key: cord-0850316-yzgovang
authors: Pinheiro de Souza, Sibele; Miyuki Asano, Karen; Lucas Sandri, Thaisa; Nunes de Barros, Iracema; José Richtzenhain, Leonardo; Eduardo Brandão, Paulo
title: Differentiation of Bovine Coronavirus (Bcov) Genotypes by a Restriction Enzyme Assay
date: 2010-09-01
journal: Braz J Microbiol
DOI: 10.1590/s1517-83822010000300034
sha: 23ffba1251c42cd4311635ab25afeba065146568
doc_id: 850316
cord_uid: yzgovang

This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.

Coronaviridae) (5, 7) , with a genome formed by a singlestranded non-segmented positive-sense RNA with 32 kb, arranged in a nucleocapsid of helical symmetry in association with the N nucleoprotein, a conserved phosphoprotein with 50-60kDa rich in basic amino acids (7, 9) . The viral envelope of BCoV is formed by a lipid bi-layer with four structural proteins (HE, S, E and M), resulting in a spiked structure (9).

The major envelope protein of BCoV is the spike (S) protein, organized as trimers that appear as 20-nm-long projections in the viral envelope with domains responsible for receptor binding, hemagglutination and induction of neutralizing antibodies. Spike protein is the most polymorphic among all coronavirus proteins and is divided in the subunits S1 and S2 (2, 3). Brandão et al. (1) reported the existence of two different genotypes of BCoV in Brazil and one of these has a deletion of 18 nucleotides in the hypervariable region of the S1 subunit of S gene.

This study aimed to evaluate the use of a restriction enzyme assay as a specific, sensitive and practical tool to differentiate genotypes of BCoV for the molecular epidemiology of BCoV-caused diseases.

A restriction enzyme selection was carried out based on the 488bp amplicon respective to the S1-coding region of BCoV described by Brandão et al. (1) . The nucleotide region of the GenBank sequences AF058942 and AY606200 referent to the above-mentioned amplicon was used, being the second sequence representative of the deleted genotype.

Using the Bioedit 7.0.5.3 software (6), the GsuI (BpmI) restriction enzyme was selected which is able to cleave the 

Molecular diversity of Brazilian strains of bovine coronavirus (BCoV) reveals a deletion within the hypervariable region of the S1 subunit of the spike glycoprotein also found in human coronavirus OC43

The coronavirus surface glycoprotein

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Comparison of bovine coronavirus isolates associated with neonatal calf diarrhea and winter dysentery in adult dairy cattle in Québec

A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae

BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT

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The authors are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for the financial support (Grant # 2007/59108-4).