The frequency of occurrence and nature of recombinant feline leukemia viruses in the induction of multicentric lymphoma by infection of the domestic cat with FeLV-945

Abstract

During feline leukemia virus (FeLV) infection in the domestic cat, viruses with a novel envelope gene arise by recombination between endogenous FeLV-related elements and the exogenous infecting species. These recombinant viruses (FeLV-B) are of uncertain disease association, but have been linked to the induction of thymic lymphoma. To assess the role of FeLV-B in the induction of multicentric lymphoma and other non-T-cell disease, the frequency of occurrence and nature of FeLV-B were examined in diseased tissues from a large collection of FeLV-infected animals. Diseased tissues were examined by Southern blot and PCR amplification to detect the presence of FeLV-B. Further analysis was performed to establish the recombination junctions and infectivity of FeLV-B in diseased tissues. The results confirmed the frequent association of FeLV-B with thymic lymphoma but showed infrequent generation, low levels and lack of infectivity of FeLV-B in non-T-cell diseases including multicentric lymphoma.

Figure 1

Southern blot analysis of genomic DNA from tumor (T) or paired normal (N) tissue from FeLV-infected cats O24, O26 and N91 bearing thymic lymphoma (TL) or multicentric lymphoma (ML) (Chandhasin et al., 2005). A. DNA samples (8 μg) were digested with KpnI and hybridized to probe B/S, a Sau3A fragment from the env gene of FeLV-B/Gardner-Arnstein specific for the major classes of endogenous FeLV that serve as substrates for recombination. The distinctive hybridizing fragment of ~3.6-kb (*) indicates recombinant FeLV-B provirus in genomic DNA (Chandhasin et al., 2005; Tsatsanis et al., 1994). B. KpnI-digested DNA samples were also hybridized to a probe representing the U3 region of the LTR of exogenous FeLV. By this analysis, clonally integrated proviruses are visualized as host-virus junction fragments in tumor DNA (Levy, Gardner, and Casey, 1984).

Figure 2

PCR amplification of the recombinant env gene of FeLV-B from the genomic DNA of naturally and experimentally infected cats. Genomic DNA samples were amplified by PCR using forward and reverse primers specific for the endogenous FeLV-related CFE-6 element and exogenous FeLV-945, respectively. A. Genomic DNA was examined from naturally occurring thymic lymphomas (1037 thy, 1110 thy and 1100 thy) and multicentric lymphomas (1345 liver, 1049 spleen, and 945 kidney). B. Genomic DNA was examined from experimentally induced thymic lymphomas (O26 thy, O24 thy, and 981-2 thy) and multicentric lymphomas (N90 liver, N91 liver, and N92 liver). Indicated by the arrow is the predicted amplification product of ~1.1-kb. Genomic DNA from uninfected FEA cells, and from canine D-17 cells infected with FeLV-B/Gardner-Arnstein were examined as negative and positive controls, respectively.

Figure 3

Nucleotide sequence of the PCR amplification products from tumor DNA of the indicated animals aligned with the sequence of endogenous FeLV-related provirus CFE-6. The previously reported sequences of FeLV-945, FeLV-A/61E and FeLV-B/Gardner-Arnstein are aligned for comparison. Nucleotide sequence differences as compared to CFE-6 are indicated; (…) indicates sequence identity with CFE-6; (____) indicates deletion relative to CFE-6; (//) indicates a break in the sequence shown. Indicated above the sequence are the positions of 34 nucleotide sequence differences in the amplification products as compared to CFE-6. Those indicated with an asterisk (*) result in amino acid change relative to CFE-6. Also indicated about the sequence are recombination sites A – G, previously reported to be preferred sites for crossover in the generation of FeLV-B (Sheets et al., 1992).

Figure 4

Schematic representation of the recombination junctions identified in the SU genes of FeLV-B viruses amplified from tumor DNA of the indicated animals. Depicted for comparison are the SU genes of exogenous viruses FeLV-A/61E (solid bar) and FeLV-945 (gray bar) and endogenous FeLV-related provirus CFE-6 (open bar). A deletion within FeLV-A/61E relative to CFE-6 is indicated (_vv). Shown diagrammatically are the positions of previously identified preferred recombination sites A – G (Sheets et al., 1992). Also indicated is the infectivity of genomic DNA for canine-D17 cells, presumably representing polytropic FeLV-B replication.