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

Authors

  • Sibele Pinheiro de Souza Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, São Paulo, SP
  • Karen Miyuki Asano Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, São Paulo, SP
  • Laura Yaneth Villarreal Buitrago Schering Plough Animal Health
  • Sheila de Oliveira Souza Silva Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, São Paulo, SP
  • Leonardo José Richtzenhain Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, São Paulo, SP
  • Paulo Eduardo Brandão Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnia, São Paulo, SP

DOI:

https://doi.org/10.11606/issn.1678-4456.bjvras.2010.26832

Keywords:

Bovine coronavirus, RT-PCR, Hemagglutinin-esterase, Genetic diversity

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.

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Published

2010-08-01

Issue

Section

UNDEFINIED

How to Cite

1.
Souza SP de, Asano KM, Buitrago LYV, Silva S de OS, Richtzenhain LJ, Brandão PE. A semi-nested RT-PCR assay targeted to hemagglutinin-esterase gene of Bovine Coronavirus. Braz. J. Vet. Res. Anim. Sci. [Internet]. 2010 Aug. 1 [cited 2024 Oct. 9];47(4):323-8. Available from: https://www.revistas.usp.br/bjvras/article/view/26832