Categorization of cats into these groups is typ- ically accomplished by immunofluorescence assays (Hardy et al., 1973b; Hoover and Mullins, 1991; Jarrett et al., 1982) as well as by FeLV p27 antigen ELISA from blood plasma (Lutz et al., 1983a); antigenemia is a marker of infection and in most but not all cats a parameter for viremia (Jarrett et al., 1982). Recently, Torres and co-workers re-examined the FeLV-host relationship and suggested the four putative categories progres- sive, latent, regressive and abortive infection based on the results of real-time Taqman ® PCR and p27 ELISA (Torres et al., 2005). Moreover, most recently, the spectrum of host response cat- egories was also refined by investigating plasma viral RNA loads in FeLV exposed cats (Hofmann-Lehmann et al., 2007; Hofmann-Lehmann et al., 2006). In spite of the common use of vaccines in veterinary prac- tices, FeLV infections are still highly prevalent. A few years ago, 7% of the Swiss pet cat population was p27- as well as 0168-1702/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2007.03.008