Molecular and phylogenetic analysis of bovine coronavirus based on the spike glycoprotein gene

Abstract

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.

Fig. 1

Map of Cuba showing the geographic distribution of the sample collection sites. The associated table indicates the quantity of samples analysed from each area and the positive RT-PCR results obtained from each of them (^∗complete S gene sequenced samples).

Fig. 2

Phylogenetic trees for BCoV sequences. Each phylogenetic marker, the best-fitted model and the gamma distribution shape parameter values used to infer phylogenies are indicated. The Cuban BCoV sequences were highlighted in blue, whereas the remaining sequences appear in red. Numbers along the branches refer to the percentages of confidence and posterior probability in the ML and BI analyses, respectively. Minor branch values were hidden. Sequences that had no close relationship with the Cuban sequences were collapsed (indicated in parentheses). (a) A: (EF445634, EU814647), B: (U06093, U06091, U06090), C: (AY606201, AY606197, AY606204, AY606195, AY606192, AY606203, AY606196, AY606199, AY606205, AY606202, M64668, AF181469, D00662, U00735, EF193075, EU401986, D00731, M80844, AF391541, AF391542, AF239307, FJ938064, DQ915164, DQ811784, FJ938063, AF239306) and D: (DQ320763, FJ425186, AY255831, AB277129, AB277121, AF058943, FJ425188, AY935639, AY935646, AY935644, AY935642, EF424619, DQ389655, DQ389657, AB277130, DQ389658, DQ389660, AY935643, DQ389653, DQ389652, DQ389656, DQ389659, DQ389634, DQ389635, DQ389640, DQ389641, DQ389639, DQ389636, DQ389654, HM573330, DQ389637); (b) A: (AF058942, D00731), B: (AB354579, M64668, EF193075, EU401989, EU401987, EU401988, U00735, D00662, AF181469, M64667), C: (FJ938063, DQ811784, AF239306, AF239307, U06093), D: (EU019216, EF445634, EU814647, EU814648), E: (DQ389637, DQ389636, DQ389640, DQ389638, DQ389656, FJ938066, AY935637, AY935638, AY935642, AY935641, AY935639, AY935644, AY935646, FJ425187, EF424619, AY935643, DQ389659, DQ389652, DQ389653, DQ389655, DQ389658, DQ389657, DQ389660, DQ389633, HM573326, HM573330, DQ389654, EU686689, EU401986, DQ389634, DQ389632, DQ389635, DQ389641, DQ389639); (c) A: (D00731, AF058942), B: (M64668, EU401989, EU401988, EU401987, AB354579, D00662, U00735, EF193075, M64667, AF181469), C: (EU814648, EU814647, EU019216, EF445634, M80844), D: (DQ811784, FJ938063), E: (AF058943, FJ425186, FJ425184, FJ425188, FJ425189), F: (HM573330, HM573326, DQ389660, DQ389657, DQ389658, DQ389655, DQ389654, AY935643, DQ389659, DQ389653, DQ389652, DQ389656, DQ389641, DQ389640, DQ389638, DQ389637, DQ389636, DQ389639, DQ389635, DQ389634, DQ389632, DQ389633, EU401986, EU686689).

Fig. 3

Molecular analysis of the deduced amino acid sequences for the entire S genes of the Cuban field and reference strains. Regions important for virus-cell interactions were highlighted: the signal peptide region (red), the hypervariable region (orange), the proteolytic cleavage region (pink), the first hydrophobic region of the S2 subunit (grey), the HR–N domain (greenish-blue) and the HR–C domain (dark green). The immuno-reactive domains S1A and S1B were outlined in red and blue, respectively; the transmembrane region was outlined in black. The particular signatures linked either with a respiratory (blue arrow) or enteric (red arrow) tropism and virulence factors (black arrow) were also indicated. The potential N-glycosylation sites were marked with black triangles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)