Determination of Infectious Bovine Viral Diarrhea Virus in Bovine Lung Lavages by a Combination of Virus Propagation in Cell Culture and Quantitative Real-Time PCR

Abstract

Material of bovine origin is often used in biotechnological applications. Bovine viral diarrhea virus (BVDV) is one of the major viral contaminants, and not only detection and inactivation but also quantification of the viral load in bovine starting material is required by the regulatory agencies. Here, we investigated combined virus propagation in cell culture and quantitative real-time PCR (qRT-PCR) for the applicability to detect and estimate low BVDV titers in bovine lung lavages, the source material for manufacturing pulmonary surfactant. qRT-PCR analyses of the crude lung lavages were performed and qRT-PCR calibration curves based on infective viral doses (TCID 50 /mL) were generated with a detection limit of 100 TCID 50 /mL. Lung lavages were inoculated on susceptible MDBK cells and cell culture samples were again analyzed by qRT-PCR. Immunofluorescence staining was performed to prove qRT-PCR results. Interestingly, initial BVDV contaminations in lung lavages were below qRT-PCR detection limit. An amplification step in cell culture enabled BVDV propagation to levels detectable by qRT-PCR. In comparison with the qRT-PCR calibration curve and control experiments with defined inoculation doses, the estimation of minor BVDV contaminations in lung lavages was possible. Both techniques can be successfully combined to estimate the viral load in dilute sample material.