RESUMEN

Bovine coronavirus (BCoV) has dual tropisms that can trigger enteric and respiratory diseases in cattle. Despite its global distribution, BCoV field strains from Brazil remain underexplored in studies investigating the virus's worldwide circulation. Another research gap involves the comparative analysis of S protein sequences in BCoV isolates from passages in cell lines versus direct sequencing from clinical samples. Therefore, one of the objectives of our study was to conduct a comprehensive phylogenetic analysis of BCoV strains identified from Brazil, including a respiratory strain obtained during this study, comparing them with global and ancestral BCoV strains. Additionally, we performed a comparative analysis between wild-type BCoV directly sequenced from the clinical sample (nasal secretion) and the cell culture-adapted strain, utilizing the Sanger method. The field strain and multiple cell passage in cell culture (HRT-18) adapted BCoV strain (BOV19 NS) detected in this study were characterized through molecular and phylogenetic analyses based on partial fragments of 1,448 nt covering the hypervariable region of the S gene. The analyses have demonstrated that different BCoV strains circulating in Brazil, and possibly Brazilian variants, constitute a new genotype (putative G15 genotype). Compared with the ancestral prototype (Mebus strain) of BCoV, 33 nt substitutions were identified of which 15 resulted in non-synonymous mutations (nine transitions and six transversions). Now, compared with the wild-type strain was identified only one nt substitution in nt 2,428 from the seventh passage onwards, which resulted in transversion, neutral-neutral charge, and one substitution of asparagine for tyrosine at aa residue 810 (N810Y).

Asunto(s)

Enfermedades de los Bovinos , Coronavirus Bovino , Filogenia , Bovinos , Brasil , Coronavirus Bovino/genética , Coronavirus Bovino/aislamiento & purificación , Coronavirus Bovino/clasificación , Animales , Enfermedades de los Bovinos/virología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Genotipo , Glicoproteína de la Espiga del Coronavirus/genética , Infecciones del Sistema Respiratorio/virología , Línea Celular

RESUMEN

Bovine coronavirus (BCoV) is an important viral pathogen associated with neonatal calf diarrhea. Our aim was to investigate the incidence of BCoV in diarrhea outbreaks in beef and dairy herds from Argentina during 1994-2010. A total of 5.365 fecal samples from diarrheic calves were screened for BCoV diagnosis by ELISA. The virus was detected in 1.71% (92/5365) of the samples corresponding to 5.95% (63/1058) of the diarrhea cases in 239 beef and 324 dairy farms. The detection rate of BCoV was significantly higher in dairy than in beef herds: 12.13% (29/239) vs. 4.32% (14/324) respectively. Phylogenetic analysis of the hypervariable S1 region of seven representative samples (from different husbandry systems, farm locations and years of sampling) indicated that BCoV strains circulating in Argentinean beef and dairy herds formed a cluster distinct from other geographical regions. Interestingly, Argentinean strains are distantly related (at both the nucleotide and amino acid levels) with the Mebus historic reference BCoV strain included in the vaccines currently available in Argentina. However, Mebus-induced antibodies were capable of neutralizing the BCoV Arg95, a field strain adapted to grow in vitro, and vice versa, indicating that both strains belong to the same CoV serotype reported in cattle. This work represents the first large survey describing BCoV circulation in Argentinean cattle.

Asunto(s)

Enfermedades de los Bovinos/virología , Infecciones por Coronavirus/veterinaria , Coronavirus Bovino/genética , Coronavirus Bovino/inmunología , ADN Viral/análisis , Filogenia , Animales , Antígenos Virales/análisis , Argentina/epidemiología , Secuencia de Bases , Bovinos , Enfermedades de los Bovinos/epidemiología , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Coronavirus Bovino/clasificación , Industria Lechera , Brotes de Enfermedades/veterinaria , Heces/virología , Femenino , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN

RESUMEN

Gastroenteritis is one of the leading causes of morbidity and mortality among young and newborn animals and is often caused by multiple intestinal infections, with rotavirus and bovine coronavirus (BCoV) being the main viral causes in cattle. Given that BCoV is better studied than equine coronaviruses and given the possibility of interspecies transmission of these viruses, this research was designed to compare the partial sequences of the spike glycoprotein (S), hemagglutinin-esterase protein (HE), and nucleoprotein (N) genes from coronaviruses from adult cattle with winter dysentery, calves with neonatal diarrhea, and horses. To achieve this, eleven fecal samples from dairy cows with winter dysentery, three from calves, and two from horses, all from Brazil, were analysed. It could be concluded that the enteric BCoV genealogy from newborn and adult cattle is directly associated with geographic distribution patterns, when S and HE genes are taken into account. A less-resolved genealogy exists for the HE and N genes in cattle, with a trend for an age-related segregation pattern. The coronavirus strains from horses revealed Betacoronavirus sequences indistinguishable from those found in cattle, a fact previously unknown.

