Quantification of avian influenza virus in tissues of mute swans using TaqMan real time qRT-PCR

This study reports on the first quantification of avian influenza virus in the organs of mute swans that died during the epizootic of avian influenza (H5N1) between January and April 2006 in the Czech Republic. The quantitative real-time Reverse Transcriptase PCR (qRT-PCR) assay based on a TaqMan probe was developed for a rapid detection and quantification of avian influenza virus RNA in clinical samples collected from mute swans. Conserved regions in the matrix protein gene of avian influenza virus served as targets for the primers and TaqMan probe design. A recombinant plasmid containing the matrix protein gene amplicon was constructed for a quantitative assay of copy numbers of the target gene. Quantification of avian influenza virus RNA was accomplished using a standard curve generated from ten-fold serial dilutions of recombinant plasmid DNA in the range of 102 to 108 copies/μl. Avian influenza virus A/Cygnus olor/Brno-cz/2006 was adapted to grow in VERO cells. In the same passage of cell cultivation, the concentration of viral RNA was determined to be 1.01 × 107 copies/ml and TCID50 was 104.2/ml. From these values the ratio of one RNA copy to 0.00157 virion capable of VERO cells infection was calculated. This ratio was used to estimate the virus concentrations in the tissues of dead mute swans.