GENERATION OF BONE MARROW CELL LINES FROM XENOPUS LAEVIS AND RANA SYLVATICA AND CHARACTERIZATION OF THEIR SUSCEPTIBILITY TO FROG VIRUS 3
Barbara Katzenback, University of Waterloo
Bibi S. H. Sokeechand, Mercedes M. Rose, William A. Ng, Maxwell P. Bui-Marinos, and Barbara A. Katzenback
Department of Biology, University of Waterloo, Waterloo, ON, Canada
In frogs, the bone marrow is the site of hematopoiesis and this process is supported by cellular and soluble signals provided by stromal cells within the hematopoietic niche. In susceptible frog species, such as the North American wood frog (Rana sylvatica), the bone marrow is one of the sites of Frog Virus 3 (FV3) replication and can lead to destruction of the hematopoietic niche. To better understand the role of bone marrow stromal cells in frog hematopoiesis and in host-virus interactions, we generated bone marrow cell lines from adult Xenopus laevis (Xela BMW3-1) and R. sylvatica (Rs BM7) using the tissue explant method. Both cell lines are adherent, stromallike and exhibit low levels of senescence-associated -galactosidase senescence. Xela BMW-3-1 and Rs BM7 are maintained at 26C and 18C, respectively, in amphibian adjusted Leibovitz’s L-15 medium supplemented with 15% FBS and 25% cell-conditioned media from previous cultures. Cells were infected with FV3 at 0, 0.002, 0.02, 0.2, 2 and 20 multiplicities of infection and monitored over 14 days to observe cellular morphology and viral replication. Xela BMW3-1 and Rs BM7 were susceptible to FV3 infection, exhibiting dose- and time-dependent cytopathic effects (loss of adherence) and increases in viral titre. These results suggest that bone marrow stromal cells from relatively FV3-resistant (X. laevis) and FV3-susceptible (R. sylvatica) frog species are equally permissive to FV3 entry and replication. Further studies are needed to characterize the growth factors produced by frog bone marrow stromal cells and cellular immunocompetence.