ABSTRACT
We report 24 bovine coronavirus (BCoV) genome sequences from Ireland. BCoV was sequenced directly from nasal swabs that had been collected during a bovine respiratory disease (BRD) outbreak among recently purchased beef suckler and pre-weaned dairy calves.
ANNOUNCEMENT
Bovine coronavirus (BCoV) is responsible for neonatal calf diarrhea, winter dysentery in adult cattle, and respiratory disease complex in cattle farms worldwide (1–3). BCoV (genus Betacoronavirus and family Coronaviridae) has a single-stranded positive-sense RNA genome of approximately 31 kb (4). We assembled BCoV genomes from sequences obtained directly from nasal swabs taken from 22 beef suckler weanling calves and 2 pre-weaned dairy calves in November 2019 (5) and March 2020 (6), respectively, after purchase from different marts and transport to the same research farm in the east of Ireland.
The study was approved by the Teagasc Animal Ethics Committee (TAEC-221-2019). Prior to nucleic acid extraction, 1× phosphate-buffered saline (PBS) was added to each nasal swab, which was then vortexed, and 300 μL of the eluate was removed and subjected to bead beating and then nuclease (RNase and DNase) treatment. A negative (blank swab with PBS) and a positive control (mixture of bovine respiratory syncytial virus [BRSV], bovine herpesvirus 1 [BoHV-1], and bovine parainfluenza virus 3 [BPI-3] bovine fetal lung cell culture isolates) was included alongside each batch of 10 sample extractions. Nucleic acids were extracted using the QIAamp MinElute virus spin kit (Qiagen, Manchester, UK) with carrier RNA substituted with 5 mg/mL linear acrylamide (Thermo Fisher). Double-stranded cDNA was synthesized using the Maxima H Minus ds-cDNA kit (Thermo Fisher) with random hexamers and purified using the GeneJet PCR purification kit (Thermo Fisher). Illumina libraries were generated using the VAHTS Universal Plus DNA library prep kit, and 150-bp paired-end sequencing was conducted on an Illumina NovaSeq.
Sequences were assembled with SPAdes (version 3.15) (7) and annotated using Prokka (version 1.14.6) (8). Adaptor sequences were removed, and reads were trimmed to remove low-quality bases using fastp (version 0.19.6). A total of 443,396,148 read pairs were generated. An average nucleotide identity of 99.78% was estimated using MEGA 11 between all 24 genome assemblies, which ranged in length from 30,879 to 31,216 bp. The BLAST nr/nt analysis (9) indicated all 24 Irish BCoV genomes were most similar to BCoV whole-genome assemblies from France (Table 1). All 24 Irish BCoV sequences shared 99% sequence coverage with their respective top BLAST nr/nt result, with the exception of two sequences, B0-128 and BB-78, which shared 100% sequence coverage.
TABLE 1.
BCoV genome information relating to animal ID, GC content, genome size, GenBank accession nos., BLASTn results, and nucleotide identity (%)
| Animal ID | GC% | Genome size (no. of nucleotides) | GenBank accession no. | SRA accession no. | Most similar BLASTn result (GenBank accession no.) | % identity of most similar BLASTn search |
|---|---|---|---|---|---|---|
| SeqDB-260 | 37 | 31,034 | ON792964 | SRR21722844 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.03 |
| SeqDB-205 | 37 | 30,957 | ON792963 | SRR21722940 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.03 |
| SeqBB-78 | 37 | 30,924 | ON792958 | SRR21530068 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.13 |
| SeqBB-129 | 37 | 31,005 | ON792962 | SRR21529975 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.14 |
| SeqBB-115 | 37 | 31,122 | ON792961 | SRR21529924 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.13 |
| SeqBB-110 | 37 | 31,122 | ON792960 | SRR21529957 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.14 |
| SeqBB-108 | 37 | 31,035 | ON792959 | SRR21530064 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.16 |
| SeqB0-88 | 36.9 | 31,061 | ON792953 | SRR21530062 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.12 |
| SeqB0-81 | 37 | 31,124 | ON792952 | SRR21530098 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.14 |
| SeqB0-79 | 37 | 31,060 | ON792951 | SRR21530099 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.11 |
| SeqB0-78 | 37 | 31,017 | ON792950 | SRR21530100 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.14 |
| SeqB0-74 | 37 | 31,078 | ON792949 | SRR21530102 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.13 |
| SeqB0-72 | 37 | 31,110 | ON792948 | SRR21530103 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.15 |
| SeqB0-70 | 37 | 31,061 | ON792947 | SRR21530105 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.09 |
| SeqB0-66 | 37 | 31,079 | ON792946 | SRR21530106 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.20 |
| SeqB0-62 | 37 | 31,216 | ON792945 | SRR21530109 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.13 |
| SeqB0-59 | 37 | 30,968 | ON792944 | SRR21530111 | Bovine coronavirus isolate ICSA16-EN (MG757139) | 98.99 |
| SeqB0-56 | 37 | 31,032 | ON792943 | SRR21530113 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.12 |
| SeqB0-54 | 37 | 31,042 | ON792942 | SRR21530115 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.17 |
| SeqB0-52 | 37 | 31,143 | ON792941 | SRR21530117 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.12 |
| SeqB0-128 | 37 | 30,879 | ON792957 | SRR21529922 | Bovine coronavirus isolate ICSA-pool-EN (MG757141) | 99.12 |
| SeqB0-122 | 37 | 31,051 | ON792956 | SRR21529926 | Bovine coronavirus isolate ICSA16-EN (MG757139) | 99.02 |
| SeqB0-116 | 37 | 31,102 | ON792955 | SRR21529928 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.10 |
| SeqB0-110 | 37 | 31,052 | ON792954 | SRR21529932 | Bovine coronavirus isolate ICSA16-LBA (MG757140) or bovine coronavirus isolate ICSA-pool-EN (MG757141) | Both 99.14 |
The 1a, rep, 2a, HE, S, E, M, and N genes were identified from the Prokka analysis (8). The amino acid (aa) sequence of the hemagglutinin-esterase (HE) gene (GenBank accession number AAA66393) from the BCoV reference strain Mebus was aligned by MUSCLE in MEGA 11 (10, 11) to the 24 BCoV HE aa sequences. Relative to the reference strain, all 10 amino acid changes in the HE gene (L5P, N49T, S158P, F181L, N214S, S237A, E239Q, V362I, S367P, and L392I) were common to all 24 Irish BCoV genome assemblies. The N214S change is of interest, as it is in the receptor binding site (aa positions 211 to 214) of HE.
Data availability.
The complete genome sequences of the BCoV/IRE/GR/2022 variants have been deposited in GenBank under the accession numbers ON792941 to ON792964.
Contributor Information
Bernadette Earley, Email: bernadette.earley@teagasc.ie.
Simon Roux, DOE Joint Genome Institute.
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Associated Data
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Data Availability Statement
The complete genome sequences of the BCoV/IRE/GR/2022 variants have been deposited in GenBank under the accession numbers ON792941 to ON792964.