Asunto(s)

Coronavirus Bovino/clasificación , Coronavirus Bovino/genética , Genes Virales , Filogenia , Animales , Brasil , Bovinos , Geografía , Caballos

RESUMEN

Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract infections in calves and feedlot cattle. In Cuba, the presence of BCoV was first reported in 2006. Since then, sporadic outbreaks have continued to occur. This study was aimed at deepening the knowledge of the evolution, molecular markers of virulence and epidemiology of BCoV in Cuba. A total of 30 samples collected between 2009 and 2011 were used for PCR amplification and direct sequencing of partial or full S gene. Sequence comparison and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100% bootstrap and 1.00 posterior probability values. The Cuban bovine coronavirus sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique.

Asunto(s)

Enfermedades de los Bovinos/virología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Coronavirus Bovino/clasificación , Coronavirus Bovino/genética , Glicoproteínas de Membrana/genética , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Bovinos , Análisis por Conglomerados , Coronavirus Bovino/aislamiento & purificación , Disentería/veterinaria , Disentería/virología , Evolución Molecular , Heces/virología , Datos de Secuencia Molecular , Filogenia , Recombinación Genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Glicoproteína de la Espiga del Coronavirus

RESUMEN

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.

Asunto(s)

Infecciones por Coronavirus/veterinaria , Coronavirus Bovino/genética , Diarrea/veterinaria , Heces/virología , Animales , Secuencia de Bases , Bovinos , Infecciones por Coronavirus/virología , Coronavirus Bovino/clasificación , Coronavirus Bovino/aislamiento & purificación , ADN Viral/análisis , Diarrea/virología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN

RESUMEN

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 percent identity for nucleotides and 96.8 percent identity for amino acids) and more similar to the BCoV-ENT strain (98.7 percent for nucleotides and 98.7 percent 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.

Asunto(s)

Animales , Bovinos , Infecciones por Coronavirus/veterinaria , Coronavirus Bovino/genética , Diarrea/veterinaria , Heces/virología , Secuencia de Bases , Infecciones por Coronavirus/virología , Coronavirus Bovino/clasificación , Coronavirus Bovino/aislamiento & purificación , ADN Viral/análisis , Diarrea/virología , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN

RESUMEN

Bovine coronavirus (BCoV) causes enteric and respiratory dis- orders in calves and dysentery in cows. In this study, 51 stool samples of calves from 10 Brazilian dairy farms were analysed by an RT-PCR that amplifies a 488-bp fragment of the hypervariable region of the spike glycoprotein gene. Maximum parsimony genealogy with a heuristic algorithm using sequences from 15 field strains studied here and 10 sequences from GenBank and bredavirus as an outgroup virus showed the existence of two major clusters (1 and 2) in this viral species, the Brazilian strains segregating in both of them. The mean nucleotide identity between the 15 Brazilian strains was 98.34%, with a mean amino acid similarity of 98%. Strains from cluster 2 showed a deletion of 6 amino acids inside domain II of the spike protein that was also found in human coronavirus strain OC43, supporting the recent proposal of a zoonotic spill- over of BCoV. These results contribute to the molecular characterization of BCoV, to the prediction of the efficiency of immunogens, and to the definition of molecular markers useful for epidemiologic surveys on coronavirus-caused diseases.

Asunto(s)

Coronavirus Humano OC43/genética , Coronavirus Bovino/genética , Genoma Viral , Glicoproteínas de Membrana/genética , Eliminación de Secuencia , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Animales , Brasil , Bovinos , Enfermedades de los Bovinos/virología , Análisis por Conglomerados , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Coronavirus Bovino/clasificación , Coronavirus Bovino/aislamiento & purificación , Heces/virología , Humanos , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/química