Improved detection of bovine viral diarrhea virus in bovine lymphoid cell lines using PrimeFlow RNA assay

Bovine viral diarrhea virus (BVDV) infections, whether as acute, persistent or contributing to co-infections, result in significant losses for cattle producers. Although, BVDV can be identified readily by real-time PCR and ELISA, detection and quantification of viral infection at the single cell level is extremely difficult. Detection at the single lymphoid cell level is important due to the immunomodulation that accompanies BVDV infection. A novel PrimeFlow RNA assay using in-situ detection of BVDV was evaluated. The model used to develop this technique included three BL-3 cell lines with different infection statuses, one not infected with BVDV, one infected with BVDV and one dual infected with BVDV and bovine leukosis virus. Using RNA probes specific for the BVDV-2a N-Pro-E-rns coding region, BVDV RNA was detected from both contaminated BL-3 cell lines by flow cytometry and fluorescent microscopy. This is the first report on in-situ detection of BVDV at the single-cell level.