A semi-nested RT-PCR assay targeted to hemagglutinin-esterase gene of Bovine Coronavirus

• Autor(a) principal: Souza, Sibele Pinheiro de
• Data de Publicação: 2010
• Outros Autores: Asano, Karen Miyuki, Buitrago, Laura Yaneth Villarreal, Silva, Sheila de Oliveira Souza, Richtzenhain, Leonardo José, Brandão, Paulo Eduardo
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Brazilian Journal of Veterinary Research and Animal Science
• Texto Completo: https://www.revistas.usp.br/bjvras/article/view/26832
• topic: Coronavírus bovino; RT-PCR; Hemaglutinina-esterase; Diversidade genética; Bovine coronavirus; RT-PCR; Hemagglutinin-esterase; Genetic diversity

Bovine 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.