Molecular analysis of the bovine coronavirus S1 gene by direct sequencing of diarrheic fecal specimens

Abstract

Bovine coronavirus (BCoV) causes severe diarrhea in newborn calves, is associated with winter dysentery in adult cattle and respiratory infections in calves and feedlot cattle. The BCoV S protein plays a fundamental role in viral attachment and entry into the host cell, and is cleaved into two subunits termed S1 (amino terminal) and S2 (carboxy terminal). The present study describes a strategy for the sequencing of the BCoV S1 gene directly from fecal diarrheic specimens that were previously identified as BCoV positive by RT-PCR assay for N gene detection. A consensus sequence of 2681 nucleotides was obtained through direct sequencing of seven overlapping PCR fragments of the S gene. The samples did not undergo cell culture passage prior to PCR amplification and sequencing. The structural analysis was based on the genomic differences between Brazilian strains and other known BCoV from different geographical regions. The phylogenetic analysis of the entire S1 gene showed that the BCoV Brazilian strains were more distant from the Mebus strain (97.8% identity for nucleotides and 96.8% identity for amino acids) and more similar to the BCoV-ENT strain (98.7% for nucleotides and 98.7% for amino acids). Based on the phylogenetic analysis of the hypervariable region of the S1 subunit, these strains clustered with the American (BCoV-ENT, 182NS) and Canadian (BCQ20, BCQ2070, BCQ9, BCQ571, BCQ1523) calf diarrhea and the Canadian winter dysentery (BCQ7373, BCQ2590) strains, but clustered on a separate branch of the Korean and respiratory BCoV strains. The BCoV strains of the present study were not clustered in the same branch of previously published Brazilian strains (AY606193, AY606194). These data agree with the genealogical construction and suggest that at least two different BCoV strains are circulating in Brazil.

Brazilian strain of bovine respiratory coronavirus is derived from dual enteric and respiratory tropism

“…Additionally, two subclades, 2A and 2B, were formed by viruses that originated from Brazil. Subclade 2B consisted of the respiratory BR-UEL11 strain, winter dysentery strains of enteric origin, and the BR-UEL 1, 2, and 3 strains obtained from diarrheic calves (Takiuchi et al, 2008). The subclade 2A consisted of several winter dysentery strains of enteric and respiratory origin and USP-1 from calf stool; however, no nucleotide deletion gaps, as observed in the USP strains of clade 1, were identified.…”

“…Nucleic acid purification was performed as described (Boom et al, 1990), and the presence of BCoV was confirmed using a semi-nested polymerase chain reaction (PCR) targeting the N gene (Takiuchi et al, 2006). For genetic analysis, a reverse transcription (RT)-PCR assay using the SPK5 pair of primers (Takiuchi et al, 2008) was used to amplify the sequence encoding the nucleotides 1256 to 1904 based on the S gene of the Mebus strain (accession number -U00735.2), which includes the polymorphic region (nucleotides 1368 -1776) (Rekik and Dea, 1994). All amplicons were visualized on an ethidium bromide-stained 2% agarose gel.…”

“…Since the first description of a Brazilian BCoV associated with calf diarrhea, most Brazilian strains of enteric origin have now been described and characterized by molecular methods (Jerez et al, 2005;Takiuchi et al, 2008;Stipp et al, 2009;Ribeiro et al, 2016); in addition, assessment of major public databases did not reveal any description of BRCoV in other South American countries. This study investigates the recent molecular evolution of BCoV by addressing the phylogenetic relationship between the enteric and winter dysentery strains based on a polymorphic region of the Brazilian BRCoV.…”

Molecular and antigenic characterization of bovine Coronavirus circulating in Argentinean cattle during 1994–2010

“…In Cuba, BCoV sequences clustered with BCoV strains from USA, suggesting a common origin for these viruses (Martinez et al, 2012). In South America, most of the information comes from studies conducted in Brazil (Asano et al, 2010;Barros et al, 2013;Brandao et al, 2008Brandao et al, , 2006Takiuchi et al, 2008). Stipp et al (2009) reported a 15.6% detection rate of BCoV in diarrheic calves from dairy and beef farms during a survey conducted in four states of Brazil.…”

Isolation in HRT-18 cells and molecular analysis of a BCoV strain from diarrheic feces of naturally infected calves

“…Despite the small size of the N gene fragment analyzed, the identity among Brazilian BCoV strains and the other BCoV strains used in this study were lower (97.6 to 98.5%) than the identity among other published sequences (98.1 to 100%) (data not shown). Since the region of the N gene amplified by the semi-nested PCR was highly conserved among coronaviruses, it is possible that other genes also present low identity; this has previously been observed in the S gene from other Brazilian BCoV strains (Takiuchi et al, 2007;2008). Thus, additional studies are necessary for molecular characterization of Brazilian BCoV strains.…”