HVMNE, a novel lymphocryptovirus related to Epstein-Barr virus, induces lymphoma in New Zealand White rabbits by Maria G. Ferrari, Emilia D. Rivadeneira, Ruth Jarrett, Liljana Stevceva, Shigeki Takemoto, Phil Markham, and Genoveffa Franchini Blood Volume 98(7):2193-2199 October 1, 2001 ©2001 by American Society of Hematology

Anti-VCA–immunoglobulin G titers Anti-VCA–immunoglobulin G titers.After HVMNE inoculation into New Zealand White rabbits, anti-VCA–immunoglobulin G titers show the seroconversion of rabbits to EBV VCA. Anti-VCA–immunoglobulin G titers.After HVMNE inoculation into New Zealand White rabbits, anti-VCA–immunoglobulin G titers show the seroconversion of rabbits to EBV VCA. Maria G. Ferrari et al. Blood 2001;98:2193-2199 ©2001 by American Society of Hematology

Abnormal findings of lymphomatous rabbit C2182 Abnormal findings of lymphomatous rabbit C2182.Lymphomatous cell infiltration in the abdominal muscles (top), lung (center), and subcutaneous mass (bottom) (original magnification, ×40). Abnormal findings of lymphomatous rabbit C2182.Lymphomatous cell infiltration in the abdominal muscles (top), lung (center), and subcutaneous mass (bottom) (original magnification, ×40). Maria G. Ferrari et al. Blood 2001;98:2193-2199 ©2001 by American Society of Hematology

Detection of HVMNE DNA by PCR and Southern blot hybridization using p536, HVMNE-specific probe, in the tissues of 3 lymphomatous rabbits.C2182 (lung, spleen, lymphoid tissues); C2174 (cultured PBMCs with and without IL-2, heart, thymus); C2178 (heart, lymph... Detection of HVMNE DNA by PCR and Southern blot hybridization using p536, HVMNE-specific probe, in the tissues of 3 lymphomatous rabbits.C2182 (lung, spleen, lymphoid tissues); C2174 (cultured PBMCs with and without IL-2, heart, thymus); C2178 (heart, lymphoid tissues, subcutaneous mass, spleen, thigh mass, PBMCs, and uterine mass). In rabbit C2174, PBMCs cultured with and without IL-2 were also analyzed. Each DNA was also assessed for amplification competence using DNA primers for cytochrome B (data not shown). Maria G. Ferrari et al. Blood 2001;98:2193-2199 ©2001 by American Society of Hematology

EBER expression.Demonstration of EBER-like reactivity in formalin-fixed, paraffin embedded sections of malignant lymphomas by in situ hybridization using labeled oligonucleotide probe. EBER expression.Demonstration of EBER-like reactivity in formalin-fixed, paraffin embedded sections of malignant lymphomas by in situ hybridization using labeled oligonucleotide probe. Panels A and C show low- and high-power views, respectively, of positive nuclear staining in the ocular tumor of lymphomatous rabbit C2175, and panels B and D show low- (original magnification, ×40) and high-power (original magnification, ×400) views of the tumor infiltrates of the heart from rabbit C2180. Maria G. Ferrari et al. Blood 2001;98:2193-2199 ©2001 by American Society of Hematology

Establishment of a primary cell-line from the PBMCs of rabbit C2174 Establishment of a primary cell-line from the PBMCs of rabbit C2174.(A) Detection, by PCR and Southern blot hybridization using HVMNE (p536)-specific probe, of HVMNE DNA in the rabbit cell-line C2174 both at 6 and 12 months after culture (lane 1, C2174 afte... Establishment of a primary cell-line from the PBMCs of rabbit C2174.(A) Detection, by PCR and Southern blot hybridization using HVMNE (p536)-specific probe, of HVMNE DNA in the rabbit cell-line C2174 both at 6 and 12 months after culture (lane 1, C2174 after 6 months of culture; lane 2, PCR-negative control; lane 3, cell line J94356 as PCR-positive control; lane 4, C2174 after 12 months of culture). (B) Electrophoretic mobility shift assay to evaluate the Jak/STAT status of the rabbit T-cell line derived from primary culture of rabbit C2174 PBMCs. Transition to IL-2 independence of the C2174PBL cell line was associated with STAT-5 activation. On the top panel, the plus and minus signs represent the use of IL-2 pulse after overnight ligand starvation. αSTAT-5 indicates the addition to the lysate of specific αSTAT-5 antibodies in lanes 2, 4, 6, 8, and 10. Resting PBMCs (lanes 1-2) and the C2174PBL cell line cultured in the presence of IL-2 (lanes 3-6) and in the absence of IL-2 (lanes 7-10) after 8 months of culture. Maria G. Ferrari et al. Blood 2001;98:2193-2199 ©2001 by American Society of Hematology

Morphologic and phenotypical characterization of the C2174PBL cells Morphologic and phenotypical characterization of the C2174PBL cells.(A,B) C2174PBL cell line cytospins after 20 months of culture; note the presence of multinucleated giant cells (Giemsa staining). Morphologic and phenotypical characterization of the C2174PBL cells.(A,B) C2174PBL cell line cytospins after 20 months of culture; note the presence of multinucleated giant cells (Giemsa staining). Original magnification of each, × 40. (C) An aliquot of the C2174PBL cultured PBMCs permeabilized and then stained with rabbit specific antibodies (CD3 FITC, CD8 FITC) and EBV-LMP1 antibody (LMP1 PE) was fixed in paraformaldehyde placed in mounting media and analyzed using a Leica DMRA microscope (using program QFISH). Original magnification, × 63. The upper portion of panel C shows a double-positive cell for LMP1 and CD3. The dent present on the upper right corner clearly shows evidence of an adjacent double-negative cell. The middle panel shows an LMP1-positive and a CD8−cell, whereas the lower panel shows a CD3+ and an LMP1-negative cell. (D) Histogram plots from the FACS analysis of the C2174 cultured PBMCs at 20 months of culture. Blue lines indicate specific isotypic control. Maria G. Ferrari et al. Blood 2001;98:2193-2199 ©2001 by American Society of Hematology