Molecular detection and analysis of Feline Leukemia Virus (FeLV) Long Terminal Repeat (LTR) sequences in neoplastic and non-neoplastic FeLV-induced diseases of domestic cats
Jackson, Marion Louise
The feline leukemia virus (FeLV) causes degenerative and proliferative hemolymphatic diseases in domestic cats. Some cats with clinicopathologic evidence of FeLV infection have no detectable FeLV antigen within tissues. To test the hypothesis that FeLV provirus is present but not expressing antigen in some cats, a region of the FeLV long terminal repeat (LTR) was detected using the polymerase chain reaction (PCR) and compared to FeLV antigen detection using immunohistochemistry (IHC) or enzyme-linked immunosorbent assay (ELISA), in tissues from cats with suspected FeLV-related disease. In the retrospective study to investigate lymphosarcomas (LSAs) from 70 cats, FeLV DNA was detected significantly more often than antigen, particularly in tumors from cats $\ge$7 yr. Conclusions were that latent or defective FeLV may be more prevalent in LSAs from older cats. Older cats may have eliminated replicating FeLV, but retained non-replicating, but LSA-inducing strains of the virus. The prospective study to investigate FeLV in peripheral blood from 68 sick cats with varying clinicopathologic evidence of FeLV-related disease, revealed no difference in the prevalence of FeLV DNA compared to antigen. The possibility exists that in test-negative cats with high suspicion of FeLV-related disease, non-replicating FeLV may have been present in tissues other than peripheral blood cells. Previous studies suggest T cell but not B cell transformation by FeLV. However in earlier reports, only mature B cell markers were used, and investigations involved mainly thymic (T cell) tumors. In this study, of 70 feline LSAs evaluated immunohistochemically using commercially available T and B cell antibodies, 67% were T cell and 27% were B cell. The B cell tumors were as likely as T cell tumors to contain FeLV DNA and antigen. Both B and T cell tumors from older cats were more likely to contain FeLV DNA than demonstrable FeLV antigen. Conclusions were that FeLV transforms B cells as well as T cells, although in older cats, LSA may be associated with non-replicating FeLV. Studies with some type C retroviruses indicate that genomic variance within the LTR relates, in part, to disease manifestation. Therefore, the nucleotide sequences of LTR PCR products from natural cases of neoplastic and non-neoplastic FeLV-related disease were determined. Phylogenetic analysis of FeLV LTR sequences from tissues of 33 infected cats, showed genomic homology among 4 of 5 antigen-negative LSAs. Previously, enhancer duplications were found to be prevalent in neoplastic FeLV diseases, however no repeat sequences were found in this study. Point mutations relative to the common form FeLV were frequent, and most involved 5 single nucleotide sites both within and peripheral to nuclear protein binding motifs. All LSAs, but particularly those not expressing antigen, contained the fewest LTR mutations compared to this region in other FeLV-related diseases. Mutations within the LTR enhancer region are unlikely to account for the lack of viral gene expression in antigen-negative LSAs. Functional studies will be required to determine how specific point mutations within the FeLV LTR relate to disease potential in infected tissues. The lack of consistent LTR changes among FeLV-induced disease groups indicates that genetic alterations within the LTR enhancer are at least not solely responsible for disease manifestation. Envelope, gag, and other regions of the viral genome, as well as cellular oncogenes and other host factors may act in concert to determine outcome in infected cats.