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

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 (TCID50/mL) were generated with a detection limit of 100 TCID50/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. 1. Introduction The genus Pestivirus of the Flaviviridae family includes the three important animal viruses classical swine fever virus (CSFV), border disease virus (BDV), and bovine viral diarrhea virus (BVDV) [1]. The latter is one of the most widespread cattle pathogens worldwide and two genotypes, each with a noncytopathic and cytopathic biotype, are known [2, 3]. Infection of nonpregnant immunocompetent animals generally causes mild indisposition like ulceration of nose, mouth, or gastrointestinal mucosa resulting in continuous salivation, coughing, and diarrhea [4]. However, when pregnant animals become infected with BVDV, then abortion or malformation of the calf may occur. When infection takes place before the fourth month of gestation, persistently infected (PI) calves may be born. These animals are immunotolerant and develop normally in the absence of serious clinical symptoms of BVDV infection and may even be selected for breeding [5, 6]. Severe mucosal disease, leading to death within weeks after breakout, can develop in PI animals after mutation of the noncytopathic BVDV into the cytopathic form. For healthy animals, PI animals represent an enormous risk of infection as they