A semi-nested RT-PCR assay targeted to hemagglutinin-esterase gene of Bovine Coronavirus
Keywords:Bovine coronavirus, RT-PCR, Hemagglutinin-esterase, Genetic diversity
AbstractBovine coronavirus (BCoV) is a non-segmented positive-sense single-stranded RNA virus whose envelope is constituted by a lipid bilayer with four structural proteins (HE, S, E and M) giving its characteristic crown-like virions appearance. Hemagglutinin-esterase (HE), is a polymorphic protein with a function of secondary receptor binder, and studies on the diversity of HE gene allow insights on BCoV evolution and host-parasite interactions. A semi-nested RT-PCR was developed for the amplification of a 441bp-long product of the HE gene of BCoV (nt 543 to 562). Optimal annealing temperatures were tested in a gradient thermocycler for the semi-nested assay and employed in the final protocol. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titer: 256) in a BCoV-free fecal suspension, with positive results up to 10-6 dilution, a high analytical sensitivity without PCR inhibition. The final semi-nested RT-PCR protocol was applied to 21 fecal samples of cows previously positive to BCoV and DNA sequencing of the 441bp amplicons of 14 of these resulted in highly-scored BCoV HE gene sequences after BLAST/n analysis. This semi-nested RT-PCR is a powerful tool for surveys of phylogenetic diversity in field strains of BCoV and for comparative studies among different genes of Coronavirus.
Download data is not yet available.
How to Cite
Souza, S. P. de, Asano, K. M., Buitrago, L. Y. V., Silva, S. de O. S., Richtzenhain, L. J., & Brandão, P. E. (2010). A semi-nested RT-PCR assay targeted to hemagglutinin-esterase gene of Bovine Coronavirus. Brazilian Journal of Veterinary Research and Animal Science, 47(4), 323-328. https://doi.org/10.11606/issn.1678-4456.bjvras.2010.26832
The journal content is authorized under the Creative Commons BY-NC-SA license (summary of the license: https://