Viral quantification of bovine viral diarrhea virus (BVDV) with Real Time PCR

BVDV is responsible of persistent infection (PI) in cattle. PI animals can develop a severe pathology, the Mucous Disease (MD). The aim of this work was to develop a Real Time PCR (qRT-PCR) in order to quantify the virus amount in different organs and apparatus from PI and MD affected animals. In order to set up a qRT-PCR a recombinant plasmid was constructed cloning a highly conserved fragment of 5’UTR region and serial dilutions were used to obtain a standard curve. Viral RNA was extracted from organs of 3 animals and it was retrotranscripted and quantified by SYBR green qRT-PCR. The Real Time assay was able to detect 10-20 copies of DNA/l. Inter-assay tests showed a coefficient of variability ranging from 1,2% to 3,8%. With the quantification of BVDV we observed a higher amount of virus in the MD affected animals respect to PI animal. This difference resulted statistically significant in some organs of respiratory and digestive apparatus, but not considering the whole amount of virus in the animals. Quantitative RT-PCR permitted to quantify the BVDV in different organs from PI and MD affected animals and resulted to be useful to study the distribution and the amount of the virus. The “not statistically significant” lower level of the total virus in PI animal could be correlated with the MD post mortem lesions evidenced in this animal that didn’t showed clinical symptoms. The method revealed to be sensitive and rapid and it is suitable for both diagnostic and pathogenesis studies.