Reação de hemi-nested RT-PCR dirigida ao gene da hemaglutinina-esterase do Coronavírus Bovino

Abstract

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.

Coronavírus bovino: Revisão

“…Os primeiros relatos sobre o vírus, o apontam como responsável pela diarreia neonatal em bezerros nos Estados Unidos da América (EUA), no ano de 1972. Nesse mesmo período iniciou-se o estudo de suas caracteristicas morfológicas, que permitiram a sua inclusão no grupo dos coronavírus (Betancourt et al, 2006;Sonza, 2006;Takiuchi et al, 2007;Souza, de et al, 2010;Júnior, 2015).…”