Brazilian Journal of Microbiology (2007) 38:699-703 ISSN 1517-8382 699 IDENTIFICATION OF A MUTATION IN THE SPIKE PROTEIN CLEAVAGE SITE IN BRAZILIAN STRAINS OF WILD-TYPE BOVINE CORONAVIRUS Elisabete Takiuchi1; Marco Antnio Bacellar Barreiros2; Alice Fernandes Alfieri1; Amauri Alcindo Alfieri1* 1Laboratrio de Virologia Animal, Departmento de Medicina Veterinria Preventiva, Universidade Estadual de Londrina, Londrina, PR, Brasil; 2Laboratrio de Microbiologia, Universidade do Vale do Itaja, Itaja, SC, Brasil Submitted: May 30, 2007; Returned to authors for corrections: September 17, 2007; Approved: September 28, 2007. ABSTRACT The spike (S) protein of coronaviruses, a type I membrane glycoprotein, is primarily responsible for entry into susceptible cells by binding with specific receptors on cells and mediating subsequent virus-cell fusion. The bovine coronavirus (BCoV) S protein is cleaved into two subunits, the N-terminal S1 and the C-terminal S2. The proteolytic cleavage site of S protein is highly conserved among BCoV strains and is located between amino acids 763 and 768 (KRRSRR). This study describes a single mutation in the S protein cleavage site of three Brazilian strains of BCoV detected in diarrheic fecal samples from calves naturally infected. The sequenced PCR products revealed that amino acid sequence of the cleavage site of our strains was KRRSSR, indicating a mutation at amino acid position 767 (R S). This amino acid substitution occurred due to a single nucleotide substitution in the sequence of DNA corresponding to the proteolytic cleavage site, CGT to AGT. This is the first description of this nucleotide mutation (C to A), which resulted in the substitution of arginine to serine in the S cleavage site. In this study we speculated the probable effects of this mutation in the proteolytic cleavage site using the murine hepatitis coronavirus (MHV) as a comparative model. Key words: BCoV, sequencing, spike protein, cleavage site, mutation *Corresponding Author. Mailing address: Departamento de Medicina Veterinria Preventiva, Centro de Cincias Agrrias, Universidade Estadual de Londrina, Londrina, PR, Brasil. Caixa Postal 6001, CEP 86051-990. Tel.: +55 43 3371 4485; Fax: +55 43 3371-4714. E-mail: alfieriuel.br Bovine coronavirus (BCoV), a member of the family Coronaviridae, order Nidovirales, belongs to group 2 of the coronaviruses which include murine hepatitis coronaviruses (MHV), porcine hemagglutinating encephalomyelitis virus (HEV), equine coronavirus (ECoV), rat coronavirus (RtCoV) and human respiratory coronavirus (HCoV-OC43) (9). BCoV is an enveloped virus with single-stranded, positive-sense RNA genome of approximately 32 kb length that encodes five major structural proteins: the nucleocapsid (N), the transmembrane (M), the hemaglutinin esterase (HE), the spike (S), and the small protein (E) (13). The S glycoprotein of BCoV is a large membrane glycoprotein of approximately 150 kDa that forms the peplomers (club-shaped structures) on the virion surface. The S protein is primarily responsible for the entry of coronavirus into susceptible cells by binding to specific receptors on cells and mediating virus-cell fusion and subsequent cell-cell fusion during infection (5). In several coronaviruses, such as infectious bronchitis virus (IBV), MHV and BCoV, as a late event in maturation, the protein is cleaved into two subunits: S1 (aminoterminal region) and S2 (carboxyterminal region) (17). Proteolytic cleavage of the S protein of these coronaviruses occurs adjacent to a sequence of basic amino acids on the carboxyterminal region of S1. In the S protein of BCoV, a predicted basic amino acid sequence (KRRSRR) is involved in the cleavage by the host cell-derived proteolytic enzyme. This sequence, highly conserved among BCoV strains, encompasses amino acids 763 to 768; the cleavage occurs between amino acids 768 and 769 (19). The cleavage of the S protein has been reported as a process related to the viral infectivity and cell fusion from other group 2 coronaviruses. Studies related to MHV, the best-studied member of the Coronavirus family, demonstrated that cleavage of S is not essential for infectivity but is associated with enhanced cell fusion (syncytia) in infected cell monolayers (7,8,20). 700 Takiuchi, E. et al. This study describes a single mutation in the S protein cleavage site of wild type Brazilian strains of BCoV detected in calves naturally infected and speculates the possible biological effects of this mutation at the proteolytic cleavage site using the MHV as a comparative model. Three BCoV positive fecal samples (BR-UEL1, BR-UEL2 and BR-UEL3) were obtained from calves up to 30 days old with clinical signs of diarrhea in a Brazilian dairy herd from Minas Gerais State (21 41 49 S; 45 18 45 W). These samples were previously identified as BCoV positive by RT-PCR assay for N gene detection (21) and negative for bovine group A rotavirus and Cryptosporidium sp by polyacrylamide gel electrop