Bovine leukemia virus (BLV) infects cattle and causes serious problems for the cattle industry, worldwide. Vertical transmission of BLV occurs via in utero infection and ingestion of infected milk and colostrum. The aim of this study was to clarify whether milk is a risk factor in BLV transmission by quantifying proviral loads in milk and visualizing the infectivity of milk. We collected blood and milk from 48 dams (46 BLV seropositive dams and 2 seronegative dams) from seven farms in Japan and detected the BLV provirus in 43 blood samples (89.6%) but only 22 milk samples (45.8%) using BLV-CoCoMo-qPCR-2. Although the proviral loads in the milk tended to be lower, a positive correlation was firstly found between the proviral loads with blood and milk. Furthermore, the infectivity of milk cells with BLV was visualized ex vivo using a luminescence syncytium induction assay (LuSIA) based on CC81-GREMG cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) in response to BLV Tax and Env expressions when co-cultured with BLV-infected cells. Interestingly, in addition to one BLV-infected dam with lymphoma, syncytia with EGFP fluorescence were observed in milk cells from six BLV-infected, but healthy, dams by an improved LuSIA, which was optimized for milk cells. This is the first report demonstrating the infectious capacity of cells in milk from BLV-infected dams by visualization of BLV infection ex vivo. Thus, our results suggest that milk is a potential risk factor for BLV vertical spread through cell to cell transmission.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis and is closely related to the human T cell leukemia virus. Since BLV infection mostly occurs via cell-to-cell transmission, BLV infectivity is generally measured by culturing BLV-infected cells with reporter cells that form syncytia upon BLV infection. However, this method is time-consuming and requires skill. To visualize the infectivity of BLV, we developed a new assay called the luminescence syncytium induction assay (LuSIA) that is based on a new reporter cell line designated CC81-BLU3G. CC81-BLU3G is stably transfected with pBLU3-EGFP, which contains the BLV long terminal repeat U3 region linked to the enhanced-green fluorescence protein (EGFP) gene. CC81-BLU3G expresses the EGFP in response to BLV Tax expression specifically, and forms fluorescing syncytia when transfected with an infectious BLV plasmid or when cultured with BLV-infected cells. Compared to the conventional assay, LuSIA was more specific and detected cattle samples with low proviral loads. The fluorescing syncytia was easily detected by eye and automated scanning and LuSIA counts correlated strongly with the proviral load of infected cattle (R = 0.8942).

Background Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease of cattle. Previously, we reported the luminescence syncytium induction assay (LuSIA), an assay for BLV infectivity based on CC81-BLU3G cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) when co-cultured with BLV-infected cells. To develop a more sensitive LuSIA, we here focused on the glucocorticoid response element (GRE) within the U3 region of the BLV long terminal repeat (LTR). Methods We changed five nucleotide sites of the GRE in a pBLU3-EGFP reporter plasmid containing the BLV-LTR U3 region promoter by site-directed mutagenesis and we then constructed a new reporter plasmid (pBLU3 GREM -EGFP) in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter. We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG). To evaluate the sensibility, the utility and the specificity of the LuSIA using CC81-GREMG, we co-cultured CC81-GREMG cells with BLV-persistently infected cells, free-viruses, white blood cells (WBCs) from BLV-infected cows, and bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells. Results We successfully constructed a new reporter plasmid harboring a mutation in the GRE and established a new reporter cell line, CC81-GREMG; this line was stably transfected with pBLU3 GREM -EGFP in which the EGFP gene is expressed under control of the GRE-mutated LTR-U3 promoter and enabled direct visualization of BLV infectivity. The new LuSIA protocol using CC81-GREMG cells measures cell-to-cell infectivity and cell-free infectivity of BLV more sensitively than previous protocol using CC81-BLU3G. Furthermore, it did not respond to BIV and BFV infections, indicating that the LuSIA based on CC81-GREMG is specific for BLV infectivity. Moreover, we confirmed the utility of a new LuSIA based on CC81-GREMG cells using white blood cells (WBCs) from BLV-infected cows. Finally, the assay was useful for assessing the activity of neutralizing antibodies in plasma collected from BLV-infected cows. Conclusion The new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-BLU3G cells and should facilitate development of several new BLV assays.

Numerical time domain modeling of the ultrasonic NDT with electromagnetic acoustic and piezoelectric transducers AIP Conf.