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

Benjamin Zeitler,Ingrid Rapp

doi:10.5402/2013/751904

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 (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.

Highlights

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 possibility to replace cell culture-based Bovine viral diarrhea virus (BVDV) detection in bovine lung lavages with quantitative real-time PCR (qRT-PCR) was evaluated during this study
As certainly infective BVDV particles must be present in the analyzed samples, the detection limit of the qRT-PCR was determined

Summary

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]. PI animals represent an enormous risk of infection as they release large amounts of virus during their lifetime In this way, BVDV guarantees preservation in the host population [5]. In countries and states where control-and-eradication campaigns do not rely on vaccination, sensitive and simple test methods must be available to detect the virus in sample material of diverse origin [9]

Methods

Results

Conclusion