Faecal samples were collected from the Cork Regional Veterinary Laboratory (CRVL) after they had tested positive for coronavirus using an immunochromatographic commercial kit (ICK), Corona Vet (Serosep, Ireland). Faecal samples were also screened for rotavirus, cryptosporidium and Salmonella. A total of 11 coronavirus positive samples were collected from neonatal calves, mean age 9 days, presenting with diarrhea between 2010 and 2011. Samples were stored at −80 °C prior to analysis.
Prior to extraction, faecal samples were homogenized in an equal volume of 0.89 % NaCl, centrifuged and filtered using a 0.20 μm pore size. The RNA was then extracted from the cell free fluid using Qiagen Viral RNA mini kit (Qiagen), following the manufacturers’ instructions. Extracted RNA was stored at −20 °C prior to analysis. Extracted RNA was tested for the presence of Coronavirus using degenerate oligonucleotide primers described previously [9], targeting a 250 bp region of the polymerase gene. A nested PCR was used to amplify the spike (S) gene [10], specifically the hypervariable region (HVR) [11].
Following analysis of this region, the most variable isolate was selected for complete characterization of the S gene using primers previously described [12, 13]. Reactions were carried out using Enhanced Avian Reverse Transcriptase kit (Sigma-Aldrich), following the manufacturers’ instructions, all reactions were carried out using a Biometra T3000 thermocycler. Amplified products were run on 1.5 % agarose gels, stained with ethidium bromide and visualized using a UV light trans-illuminator. Bands containing positive samples were cleaned using Roche High Pure PCR clean kit (Roche) and sequenced using a commercial service (MWG Eurofins, Germany).
Resulting sequence data was then analysed and edited using Bioedit v7.0.9.0 [14] and online BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi), to identify homologous strains. Sequence alignment was carried out using Clustal W in Bioedit [14], and a sequence alignment profile generated (Fig. 1). For analysis of the whole S gene, contigs were assembled using DNAstar program Seqman. The phylogenetic trees for the S gene were constructed using Maximum likelihood (ML) in MEGA5.1 [15] with a GTR model, plus gamma distribution and invariant sites with 1000 bootstrap replicates (Figs. 2 and 3). In ML trees shown, all strains are displayed with accession numbers.
Partial nucleotide sequences are available for Polymerase gene (JN204179, JN204180), Spike (KF272908, KF272910, KF272912, KF272914). The complete gene sequence of the spike protein of Irish BCoV strain RVLC7 was registered in Genbank under the accession number-KF272919.