Unintegrated bovine leukemia virus DNA: association with viral expression and disease.

The correlation between bovine leukemia virus (BLV) unintegrated DNA, viral expression, and stage of disease was determined in cattle naturally infected with BLV. The concomitant presence of unintegrated BLV DNA with viral transcriptional activity was observed in 53% (18 of 34) of hematologically normal, BLVseropositivecattleandin100%(10of10)ofBLV-seropositivecattlewiththepreneoplasticsyndromepersistent lymphocytosis. In vitro studies suggested that accumulation of unintegrated BLV DNA resulted from a process of reinfection rather than intracellular reverse transcription of newly synthesized BLV RNA. Interestingly, unintegratedBLVDNAwasnotdetectedintumorcellsfromcattlewithBLV-associatedlymphocyticleukemia/ malignant lymphoma despite viral transcriptional activity in 100% (eight of eight) of these cattle. Thus, the presence of unintegrated BLV DNA differentiated nonneoplastic from neoplastic conditions in BLV-infected cattle. These results demonstrate that unintegrated viral DNA serves as a marker of disease progression in BLV-infected cattle but is not necessarily associated with induction or maintenance of the neoplastic state. Bovine leukemia virus (BLV) is a B-lymphocytotropic retrovirus closely related by genomic organization and disease progression to human T-cell leukemia virus type 1 (HTLV-1) (for reviews, see references 7 and 12). Infection by BLV is characterized by a long clinical latency and a persistent immune response associated with low-level or transient viral expression (9, 11). Approximately 65% of infected cattle remain clinically and hematologically normal, whereas ;30% develop apersistentnonneoplasticproliferationofBlymphocytes(persistent lymphocytosis [PL]). In less than 5% of infections, a fatal, monoclonal, neoplastic transformation of B lymphocytes appears, resulting in lymphocytic leukemia (LL)/malignant lymphoma (ML). Because the risk of developing LL/ML is greater in cattle with PL than in BLV-seropositive cattle with normal hematological parameters, PL should be considered a preneoplastic condition (15). ThechronicnatureofBLVinfectionsreflectsthecapacityof BLV to integrate within host chromosomal DNA. As is typical of most retroviruses, integrated proviral DNA serves as a template for the synthesis of viral gene products. In addition to integrating within the host genome, the provirus can exist in both unintegrated linear and circular forms (4). Although limited gene expression from unintegrated retroviral DNA has been reported (29‐31), these proviral species are generally believed to be transcriptionally silent remnants of the viral replication cycle (3, 18, 22). Inseveralretroviralsystems,highlevelsofunintegratedviral DNA have been correlated with superinfection, the onset of disease, and cytopathicity (19, 20, 24). Although unintegrated viral DNA has not been reported in cattle naturally infected with BLV, these obligate intermediates of retroviral reverse transcription (4, 8) could serve as markers for BLV infection and disease progression. The purpose of this study was to determine if unintegrated viral DNA was present in lymphocytes of BLV-infected cattle and, if so, to correlate its presence with viral expression and stage of disease. The circular form of the BLV provirus was chosen as a marker for unintegrated viral DNA on the basis of our ability to specifically detect this form in the presence of both integrated and linear unintegrated viral DNA. The use of this animal model, with cattle in well-defined stages of infection and disease, may provide insight into the progression of analogous retroviral infections in humans.